Experimental Biology and Medicine

Since its initial characterization in 1988, over 18,236 papers, including 2,485 reviews, have been published in the endothelin (ET) field. Over this period, several generations of selective and mixed (dual) ET receptor antagonists (ERAs), from peptidic backbones to orally active potent (subnanomolar) small molecular compounds, have been developed. These agents have been studied in many experimental animal models of various pathological conditions (cardiovascular, respiratory, and neuro-immunological). Continued basic research has led to a better understanding of the complex interactions between the ET axis and other biologic systems in human pathophysiology. The first clinical trial involved patients with idiopathic pulmonary arterial hypertension and led to approval of bosentan (Tracleer) for use in the United States and Europe in 2002. Since then, bosentan, the only currently approved dual (mixed) ERA, has been used in numerous other clinical trials. In addition, more selective ET_A receptor antagonists (ambrisentan, atrasentan, avosentan, clazosentan, darusentan, and sitaxsentan) are undergoing clinical trials. Here we outline the ERAs undergoing development and summarize the standing of completed and ongoing trials at the time of the Ninth International Conference on Endothelin and even thereafter. This review is intended to provide a useful reference for those interested in the current state of clinical trials involving ERAs, and to identify lessons that might apply to the design of future trials.

Circulating plasma endothelin (ET)-1 concentrations are substantially elevated, and correlate with the hemodynamic severity and New York Heart Association (NYHA) class, in patients with chronic heart failure (CHF). In early preclinical studies involving different models of experimental heart failure, ET antagonists reduced cardiac pressures, increased cardiac output, and prolonged survival. ET receptor antagonists also impressively improved systemic and pulmonary hemodynamics in patients with CHF, without causing neurohormonal activation. However, recent clinical trials, including the ENABLE (Endothelin Antagonist Bosentan for Lowering Cardiac Events in Heart Failure) and EARTH (Endothelin A Receptor Antagonist Trial in Heart Failure) studies, have shown neutral effects in terms of mortality and symptoms. This paper describes the possible reasons why benefit was not seen in these clinical studies, and suggests what lessons can be learnt from the way the studies were undertaken to apply to future studies.

Expression of the endothelin (ET)-1 gene is subject to complex regulation by numerous factors, among which the cytokine transforming growth factor-β (TGF-β) is one of the most important. TGF-β action is based on the activation of the Smad signaling pathway. Smad proteins activate transcription of the gene by cooperation with activator protein-1 (AP-1) at specific sites on the ET-1 promoter. Smad signaling pathway is initiated by binding of the cytokine to a heteromeric complex of type I and type II receptors. Signal is then propagated to the nucleus by specific members of the Smad family. Most cell types contain a type I receptor known as ALK5. However, endothelial cells are unique because they coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they actually activate different Smad-mediated expression programs that lead to specific endothelial phenotypes. TGF-β/ALK5/Smad3 pathway is associated to a mature endothelium because it leads to inhibition of cell migration/proliferation. Conversely, TGF-β/ALK1/Smad5 activates both processes and is more related to the angiogenic state. We have analyzed the TGF-β receptor subtype requirements for the activation of the ET-1 gene. For that purpose, we have overexpressed type I receptor and Smad isoforms in endothelial cells and analyzed the effect on ET-1 expression. Our experiments indicate that TGF-β induces ET-1 expression preferentially through the activation of the ALK5/Smad3 pathway and, therefore, the expression of the vaso-constrictor may be associated to a quiescent and mature endothelial phenotype.

Endothelin-1 (ET-1) is a potent endothelial-derived vasocon-strictor and cellular mitogen. Perturbations in ET-1 levels have been observed in a number of cardiovascular and renal disorders. Steady-state ET-1 mRNA expression is regulated in the vascular endothelium by an inducible promoter and a constitutively short mRNA half-life. Recent studies have identified mRNA stabilization as a pathophysiologically relevant mechanism of ET-1 induction in vascular endothelial cells. However, mechanistic studies on posttranscriptional pathways in physiologically relevant postconfluent primary endothelial cell monolayers have remained challenging because of endothelial resistance to DNA-based gene transfer and expression. To overcome these challenges, we developed an RNA transfection method to study ET-1 posttranscriptional regulation. Reporter transcripts transfected into either preconfluent or postconfluent primary endothelial cells were rapidly and robustly expressed. RNA transfection reconstituted poly(A)-tail–dependent protein expression and ET-1 3′-UTR–dependent mRNA destabilization, suggesting that the transfected RNA accessed endothelial cell posttranscriptional pathways. Because RNA transfection uncouples transcription from expression, the influence of the ET-1 3′-UTR on posttranscriptional expression kinetics could also be monitored. Taken together, our results suggest that RNA transfection is a versatile tool to investigate ET-1 posttranscriptional regulation in endothelial cell culture models.

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).

Endothelin-converting enzyme (ECE)-1 is a metalloenzyme with four subisoforms, which differ only in their amino-terminal domain. ECE-1a and c are the most common isoforms and are found at the plasma membrane and in the Golgi complex, whereas ECE-1b displays lysosomal localization. We have recently shown that ECE-1a but not ECE-1b also colocalizes with nuclear membrane markers, and that maintenance of cells in high glucose (25 mM) promotes relocalization of ECE-1a from the membrane to the intracellular compartment. To investigate the mechanisms involved in this process, we conducted a search for potential phosphorylation sites, which yielded a different number of putative sites for protein kinase (PK)-C and PKA in the amino-terminal region. Stimulation of Chinese hamster ovary (CHO) cells expressing a green fluorescent protein (GFP)-tagged human ECE-1a or ECE-1b with 100 nM phorbol myristate acetate (PMA) resulted in phosphorylation of ECE-1a, as determined by immunoprecipitation with an antibody to GFP followed by immunoblotting with an antibody to phosphoserine. Stimulation of cells with PMA also promoted intracellular relocalization, as seen in cells grown under high-glucose conditions. Incubation of cells grown in 25 mM glucose with the PKC inhibitor, calphostin C (100 nM), partially prevented the relocalization of ECE-1a from the plasma membrane to intracellular compartments. Stimulation of cells with 100 nM forskolin caused phosphorylation of ECE-1b and not ECE-1a, which is consistent with the lack of a putative PKA site in the ECE-1a amino-terminal sequence. Although phosphorylation is not required for ECE-1 enzymatic activity, these results suggest that ECE-1 isoforms are phosphorylated and that phosphorylation might play an important role in the regulation of intracellular trafficking of ECE-1 subisoforms.

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metalloprotease responsible for production of vasoactive endothelin (ET)-1 from inactive big ET-1. ECE-1 exists as four separate isoforms, ECE-1a, b, c, and d, which differ only in their amino-terminal regions. We investigated the expression and localization of the ECE-1 isoforms in primary human umbilical vein endothelial cells (HUVECs) and EAhy926 cells. Reverse transcriptase polymerase chain reaction showed expression of all four isoforms in both cell lines, with ECE-1d seeming, at least qualitatively, to be the predominant isoenzyme. Isoform-specific polyclonal antibodies were used to investigate isoform protein expression. ECE-1a, b, and c protein was detected in EAhy926 cells by immunoblotting; only ECE-1a and ECE-1c were detected in HUVECs. Using immunofluorescence microscopy analysis, both HUVEC and EAhy926 cells showed nuclear immunoreactivity with a monoclonal antibody recognizing all ECE-1 isoforms. The ECE-1a antibody also showed nuclear immunoreactivity in both cell lines; this seemed to colocalize with nucleolin. The ECE-1b antibody showed nuclear immunoreactivity in EAhy926 cells, but no overlap with nucleolin was seen. Intracellular immunoreactivity was seen in both cell lines using the ECE-1c antibody; this showed some colocalization with concanavalin A (an endoplasmic reticulum marker). von Willebrand Factor was used as a marker for Weibel-Palade bodies in HUVECs, but no colocalization with ECE-1 was seen during this study. The data presented here sheds new light on the localization of ECE-1a, b, and c in cultured human endothelial cells, which may further understanding of the ET system and aid design of therapeutic ECE inhibitors.

Endothelin-converting enzyme (ECE)-1 cleaves big endothelins, as well as bradykinin and β-amyloid peptide. Several isoforms of ECE-1 (ECE-1a, 1b, 1c, and 1d) have been identified to date, they differ only in their amino terminus and share the catalytic domain located in the C-terminal end. In addition to full-length ECE-1 forms, we identified novel, alternatively spliced messenger RNAs (mRNAs) of ECE-1b, 1c, and 1d. These splice variants (SVs) lack exon 3′, which codes for the transmembrane (TM) region and is present in full-length forms. SV mRNAs were highly expressed in endothelial cells (EC) derived from macrovascular and microvascular beds. Analyses of ECE-1d and its SV forms in stably transfected human embryonic kidney (HEK)-293 cells revealed that both proteins were recognized by antibodies to C-terminal ECE-1, but an antibody to the N-terminal only bound ECE-1d. The novel protein, designated ECE-1sv, has an apparent molecular weight of 75 kDa. ECE-1sv lacks the TM sequence (or signal peptide) and, therefore, is expected to remain cytosolic. Presence of ECE-1sv in different cellular compartments than the full-length forms of the ECE-1 may suggest a distinct physiologic role for these proteins.

Endothelins (ETs) and sarafotoxins (SRTXs) are active isopeptides that have very similar structures and functions. All isoforms interact with two specific G-protein–coupled receptors, ET_A and ET_B. To characterize functional vascular ET receptors in the poisonous snake, Bothrops jararaca, cumulative concentration-response curves to ETs and SRTXs were performed in isolated aortic rings, in the absence and presence of selective ET receptor antagonists. Vascular expression of ET receptor messenger RNA (mRNA) was evaluated by reverse transcriptase (RT) polymerase chain reaction (PCR) analysis, and a fragment of the ET_A receptor was cloned and sequenced. In vivo, ET-1 induced a dose-dependent biphasic response on anesthetized B. jararaca snakes. In vitro, ET-1, SRTX-b, ET-3, SRTX-c, and IRL-1620 induced concentration-dependent vasoconstriction, with a potency order suggesting the presence of typical ET_A receptors. BQ-123, a selective ET_A antagonist, inhibited contractions induced by ET-1 and SRTX-b with expected negative log of the dissociation constant, K_B, (pK_B) values for mixed ET_A/ET_B receptor populations. The nonselective ET_A/ET_B receptors antagonist, PD-142893, produced similar inhibition. The ET_B antagonist, IRL-1038, potentiated contractile responses to SRTX-c. ET-1 and SRTX-c responses were also potentiated when aortic rings were pretreated with N_ω-nitro-L-arginine methyl ester (L-NAME) plus indomethacin. Processing of the B. jararaca aortic first-strand complementary DNA, by RT-PCR with primers designed from the Gallus gallus ET_A receptor sequence, enabled isolation, purification, cloning, and sequencing of a single band. The partial sequence of the B. jararaca ET_A receptor showed a very high sequence similarity with ET_A receptor sequences from chicken, rat, human, and Xenopus. In conclusion, vascular responses to SRTXs/ETs in the B. jararaca aorta are mediated predominantly, but not exclusively, by typical ET_A receptors.

The endothelin (ET) receptor system has been shown to play a role in a number of vascular diseases. We have synthesized ¹⁸F-and ¹¹C-labeled radioligands to enable in vivo imaging of the fundamental processes involved in ET receptor pharmacology in normal and diseased tissue using positron emission tomography (PET). One aim is to elucidate the proposed role of the ET_B subtype as clearing receptor, removing ET-1 from the circulation, and whether this is an important mechanism to limit the detrimental effects caused by upregulated ET-1 in disease. To image ET_B receptors we have labeled the selective agonist BQ3020 with ¹⁸F. In vitro characterization verified that [¹⁸F]-BQ3020 bound with a single subnanomolar affinity (K_D == 0.34 ± 0.10 nM, B_max == 9.23 ± 3.70 fmol/mg protein) to human left ventricle. Binding of [¹⁸F]-BQ3020 to human kidney was inhibited by ET-1 and unlabeled BQ3020 but not by the ET_A selective antagonist FR139317, confirming that selectivity for the ET_B receptor was retained. In vitro autoradiography revealed, as expected, high levels of ET_B receptor densities in lung and kidney medulla, whereas kidney cortex and heart showed lower levels of ET_B receptor densities. Furthermore, a high level of [¹⁸F]-BQ3020 binding was found to colocalize to macrophages in atherosclerotic coronary arteries. MicroPET studies demonstrated high uptake of [¹⁸F]-BQ3020 in ET_B receptor–rich tissue, including lung, liver and kidney. The in vivo biodistribution of [¹⁸F]-BQ3020 was comparable to that previously obtained for [¹⁸F]-ET-1, supporting our hypothesis that the ET_B receptor plays a significant role in the uptake of ET-1. In conclusion, [¹⁸F]-BQ3020 has retained high affinity and selectivity, allowing imaging of ET_B receptor distributions in vitro and in vivo in human and animal tissue. Furthermore, in vitro data suggest that [¹⁸F]-BQ3020 potentially can be used to image atherosclerotic lesions in vivo using PET.

We have previously shown that in homozygous endothelin (ET)_B^{− /−} deficient mice, ET_A receptor density is significantly downregulated in the brain by 45%. In these mice, plasma ET-1 levels are elevated. Our aim was to use quantitative autoradiography to establish the distribution of ET receptor subtypes in peripheral tissues from wild-type mice and to measure the density of the ET_A subtype in ET_B^{− /−} knockout animals. Our second aim was to test whether deletion of ET_B receptors, which is associated with elevated plasma levels of ET-1, would also reduce ET_A expression in the periphery. In longitudinal sections from wild-type mice, the highest densities of ET_A receptors localized to major organs including the ventricle of the heart, lung, and liver parenchyma. High densities of ET_A receptors were detected in the smooth muscle layer of the vasculature such as intrarenal vessels as well as the smooth muscle layer and epithelial cells of the gastrointestinal tract. In these tissues, the ET_A subtype was more abundant, representing between 60% and 100% of the ET receptors. ET_B receptors predominated in the medulla of kidney, with high densities also localizing to glomeruli within the cortex and to the sinusoids from the liver. Lower densities of ET_B receptors were also present in the lung, heart, liver, and the smooth muscle layer of the gastrointestinal tract. In ET_B^{− /−} knockout mice, ET_B receptors were not detected as expected by either ligand binding or immunocytochemistry. The pattern of ET_A receptor distribution in the ET_B^{− /−} knockout mice was similar to the controls, but the density of ET_A receptors was significantly reduced in the lung by 39%. Diminished responses to the endogenous agonist after repeated stimulation are an important feature of G-protein signaling, preventing potential damage to the overstimulated cell, and it is likely that downregulation occurs in response to higher circulating levels of ET-1.

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ET_A and ET_B receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ET_A and ET_B receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ET_A receptors, 83 ± 2% of these receptors colocalized with pan cadherin. In ET_B receptor–transfected cells, 54 ± 2% of the receptor colocalized with pan cadherin. When ET_A and ET_B receptors were cotransfected, 97 ± 1% of ET_B receptors colocalized with ET_A receptors and 84 ± 2% of ET_B receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ET_B receptor agonist) increased [Ca²⁺]_i in cells transfected with ET_A or ET_B receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ET_B receptors desensitized faster (t_1/2 = 21 ± 1 sec) than ET_A receptors (t_1/2 = 48 ± 1 sec). S6c-induced increases in [Ca²⁺]_i desensitized in cells expressing ET_B receptors only (t_1/2 = 17 ± 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ET_A receptors localize to the cell membrane, whereas ET_B receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ET_B receptors desensitize faster than ET_A receptors, but receptor coexpression eliminates desensitization. Finally, ET_A and ET_B receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.

Endothelin-1 (ET-1) effects in human glomerular mesangial cells (GMC) include proliferation, contraction, and extracellular matrix synthesis. Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human glomerulae. Working in concert with Pyk2, adaptor protein CrkII and a recently discovered guanidine exchange factor for certain small GTPases BCAR3 can be involved in ET-1 signaling in the kidney. Signaling through CrkII and BCAR3 might be critical in some proliferative kidney pathologies. The current study was designed to determine the possibility of CrkII and BCAR3 interaction in response to ET-1 in human GMC and the role of Pyk2 in the association of these proteins. Using adenovirus-mediated transfer of genes encoding either green fluorescent protein (control) or dominant interfering Pyk2 construct, we demonstrated that CrkII and BCAR3 can be coprecipitated from unstimulated and ET-1 stimulated GMC; ET-1 treatment time-dependently increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to a significant decrease in CrkII/BCAR3 association both basal and stimulated.

Endothelin-1 (ET-1) acts on two different G protein–coupled receptors, namely the endothelin A (ET_A) and the endothelin B (ET_B) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ET_A and ET_B receptors to stimulate extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, we analyzed the role of the extracellular N terminus for ERK1/2 activation, because the ET_B receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ET_A receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ET_B receptor showed a biphasic ERK1/2 activation. A truncated mutant ET_B receptor, lacking the proteolytically cleaved N terminus (Δ2-64 ET_B) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 Δ2-64 ET_B cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ET_B receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ET_B receptor in which the N terminus was replaced by the N terminus of the ET_A receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ET_B receptor is necessary for the second phase of ERK1/2 activation. However, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation.

β₁Pix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor (GEF) for the Rho family small G protein Cdc42/Rac. On stimulation with extracellular signals, GEFs induce the exchange of guanosine diphosphate to guanosine triphosphate, resulting in the activation of the small guanosine 5C-triphosphatases. This activation enables the signal to propagate to downstream effectors. Herein, we show that G_sα stimulation by cholera toxin increased Cdc42 activation by endothelin-1 (ET-1), whereas pertussis toxin had no effect. H-89, a protein kinase A (PKA) inhibitor, strongly inhibited Cdc42 activation by ET-1. Moreover, the overexpression of β₁Pix enhanced ET-1–induced Cdc42 activation. The essential role of β₁Pix in ET-1–induced Cdc42 activation was evidenced by the blocking of Cdc42 activation in cells expressing β₁Pix mutant lacking the ability to bind PAK (β₁Pix SH3m[W43K]) or mutant lacking GEF activity (β₁PixΔDH). The overexpression of mutant lacking the pleckstrin homology domain β₁PixΔPH, which is unable to bind phospholipids, had no effect on Cdc42 activation. These results demonstrate that β₁Pix, along with PKA, plays a crucial role in the regulation of Cdc42 activation by ET-1.

Low-density lipoproteins (LDLs) represent the most important treatable risk factors for coronary artery disease. Although it has been previously shown that hypercholesterolemia stimulates the endothelin system, the effects of increased levels of LDL on endothelial endothelin receptors have not been previously studied. In particular, the influence of native and oxidatively modified LDLs (nLDLs and oxLDLs) and the regulatory mechanisms in endothelial cells are currently unknown. Human endothelial cells almost exclusively express the endothelin receptor type B (ET_B). Therefore, the effect of nLDL and oxLDL on the expression of ET_B was studied in primary cultures of human umbilical vein endothelial cells (HUVEC). HUVEC were stimulated by nLDL and oxLDL in a time-dependent (1–12 hrs) and dose-dependent (25–100 μg/ml) manner. To analyze signal transduction pathways involved in the regulation of ET_B, protein kinase C (PKC) was inhibited using 100 nM Ro-31-8220. The mRNA expression of ET_B was determined by quantitative reverse transcription–polymerase chain reaction and ET_B protein expression by Western blot. Native LDL induced ET_B mRNA after 1 hr (100 μg/ml, 199 ± 35%, n = 15, P < 0.05 vs. control). Stimulation of HUVEC with oxLDL increased ET_B mRNA expression (1 hr, 100 μg/ml oxLDL: 308 ± 48%, n = 15, P < 0.05 vs. control) as well. Induction of ET_B was also found on the protein level. nLDL was even more potent than oxLDL in inducing ET_B protein expression. Induction of ET_B expression by oxLDL is mediated by PKC. These data demonstrate that low-density lipoproteins even independent of oxidative modification are potent inducers of ET_B receptors at the mRNA and protein level in HUVEC. Given the nitric oxide-releasing capacity of endothelial ET_B receptors, this effect may represent a possible vasoprotective mechanism.

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-α–stimulated ET-1 release from PAECs in a dose-dependent manner (1–10 μM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 μM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1–related diseases.

Although endothelin (ET)-1 is one of the strongest known vasoconstrictors in most species, we and others have previously found that it is only weakly effective in the mouse aorta. The aim of this study was to further investigate vasoactive effects of ET-1 in vascular beds generally known to be particularly sensitive to ET-1, such as the renal artery. Experiments were performed to determine the vasoconstrictor responses in the thoracic aorta, and in the carotid, femoral, and renal arteries. Isolated vascular rings of C57BL/6 adult male mice (35–40 weeks of age) were exposed to ET-1 (0.01–300 nM) in the presence of the nitric oxide synthase inhibitor l-NAME (0.3 mM) to exclude effects of nitric oxide. Vessels from different vascular beds demonstrated distinct patterns in potency of the contractions to ET-1 and the dynamics of the responses. The maximal contraction to ET-1 was strong and significantly greater in the femoral (105 ± 7% KCl) and renal artery (62 ± 7% KCl) than in the carotid artery or the aorta (P < 0.05). The dynamics of the contractile response to ET-1 varied between the different vessels: the renal artery showed a rapid vasoconstriction, followed by a near complete loss of tension, whereas in the aorta, carotid, and femoral artery, vasoconstriction was more sustained. In conclusion, the data demonstrate that mouse femoral and renal arteries exhibit strong contractions in response to ET-1 compared with aorta and carotid artery, and that contractile dynamics differ markedly between arterial vascular beds. These findings may be important for studying the effects of endothelin in mouse models of human disease.

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin (ACTH), α-melanocyte–stimulating hormone (MSH), and the endogenous opioid, β-endorphin (EP). ACTH-dependent Cushing’s syndrome is characterized by ACTH overproduction and is associated with an increased risk of cardiovascular disease. Endothelial dysfunction has been recognized as an early marker of cardiovascular disease. However, the mechanism underlying endothelial dysfunction by ACTH overexpression in Cushing’s patients remains elusive. Endothelial cells, the primary cells producing endothelin (ET)-1, are both the source and target of POMC-derived peptides. In the present study, we generated adenovirus vectors (Ad) encoding POMC (Ad-POMC) and green fluorescent protein (GFP; Ad-GFP) to investigate whether POMC gene transfer altered the ET-1 homeostasis and angiogenic functions in human EA.hy926 endothelial cells. Via adenovirus gene delivery, the POMC-transduced EA.hy926 cells released significantly elevated ACTH and β-EP levels (P < 0.001). In addition, POMC gene delivery significantly decreased the ET-1 release (P < 0.001) without affecting the ET-1 messenger RNA (mRNA) level. Despite no effect on the secretion of matrix metalloproteinases (MMPs) and cell proliferation, POMC gene delivery significantly inhibited the migration (P < 0.01) and tube-forming capability (P < 0.01) of endothelial cells. Moreover, the POMC-induced inhibition of tube formation could be partially reversed by adding exogenous ET-1 (P < 0.05). In summary, the attenuated ET-1 release and angiogenic processes by POMC overexpression may contribute to endothelial dysfunction, thereby providing a link between Cushing’s syndrome and cardiovascular diseases.

Strength exercise training induces a decrease in arterial distensibility, whereas endurance exercise training causes an increase in arterial distensibility. Endothelin-1 (ET-1), which is produced by vascular endothelial cells, has potent vasoconstrictor and proliferative activity on vascular smooth muscle cells. We hypothesized that endogenous ET-1 participates in alteration of arterial distensibility by different exercise training types (i.e., strength and endurance exercise training). The purpose of the present study was to investigate plasma ET-1 concentration and arterial distensibility in strength- and endurance-trained athletes. Subjects were male strength-trained athletes (discus, hammer, or javelin throwers; 22.2 years; SA), male endurance-trained athletes (long- or middle-distance runners; 20.7 years; EA), and sedentary healthy men (20.6 years; sedentary control, SC). Maximum hand-grip strength was markedly greater in SA compared with EA and SC (55.3 vs. 41.1 vs. 40.5 kg, P < 0.05). Maximum oxygen uptake was markedly greater in EA than in SA and SC (60.9 vs. 43.1 vs. 43.6 ml/kg/min, P < 0.05). Arterial pulse wave velocity (PWV), which is an index of arterial distensibility, was significantly higher in SA than in EA and SC (688 vs. 529 vs. 601 cm/sec, P < 0.05). In EA, PWV was significantly lower in comparison to that in SC (P < 0.05). Thus arterial distensibility was lower in SA than in EA and SC and higher in EA than in SC. Plasma ET-1 concentration was significantly higher in SA compared with EA and SC (1.64 vs. 1.12 vs. 1.24 pg/ml, P < 0.05). Plasma ET-1 concentration tended to be lower in EA than in SC. These results suggest that the difference in plasma ET-1 level may participate in the mechanism underlying different adaptation of arterial distensibility between strength- and endurance-trained athletes.

Endothelin-converting enzyme (ECE-1) is a critical enzyme in the production of the potent vasoconstrictor peptide endothelin (ET-1). It has previously been shown that the levels of both ET-1 and ECE-1 are raised in atherosclerosis, but the possible relevance of the isoforms of ECE-1 in these changes has not yet been investigated. The aim of this study was to examine the expression of the ECE-1a and ECE-1c isoforms in human atherosclerotic pathologies. Immunohistochemical analysis was carried out on sections from atherosclerotic and non-atherosclerotic vascular tissue using a combination of ECE-1 isoform-specific antibodies, anti–α-actin antibodies to identify smooth muscle cells (SMC) and anti-CD68 antibodies to identify macrophages. ECE-1 isoform expression was also examined in cultured SMC and in macrophages isolated from human blood. Results indicated differences in isoform expression in athero-sclerotic lesions, with distinct patterns of staining for ECE-1a and ECE-1c. ECE-1c immunoreactivity was seen in macrophages, and also correlated with actin staining. ECE-1a was also localized to macrophages and SMC. Results of this study suggest that these local changes influence the expression patterns of the ECE-1 isoforms within individual cell types. Correlation of these isoform expression patterns with the stage of atherosclerosis could provide novel indicators of disease progression.

Elevated plasma and tissue endothelin (ET)-1 levels in patients with critical limb ischemia (CLI) has been described. Here the effect of a period of acute ischemia and subsequent reperfusion on plasma ET-1 and tissue ET-1/ET receptors in skeletal muscle biopsies from CLI patients undergoing femoro-distal bypass surgery was studied. Peripheral and “local” blood and muscle biopsies were obtained from patients undergoing femoro-distal bypass surgery, at the start of the procedure (control), after a period of vascular clamping (ischemia), and after clamp release (reperfusion). Plasma ET-1 was determined by enzyme-linked immunosorbent assay. Tissue ET-1 was assessed by counting ET-1 immunostaining cells per unit area, and ET_A/ET_B receptors were identified on sections by in vitro autoradiography in which binding was quantitatively assessed by densitometry. There was no significant effect of ischemia or reperfusion on plasma ET-1 levels or on ET_A/ET_B receptor binding. However, tissue ET-1 increased during both acute ischemia and reperfusion (P < 0.05). A high proportion of positive ET-1 immunostaining was associated with microvessels and also exhibited a similar distribution to macrophages. Previously, it has been shown that both plasma ET-1 and tissue ET-1/ET receptors are increased in CLI patients compared with atherosclerotic controls. Also, increased muscle ET-1 levels have been described in acute ischemia caused by tourniquet application in nonischemic patients undergoing total knee replacement. In CLI patients, in whom ET-1 is already upregulated, this further increase may exacerbate existing pathologic processes and contribute to ischemia-reperfusion injury. ET-1 antagonists may therefore be useful adjuncts in CLI and other surgical procedures in which ischemia-reperfusion damage occurs.

We have discovered that endothelin-1 (ET-1) vasoconstriction is significantly enhanced in aortas of young (8–16-week-old) apolipoprotein E–deficient (ApoE^−/−) mice devoid of atherosclerotic lesions (maximum response expressed as a percentage of the mean response to 100 mM KCl (E_MAX) = 55.7% ± 19.5% KCl, n = 5) compared to age-matched C57BL/6/J control animals (E_MAX = 12.6% ± 2.5% KCl, n = 8), indicating that alterations in the endothelin system may contribute to disease progression, at least in this animal model. There was no difference in the potency of ET-1 to contract aorta from the two groups (C57BL/6/J pD₂ = 8.74 ± 0.30; ApoE^−/− pD₂ = 8.50 ± 0.15, P > 0.05). This increased response was specific to ET-1, as it was not observed with phenylephrine or U46619, nor was it due to a non–receptor mediated increase in contractile sensitivity, as there was no change in response to KCl between the two groups. [¹²⁵I]ET-1 bound with subnanomolar affinity (K_D) to aorta (K_D == 0.018 ± 0.002 nM, n == 4) and, with an order of magnitude lower affinity, to heart (K_D == 0.47 ± 0.05, n == 5) of C57BL/6/J mice with binding densities (B_MAX) of 9.3 ± 2.4 fmol mg⁻¹protein and 100 ± 14 fmol mg⁻¹ protein, respectively. Alterations in vascular reactivity to ET-1 could not be explained by increased endothelin receptor density or affinity, as these were not altered in aorta (K_D == 0.011 ± 0.003 nM; B_MAX == 10.1 ± 3.9 fmol mg⁻¹, n == 4) and heart (K_D == 0.43 ± 0.04 nM; B_MAX == 115 ± 26 fmol mg⁻¹, n == 6) of ApoE^−/− animals. The ratio of ET_A to ET_B receptors in heart of control and ApoE^−/− mice was similar, comprising 89% and 85% ET_A receptors, respectively. In isolated aorta from ApoE^−/− mice on the Western diet, which more closely resembled more advanced stages of the disease in man, the augmented ET-1 vasoconstrictor response was maintained (E_MAX == 25.2% ± 6.8% KCl, n == 9); however, it was completely prevented in animals that had received 10 weeks of oral atorvastatin (30 mg kg⁻¹ day⁻¹) (E_MAX == 4.0% ± 1.5% KCl, n == 5), a concentration that was chosen because it did not affect plasma cholesterol and triglyceride levels. Therefore, this protective prevention of enhanced ET-1 vasoconstriction in ApoE^−/− mice by atorvastatin was independent of its lipid-lowering properties.

Vascular endothelin (ET)-1 is upregulated in several forms of salt-induced hypertension. It is unclear to what extent these effects are primary or secondary to endothelial damage. We hypothesized that a high-sodium diet (HNa) increases vascular ET-1 production independent of arterial blood pressure changes. We investigated the effect of chronic HNa with and without ET_A blockade on circulating and aortic ET-1 protein levels as well as aortic expression of ET-1 and ET _A messenger RNA (mRNA) in inbred Wistar-Kyoto (WKY) and congenic ET_B-deficient rats. Comparing WKY rats fed a low-sodium diet (LNa) with those fed HNa for 3 weeks, aortic wall ET-1 protein is significantly increased in response to HNa (331 ± 43 pg/g tissue for LNa vs. 557 ± 34 pg/gm tissue for HNa). HNa also increased aortic wall ET-1 mRNA levels by 40%, as determined by quantitative reverse transcriptase polymerase chain reaction. We then compared rats chronically treated with the ET_A-selective antagonist, ABT-627, while receiving either LNa or HNa. There were no differences in arterial blood pressure (mean arterial pressure 89 ± 1 mm Hg for WKY on LNa; 90 ± 3 for WKY on HNa; 91 ± 2 for ET_B-deficient/ABT-627–treated on HNa) or heart rate. However, aortic wall ET-1 protein levels were 4-fold higher in the HNa group. Further, HNa increased aortic wall ET-1 mRNA (~1.5- to 3-fold) and ET _A mRNA (~2- to 7-fold), independent of activation of ET_B. Therefore, the expression of ET-1 mRNA by the aortic wall is increased in response to chronic high dietary sodium in WKY rats in the absence of changes in arterial blood pressure.

Evidence for endothelin (ET) involvement in the control of fluid volume balance and arterial pressure has been derived in part from the observations that rats lacking the ET_B receptor develop hypertension when placed on a high-salt (HS) diet. The present study was designed to determine the effect of superoxide on salt-induced hypertension in male and female ET_B-deficient (sl/sl) and wild-type control (wt) rats. After 14 days on a HS (8% NaCl) diet, female sl/sl rats had significantly elevated arterial pressure (183 ± 2 mm Hg, tail cuff) compared with female wt rats (134 ± 2 mm Hg). The response to a HS diet was lower in male sl/sl rats (166 ± 6 mm Hg) yet was significantly greater than that in male wt controls (135 ± 3 mm Hg). Separate groups of male and female sl/sl and wt rats were given tempol (1 mM in drinking water) during HS treatment. Arterial pressures were 149 ± 5 mm Hg in male and 143 ± 3 mm Hg in female sl/sl rats treated with tempol, values that were similar to those of controls on a normal salt diet. After 14 days, however, male and female sl/ sl rats recovered from the blood pressure–lowering effects of tempol. On Day 15, arterial pressures in female sl/sl rats on a HS diet were 160 ± 6 mm Hg and 177 ± 6 mm Hg in tempol-treated and untreated groups, respectively. In male sl/sl rats, arterial pressures were 155 ± 3 mm Hg and 165 ± 5 mm Hg in tempol-treated and untreated groups, respectively. On Day 15, no differences among groups with or without tempol were observed in plasma thiobarbituric acid–reactive substance (TBARS) concentrations or in urinary excretion of TBARS. Plasma ET-1 concentrations were significantly higher in female vs. male sl/sl rats. These results indicate that the early stages of salt-dependent hypertension produced by ET_B receptor deficiency are dependent on superoxide and that the elevated pressure in the female rats may be due to elevated circulating levels of ET-1.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin (ET) converting enzyme-1 (ECE-1), with respective IC₅₀ values of 22, 2, and 55 nM. The aim of the present study was to establish the hemodynamic profile of Zucker diabetic fatty (Zdf)-Fatty rats, a high-fat diet gene-prone model developing spontaneous Type 2 diabetes (T2D) and the effects of CGS 35601. Male Zdf-Fatty (14 weeks, n = 17–23), Zdf-Lean (14 weeks, n = 8–10), and Wistar (14 weeks, n = 9–10) rats on distinct diets were implanted with a catheter in the left carotid and placed individually in a metabolic cage for 30 days. The hemodynamic profile and some metabolic biomarkers were assessed daily. After a 7-day stabilization period, the Zdf-Fatty rats were divided into two groups: Group 1, controls (n = 7–10) receiving vehicle-saline (250 μl/hr) and Group 2, (n = 10–13) receiving increasing doses of CGS 35601 (0.1, 1, and 5 mg/kg/day × 6 days each, intra-arterially) followed by a 5-day washout period. Mean arterial blood pressure (MABP) of young Zdf-Fatty rats was compared with age-matched Zdf-Lean and Wistar rats, which were found similar. MABP decreased by 5.9% (from baseline at 102 ± 5 to 96 ± 4 mmHg), 12.7% (to 89 ± 6 mmHg) and 21.6% (to 80 ± 4 mmHg), at 0.1, 1, and 5 mg/kg/day, respectively, in CGS 35601-treated Zdf-Fatty rats. Systolic and diastolic blood pressures were similarly reduced. The heart rate was not affected. Hyperglycemic status and insulin-resistance were not modulated by short-term treatment. CGS 35601 presented an excellent short-term safety profile. This novel molecule and class of VPI may be of interest for lowering vascular tone. Further long-term studies, once cardiovascular and renal complications have developed in this T2D rat model are warranted to define the efficacy of this class of VPI.

We previously reported that CGS 35601, a potent triple inhibitor of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme 1, completely normalized mean arterial blood pressure (MABP) in 36-week-old spontaneously hypertensive rats, a normal renin model. The aim of the present study was to determine the effects of this triple vasopeptidase inhibitor (VPI) on the hemodynamic profile of instrumented, conscious, and unrestrained Dahl salt-sensitive (DSS) rats, a gene-prone, high-salt diet–induced low-renin hypertension model. Male DSS rats (mean weight [±SEM], 385 ± 10 g) were fed a normal diet (Group 1) or a high-salt diet (Groups 2 and 3; 8% NaCl in food) for 6 weeks and then instrumented with a carotid catheter and placed individually in metabolic cages for 30 days. The hemodynamic, hematological, and biochemical profiles were assessed daily. Dose-dependent treatment started after a 7-day stabilization period in Groups 1 and 2 (vehicle dosage, 250 μl/hr) and Group 3 (CGS 35601 dosages of 0.1, 1, and 5 mg/kg/day for 6 days per dose by means of constant intra-arterial infusion), followed by a 5-day washout period. Two additional groups included normotensive Wistar rats (Group 4) and DSS rats that received a double high-salt solid (8% NaCl) and liquid (1% NaCl) diet (Group 5).The MABP in rats receiving CGS 35601 decreased in a dose-dependent fashion toward the baseline level observed in DSS rats receiving a normal diet. The heart rate was unaffected. The hemodynamic profile returned to normal during the washout period. This novel triple VPI is a potent and effective antihypertensive agent with a safe short-term profile that may be of interest for treating hypertension and other cardiovascular diseases. Other hypertensive rat models are being tested.

The objective of this study was to investigate whether circulatory and hormonal changes during xenon plus remifentanil or isoflurane plus remifentanil anesthesia are altered by endothelin-A (ET_A) receptor blockade. Eight beagle dogs were studied in four protocols (n = 7 each). After a 30-min awake period, anesthesia was induced with 8 mg/kg propofol, administered intravenously (iv), and maintained with either 0.8% ± 0.01% (vol/vol) isoflurane plus 0.5 μg/kg/min remifentanil (Protocol 1) or 63% ± 1% (vol/vol) xenon plus 0.5 μg/kg/min remifentanil (Protocol 2) for 1 hr. Protocols 3 and 4 were preceded by ET_A blockade with ABT-627 (Atrasentan; iv bolus of 1 mg/kg, then 100 μg/kg/h continuously). Irrespective of Atrasentan administration, the mean arterial blood pressure (MAP) ranged between 92 and 96 mm Hg in the awake state and fell to 67 ± 3 mm Hg in controls (mean ± SEM) and to 64 ± 2 mm Hg in the Atrasentan group during isoflurane plus remifentanil anesthesia, whereas MAP remained constant during xenon plus remifentanil anesthesia. A decrease in heart rate was observed during either kind of anesthesia, but bradycardia was most prominent during xenon plus remifentanil anesthesia. In the control groups, and in the Atrasentan-treated dogs, a decrease in cardiac output and an increase in systemic vascular resistance were more prominent during xenon plus remifentanil than during isoflurane plus remifentanil anesthesia. Hormonal alterations during anesthesia remained unaffected by ET_A receptor blockade. Angiotensin II and vasopressin increased in all protocols, and adrenaline and noradrenaline concentrations rose only during xenon plus remifentanil anesthesia. We conclude that the hemodynamic and hormonal adaptation after xenon plus remifentanil and isoflurane plus remifentanil anesthesia does not depend on the endothelin system, because it is unaffected by ET_A receptor inhibition. Therefore, the use of Atrasentan does not impair cardiovascular stability during xenon- or isoflurane-based anesthesia in our dog model. However, the way anesthesia is performed is of crucial importance for hemodynamic and hormonal reactions observed during research in animals because the release of vasopressin and catecholamines may be intensified by xenon plus remifentanil anesthesia.

Endothelin (ET) levels are elevated in congestive heart failure secondary to myocardial infarction (MI) and correlate well with the severity of pulmonary hypertension (PH), suggesting that the ET peptide could contribute to the pathophysiology of venous PH. Alterations of pulmonary vasoreactivity to ET after MI and the respective roles of the ET_A and ET_B receptors (ET_A-R and ET_B-R) have never been evaluated, to our knowledge. MI was induced in rats. Three weeks later, small pulmonary resistance arteries were mounted on a microvascular myograph. Cumulative concentration-response curves to ET-1 and sarafo-toxin 6c (S6c) were performed. Response to ET was also assessed in the presence of ET-R antagonists. Heterodimeriza-tion of receptors was evaluated by immunoprecipitation of the ET_B-R, followed by western blotting for the expression of the ET_A-R. Maximal vasoconstriction and sensitivity to ET-1 were similar in sham and MI with values of 88 ± 3.9% and 80 ± 3.8%, respectively. The response to S6c was similarly less in both sham (67 ± 5.7%) and MI groups (60 ± 6.6%). When administered alone, the ET_A-R antagonist (10 nM A-147627.1) and the ET_B-R antagonist (1 μM A-192621.1) had no significant effect. However, their combination markedly reduced vaso-constriction (52 ± 5.3%; P < 0.001). The endothelial and medial distribution of ET-Rs was similar in sham and MI groups. In vitro studies demonstrated co-immunoprecipitation of the ET_A-R and ET_B-R. Vasoconstriction of isolated resistance pulmonary arteries to ET agonists is not altered after MI. Dual antagonism results in optimal blockade of vasoconstriction, possibly because the ET_A-R and ET_B-R can form functional heterodimers.

Angiotensin (AT) II, endothelin (ET)-1, and atrial natriuretic peptide (ANP) play an important role in cardiovascular regulatory processes under physiologic and pathophysiologic conditions. All of these agents are present in the pericardial fluid, and alteration of their pericardial concentrations mirror changes in the myocardial interstitium. Moreover, the composition the pericardial fluid may also reflect the myocardial interaction of these agents. The local myocardial effects of AT II on cardiac ET-1 and ANP production, as well as on cardiovascular function, was studied by intrapericardial (ip) administration of AT II (0.125–1.0 μg/kg) to the in situ dog heart (n = 8). Big ET, ET-1, and ANP [1–28] fragment concentrations were measured by enzyme-linked immunosorbent assay in pericardial infusate samples and in peripheral blood before and after an AT II treatment of 15 mins. Systemic blood pressure (BP), heart rate (HR), and left ventricular contractility (dP/dt) were also recorded. In our studies, the pericardial big ET (but not ET-1) concentration was increased to a maximum value of 139 ± 28 versus 74 ± 12 pg/ml (control; P < 0.02) with ip AT II administration, with parallel elevations of the pericardial ANP levels (36.8 ± 7.2 vs. 24.4 ± 3.6 ng/ml; P < 0.05). The ip administration of AT II did not influence HR, and it elicited moderate changes in BP (BP_max, +14 ± 2 mm Hg, P < 0.001; dP/dt_max, +10 ± 3%, P < 0.02). The plasma levels of big ET, ET-1, and ANP did not change significantly. The results suggest that AT II promotes production of big ET and ANP in the heart. However, no detectable conversion of big ET-1 to ET-1 was observed within 15 mins. The myocardial formation of big ET-1 and ANP occurred, at least in part, independently of the changes in cardiovascular function.

Radiofrequency catheter ablation or modification of the atrio-ventricular junction is an effective therapy of drug refractory supraventricular tachyarrhythmias (ST). Higher endothelin (ET) levels were observed during nonsustained STs. We aimed to examine the effect of sustained STs and the applied rate-control therapy on plasma ET levels. Twenty-two patients (12 men; mean age, 64.4 ± 13.2 years; ejection fraction, 41.8 ± 11.2%; New York Heart Association (NYHA) class I: 3 cases, NYHA II: 11 cases, and NYHA III: 8 cases) suffering of atrial fibrillation (n = 11), atrial flutter (n = 7), atrial paroxysmal tachycardia (n = 3), or sinus tachycardia (n = 1) were studied, having coronary artery disease (n = 8), dilative cardiomyopathy (n = 5), or no underlying diseases (n = 9). All groups went under catheter ablation (same protocol, duration: 35 ± 10.3 mins; rate before ablation, 100–170/min in every case; after ablation, 70–80/min in Groups I and II and 70–90/min in Group III). A pacemaker (PM) was implanted 2 months before ablation in Group I (n = 9) and during ablation in Group II (n = 7). No PM was implanted in Group III (n = 6). A control group (n = 13; 7 men; mean age, 66.15 ± 6.7 years) with sinus rhythm got a PM without ST and ablation. Blood samples were collected from the cubital vein immediately before (control), and 5 mins and 24 hrs after ablation. Plasma ET-1 and big ET-1 levels were measured after immunoprecipitation with Western blot analysis. There were no differences between plasma ET-1 levels in the ST groups and the control group (Groups I, II, and III vs. control group: 0.66 ± 0.04 fmol/ml, 0.93 ± 0.12 fmol/ml, and 0.68 ± 0.05 fmol/ml vs. 0.50 ± 0.05 fmol/ml, respectively; P < 0.05). Comparing the control, 5-min, and 24-hr samples, ET-1 levels decreased significantly after supraventricular tachycardia ablation in Groups I and III (control vs. Group I, 5 mins and 24 hrs: 0.66 ± 0.04 fmol/ml vs. 0.50 ± 0.04 fmol/ml and 0.29 ± 0.05 fmol/ml; control vs. Group III, 24 hrs: 0.68 ± 0.05 vs. 0.34 ± 0.05 fmol/ml; P < 0.05). No plasma big ET-1 changes were measured in any of the groups. The rapid decrease of ET levels after catheter ablation suggests that a high ventricular rate can be a trigger of ET production. PM implantation procedure seems to interfere with the ET decrease in ST patients.

Although experimental prevention studies have suggested therapeutic potential of endothelin (ET) antagonists for the treatment of heart failure, the results of clinical trials using ET antagonists on top of standard heart failure medications have been largely disappointing. This experimental study investigated the effects of chronic ET_A receptor blockade in long-term survivors of myocardial infarction who had developed stable chronic heart failure in the absence of other treatments. Systolic blood pressure, heart rate, organ weights of the right atrium and ventricle, and the lungs were determined, and tissue ET-1 peptide levels were measured in cardiac tissue, lung, and aorta. The results show that chronic blockade of ET_A receptors stabilizes systolic blood pressure and reverses the heart failure–induced weight increases of right heart chambers and lung. The changes observed occurred independently of tissue ET-1 concentrations and heart rate, suggesting mechanisms independent of local cardiac or pulmonary ET-1 synthesis, which are yet to be identified.

Left ventricular assist device (LVAD) implantation and heart transplantation (HTx) are established therapeutic approaches in the treatment of end-stage heart failure. The postoperative humoral responses to the two treatments have not yet been compared. All patients were treated with inhaled nitric oxide (iNO) on weaning from cardiopulmonary bypass as they presented with pulmonary hypertension. We investigated atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cGMP, endothelin (ET)-1, big endothelin (big ET), and hemodynamic parameters after LVAD implantation (15 patients; age 51 ± 8 years) or HTx (10 patients; age 53 ± 6 years) preoperatively, on cardiopulmonary bypass and postoperatively up to 72 hrs after cessation of iNO. Preoperatively, cardiac index (CI), pulmonary artery pressure, pulmonary capillary wedge pressure (PCWP), central venous pressure (CVP), and mean atrial pressure (MAP) were similar for both groups. Similarly, ANP, BNP, cGMP, ET-1, and big ET were comparable before surgery. Seventy-two hours after weaning from iNO, the administered epinephrine dose was higher in the HTx group (P = 0.003); whereas the CVP (P = 0.04) and pulmonary vascular resistance (PVR; P = 0.03) were lower. The following humoral parameters differed markedly: ANP (preoperatively: LVAD, 99 ± 123 pg/ml; HTx, 197 ± 199 pg/ml; P = 0.14; vs. 72 hrs after iNO: LVAD, 110 ± 106 pg/ml; HTx, > 640 ± 0 pg/ml; P = 0.003) and cGMP (preoperatively: LVAD, 4.4 ± 5.8 pg/ml; HTx, 5.0 ± 3.0 pg/ml; P = 0.35; vs. 72 hrs after iNO: LVAD, 8.0 ± 10.8 pg/ml; HTx, 26.2 ± 15.8 pg/ml; P = 0.02). Although the hemodynamic effects of both LVAD implantation and HTx in the treatment of end-stage heart failure are comparable, except for the effects on CVP and PVR, the humoral responses with respect to ANP and cGMP were strikingly different. These effects are independent of volume status, iNO, and ETs, suggesting a physiologic response to maintain circulatory homeostasis.

It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH_i) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH_i changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH_i by 0.05 ± 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1–mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH_i responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)–fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-ε-YFP–expressing myocytes, the ET-1–mediated positive inotropic response was greatly diminished to 13 ± 15%, but alkalinization was still observed. Expression of dn-PKC-δ-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-δ and PKC-ε caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH_i in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-ε by mechanisms other than intracellular alkalinization.

Endothelin (ET)-1 is produced by endothelial cells and cardiac myocytes. ET-1 has positive inotropic and chronotropic effects on the heart and causes myocardial cell hypertrophy. Exercise training induces a physiologic cardiac hypertrophy. To study whether myocardial ET-1 is involved in the formation of exercise training–induced cardiac hypertrophy, we investigated time-course alterations of myocardial ET-1 gene expression and ET-1 peptide level in the heart of rats during a formative process of exercise training–induced cardiac hypertrophy. We used the hearts of rats that had been exercise-trained for 4 weeks (4WT) or 8 weeks (8WT) and sedentary control rats for 4 weeks (4WC) or 8 weeks (8WC). Exercise-trained rats performed treadmill running for 5 days/week (60 mins/day). Left ventricular mass index and wall thickness and stroke volume index, measured using echocardiography, in the 8WT group were significantly greater than in the 8WC group, although there were no differences between the 4WC and 4WT groups in these parameters. These results indicated that the 8WT rats developed physiologic cardiac hypertrophy, whereas the 4WT rats did not yet have cardiac hypertrophy. Myocardial ET-1 gene expression and tissue ET-1 concentration in the heart were significantly higher in the 8WT group than in the 8WC group, whereas these values did not differ between the 4WC and 4WT groups. The present study suggests that an alternation of myocardial ET-1 production corresponds with the formation of exercise training–induced cardiac hypertrophy. Therefore, the exercise training–induced change in myocardial ET-1 production may participate in a mechanism of exercise training–induced cardiac adaptation (e.g., cardiac hypertrophy).

This study investigated how the endocardial endothelium (EE) and particularly endothelial type B (ET_B) receptors influence the effects of endothelin-1 (ET-1) on diastolic distensibility. ET-1 (0.1, 1, and 10 nM) was tested in rabbit papillary muscles (Krebs-Ringer; 1.8 mM CaCl₂, 35°C) (i) with intact EE (n = 10), (ii) with damaged EE (0.5% Triton X-100, n = 11), and (iii) in the presence of RES-701-1 (selective endothelial ET_B1 receptor antagonist, 1 μM, n = 6). Additionally, increasing doses (0.1 nM to 1 μM) of Sarafotoxin S6c (SRTXc, a selective ET_B receptor agonist) and IRL-1620 (a selective endothelial ET_B1 agonist) were studied (i) in muscles with intact EE (n = 7 and n = 6, respectively) and (ii) after damaging the EE (n = 8 and n = 7, respectively). In papillary muscles with intact EE, ET-1 induced dose-dependent positive inotropic and lusitropic effects. At 10 nM, active tension (AT) increased 78% ± 17%, maximum velocity of tension rise (dT/dt_max) increased 82% ± 10%, and maximum velocity of tension decline (dT/dt_min) increased 77% ± 17%. These effects were maintained when ET-1 was given after damaging the EE (AT increased 70% ± 12%, dT/dt_max increased 93% ± 14%, and dT/dt_min increased 56% ± 14%), but were significantly reduced in the presence of RES-701-1 (AT increased 30% ± 6%, dT/dt_max increased 37% ± 7%, and dT/ dt_min increased 29% ± 9%). ET-1 reduced resting tension (RT) and increased diastolic distensibility by 3% ± 1%, 5% ± 1%, and 9% ± 2% (at 0.1, 1, and 10 nM, respectively) in muscles with intact EE. This effect was abolished after damaging the EE or in the presence of RES-701-1. In muscles with intact EE, SRTXc had no significant effects, whereas, when given after damaging the EE, SRTXc (1 μM) increased inotropy and lusitropy (AT increased 116% ± 24%, dT/dt_max 110% ± 28%, and dT/dt_min 88% ± 19%) without affecting RT. IRL-1620 dose-dependently decreased AT, dT/dt_max, and dT/dt_min in muscles with intact EE—effects that were abolished after EE damage. No significant effects were elicited by IRL-1620 in RT. ET-1–induced increase in myocardial distensibility, previously shown to be mediated by ET_A receptor stimulation, requires an intact EE and active endothelial ET_B1 receptors.

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key down-stream substrate of the endothelin signaling pathway and plays a role in regulating protein function at the membrane-cytoskel-etal interface. However, the dynamic properties of distinct pools of PIP₂ are poorly understood, especially for PIP₂ that is bound to cytoskeletal proteins. We investigated the effects of endothelin-1 (ET-1) stimulation on protein-bound PIP₂ in cardiac muscle. Isolated rat myocytes and homogenized mouse ventricles were exposed to 10 nM ET-1 for varying time periods and protein-bound PIP₂ was analyzed using an anti-PIP₂ antibody and Western blotting. Several cytoskeletal proteins were found to contain tightly bound PIP₂, including profilin-1 (~15 kDa), capZ (~32 kDa), gCap39, (~39 kDa) and α-actinin (~106 kDa). Interestingly, ET-1 pretreatment reduced the amount of PIP₂ bound to profilin-1 by 46% after 15 mins, followed by a recovery to near basal levels after 60 mins. ET-1 had no effect on capZ-, gCap39-, or α-actinin–bound PIP₂ levels. To further explore the dynamics of PIP₂ binding, brefeldin-A (BFA) was used to disrupt PIP₂ binding to ADP-ribosylation factors and to impair receptor internalization. Pretreatment with 1 μM BFA increased the PIP₂ signal on profilin-1 ×54% after 15 mins, followed by a decline to subbasal levels after 60 mins. Like ET-1, BFA had no effect on levels of PIP₂ bound to capZ or to α-actinin. Taken together, the data indicate that profilin-1 binds PIP₂ dynamically and may serve as a key regulator of the balance between cytoskeletal integrity and PIP₂ availability for Ca²⁺/PKC signaling in the heart.

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro–ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1–induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1–administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco’s modified Eagle’s medium and Ham F12 supplemented with 0.1% fatty acid–free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1–treated (0.1 nM) group, and ET-1–treated group pretreated with EPA (10 μM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro–ET-1 mRNA expression was 53% upregulated in ET-1–induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1–administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET_A and ET_B, tended to be increased in the ET-1–treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro–ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1–induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.

Inotropic effects of selective ET_B receptor stimulation depend on the functional integrity of the endocardial endothelium (EE), which is negative when it is intact and positive when it is damaged. These results have been attributed to the existence of two subtypes of ET_B receptors in the heart: (i) ET_B1, located on the EE, decreases inotropy; (ii) ET_B2, located on myocardial cells, increases inotropy. In the present study we investigated the functional integrity of the EE in a heart failure (HF) model (doxorubicin-induced cardiomyopathy) by evaluating the contractile response to ET_B1 receptor stimulation. New Zealand White rabbits were treated with doxorubicin (DOX-HF, 1 mg/kg, iv, twice weekly for 8 weeks) or with saline. Contractile effects of increasing doses of a selective agonist of endothelial ET_B receptors, IRL-1620 (10⁻⁹ to 10⁻⁶ M), were studied in papillary muscles (Krebs-Ringer: 1.8 mM CaCl₂, 35°C) from control (n = 10) and DOX-HF rabbits (n = 7). Isotonic and isometric twitches were recorded and analyzed. Reported parameters included active tension (AT) and maximum velocities of tension rise (dT/dt_max) and decline (dT/dt_min). On echocardiography, DOX-HF rabbits had increased left ventricular (LV) end-diastolic and end-systolic diameters and reduced ejection fraction (52% ± 2% vs. 61% ± 1%). Contrary to control papillary muscles, DOX-HF muscles showed a steady decrease in contractility between 1 and 4 Hz. In the control group, IRL-1620 induced dose-dependent negative inotropic and lusitropic effects that decreased at 10⁻⁶ M: 26% ± 3%, AT; 17% ± 3%, dT/dt_max; and 16% ± 5%, dT/dt_min. In the DOX-HF group, these effects were significantly reduced. At the same concentration, IRL-1620 decreased AT (8% ± 3%) and dT/dt_max (8% ± 3%), without significantly affecting dT/dt_min. This study showed an impaired response to endothelial ET_B receptor stimulation, providing for the first time strong evidence of the occurrence of EE dysfunction in the failing heart and further highlighting the potential use of ET_B receptor stimulation as a marker of EE function.

Endothelin regulates cytokine expression in vitro and in vivo. This study investigated the effects of chronic allograft rejection on hepatic endothelin-converting enzyme-1 (ECE-1) gene expression and endothelin-1 (ET-1) plasma clearance. Using the Lewis-F344 minor histocompatibility mismatch model of heterotopic cardiac transplantation, hepatic ECE-1 gene expression was measured by real-time polymerase chain reaction and host plasma clearance of ET-1 was measured 8 weeks after transplantation in the absence of immunosuppression. In animals undergoing allograft rejection, hepatic ECE-1 gene expression increased 2-fold (P < 0.05), whereas no effect of rejection on ET-1 clearance from plasma was observed. In summary, upregulation of ECE-1 gene expression occurs in the liver of the host during chronic allograft rejection. Because the liver represents both a key organ for cytokine production and for endothelin metabolism, increased hepatic ECE-1–mediated ET-1 synthesis may contribute to host responses and cytokine production during allograft rejection.

Cardiovascular complications are an important feature of diabetes mellitus (DM). Abnormal and decreased coronary collateral development has been implicated in the pathogenesis of cardiac complications in DM. More recently, decreased expression of vascular endothelial growth factor (VEGF) and its receptors has been found in diabetic heart. To our knowledge, no study has focused on the therapeutic improvement associated with VEGF in diabetic heart. DM was induced by intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats, while control rats received only citrate buffer. After 1 week, the streptozotocin-treated rats were randomly divided into two groups: one group received the selective endothelin (ET) type A receptor antagonist TA-0201 at a dose of 1 mg/kg/day for 2 weeks by osmotic mini-pump, and the vehicle group received saline only. The plasma glucose level was 504 ± 75 mg/dl in the diabetic rats and was unchanged by treatment with ET antagonist. The body weight was decreased in the diabetic rats compared with the control rats, but the left ventricular (LV)–body weight ratio was increased in the diabetic group and was unaffected by treatment with ET antagonist. mRNA expression of VEGF and its receptors (Flt-1 and Flk-1) in the LV tissues was assessed using real-time polymerase chain reaction. VEGF expression was significantly decreased in diabetic heart and was greatly improved by treatment with ET antagonist. The expression of VEGF receptors was down-regulated in early diabetic heart but was not recovered by treatment with ET antagonist. ET and its receptor A might have differential regulation on the gene expressions of VEGF and its receptors in early diabetic heart.

The effects of calcium channel blockers (CCBs) on complications associated with diabetes mellitus (DM) have been well studied in clinical and basic science investigations. Cardiovascular complications are a common feature of type 2 DM, and insulin resistance is an early clinical manifestation of type 2 DM. CCBs are widely used to treat cardiovascular diseases in patients with DM. In this study, we used a spontaneous type 2 diabetic rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats, at a highly insulin-resistant stage with modest hyperglycemia. We examined cardiac expression of transforming growth factor–β₁ (TGFβ₁) and endothelin-1 (ET-1) in male OLETF rats. At 8 weeks of age, OLETF rats were treated for 12 weeks with the long-acting CCB benidipine (1 mg/kg/day or 3 mg/kg/day, po, n = 12), with hydralazine hydrochloride (3 mg/kg/day, po, n = 12), or with vehicle (OLETF, n = 12), and male age-matched genetic control Long-Evans Tokushima Otsuka (LETO, n = 12) rats were used. Blood pressure was significantly higher in OLETF rats than in LETO rats, and benidipine treatment at both dosages in OLETF rats for 12 weeks did not significantly reduce blood pressure, whereas hydralazine treatment significantly lowered blood pressure in OLETF rats. Hydralazine and both dosages of benidipine significantly reduced upregulated cardiac ET-1 levels in OLETF rats. Plasma and cardiac TGFβ₁ levels were remarkably higher in OLETF rats compared with LETO rats and were normalized by treatment with benidipine (3 mg/kg/day). Our results suggest that CCBs are effective in normalizing upregulated cardiac TGFβ₁ and ET-1 levels at the insulin-resistant stage in OLETF rats, which may improve cardiac morphology and function in this rat model without altering blood pressure and plasma glucose levels. In contrast, hydralazine treatment also normalizes cardiac ET-1 levels while significantly reducing blood pressure.

Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1–induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1–induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid–free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 μM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1–treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1–induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.

Endothelin-1 (ET-1) has been implicated in hypertension, heart failure, atherosclerosis, and pulmonary hypertension. In all these conditions, plasma immunoreactive ET-1 levels are elevated, and tissue ET-1 expression is increased. Clinical trials have demonstrated potentially important benefits of ET antagonism among patients with essential hypertension, pulmonary hypertension, and heart failure. It is unknown whether ET antagonism affects the production of ET-1 in stroke-prone spontaneously hypertensive rat (SHRSP) heart at the typical hypertensive stage. The objective of this study was to investigate the effects of ET blockade on the expression levels of plasma and cardiac ET-1 in SHRSPs. SHRSPs were treated for 3 months with SB209670 (ET_A/ET_B dual receptor antagonist) or with saline (vehicle) commencing at the prehypertensive stage (age 6 weeks). Plasma and left ventricular ET-1 peptide levels were measured using enzyme-linked immunoabsorbent assay. Compared with age-matched control Wistar-Kyoto rats, peptide levels of ET-1 were significantly upregulated in vehicle-treated SHRSP heart; this upregulation was reversed by long-term ET antagonism. Plasma ET-1 levels were also significantly increased in vehicle-treated SHRSPs and were normalized by ET antagonism. mRNA expression of preproET-1, which is the source of ET-1 peptide production, was significantly increased in vehicle-treated SHRSP heart and was normalized by ET antagonism. Marked cardiac hypertrophy and fibrosis at the histologic level in SHRSPs were ameliorated by ET antagonism, and left ventricular hypertrophy as seen on echocardiography in SHRSPs was suppressed by ET blockade. After ET antagonism, systolic blood pressures were reduced in SHRSPs; diastolic blood pressures were unchanged. The reversal effect of the upregulated ET system in SHRSP heart by ET antagonism might be independent of blood pressure change. By suppressing the upregulated ET system, ET antagonism might be beneficial in arresting cardiac remodeling.

Vascular tone is regulated through the actions of locally produced agents. Among the vasoconstrictors, the most potent agent is endothelin (ET), which exerts its vasoconstrictor actions principally through ET type A (ET_A) receptors. Of the vasodilators, nitric oxide (NO) seems to be the most important contributor to the acute regulation of vascular tone. Vasculopathy is an important feature of diabetes mellitus (DM). Endogenous ET-mediated vasoconstrictor tone is augmented in diabetic states, and conflicting results persist concerning the NO system in diabetes. The present study investigated the expressions of inducible NO synthases (iNOS) and endothelial NOS (eNOS) in the heart of diabetic animals and the effects of a selective ET_A receptor antagonist on these alterations. Type I diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats, while control (Con) rats received only citrate buffer. After 1 week, the streptozotocin-administered rats were randomly divided into two groups: the selective ET_A receptor antagonist–administered group (DM+TA-0201, 1 mg/kg/day, by osmotic minipump for 2 weeks) and the DM+vehicle group (comprising the diabetic rats that received saline). The random blood glucose level was 405 ± 103 mg/dl in DM animals, and this level was unchanged by ET antagonism. Body weight was more greatly decreased in DM rats than in Con rats, but the left ventricle to body weight ratio was increased in the DM group and was unaffected by ET antagonism. Protein expressions of eNOS and iNOS were assessed in the left ventricular tissues. eNOS expression was significantly increased in DM heart and was greatly inhibited by the treatment with ET antagonist. The expression of iNOS was also increased in early DM heart but was reversed by the ET antagonist. Thus, endothelin antagonism might be beneficial for DM heart by reversing the upregulated eNOS and iNOS expressions.

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1–induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1–administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)–treated group, and the ET-1 group pretreated with EPA (10 μM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1–induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1–induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1–administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.

Endothelin (ET)-1_1–21 is known to play an important role in the pathogenesis of acute ischemic arrhythmia. In the present study, we attempted to determine whether administration of ET-1_1–31 would result in arrhythmia in perfused isolated rat hearts. Forty-eight Sprague-Dawley rats weighing ~250–350 g were randomized into 6 groups. Heart was isolated and perfused in a Langendorff mode. The effects of ET-1_1–31 on arrhythmia, heart rate, coronary flow, and heart function were analyzed. Perfusion with 1 nM ET-1_1–31 resulted in frequent ventricular ectopic beats (VEBs) and ventricular tachycardia (VT). Overall VEB was 128.0 (~66.0–1015.0), and the arrhythmia score (AS) was 2.18 ± 0.87; both were significantly higher than those of the control group (P < 0.01). Pretreatment with perfusion of 10 nM of the ETA-receptor antagonist BQ₁₂₃ markedly attenuated the occurrence of VEB and VT induced by ET-1_1–31. AS in 10 nM BQ₁₂₃ group was significantly lower than that in 1 nM ET-1_1–31 group (P < 0.01). The arrhythmia induced by 1 nM ET-1_1–31 was partially but significantly reduced by phosphoramidon (1 μM), a neutral endopeptidase/ET-converting enzyme inhibitor. ET-1_1–31 per se caused arrhythmia in perfused isolated rat hearts. This arrhythmogenic action is in part mediated by ET_A receptor and may be attributed mainly to the conversion of ET-1_1–31 to ET-1_1–21.

Sildenafil, an oral phosphodiesterase Type 5 inhibitor, has vasodilatory effects through a cGMP-dependent mechanism. We previously showed that aortic banding could result in left ventricular overloading and pulmonary hypertension (PH). In this study, we investigated whether early administration of sildenafil, either immediately after or 2 weeks after aortic banding, could ameliorate the development of PH and alter gene expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS), and alter the levels of cGMP in rats undergoing an ascending aortic banding. Rats (n = 32) were divided into sham-operated and banding groups with or without treatment. The banded rats were further divided into three groups: (i) receiving saline on Days 1–28 (AOB₂₈; n = 8), (ii) receiving saline on Days 1–14 followed by treatment with 50 mg/kg/day sildenafil on Days 15–28 (AOB₂₈/Sil_15–28; n = 8), and (iii) receiving 50 mg/kg/day sildenafil on days 1–28 (AOB₂₈/Sil_1–28; n = 8). The sham-operated rats were administrated saline on Days 1–28 (n = 8).

Four weeks after banding, there was a significant development of PH with pulmonary vascular remodeling. Although both sildenafil-treatment groups had significant increases in cGMP and had reductions in the thickening in the medial layer of pulmonary arteriole, notable attenuation of PH occurred only in the AOB₂₈/Sil_1–28 group. PreproET-1 and eNOS messenger RNA (mRNA) expressions were measured by competitive reverse transcription polymerase chain reaction, and eNOS protein was determined by Western blotting. Sildenafil did not alter the elevated ET-1 or preproET-1 mRNA in banded rats. Interestingly, pulmonary eNOS increased in the AOB₂₈/Sil_1–28 group. In conclusion, early treatment with sildenafil inhibited the rise in pulmonary arterial pressure and pulmonary vascular remodeling in PH secondary to heart failure, and cGMP, but not ET-1, might be involved. Clinically, early repeated administration of sildenafil may offer an alternative in protecting against PH in heart failure.

Pressure overload in the left ventricle of the heart follows a chronic and progressive course, resulting in eventual left heart failure and pulmonary hypertension (PH). The purpose of this research was to determine whether a differential pulmonary gene change of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS) occurred in adult rats with left ventricular overload. Eight groups of eight rats each were used (four rats with banding and four rats with sham operations). The rats underwent ascending aortic banding for 1 day, 2 weeks, 4 weeks, and 12 weeks before sacrifice. Significant medial hypertrophy of the pulmonary arterioles developed in two groups (4 and 12 weeks). Increased pulmonary arterial pressures were noted in three groups (1 day, 4 weeks, and 12 weeks). The aortic banding led to significant increases in pulmonary preproET-1 messenger RNA (mRNA) at 1 day and 12 weeks, and in pulmonary eNOS mRNA at 1 day and 12 weeks. In addition, there was increased pulmonary eNOS content at 1 day and 12 weeks in the banded rats, and increased lung cGMP levels were observed at 1 day. Increased lung ET-1 levels were also noted at 1 day (banded, 310 ± 12 ng/g protein; sham, 201 ± 12 ng/g protein; P < 0.01), 4 weeks (banded, 232 ± 12 ng/g protein; sham, 201 ± 12 ng/g protein; P < 0.01) and 12 weeks (banded, 242 ± 12 ng/g protein; sham, 202 ± 12 ng/g protein; P < 0.01). This indicates that the upregulated expression of ET-1 developed at least 4 weeks before eNOS expression in the course of PH, and, thus, medication against ET-1 could play a crucial role in treating PH with cardiac dysfunction secondary to aortic banding.

Pulmonary hypertension (PH) usually develops secondary to left ventricular (LV) dysfunction; therefore, it is also called retrograde PH. To investigate our hypothesis that PH is at least partially reversible, as in some congenital heart diseases, in a rat model we investigated whether release of constriction could attenuate pulmonary vascular remodeling and change the expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS). We used rats with LV dysfunction produced by an ascending aortic banding. In this study, there were four groups enrolled: 4-weeks banded (AOB_1–28; n = 7), 7-weeks banded (AOB_1–49; n = 7), debanded groups (AOB_1–28/DeB_29–49; n = 7), and rats receiving a sham operation (n = 7). Subsequently, there was significant attenuation of medial hypertrophy in pulmonary arterioles and reversal of PH in the AOB_1–28/DeB_29–49 group (sham, 19 ± 1.3 mm Hg; AOB_1–28, 31 ± 2.7 mm Hg; AOB_1–49, 32 ± 2.7 mm Hg; and AOB_1–28/DeB_29–49, 20 ± 1.3 mm Hg). PreproET-1 mRNA and eNOS mRNA were measured by competitive reverse transcriptase (RT) polymerase chain reaction (PCR), and eNOS was measured by Western blotting. Compared with the banded groups, debanding significantly decreased pulmonary preproET-1 mRNA, pulmonary ET-1 (sham, 210 ± 12 pg/g protein; AOB_1–28, 242 ± 12 pg/g protein; AOB_1–49, 370 ± 49 pg/g protein; and AOB_1–28/DeB_29–49, 206 ± 1.9 pg/g protein), and plasma ET-1 levels (sham, 10.1 ± 1.5 pg/ml; AOB_1–28, 13.4 ± 2.0 pg/ml; AOB_1–49, 15.4 ± 2.0 pg/ml; and AOB_1–28/DeB_29–49, 10.3 ± 0.9 pg/ml protein). Debanding could not, however, alter pulmonary eNOS, eNOS mRNA, or cGMP. These findings suggest that pulmonary vascular remodeling, increased pulmonary arterial pressure, and upregulation of ET-1 gene expression are all reversible. We infer that it is the upregulated gene expression of ET-1, not eNOS, that is closely related to the development of the PH secondary to 4 weeks of aortic banding.

Inhalation of endothelin (ET)-A receptor antagonists has been shown to improve gas exchange in experimental acute lung injury (ALI) but may induce side effects by increasing circulating ET-1 levels. We investigated whether the inhaled ET_A receptor antagonist, LU-135252, at low doses, improves gas exchange without affecting ET-1 plasma concentrations and lung injury in an animal model of ALI. Twenty-two piglets were examined in a prospective, randomized, controlled study. In anesthetized animals, ALI was induced by surfactant depletion. Animals received either LU-135252 at a dose of 0.3 mg/kg during 20 mins (LU group; n = 11), or nebulization of saline buffer (control group; n = 11). The Mann-Whitney U test was used to compare groups (P < 0.05). In the LU group, arterial partial pressure of oxygen (PaO₂) and mean pulmonary artery pressure (MPAP) improved compared with the control group (PaO₂, 319 ± 44 mm Hg vs. 57 ± 3 mm Hg; MPAP, 32 ± 2 mm Hg vs. 41 ± 2 mm Hg; values at 6 hrs after induction of ALI; P < 0.05). Mean arterial pressure and cardiac output were not different between groups. ET-1 plasma concentrations increased from 0.96 ± 0.06 fmol/ml after induction of ALI to a maximum of 1.17 ± 0.09 fmol/ml at 3 hrs after ALI onset in the LU group and did not differ significantly from the control group (1.21 ± 0.08 fmol/ml, not significant). On histologic examination, we found no differences in total lung injury score between groups. However, the LU group revealed significantly reduced interstitial inflammation and hemorrhage (P < 0.05 vs. control group). In this animal model of ALI, inhalation of LU-135252 at a dose of 0.3 mg/kg induced a significant and sustained improvement in gas exchange, whereas there were no changes in ET-1 plasma concentrations. Furthermore, our data indicate a trend toward decreased pulmonary inflammation in the group receiving the inhaled ET_A receptor antagonist

The dual endothelin receptor antagonist, bosentan, and the phosphodiesterase inhibitor, sildenafil, are efficacious in experimental and clinical pulmonary hypertension (PHT). The effects of bosentan, sildenafil, and their combination were evaluated in rats with monocrotaline (MCT)-induced PHT. A first group consisted of control rats with no MCT injection. Four other groups of rats received MCT subcutaneously and were assigned to receive no treatment, 300 mg/kg/day bosentan as food admix, 100 mg/kg/day sildenafil in drinking water, or their combination for 4 weeks. The doses of bosentan and sildenafil were the maximally effective doses based on a dose-range–finding study. Mortality was 0%, 53%, 11%, 11%, and 0%, respectively, in the five different groups. Bosentan and sildenafil significantly attenuated the increase in mean pulmonary arterial pressure, and the combination had an additional effect. Similarly, bosentan, sildenafil, and, to a greater extent, their combination significantly reduced right ventricular (RV) hypertrophy. Bosentan, but not sildenafil, decreased norepinephrine and BNP plasma concentrations, reduced kidney weight, and normalized systemic hemodynamics. In conclusion, bosentan and sildenafil are efficacious in rats with chronic PHT, and their combination shows an additional effect for decreasing pulmonary arterial pressure, reducing plasma catecholamines, maintaining body weight, and reducing mortality.

Beneficial effects of inhaled nitric oxide (iNO) on arterial oxygenation in acute lung injury (ALI) suggest the presence of vasoconstriction in ventilated lung regions and this may be influenced by endothelin-1 (ET-1). We studied a possible interaction between ET-1 and iNO in experimental ALI. Sixteen piglets were anesthetized and mechanically ventilated (inspired O₂ fraction, 1.0). After induction of ALI by surfactant depletion, animals were randomly assigned to either inhale 30 ppm NO (iNO group, n=8), or to receive no further intervention (controls, n=8). Measurements were performed during the following 4 hrs. In all animals, induction of ALI significantly decreased arterial oxygen tension (PaO₂) from 569 ± 15 (prelavage) to 58 ± 3 mm Hg. Inhaled NO significantly increased PaO₂ when compared with controls (iNO group: 265 ± 51 mm Hg; controls: 50 ± 4 mm Hg, values at 4 hrs, P < 0.01). Prelavage ET-1 plasma levels were comparable between groups (iNO: 0.74 ± 0.03, controls: 0.71 ± 0.03 fmol/ml, NS). During the protocol, the ET-1 levels increased and were different at 3 hrs (iNO: 0.93 ± 0.06, controls: 1.25 ± 0.09 fmol/ml; P < 0.05). PaO₂ changes induced by iNO revealed a moderate and significant correlation with ET-1 plasma levels (R = 0.548, P = 0.001). Our data suggest that endogenous ET-1 production influences the efficacy of iNO in ALI. Furthermore, iNO reduced ET-1 plasma levels, possibly indicating anti-inflammatory properties of iNO in the early phase of ALI.

Perturbation of vascular homeostasis is an important mechanism related to the acute health effects of inhaled pollutants. Inhalation of urban particulate matter and ozone by rats has been shown to result in increased synthesis of the potent vasoactive peptide endothelin (ET)-1 in the lungs, with spillover into the circulation. In the present work, we have analyzed the interrelationships between responses of the three major endothelin isoforms, ET-1_[1–21], ET-2_[1–21], and ET-3_[1–21], to inhaled pollutants at the peptide and gene expression levels. Fisher-344 rats were exposed for 4 hrs by nose-only route to clean air, urban particles EHC-93 (0, 50 mg/m³), ozone (0, 0.8 ppm), or ozone and particles together. Circulating levels of both the ET-1 _[1–21] and ET-3_[1–21] peptides were increased immediately after exposure to particulate matter or ozone. While expression of preproET-1 mRNA in the lungs increased, expression of preproET-3 mRNA decreased immediately after exposure. PreproET-2 mRNA was not detected in the lungs, and exposure to either pollutant did not affect plasma ET-2 levels. Co-exposure to ozone and particles, while altering lung preproET-1 and preproET-3 mRNA levels in a fashion similar to ozone alone, did not cause changes in the circulating levels of the two corresponding peptides. Thus, de novo synthesis of ET-3 in the lungs is not responsible for the increase of circulating plasma ET-3 after inhalation of pollutants, which implies regulation of preproET-3 at a remote site and, hence, systemic impacts of the pollutants. Upregulation of preproET-1 coupled with down-regulation of preproET-3 in the lungs of animals exposed to air pollutants implies a mismatch of local ET-1/ET_A receptor–mediated vasoconstriction and ET-3/ET_B receptor–mediated vasodilation.

Endothelin-1 (ET-1) is increasingly recognized as a proinflammatory mediator in various diseases, such as atherosclerosis and acute respiratory distress syndrome (ARDS). Angiopoietin-1 (Ang-1), a ligand of the endothelial receptor Tie2, inhibits endothelial apoptosis, reduces vascular leakage, and suppresses the induction of inflammatory markers, indicating that it has diverse vasoprotective, anti-inflammatory actions. Thus, we examined the effects of Ang-1 on ET-1 production in vitro and in vivo and investigated cell-based gene transfer of Ang-1 in a rat model of lipopolysaccharide (LPS)-induced ARDS. Cultured human endothelial cells were treated with recombinant Ang-1 with or without tumor necrosis factor-α (TNF-α) (100 U/ml). ET-1 release into the culture medium after 24 hrs was determined by enzyme-linked immunosorbent assay. Levels of preproendothelin-1 (ppET-1) mRNA were measured by quantitative reverse transcription–polymerase chain reaction. Fisher344 rats were subjected to cell-based gene transfer to the lung circulation by injecting syngeneic fibroblasts transfected with Ang-1 cDNA or a null plasmid vector. After 24 hrs, LPS (100 μg/kg body wt) was instilled intratracheally to induce pulmonary inflammation. Bronchoalveolar lavage was performed 6 hrs later, and lungs were harvested for histologic and molecular analyses. ET-1 release from cultured endothelial cells was dose-dependently reduced by Ang-1, which also prevented induction of ET-1 release by TNF-α (P < 0.05). RNA expression of ppET-1 was similarly reduced. In LPS-challenged lungs, ppET-1 RNA was induced 3.4-fold, and ET-1 protein in lavage fluid was increased 5.6-fold (P < 0.05). Ang-1 gene transfer attenuated the LPS-induced increases in ppET-1 RNA and lavage ET-1 protein by 34% and 33%, respectively (P < 0.05). The downregulation of ET-1 correlated with the amelioration of pulmonary inflammation, as indicated by reductions in leukocyte infiltration (by 43%) and intra-alveolar septal thickening (by 40%). These results show that ET-1 transcript and protein levels are downregulated by Ang-1 in both in vitro and in vivo systems and that cell-based Ang-1 gene transfer markedly ameliorated inflammation in vivo in an experimental model of ARDS. Thus, cell-based gene transfer of Ang-1 may provide a novel treatment strategy for ARDS by attenuating vascular inflammation via suppression of ET-1.

Septic shock is characterized by hypotension and a hyporeactive response to vasopressor agents. The pathogenesis is due to vascular leaks and an increased synthesis of cytokines and nitric oxide (NO). The present study examined the time-dependent alterations of endothelin-1 (ET-1) and the expression of NO synthase (NOS) in lung tissue in a septic rat model. Normal Sprague-Dawley (SD) rats aged 10 weeks received 15 mg/kg lipopolysaccharide (LPS) and then were sacrificed at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. Both systolic and diastolic pressure decreased in SD rats after LPS administration. Time-dependent onset of features of acute lung injury, such as the infiltration of inflammatory cells and thickening of alveolar septa, were seen in rats that received LPS. A 2.8-fold increase in the expression of preproET-1 level was observed in lung tissue 6 hrs after LPS administration. The expression of endothelial NOS (eNOS) was also altered in lung tissue in a time-dependent fashion. After the administration of LPS, there was a 16-fold increase in the expression of eNOS mRNA. The peak expression of inducible NOS (iNOS) in lung tissue specimens obtained from rats that received LPS was 45-fold higher than that in control rats. ET-1 is a potent vasoconstrictor and thereby may play an important role in the pathogenesis of acute lung injury in a septic rat model. The increased expression of NOS may result in excess NO production and may also play a role in the pulmonary complications of endotoxemia.

Endothelin (ET)-1 is involved in the pathophysiology of various renal disorders, promoting renal cellular proliferation and extracellular matrix protein accumulation, and, thus, diminishing fundamental renal function, including filtration. To determine whether ET-1 and ET-2 play a role in feline chronic renal failure, we analyzed the messenger RNA (mRNA) expression of the prepro-ET (PPET )-1 and PPET-2 genes in affected cat kidney after molecular cloning of full-length PPET-2 complementary DNA (cDNA). Conceptual analysis of the primary structure of cat PPET-2 based on the cloned sequence demonstrated that the putative regions corresponding to a mature peptide and peptidase processing sites are present in cat PPET-2. Homology analysis showed that the similarity of the cat PPET-2 amino acid sequence with those from human, mouse, rat, rabbit, dog, ferret, cow, and horse was 73.0%, 68.6%, 69.1%, 76.4%, 81.2%, 83.1%, 76.3%, and 79.2%, respectively. Analysis of PPET-1 and PPET-2 mRNA in cat by reverse transcription polymerase chain reaction showed upregulated expression of both genes in kidneys affected by renal failure.

It is well established that activation of endothelin B (ETB) receptor induces natriuresis and diuresis and thus reduces blood pressure. However, the site of action of ETB receptor is debatable. The present study was undertaken to address the role of renal medullary ETB receptor in renal excretory function. In volume-expanded Sprague-Dawley rats, infusion of the ETB antagonist A192621 at 0.5 mg/kg/hr to the renal medulla induced an immediate and significant reduction of urine flow rate that was 87.5% ± 7.1%, 68% ± 20%, and 58.3% ± 15.5% of the control value at 10, 30, and 60 mins, respectively (n=5, P < 0.05 at each time point). Following intramedullary infusion of A192621, urinary sodium excretion remained unchanged during the first 20 mins but started to decline thereafter with a maximal effect at 60 mins. Changes in urinary excretion of potassium and chloride followed the same pattern of changes as for urinary sodium. In contrast, urinary osmolality gradually and significantly increased (control: 419 ± 66; A192621 at 60 mins: 637 ± 204 mOsm/kg H₂O, P < 0.05). Over a 60-min period of intramedullary infusion of A192621, none of the hemodynamic parameters examined, including mean arterial pressure, renal blood flow, or medullary blood flow, were affected. These data suggest that: (i) intramedullary blockade of ETB receptor produces antidiuresis and antinatriuresis independently of hemodynamic changes, and (ii) the immediate response to intramedullary blockade of ETB receptor is the reduction of water excretion followed by the reduction of sodium excretion.

Adenosine triphosphate (ATP) and endothelin (ET)-1 inhibit vasopressin-stimulated water reabsorption in the inner medullary collecting duct (IMCD). Because both ATP and ET-1 are released by the IMCD and can act in an autocrine manner to regulate IMCD water transport, we sought to determine whether these factors can modulate the other’s production. To begin such studies, the effect of ATP on IMCD ET-1 production was examined. ATP caused a dose-dependent inhibition of ET-1 release and inhibited ET-1 mRNA levels in primary cultures of rat IMCD cells. This effect was first evident after 4 hrs of exposure to ATP and persisted for at least 24 hrs. The 50% inhibitory concentration for ATP inhibition of ET-1 production was approximately 1 μM, and the maximal response was observed at 25–100 μM. ATP acted, at least in part, through the P2Y2 receptor because its effect was mimicked by UTP, but not by the P2X agonist, α,β-methylene–ATP. N-methyl-l-arginine, or indomethacin, did not block the ATP inhibitory effect. In summary, these data demonstrate that ATP inhibits IMCD ET-1 protein and mRNA accumulation, that this is mediated via P2Y receptors, and that the ATP effect is independent of cyclooxygenase or nitric oxide synthase metabolites. These findings suggest that although ATP and ET-1 both antagonize vasopressin action in the IMCD, they may have a complex interaction that ultimately determines the degree to which they each participate in modulating collecting duct function.

We investigated the effects of three different daily doses (10 mg, 20 mg, and 40 mg) of atorvastatin, a relatively new and potent statin, on plasma endothelin (ET)-1 and highly sensitive C-reactive protein (CRP) levels in type 2 diabetic subjects. Twenty-nine type 2 diabetic patients with dyslipidemia were enrolled and randomly assigned to receive atorvastatin orally at 10 mg (A10; n = 10), 20 mg (A20; n = 10), or 40 mg (A40; n = 9) daily for 12 weeks. Levels of plasma total cholesterol and low-density lipoprotein (LDL)-cholesterol (C) in all three studied groups were significantly decreased after treatment with atorvastatin for 12 weeks (all groups, P < 0.001). However, the greatest LDL-C lowering effect and the highest percentage of subjects achieving the National Cholesterol Education Program’s Adult Treatment Panel III (NCEP-ATP III) LDL-C goal were observed in the A20 group. All diabetic subjects had a higher plasma ET-1 concentration (A10, 1.02 ± 0.37 pg/ml, mean ± SD; A20, 1.17 ± 0.55 pg/ml; and A40, 0.87 ± 0.45 pg/ml) than that of age- and sex-matched normal control subjects (0.64 ± 0.15 pg/ml; all groups, P < 0.001). Plasma ET-1 levels showed a borderline significant decrease at the end of study, by 22% in diabetic subjects treated with 10 mg atorvastatin (P=0.05 compared with baseline), and by 30% in subjects treated with 20 mg atorvastatin (P = 0.06, compared with baseline). Paradoxically, the 40-mg dose of atorvastatin provided an increase of 2% in plasma ET-1 levels at the end of study, which is significantly different (P < 0.05) and marginally significant (P = 0.057) from the levels of the 10- and 20-mg doses, respectively. Similarly, although insignificantly, plasma concentrations of CRP also tended to decrease by 12% and 48%, and paradoxically increased by 18% in diabetic patients treated with 10 mg, 20 mg, and 40 mg atorvastatin, respectively. The clinical significance of these biphasic lipid-independent statin effects is unknown and the present study suggests that 20 mg atorvastatin may have the best benefits in treating diabetic patients with dyslipidemia.

Vascular dysfunction characterized by a hyperreactivity to vasoconstrictors and/or impaired vascular relaxation contributes to increased incidence of cardiovascular disease in diabetes. Endothelin (ET)-1, a potent vasoconstrictor, is chronically elevated in diabetes. However, the role of ET-1 in resistance versus larger vessel function in mild diabetes remains unknown. Accordingly, this study investigated vascular function of third-order mesenteric arteries and basilar arteries in control Wistar and Goto-Kakizaki (GK) rats, a model of mild Type 2 diabetes. Six weeks after the onset of diabetes, contractile responses to 0.1–100 nM ET-1 and relaxation responses to 1 nM–10 μM acetylcholine (ACh) in vessels preconstricted (baseline + 60%) with serotonin (5-HT) were assessed by myograph studies in the presence or absence of a nitric oxide synthase (NOS) inhibitor, N-nitro-l-arginine (L-NNA). Maximum contractile response to ET-1 was augmented in mesenteric vessels (155 ± 18% in GK vs. 81 ± 6% in control; n = 5–7) but not in the basilar artery (134 ± 29% in GK vs. 107 ± 17% in control; n = 4 per group). However, vascular relaxation was impaired in the basilar arteries (22 ± 4% in GK vs. 53 ± 7% in control; n = 4 per group) but not in mesenteric arteries of GK rats. Inhibition of NOS decreased the relaxation response of basilar arteries to 15 ± 8% and 42 ± 5% in GK and control rats, respectively; whereas, in resistance vessels, corresponding values were 56 ± 7% and 89 ± 3% (vs. 109 ± 2% and 112 ± 3% without NOS blockade), indicating the involvement of different vasorelaxation-promoting pathways in these vascular beds. These findings provide evidence that the ET system is activated even under mild hyperglycemia and that it contributes to the hyperreactivity of resistance vessels, therefore, the ET system may play an important role in elevated blood pressure in Type 2 diabetes.

Fibronectin (FN), a key extracellular matrix protein, is upregulated in target organs of diabetic angiopathy and in cultured cells exposed to high levels of glucose. FN has also been reported to undergo alternative splicing to produce the extra domain-B (ED-B) containing isoform, which is exclusively expressed during embryogenesis, tissue repair, and tumoral angiogenesis. The present study was aimed at elucidating the role and mechanism of endothelins (ETs) in FN and ED-B FN expression in diabetes. We investigated vitreous samples for ED-B FN expression from patients undergoing vitrectomy for proliferative diabetic retinopathy. Our results show increased FN and ED-B FN expression in the vitreous of diabetic patients in association with augmented ET-1. Using an antibody specific to the ED-B segment of FN, we show an increase in serum ED-B FN levels in patients with diabetic retinopathy and nephropathy. We further examined retinal tissues, as well as renal and cardiac tissues, from streptozotocin-induced diabetic rats. Diabetes increased FN and ED-B FN in all three organs, which was prevented by ET antagonist bosentan. To provide insight into the mechanism of glucose-induced and ET-mediated ED-B FN upregulation, we assayed endothelial cells (ECs). Inhibition of mitogen-activated protein kinase with pharmacological inhibitors and protein kinase B with dominant negative transfections prevented glucose- and ET-1–mediated FN and ED-B FN expression. Furthermore, treatment of cells exposed to high levels of glucose with ET antagonist prevented the activation of all signaling pathways studied and normalized glucose-induced ED-B FN expression. We then determined the functional significance of ED-B in ECs and show that ED-B FN is involved in vascular endothelial growth factor expression and cellular proliferation. These studies show that glucose-induced and ET-mediated FN and ED-B FN expressions involve complex interplays between signaling pathways and that ET may represent an ideal target for therapy in chronic diabetic complications.

To determine whether renal expression of endothelin-converting enzymes (ECEs) and endothelin (ET) is affected in the early stages of autoimmune diabetes mellitus and whether ET_A receptors are involved, prediabetic nonobese diabetic (NOD) and control mice were treated with the ET_A receptor antagonist BSF461314 (a follow-up compound of darusentan) or with placebo. Blood samples were analyzed for glucose levels, and renal gene expression of ECE-1, ECE-2, and prepro-ET-1 was determined using real-time polymerase chain reaction. Renal morphology was assessed using standard histologic techniques. ECE-1, ECE-2, and prepro-ET-1 mRNA was detected in the kidneys of NOD and control mice. Despite normal renal histology, expression of ECE-1 and prepro-ET-1 was reduced in NOD mice by approximately 50% compared with controls (P < 0.01); ECE-2 was markedly decreased by almost 90% compared with controls (P < 0.001). Treatment with BSF461314 for 6 weeks delayed the onset of diabetes (P < 0.05) and increased expression of all three genes (P < 0.05) in NOD mice only. Hyperglycemia at an early stage of autoimmune diabetes is associated with transcriptional downregulation of ECE-1, ECE-2, and prepro-ET-1 in the kidney. Blockade of ET_A receptors inhibits diabetes-associated gene regulation and delays the onset of diabetes, suggesting its therapeutic potential for the treatment of autoimmune forms of diabetes.

Erectile dysfunction (ED) affects approximately 50% of male patients with diabetes mellitus (DM) and is possibly due to the vascular and neuropathic complications of DM. Recently, apoptosis has been regarded as a downstream event in ED. More recently, the importance of alterations in apoptosis-related molecules in the mechanism of DM-induced ED has begun to be appreciated. Endothelin-1 (ET-1) plays a role via ET_A and ET_B receptors in the regulation of cavernosal smooth-muscle tone in penile tissues. We found that the ET-1 level in the penis of rats with DM was higher than that in the penis of control animals. The present study investigated a rat model in which DM was induced by a 3-week regimen of streptozotocin (STZ) to assess the expression of several apoptosis-related molecules in penile tissue and, concomitantly, the effects of ET antagonism on these changes. Male Sprague-Dawley rats (weight [±SD], 450 ± 26 g) received a citrate saline vehicle or STZ (65 mg/kg ip). DM was confirmed by the presence of hyperglycemia. Diabetic animals were further separated into two treatment groups 1 week after onset of disease: one group received ET_A/B dual receptor antagonist (SB209670) by means of osmotic minipump at a dosage of 1 mg/day, and the other group received saline. Rats in both groups were treated for 2 weeks and then sacrificed. Plasma glucose levels (±SD) in rats with DM were significantly higher than those in rats without DM (506 ± 70 vs. 111 ± 11 mg/dl). In the penile tissue of rats with DM, a 35% decrease in the expression of Bcl-2 protein (an important antiapoptotic marker detectable by immunoblotting) was seen, and ET_A/B dual antagonist was observed to significantly counteract this decrease. Real-time polymerase chain reaction revealed that the expression of Bcl-2 mRNA was consistent with Bcl-2 protein expression. Levels of Bax and caspase-3, two important proapoptotic markers, were not significantly altered in the present study. Thus, we conclude that, in the penis of rats with early stage DM, the protection against apoptosis has decreased but can be improved by ET antagonism.

The objective of this study was to determine the change of plasma endothelin (ET)-1 concentrations and insulin resistance index after therapy for hyperthyroidism. We studied 20 patients with hyperthyroidism (15 women and 5 men; age, 34.0 ± 2.8 years), and 31 patients with euthyroid goiters as controls (27 women, 4 men; age, 37.0 ± 2.4 years). All hyperthyroid patients were treated with antithyroid drugs. The patients received evaluations before and after normalization of thyroid function. The evaluations included body mass index (BMI), body fat, and measurement of circulating concentrations of thyroid hormones, glucose, insulin, and ET-1. Hyperthyroid subjects had higher plasma ET-1 concentrations than the control group (P < 0.001). No significant differences in serum glucose and insulin concentrations or insulin resistance index estimated by the R value of the homeostasis model assessment (HOMA-R) were noted between the groups. Plasma ET-1 concentrations decreased after correction of hyperthyroidism compared with pretreatment (P = 0.006). Serum glucose concentrations decreased after correction of hyperthyroidism (P = 0.005). Moreover, both body weight–adjusted insulin concentrations and the HOMA-R index were also decreased after correction of hyperthyroidism compared with pretreatment (P = 0.026 and P = 0.019, respectively). Pearson’s correlation revealed that plasma ET-1 levels positively correlated with serum triiodothyronine (T3) and free thyroxine (FT4) levels. Serum insulin levels and the HOMA-R index positively correlated with BMI and body fat. The HOMA-R index also positively correlated with serum T3 and FT4 levels. Neither insulin levels nor the HOMA-R index correlated with ET-1 levels.

Hyperthyroidism is associated with higher plasma ET-1 concentrations. In addition, correction of hyperthyroidism is also associated with a decrease of plasma ET-1 levels as well as the insulin resistance index calculated by HOMA-R.

Obesity is associated with endothelial dysfunction that may contribute to the development of diabetes, hypertension, and atherosclerosis. Endothelin-1 (ET-1), which is produced mostly by vascular endothelial cells, has potent vasoconstrictor and proliferative activity in vascular smooth muscle cells and, therefore, has been implicated in regulation of vascular tonus and the progression of atherosclerosis, suggesting that ET-1 may be important in endothelial dysfunction. We studied whether diet-induced weight loss (i.e., lifestyle modification) affects plasma ET-1 concentration in obese individuals. We measured plasma ET-1 concentration in seven obese men (age: 48 ± 4 years old, body mass index: 27.7 ± 0.5 kg/m²) before and after a 3-month, diet-induced weight reduction program (i.e., lifestyle modification program). Caloric restriction reduced body weight from 78 ± 3 to 68 ± 2 kg (P < 0.001) and resulted in 12.1 ± 1.2% reduction in body mass index (24.3 ± 0.3 kg/m², P < 0.0001). After the weight reduction program, systolic and diastolic blood pressure significantly decreased (128 ± 7 vs. 115 ± 4 mm Hg, P < 0.05 and 88 ± 4 vs. 77 ± 2 mm Hg, P < 0.01, respectively). The plasma level of ET-1 significantly decreased after the program (5.1 ± 0.4 vs. 4.0 ± 0.3 pg/ml, P < 0.05). The percentage systolic blood pressure reduction and percentage plasma ET-1 concentration reduction was in a linear relationship (r = 0.86, P < 0.05). Furthermore, the relationship between percentage weight reduction and percentage plasma ET-1 concentration reduction was linear (r = 0.87, P < 0.05). We conclude that weight loss by low-calorie diet (i.e., lifestyle modification) reduces plasma ET-1 concentration in obese individuals. This reduction may contribute to the improvement of obesity-induced endothelial dysfunction.

In recent years endothelin-converting enzyme (ECE-1) has been suggested to play an important role in amyloid-β peptide metabolism as one of the amyloid-degrading enzymes. In this connection, the analysis of the levels of expression and distribution of ECE-1 in the brain under normal and pathologic conditions could be important in neurodegeneration and pathogenesis of Alzheimer disease. In our previous studies, we have demonstrated that expression of ECE-1 was significantly reduced in the cortex of adult rats after 15 mins of global ischemia. It was also significantly reduced in the striatum of rats subjected to prenatal hypoxia. In the present study, we analyzed effects of hypoxia and oxidative stress on ECE-1 in human neuroblastoma NB7 cells and effects of the cholinergic agonist carbachol and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). We have found that chronic (24 hrs) hypoxia and oxidative stress resulted in 30% and 20% decrease in expression of ECE-1 at the protein level, respectively, although at the level of ECE-1 mRNA there were no statistically significant changes. Serum withdrawal from the incubation medium as well as addition of carbachol or PMA for 24 hrs also led to a significant reduction of the levels of ECE-1 protein in NB7 cells. Further study of the downstream signaling cascades involved in downregulation of ECE expression in NB7 cells and primary neuronal cells might provide us with new insights into possible therapeutic strategies for prevention or treatment of Alzheimer disease in elderly patients and those who suffer from stroke or cerebrovascular disorders.

Though cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been recognized for over half a century, it remains a major complication in patients with SAH. Clinical studies have shown that elevated levels of endothelin-1 (ET-1) are present in the cerebrospinal fluid of patients with SAH, suggesting that ET-1–mediated vasoconstriction contributes to vascular constriction after SAH. Administration of estrogen promotes vasodilation in humans and in experimental animals, in part by decreasing the production of ET-1. This study evaluated the influence of 17β-estradiol (E2) on the production of ET-1 and cerebrovasospasm in an experimental SAH 2-hemorrhage model in rat. A 30-mm Silastic tube filled with E2 in corn oil (0.3 mg/ml) was subcutaneously implanted in male rats just before SAH induction. The degree of vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Plasma samples collected before death were assayed for ET-1. The protective effect of E2 in attenuating vasospasm achieved statistical significance when compared with the SAH only or SAH plus vehicle groups (P < 0.01). Concentrations of ET-1 were higher in the SAH only and SAH plus vehicle groups than in controls (P < 0.001). Serum levels of ET-1 in the SAH plus E2 and E2 only groups were significantly lower than those in the SAH only and SAH plus vehicle groups (P < 0.001). There was no significant difference between ET-1 levels in the healthy control and SAH plus E2 groups. A significant correlation was found between the cross-sectional areas of basilar artery and ET-1 levels (P < 0.001). The beneficial effect of E2 in attenuating SAH-induced vasospasm may be due in part to decreasing ET-1 production after SAH. The role of E2 in the treatment of cerebral vasospasm after SAH is promising and is worthy of further investigation.

During severe sepsis, several immunological defense mechanisms initiate a cascade of inflammatory events leading to multiorgan failure, including septic encephalopathy and ultimately death. Endothelin-1 (ET-1) has recently been investigated in different cerebral pathologies. Some reports suggest the involvement of ET-1 in sepsis. However, no study to date has reported the alterations in expression of the genes encoding preproET-1 and ET receptors in the frontal cortex of the septic brain. Male Sprague-Dawley (SD) rats 8 weeks of age were administered either saline or 15 mg/kg lipopolysaccharide (LPS) at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. The rats were sacrificed with ether, and the brain tissues were harvested. Systolic and diastolic blood pressure decreased 1 hr after LPS administration and then gradually returned to normal, without any change in the heart rate. We confirmed the induction of endotoxemia in the brains of SD rats by measuring the expression of nitric oxide synthase (NOS) mRNA induced in the cerebrum. The expression of inducible NOS (iNOS) mRNA in the brains of SD rat after LPS administration was 30-fold higher than that in the brains of control rats. mRNA expression of preproET-1 in the frontal cortex of SD rats after LPS administration was 2-fold higher than that in control rats. A time-dependent increase in the expression of the gene encoding the ET_A receptor (vasoconstrictive property) after LPS administration was observed in SD rat brain, whereas expression of the gene encoding the ET_B receptor (vasodilatatory property) showed an initial upregulation and then gradually decreased as sepsis progressed. In conclusion, we report for the first time that expressions of the genes encoding ET-1 and ET receptors are altered in the endotoxemic brain and that these alterations are time-dependent in SD rats. The alterations in the ET system in brain tissue observed in the present study may contribute to the understanding of the pathophysiological changes in the endotoxemic brain.

The substantial role of endothelin-1 (ET-1) in the development of cerebral vasospasm (CVS) after subarachnoidal hemorrhage (SAH) has been demonstrated by numerous experimental and, recently, clinical investigations. Whether the expression or function of the ET(B) receptor is altered in CVS is still unclear, however. The aim of the present study was, therefore, to characterize the cerebroarterial ET(B) receptor function during CVS. Experimental CVS was induced by the rat double-hemorrhage model. Reduction of the cerebral blood flow (CBF) was confirmed by magnetic resonance perfusion-weighted imaging. Animals were sacrificed on days 3 (d3) and 5 (d5) after CVS induction. The basilar arteries (BA) were dissected, cut into ring segments, and prepared for measurement of isometric force in an organ bath. Concentration-effect curves (CECs) were constructed by cumulative application of ET-1, acetylcholine (Ach), or sarafotoxin S6c (S6c). Segments with (E+) endothelial function were used. CECs were compared by the maximum effect (E_max), the pD_2, and the shift calculated on the pD₂ level. The pD₂ is the negative decadic logarithm of the concentration producing the half maximal effect (−log₁₀EC₅₀). After SAH, the relative regional CBF in the d3 and d5 groups was reduced to 63% and 32%, respectively, of the CBF in controls. ET-1 induced a dose-dependent contraction of segments with and segments without CVS. In E+ segments, the E_max for ET-1 was not significantly changed after SAH (mean values [ ± SEM] of 104% ± 4% for the control group, 106% ± 4% for the d3 group, and 104% ± 3% for the d5 group). The CECs, however, were significantly shifted to the left versus the control by factors of 2.4 in the d3 group and 3.6 in the d5 group. Relaxation by S6c was significantly reduced after SAH (E_max: 73% ± 11% in the control group, 21% ± 13% in the d3 group, and 13% ± 8% in the d5 group), whereas relaxation associated with Ach was not significantly changed (E_max: 45% ± 7% in the control group, 56% ± 6% in the d3 group, and 43% ± 6% in the d5 group). Significant contraction by S6c was not observed in E+ and E − segments in any of the study groups. The present data indicate the loss of the ET(B) receptor–mediated relaxation of the cerebral arteries in cases of CVS, which is independent of the endothelial nitric oxide synthase level.

Endothelin-1, a potent vasoconstrictive peptide, has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). The goal of this study was to evaluate the effect of continuous intravenous infusion of a highly selective endothelin-converting enzyme-1 inhibitor, CGS 35066, on the prevention and reversal of cerebral vasospasm following SAH. New Zealand white rabbits were subjected to SAH by injecting autologous arterial blood into the cisterna magna. Infusion of CGS 35066 at dosages of 1, 3, or 10 mg/kg/ day was initiated either 1 hr and 24 hrs later in the prevention and reversal protocols, respectively. Animals were sacrificed by perfusion-fixation 48 hrs after SAH induction. The cross-sectional areas of basilar arteries were measured using computer-assisted videomicroscopy. Ultrastructural changes in basilar arteries were determined using electron microscopy. CGS 35066 significantly prevented and reversed the arterial narrowing after SAH in all three groups. The mean cross-sectional areas of arteries from animals in both the prevention and reversal protocol groups that received 10 mg/kg/day of CGS 35066 did not differ significantly from those of the healthy controls. Histological studies of the basilar artery in the 10 mg/kg/day treatment group did not show pathomorphological changes, such as corrugation of the endothelium seen at 2 days after SAH induction or vacuole formation in the endothelial cells noted in the vehicle-treated SAH group. These findings suggest that CGS 35066 is a promising therapeutic agent for the prevention and reversal of cerebral vasospasm after SAH. It also prevents the pathological changes in vascular walls due to SAH.

Endothelin-1 (ET-1) acts at selected brain loci to elicit a pressor response and secretion of vasopressin (AVP). Glutamatergic receptors of the N-methyl-D-aspartate (NMDA) subtype mediate ET-1–induced AVP secretion in vitro, but the role of glutamatergic receptors in the pressor response and the secretion of AVP in vivo has not been studied. We hypothesized that both the pressor response and AVP secretion in response to ET-1 microinjection into subfornical organ (SFO) would be suppressed by ionotropic glutamatergic receptor antagonists in the paraventricular nucleus (PVN). Sinoaortic denervated male Long Evans rats were equipped with intracerebral cannulae directed into the SFO and the magnocellular region of the PVN bilaterally. Experiments were performed 5 days later in conscious rats. Direct injection of 5 pmol ET-1 into the SFO resulted in a 20 ± 3 mm Hg increase in mean arterial pressure (MAP) (± SE) and a 14.1 ± 0.3 pg/ml increase in the mean plasma AVP level (± SE) (P < 0.001 vs. artificial CSF) that was blocked by selective ET_A inhibition. Neither the pressor response nor the increase in plasma AVP in response to ET-1 was altered despite prior injection of the NMDA blocker diclozipine (5 μg, MK801) into PVN bilaterally. In contrast, bilateral PVN injection with 6-cyano-7-nitroquinoxaline-2,3-dione (40 nmol, CNQX) prevented the pressor response (MAP ± SE, − 4 ± 4 mm Hg) and also inhibited AVP secretion (mean AVP level ± SE, 0.16 ± 0.50 pg/ ml) (P < 0.001 vs. vehicle in PVN after injection of ET-1 into SFO). These findings support the conclusion that both the pressor response and AVP secretion in response to ET-1 acting at the SFO are mediated by a non-NMDA, most likely an aminopropionic acid glutamatergic receptor within the PVN.

Trabecular meshwork (TM) cells are now considered to play an active role in the aqueous outflow mechanism because they exhibit smooth muscle–like contractile properties. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been proposed to play a role in the local regulation of aqueous outflow and intraocular pressure (IOP) control. We propose an in vitro culture model as a method for the study of ET-1–induced human TM (HTM) cell contractility and for the study of whether pre-incubation with flunarizine, a calcium-channel blocker, can inhibit the action of ET-1. Experiments were performed on semiconfluent HTM cells (primary cultures established from normotensive human donor eyes) at the second passage, with phosphate-buffered saline (PBS) as a control. The contractile status of the cells was evaluated by a morphometric analysis of cell area, assuming that HTM cells in culture are able to reduce their area as a consequence of cytoskeletal contraction, rather than regulatory volume decrease. After incubation with 10 μM ET-1 for 5 mins, we observed a reduction of HTM cell area with respect to PBS-treated cells: 2425 ± 876 μm² versus 3125 ± 987 μm² (P < 0.001); and cells exhibited a retraction in shape and a reduction in number of indented profiles. Administration of ET-1 at progressively lower doses produced a corresponding lower reduction of HTM cell area, suggesting a dose-response effect of ET-1. Pre-incubation with 10 μM flunarizine strongly inhibited the ET-1 effect on HTM cell contraction: 2806 ± 865 μm² versus 2910 ± 846 μm² (P = not significant). Our data indicate that ET-1 induced a statistically significant reduction in the area of HTM cells versus controls, and that ET-1 can directly influence the aqueous outflow. Moreover, we observed that flunarizine inhibited the effect of ET-1 on the HTM cells.

The retinal ischemia-reperfusion model is used in the study of transient ischemia–related diseases, such as central retinal artery occlusion, angle-closure glaucoma, and others. There are two methods for experimentally producing an ischemia-reperfusion model in the rat retina: (i) the intraocular pressure is greatly raised by increasing the height of the infusion bottle connected with the needle in the anterior chamber; or (ii) the blood vessel that accompanies the optic nerve in retina is ligated. However, each method has some drawbacks. For example, in the first method, the needle must be fixed in the anterior chamber for 1 hr, thus, the technique is not stable and mechanical damage to ocular structures sometimes occurs. In the second method, because of the unavoidable involvement of the optic nerve, damage to the nerve induces retinal changes unrelated to ischemia. In this study, we injected endothelin (ET)-1 under the conjunctiva of the eyeball (subconjunctival injection), and evaluated whether a retinal ischemia-reperfusion model could be generated by this method, simply and noninvasively. We injected 4 × 10⁻⁵ M ET-1 solution into the right eye of the rat and injected a control vehicle (artificial tears) into the left eye. From 5–60 mins after the injection, 50 mg/ml fluorescein isothiocyanate (FITC)-dextran was injected to the left ventricle of heart. Then, the retina was removed and flat mounted. We compared the perfusion conditions of the FITC-dextran to each retina in the right and left eye. There was a complete perfusion of FITC-dextran in the retinal main artery, vein, and the capillary vessels in all of the control eyes. However, perfusion could not be completely observed in the ET-1 injected eye from 5–35 mins after injection; afterwards, the flow was returned. This method of subconjunctival injection of ET-1 is, thus, a feasible technical option for producing a retinal ischemia-reperfusion model in rat.

Diabetic retinopathy (DR), one of the most serious causes of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in retina. Recently, leukocyte adhesion (leukostasis) is blamed for the occlusion of retinal capillary vascularity, which ultimately contributes to the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in the diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is deeply linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study yet has reported whether ET-1 dual receptor antagonist could alter the upregulated VEGF and ICAM-1 levels in the diabetic retina. The present study investigated the effect of ET_A/B dual receptor antagonist (SB209670; 1 mg/rat/day) on the expression of VEGF and ICAM-1 in the diabetic rat retina. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 65 mg/kg) in Sprague-Dawley rats, whereas control rats (non-DM control) received only citrate buffer. After 1 week, the STZ-administered rats were randomly divided into two groups: one group (DM+SB209670) received ET_A/B dual receptor antagonist for 2 weeks, and a vehicle group (DM+vehicle) was treated only with saline. After the treatment period, the retinas were removed from the eyeballs. In DM+vehicle group, the VEGF expression of the retinas was significantly increased (32.8 pg/mg) in comparison to that in the non-DM control group (26.2 pg/mg); this upregulation of VEGF was reversed in the DM+SB209670 group (28.6 pg/mg). The expression of retinal ICAM-1 was increased in the DM+vehicle group (152.2 pg/mg) compared with the non-DM control group (121.6 pg/mg). However, SB209670 treatment did not alter the expression of retinal ICAM-1 level (154.8 pg/ml) in DM rats. Thus we conclude that an ET_A/B dual receptor antagonist could reverse the expression level of VEGF in the diabetic retina while failing to normalize the upregulated ICAM-1 expression.

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.

Altered activity of retinal endothelin-1 (ET-1) and nitric oxide may play a causal role in the hemodynamic and histopathological changes of diabetic retinopathy. This study evaluated the therapeutic potential of long-term selective blockade of the ET-1_A receptor (ETRA) to prevent the development of retinopathy in a genetic mouse model of nonobese type 1 diabetes (NOD). Mice with NOD that received subcutaneous implantation of insulin pellets and wild-type control mice were treated for 4 months with the selective ETRA antagonist LU208075 (30mg/kg/day) via drinking water. At the end of the study, blood glucose levels were evaluated, and animals were anesthetized and perfused intracardially with FITC-labeled dextran. Retinas were removed and either fixed in formalin for confocal microscope evaluation of retinal vascular filling or transferred to RNALater for quantitative reverse transcriptase–polymerase chain reaction to evaluate expression of NOS-3, NOS-1, ET-1, ETRA, ETRB, and the angiogenic factor adrenomedullin. Compared with wild-type controls, expression of ET-1, ETRA, ETRB, and adrenomedullin in mice with NOD were markedly upregulated in the retinas of nontreated mice (cycle time values relative to GAPDH [ΔCt], 14.8 vs. 13.7, 18.57 vs. 17.5, 10.76 vs. 9.9, and 11.7 vs. 9.1, respectively). Mean integral fluorescence intensity (MIFI) of retinal vascular filling was reduced from normal values of 24 to 12.5 in nontreated animals. LU208075 treatment normalized the upregulated expression of ET-1 and adrenomedullin, as well as the deficit in MIFI, but did not affect the increased ETRA and ETRB expression or the elevated plasma glucose levels found in nontreated animals. NOS isoform expression was essentially unchanged. ETRA antagonists may provide a novel therapeutic strategy to slow or prevent progression of retinal microvascular damage and proliferation in patients for whom there is clear evidence of activation of the ET-1 system.

Endothelin (ET)-1 can influence cancer invasion and metastasis by exerting an autocrine (epithelial) or paracrine (stromal) influence on growth. ET-1 is generated from big ET-1 by endothelin-converting enzyme (ECE)-1, which has four recognized isoforms, ECE-1a, ECE-1b, ECE-1c, and ECE-1d, differing only in their amino-terminal regions. This study investigated the expression and localization of the ECE-1 isoforms in prostate cancer (PC). The epithelial cell lines used were androgen-sensitive LNCaP, androgen-independent PC-3 and Du145, and nonmalignant transformed PNT1-a, PNT2-C2, and P4E6 prostate cells. Primary cells derived from malignant and benign tissue from radical prostatectomies were also exploited. Previously, we reported increased ECE-1 expression in androgen-independent PC cell lines, as compared with androgen-sensitive cells. Our present data show that transcripts for all ECE-1 isoforms were present in all epithelial cell lines analyzed. However, only the ECE-1c protein was detectable in PC-3, Du145, PNT2-C2, and PNT1-a cells. ECE-1c localized to both the cell surface and intracellular compartments in individual cell lines. In primary stromal cells, all individual ECE-1 isoforms were expressed at the mRNA level, with the exception of ECE-1a. ECE-1b and ECE-1c protein levels were higher in malignant stromal cells, as compared with benign cells. In stroma, ECE-1c protein was localized to the cell surface, with filamentous immunoreactivity throughout the cell, whereas ECE-1b immunoreactivity was punctate throughout the cytoplasm. The upregulation of the ECE-1c isoform in PC cell lines is being investigated further.

Endothelin (ET)-1 is an autocrine/paracrine growth factor or an antiapoptotic factor in human cancers, and blockade of ET-1 receptors can sensitize human tumor cells to apoptosis. The role of the ET-1 axis in the proliferation and/or apoptosis of melanoma cells and in their response to the alkylating agent, dacarbazine (DTIC), used in clinical treatment of human melanoma were investigated in five human melanoma cell lines obtained form surgical resection specimens. Melanoma cells expressed the messenger RNAs (mRNAs) for the components of the ET-1 axis. ET-1 binding was mediated by ET_B but was inhomogeneous among melanoma cells. Exogenous ET-1 did not induce human melanoma cell proliferation. Bosentan, a dual ET_A/B-receptor antagonist, decreased melanoma cell viability and DNA synthesis and induced melanoma cell apoptosis in defined human melanoma cells. Bosentan potentiated Fas ligand–induced apoptosis only in one melanoma cell line. Variants of ET _B were determined using reverse transcriptase (RT) polymerase chain reaction (PCR) and primers spanning the whole sequence of the ET _B gene. ET _B variants were demonstrated only in one of the five cell lines, corresponding to the absence of ET-1 binding by these cells. Bosentan did not inhibit the effects of alkylating agents, and the effects of bosentan and alkylating agents were additive in melanoma cells. In conclusion, exogenous ET-1 is not a growth factor for human melanoma cells, but blockade of ET receptors decreases proliferation, induces apoptosis, and potentiates the effects of anticancer agents in defined melanoma cells, suggesting that combination therapy of ET-receptor antagonists with alkylating agents may improve their efficacy.

Endothelin (ET)-B receptors are expressed in human breast carcinoma. We previously demonstrated that intravenous administration of the ET_B receptor agonist, IRL-1620, to tumor-bearing rats, increased blood perfusion and enhanced delivery of paclitaxel to breast tumor tissue. The present study was conducted to determine whether IRL-1620 alters the pharmacokinetics of paclitaxel. Breast tumor–bearing rats were given 0.3 ml/kg saline or 3 nmol/kg IRL-1620 by intravenous (iv) administration. Fifteen minutes after saline or IRL-1620, 40 μCi/rat ³H-Paclitaxel was administered iv and serial plasma samples were collected until 24 hrs. ³H-Paclitaxel radioactivity in the plasma samples was measured by liquid scintillation counting. Data were fit to a three-compartment model and pharmacokinetic parameters were generated using WinNonlin software. IRL-1620 did not produce any change in the plasma paclitaxel pharmacokinetics of tumor-bearing rats. The AUC_0–∞ (9.43 ± 3.18 μg-hr/ml), clearance (0.69 ± 0.17 l/hr/kg), volume of distribution (10.31 ± 4.54 l/kg), and half-life (1.0 ± 0.32 hrs) of paclitaxel were similar between rats treated with saline or IRL-1620. In conclusion, the ET_B receptor agonist, IRL-1620, does not alter paclitaxel plasma pharmacokinetics and, therefore, could be used to augment the delivery of paclitaxel to the tumor tissue.

The green tea polyphenol, epigallocatechin-3-gallate (EGCG), has been shown to prevent cancer; however, a precise mechanism responsible for tumor growth inhibition has not yet been clearly described. The endothelin (ET) A receptor (ET_AR)/ET-1 autocrine pathway is overexpressed in ovarian carcinoma and triggers tumor growth, neoangiogenesis, and invasion. These latter tumor-promoting effects are mediated through the activation of cyclooxygenase (COX)-1– and COX-2–dependent pathways by ET-1. In the present study, pretreatment of HEY and OVCA 433 ovarian carcinoma cell lines with green tea and EGCG inhibited ET-1/ET_AR expression, ET_AR-mediated COX-1/2 mRNA expression, and COX-2 promoter activity. These effects were associated with a significant reduction in the COX-1/2–derived prostaglandin E₂ (PGE₂) production. These results provide a novel insight into the mechanism by which EGCG, by affecting ET_AR-dependent COX-1/2 pathways may inhibit ovarian tumors suggesting that EGCG may be useful in preventing and treating ovarian carcinoma in which activation of ET_AR by ET-1 plays a critical role in tumor growth and progression.

In a range of human cancers, tumorigenesis is promoted by activation of the endothelin A receptor (ET_AR)/endothelin-1 (ET-1) axis. ET-1 and ET_AR are overexpressed in primary and metastatic ovarian carcinomas, and high levels of ET-1 are detectable in patient ascites, suggesting that ET-1 may promote tumor dissemination. Moreover, in these tumors, engagement of ET_A receptor by ET-1 triggers tumor growth, survival, angiogenesis, and invasiveness. Thus, ET-1 enhances the secretion of matrix metalloproteinases, disrupts intercellular communications, and stimulates cell migration and invasion. Therefore, we investigated the role of the ET-1/ET_AR autocrine axis in promoting epithelial to mesenchymal transition (EMT) in ovarian tumor cells, a key event in cancer metastasis, in which epithelial cells depolarize, disassemble cell-cell contacts, and adopt an invasive phenotype. Here, we examine the potential role of ET-1 in regulating cell morphology and behavior and epithelial and mesenchymal proteins employing an in vitro 3-D culture system. We found that in 3-D serum-free collagen I gel cultures, HEY and OVCA 433 ovarian carcinoma cells undergo fibroblast-like morphologic changes between 3 and 5 days of ET-1 treatment. In these cells, ET-1 induces loss of adherens and tight-junction protein expression, E-cadherin, β-catenin, and zonula occludens-1, and gain of N-cadherin and vimentin expression. These results confirm the ability of ET-1 to promote EMT, a metastable process involving sustained loss of epithelial markers and gain of mesenchymal markers. Collectively, these findings provide evidence of a critical role for the ET-1/ET_AR axis during distinct steps of ovarian carcinoma progression, thus underlining this axis as a potential target in the treatment of ovarian cancer.

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinomas. In these tumors, the presence of ET-1 correlates with tumor grade, enhanced neovascularization, and with vascular endothelial growth factor (VEGF) expression. ET-1 acts as an autocrine factor selectively through ET_A receptor (ET_AR), predominantly expressed in ovarian carcinoma cells resulting in increased VEGF production and VEGF-mediated angiogenic effects. Previous results demonstrated that in ovarian carcinoma cells, activation of the ET-1/ET_AR axis promotes cell proliferation, neovascularization, and invasion, which are the principal hallmarks of tumor progression. The present study was designed to investigate the in vitro effects of trans, trans-2(4-methoxydhenyl)-4-(1–3-benzodiazol-5-yl)-1-(dibutylaminocarbonylmethyl)-pyrrolidine-3-carboxylic acid (ZD4054), an orally active specific ET_AR antagonist, on the ET-1–induced mitogenic effect in OVCA 433 and HEY ovarian carcinoma cell lines secreting ET-1 and expressing ET_AR and ET_BR mRNA. We show that ET_AR blockade by ZD4054 inhibits ET-1–induced mitogenic effects, while the ET_BR antagonist, BQ 788, is ineffective. In conclusion, our data demonstrate that ZD4054 is capable in inhibiting the proliferative activity of ET-1, indicating that this specific ET_AR antagonist may be a potential candidate in developing novel treatment of ovarian carcinoma.

Trigeminal neuropathic pain, which is associated with marked orofacial mechanical allodynia, is frequently refractory to currently available drugs. Because endothelins (ETs) can contribute to nociceptive changes in animal models of inflammatory, cancer, and diabetic neuropathic pain, the present study evaluated the influence of ET_A and ET_B receptor antagonists on orofacial mechanical allodynia in a rat model of trigeminal neuropathic pain. Unilateral constriction (C) of the infraorbital nerve (ION) caused pronounced and sustained bilateral mechanical allodynia, evaluated by application of von Frey hairs to the vibrissal pad. Mechanical allodynia on postoperative days 12–15 after nerve injury was abolished for up to 90 mins by subcutaneous administration of 2.5 mg/kg morphine, but was fully refractory to intravenous (iv) administration of 10 mg/kg of the dual ET_A plus ET_B or selective ET_A receptor antagonists, bosentan and atrasentan, respectively. In sharp contrast, iv administration of 20 mg/kg of the selective ET_B receptor antagonist, A-192621, caused a net 61 ± 15% reduction of mechanical threshold, lasting 2 hrs. Co-injection of atrasentan plus A-192621 did not modify ION injury-induced mechanical allodynia. Injection of 10 pmol ET-1 into the upper lip of naive rats caused ipsilateral mechanical allodynia lasting up to 5 hrs. Thus, ET_B receptor–mediated mechanisms contribute to orofacial mechanical allodynia induced by CION injury, but, some-how, functional ET_A receptors are required for expression of the antiallodynic effect of ET_B receptor blockade.

In addition to causing overt nociception, intraplantar (ipl) endothelin (ET)-1 injection into the rat hind paw induces hyperalgesia to mechanical stimuli, mediated via local ET_B receptors coupled to protein kinase (PK) C, but not PKA. The present study further examines the intracellular signaling mechanisms underlying this effect of ET-1. ET-1 (30 pmol) or phospate-buffered saline (PBS) was injected ipl in rats and the threshold of responsiveness to mechanical stimulation was assessed repeatedly each hour up to 8 hrs and 24 hrs, using the dynamic plantar aesthesiometer test, which detects the minimal pressure required to evoke paw withdrawal. Different groups were treated, 15 mins before ET-1 administration, with ipsilateral injection of selective inhibitors of either phospholipase (PL) A₂ (1 nmol PACOCF3), PLC (30 pmol U73122), PKC (1 nmol GF109203X), p38 mitogen-activated protein kinase (MAPK; 30 nmol SB203580), extracellular signal-regulated kinase (ERK1/2; 30 nmol PD98059), c-Jun N-terminal kinase (JNK; 30 nmol SP600125), or vehicle, to assess their influence on the hyperalgesic response. The mechanical hyperalgesia caused by ET-1 started 2 hrs after injection, peaked at 5 hrs (PBS, 29 ± 0.5 g; ET-1, 17 ± 1.3 g) and lasted up to 8 hrs. The inhibitors of PLC, PKC, p38 MAPK, ERK1/2, and JNK caused long-lasting reductions of the mechanical hyperalgesia (inhibitions at 4 hrs of 100%, 90%, 97%, 90%, and 100%, respectively), but the PLA₂ inhibitor reduced hyperalgesia only at 4 hrs (by 58%). Thus, mechanical hyperalgesia triggered by ET-1 in the rat hind paw depends importantly on signaling pathways involving PLC, PKC, p38 MAPK, ERK1/2, and JNK, whereas the contribution of PLA₂ is relatively minor.

Endothelin (ET)-1 evokes a burning pruritus sensation when injected intradermally in humans and nocifensive behavior when injected into the hind paw of rodents. Because pain and pruritus are clearly distinct nociceptive sensory modalities in humans, the current study evaluates the potential of ET-1 to elicit scratching behavior in mice. Mice received an intradermal injection of 1–30 pmol ET-1; 10 μg of the mast cell degranulator compound, 48/80; 100 nmol histamine; or vehicle into the scruff, and the number of scratching bouts displayed during the first 40 mins was recorded. ET-1 caused dose-dependent scratching bouts, which, like the responses to histamine and compound 48/80, occurred mainly during the first 5 to 10 mins of injection, but fewer episodes were also seen up to 35 mins. The effect of ET-1 was maximal at 10 pmol (total 40 ± 7 bouts), a value similar to that caused by histamine (52 ± 5 bouts) and compound 48/80 (53 ± 6 bouts). The selective ET_B receptor agonist, IRL-1620 (10 pmol), was not pruritic per se, and actually inhibited responses to histamine and ET-1. Pruritus induced by ET-1 was inhibited by the ET_A receptor antagonists, 10 nmol BQ-123 (co-injected; net inhibition, 87%) and 10 mg/kg atrasentan (intraperitoneal administration; net inhibition, 83%), or the ET_B receptor antagonist, 20 mg/kg A-192621 (intraperitoneal administration; net inhibition, 64%), but the response was augmented by co-injection of the ET_B receptor antagonist, 3 nmol BQ-788 (net potentiation, 234%). Responses to compound 48/80 or responsiveness of vehicle-treated mice were unaffected by these antagonists. Thus, ET-1 displays potent pruritic actions in the mouse mediated to a substantial extent via local ET_A receptors. The findings with IRL-1620 and BQ-788 suggest that local ET_B receptors exert an antipruritic role, but, for reasons still unknown, the results obtained using systemic A-192621 injection are at variance with this view.

Long-term use of morphine in pain management leads to adverse effects, such as development of antinociceptive tolerance. We have previously shown the involvement of central endothelin (ET) mechanisms in morphine analgesia and development of tolerance in vivo. The present study was conducted to investigate the in vitro mechanism of interaction of the ET_A receptor antagonist, BMS182874, and morphine during acute and chronic morphine tolerance in SH-SY5Y cells. SH-SY5Y cells were exposed to acute and chronic treatment with vehicle, morphine, ET-1, BMS182874, or morphine plus BMS182874. Activation of G-protein–coupled receptors in SH-SY5Y cells was determined using [³⁵S]GTPγS binding assays. Acute morphine treatment produced a concentration-dependent increase in GTP binding. Median effective concentration (EC₅₀) values were significantly decreased after acute morphine treament, suggesting sensitization of opioid receptors. Chronic morphine treatment produced a lower maximal response of GTP binding compared with both control (vehicle treated) and acute morphine treatment, indicating uncoupling of G-proteins. Acute and chronic exposure of cells to ET-1 did not affect changes in ET-1–induced GTP binding. BMS182874 treatment alone (acute or chronic) did not produce G-protein activation. However, in cells chronically cotreated with 10 μM morphine and 1 μM BMS182874, morphine-induced GTP stimulation was significantly higher than control (vehicle treated). The EC₅₀ value after control treatment was 414 nM, and was significantly increased in chronically morphine-treated cells (>1000 nM ). However, the EC₅₀ value in cells receiving a chronic treatment of BMS182874 and 63 nM morphine was significantly reduced compared with control (vehicle treated) and chronic morphine treatment. ET_A antagonists significantly enhance the coupling of G-protein to opioid receptors. Therefore, we propose that restoration of morphine antinociception by ET_A antagonists in morphine-tolerant animals is likely via a G-protein mediated mechanism.

The involvement of central endothelin (ET) receptors in neonatal morphine tolerance has been demonstrated. The present study investigates the role of central ET receptors in morphine withdrawal in neonatal rats. The aim was to determine whether activation of G-proteins coupled to opioid and ET receptors by morphine and various ET receptor modulators is affected during morphine withdrawal in neonatal rats. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets during 7 days. On Day 8, pellets were removed and rats were allowed to undergo withdrawal for 24 hrs. Rat pups were delivered by cesarean section. G-protein stimulation induced by morphine; ET-1; the ET_A receptor antagonist, BMS182874; and the ET_B receptor agonist, IRL1620, were determined in the brain of neonatal rats undergoing morphine withdrawal by [³⁵S]GTPγS binding assay. Morphine produced higher (P < 0.05) maximal stimulation of G-protein in the morphine-withdrawal group (83.60%) compared with the placebo group (66.81%). ET-1–induced G-protein stimulation was also altered, and the median effective concentration (EC₅₀) during morphine withdrawal (170.60 nM) was significantly higher than placebo (62.5 nM; P< 0.05). The maximal stimulation induced by the ET_A receptor antagonist, BMS182874, in the morphine-withdrawal group (86.07%; EC₅₀ = 31.25 nM) was significantly higher than in the placebo group (EC₅₀ > 1000 nM). The ET_B agonist, IRL1620, induced G-protein stimulation was similar in placebo (73.43%, EC₅₀ = 13.26 nM) and morphine-withdrawal groups (75.08%, EC₅₀ = 11.70 nM), respectively. To our knowledge, this is the first report indicating involvement of central ET_A receptors in neonatal morphine withdrawal.

Endothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to painful stimuli. The cellular mechanisms of the ET-1–mediated effects are only poorly understood. TRPV1, the heat-, proton-, and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released by painful stimuli and during inflammation, is a potential target for the action of ET-1. In immunocytochemistry of rat lumbar dorsal root ganglion using TRPV1- and ET_A receptor–specific antibodies, both proteins were found to be co-expressed in small sensory neurons. To provide evidence that ET-1 can modulate TRPV1 activity via the ET_A receptor, we used HEK 293 cells transiently co-expressing a fusion protein of TRPV1 and the yellow fluorescent protein (TRPV1-YFP) and the ET_A receptor. In whole-cell patch clamp recordings of HEK293 cells co-expressing TRPV1-YFP and the ET_A receptor, capsaicin (10 nM) elicited small currents, which were markedly potentiated when capsaicin (10 nM) and ET-1 (100 nM) were applied simultaneously. The data indicate that ET-1 potentiates TRPV1 activity via the ET_A receptor and that this process is likely to play a crucial role in the pain-producing and pain-potentiating effects of ET-1. Thus, ET_A receptor antagonists may be of importance in painful states with increased circulating ET-1 levels, as found in cancer and in chronic inflammation.

Subcutaneous endothelin-1 (ET-1; 200 μM, 2 nmoles/paw) injected into the rat hind paw, has been shown to cause robust hind paw flinching (HPF) and paw licking, and to induce impulses selectively in primary nociceptors. Here we report that a much lower [ET-1] sensitizes the paw to a nocifensive withdrawal response to tactile stimulation (by von Frey hairs, VFH), a sensitization that involves local TRPV1 receptors. Injection of 10 μM ET-1 (0.1 nmole/paw) causes only marginal HPF but rapidly (20 mins after injection) lowers the force threshold for paw withdrawal (PWT) to VFH, to ~30% of pre-injection baseline. Such tactile allodynia persists for 3 hrs. In rats pre-injected with the TRPV1-antagonists capsazepine (CPZ; 1.33 mM) or 5′-iodoresiniferatoxin (I-RTX; 0.13 μM), 15 min before ET-1, a fast initial drop in PWT, as with ET-1 alone, occurs (to 40% or to 19% of baseline, respectively), but this earliest reduction then regresses back to the pre-injection PWT value more rapidly than with ET-1 alone. The recovery of allodynia from the maximum value is about two times faster for ET-1+CPZ and about 4 times faster for ET-1+ I-RTX, compared with that from ET-1 +vehicle (t_½ = 130, 60, and 250 mins, respectively). In contrast, spontaneous pain indicated by overt HPF from ET-1 is not attenuated by TRPV1 antagonists. Tactile allodynia is similarly abbreviated by antagonists of both ET_A (BQ-123, 32 nmoles/paw) and ET_B (BQ-788, 30 nmoles/paw) receptors, whereas HPF is abolished by this ET_A antagonist but enhanced by the ET_B antagonist. We conclude that low ET-1 causes tactile allodynia, which is characterized by a different time-course and pharmacology than ET-1-induced nociception, and that local TRPV1 receptors are involved in the maintenance of this ET-1-induced allodynia but not in the overt algesic action of ET-1.

Endothelin (ET)-1 is an angiogenic factor that, among others, is secreted by endothelial cells during development of several neoplasias. In particular, Kaposi sarcoma (KS) skin lesions show overexpression of the ET-1 system. Spindle cells, which characterize tumor lesions, are of endothelial origin and during disease are infected by human herpesvirus 8 (HHV-8). The majority of these cells are latently infected, suggesting that latent genes are sufficient for maintenance of viral infection and development of KS. The establishment of a reliable infection system is required to better understand the role of viral and cellular angiogenic factors involved in KS progression. For this purpose, we used human microvascular endothelial cells (HMEC-1) to establish an ET-1–producing model of infection with HHV-8. Viral particles purified from BCBL-1 cells were used to infect HMEC-1 monolayer, and infection was assessed by polymerase chain reaction, reverse transcription polymerase chain reaction, and confocal microscopy. Mitochondrial activity and cell viability, measured at 24, 48, and 72 hours after infection by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was reduced in HHV-8–infected cells compared with control. In contrast, 1 week after infection, HHV-8–positive cells showed higher mitochondrial functionality. Endothelin production was measured in culture media collected at 24, 48, and 72 hours after infection. The levels of endothelin precursor big endothelin-1 was increased 3 days after infection, although big ET-1 and ET-1 production did not differ significantly between infected and uninfected cells. These results indicate this model as a useful tool to further characterize the effects of HHV-8 in the early and late phases of infection, and to determine its ability to interfere with the endothelin system.

Cerebral malaria (CM) remains a deadly complication of Plasmodium falciparum infection, and children are at high risk of developing encephalopathy as a result of CM. This is probably a consequence of the activation of many of the inflammatory cytokines as well as the glial cells and the vascular endothelium in the brain. We have previously demonstrated that there is a striking reduction in cerebral blood flow by magnetic resonance imaging when mice are infected with Plasmodium berghei ANKA (PbA), and we now demonstrate a possible role for endothelin (ET-1) in the pathogenesis of CM. The brains of female C57BL/6 mice with PbA infection were examined at Day 5 for the expression of ET-1, endothelin converting enzyme (ECE), and the endothelin receptors A and B (ET_A and ET_B) by both reverse transcription–polymerase chain reaction (RT-PCR) and quantitative real-time PCR. ET-1 and ECE mRNA expression was markedly increased by RT-PCR in PbA-infected mice. Real-time quantitative PCR demonstrated a 3-fold increase in ET-1 (P < 0.05) and a significant increase in ET_A and ET_B expression (P < 0.05) in PbA-infected mice. Histopathology bof PbA-infected mice demonstrated a transformation in the morphology of microglial cells and clustering of these cells consistent with activation. Though the full impact of ET-1 on CM remains to be elucidated, these findings demonstrate that in the murine model, there is a significant increase in ET-1 and its components, which is associated with the vasculopathy and immunopathology of CM.

Sepsis involves a heterogeneous class of syndromes, and septic shock, a severe form of sepsis, is associated with the development of progressive damage in multiple organs. The present study examined the time-dependent alterations of endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) levels in liver tissue in a septic rat model. Healthy male Wistar rats aged 15 weeks received 15 mg/kg lipopolysaccharide (LPS) and were sacrificed at different time points (1, 3, 6, and 10 hrs after treatment). Rats that did not receive LPS were considered to be controls. A 28-fold increase in the ET-1 level was observed in liver tissue 10 hrs after LPS administration. VEGF was also altered in hepatic tissue in a time-dependent manner. A gradual increase of VEGF expression in liver tissue after LPS administration was observed. Expression of Flt-1, the vascular permeability receptor of VEGF, was also increased in liver tissue after LPS administration. ET-1 is a potent vasoconstrictor and, therefore, may play a role in the regulation of hepatic perfusion in a sepsis model. On the other hand, VEGF may be involved in capillary leakage in liver tissue after LPS administration. The present findings suggest that there might be a loss of balance between the ET-1 and VEGF levels in the septic liver at different time points, which could contribute to the pathogenesis of acute liver injury in endotoxemia.