Experimental Biology and Medicine

The STAT transcription factors are mediators of signal transduction of a variety of factors, including interferons (IFNs), interleukins, growth factors, and peptide hormones. Subsequent to activation, STATs are translocated to the nucleus apparently through the well-described importin/Ran system, where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, thus raising the question of how they are imported into the nucleus. Of considerable interest is the observation that ligands and/or receptors that signal through STATs contain putative NLSs and, where examined, either ligand or receptor undergoes nuclear translocation. We hypothesize that ligands and/or their receptors serve as vehicles for the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFN_γ as a model system, we provide a possible mechanism for how this direct role is fulfilled. A functional NLS has been identified in a C-terminal domain of IFN_r This domain and the NLS contained within are crucial for the biological properties of IFN_γ in that a peptide encompassing this domain is sufficient to induce an antiviral state. Further, this domain binds specifically to a membrane-proximal region internal cytoplasmic domain of the α subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.

Rapid progress has occurred recently in understanding the origin, chemistry, synthesis, control, and actions of endogenous materials that may be ligands for the cardiac glycoside binding site on the mammalian sodium pump (Na, K-ATPase). The present paper reviews this progress and examines in detail the evidence supporting ouabain-like and bufodienolide-like compounds as functioning in endogenous control of sodium pump activity, renal sodium excretion, blood pressure, and cardiovascular contractility. Other novel compounds identified in this search as potentially influencing natriuresis and blood pressure are also discussed.

Nicotine, a major component of tobacco and cigarette smoking is an addictive agent and has been characterized as a drug of abuse by the US Surgeon General (1–3). The economic burden due to abuse of this drug is substantial because of the well-documented pathophysiological effects of nicotine on organs in the cardiovascular, respiratory, hepatic, renal, and nervous systems (3). Association of nicotine through cigarette smoking with the increased incidence of pancreatitis and pancreatic cancer has also been reported (4–18). A survey on the association between cigarette smoking and pancreatic cancer showed that cigarette smokers had a significant 70% higher risk of pancreatic cancer in comparison to nonsmokers (8–16). When compared with nonsmokers, subjects who smoke filtered cigarettes had a 50% elevated risk. The proportion of pancreatic cancer attributable to cigarette smoking was 29% in blacks and 26% in whites (10). Most of the data that link cigarette smoke/nicotine to pancreatic diseases were gathered in humans. Recent studies with animals have also shown that nicotine or its metabolites could induce pathological and functional changes in the pancreas (17–25). This review will present and discuss the current understanding of the pathophysiology induced by nicotine in the exocrine pancreas and the possible mechanism of action of nicotine.

Effects of Nicotine and its Metabolites on the Structural and Functional Changes of the Exocrine Pancreas

Cotinine and nornicotine are natural metabolites of nicotine. About 80%-90% of the dose of nicotine consumed can be accounted for in human urinary metabolites. In rodents, hepatic nicotine metabolism is found to involve cytochrome p450s that catalyze the first step of this pathway (26, 27). It has been demonstrated that pancreatitis could be induced in mice fed a caerulein and choline-deficient ethionine (CDE) supplemented diet (28–33).

Dehydroepiandrosterone (DHEA) is the major adrenal steroid of young adults; however, its physiologic functions, if any, are not known. The purpose of this review is to evaluate the current literature in which DHEA was administered to either humans or experimental animals to discern what these functions might be. Reports are divided into five areas: neurologic, immunologic, cardiovascular, oncologic, and metabolic. Particular attention is paid to the dosage and route of administration. This type of analysis shows that at the lowest doses, DHEA has effects on neurologic and immunologic tissues, suggesting that these two sites may be physiologic targets. DHEA also affects cardiologic and metabolic functions as well as tumor growth, but such actions require higher doses and may reflect'pharmacologic'activities. It is proposed that DHEA's pattern of activity represents a new class of steroid hormones, the “Regnantoids.” Further progress in the endocrinology of this family of steroids may only come when synthetic, long-acting analogs of DHEA are available for in vitro studies to allow correlations between hormone action and receptor binding.

Essential hypertension is characterized by a near normal cardiac output but an increase in total peripheral resistance. In turn, total peripheral resistance is controlled directly by the diameter of the small arteries and arterioles like those in the kidney. The dynamic regulation of renal vessel diameter is governed by the contractile state of the vascular smooth muscle cells that line the vessel walls. This review addresses the role of a number of different ion channels to initiate and maintain the contractile state of the vascular smooth muscle cells in hypertension and the potential prevention of hypertension through gene therapy. These specific channels include Ca²⁺, K_Ca, K_v, and Cl~ channels. In hypertension, it has been reported that increased activity of Ca²⁺ channels and decreased activity of K_v channels are responsible for the increased contractile tone and resting membrane potential observed in dissociated vascular smooth muscle cells from the spontaneously hypertensive rat. In contrast, increased activity of K_Ca channels in vascular smooth muscle cells of the SHR has been hypothesized to dampen or brake the activity of Ca²⁺ and K_v channels. Finally, recent evidence suggests that introducing angiotensin II type-1 receptor antisense into prehypertensive rat pups prevents the onset of pathophysiological alterations observed in hypertension including K⁺ channel alterations. These results suggest that gene therapy may be a useful pharmacological and physiological tool to combat hypertension.

The coitally induced gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge in the rabbit is preceded by an enhanced secretion of hypothalamic norepinephrine (NE). To investigate if adrenergic receptors are essential for the GnRH/LH surge, we administered a specific α₁ receptor blocker, prazosin, into either the arcuate nucleus-median eminence (AME) or the third cerebroventricle (3rd V) of tethered, freely moving intact female rabbits via push-pull perfusion (PPP). Dual cannulae for PPP and drug infusion were placed stereotaxically either into the AME or AME and 3rd V of each rabbit after insertion of a permanent femoral venous catheter for serial blood sampling. During an experiment, continuous PPP samples and 10-min intermittent blood samples were collected for 5–6 hr. Females received either prazosin or control medium (artificial cerebrospinal fluid into an AME or saline into a 3rd V cannula) for 4 hr, beginning 1 hr before coitus. Intraventricular infusion of prazosin significantly (P < 0.05) suppressed both the postcoital GnRH and the LH surges. Administration of prazosin into the AME also attenuated the magnitude of the postcoital GnRH surge (P < 0.05) whereas postcoital LH values were not decreased below that of the control group (P > 0.05). The results suggest that α₁ receptors are physiologically active in the initiation of the postcoital GnRH release. These findings, along with our earlier report of enhanced postcoital NE secretion, reinforce the hypothesis that NE plays an essential role in the preovulatory GnRH/LH surge in rabbits.

Dietary effects of dehydroepiandrosterone sulfate (DHEAS) supplementation on tissue antioxidants and lipids were investigated in retrovirus infected mice. DHEA is a powerful antioxidant and immunomodulator whose production declines with age. For this study, twenty-four female, 15-month-old C57BL/6 mice were left uninfected while twenty-four were infected with LP-BM5 murine leukemia virus, causing murine AIDS. The retroviral infection caused immune dysfunction and loss of hepatic and cardiac vitamins E and A, resulting in increased lipid peroxides. Treatment with DHEAS at 0.01 or 0.005% in drinking water for 10 weeks post-infection significantly (P < 0.05) lowered lipid peroxidation in both heart and liver tissues. Treatment with DHEAS also largely prevented loss of the antioxidants, such as vitamin E and A, and prevented loss of phospholipid in the hearts and livers of the old uninfected as well as infected mice. This study suggests that DHEAS supplementation reduces damage associated with elevated oxidation due to aging and retrovirus infection.

Reduced dietary calories can delay the onset and diminish the severity of murine autoimmunities of numerous inbred and hybrid mutant strains. We sought to determine whether the precipitous, autoimmune, crescentic glomerulonephritis of recombinant inbred SCG/Kj mice could be abrogated similarly by calorie restriction. Weanling SCG/Kj mice develop hematuria and proteinuria, and 50% die as 16-week-old young adults. In this study, 113 4-week-old SCG/Kj mice were fed either ad libitum a milled chow (Group A, n = 50), or a semipurified diet (Group B, n = 29), or were fed a calorie-restricted semipurified diet (Group C, n = 34), so that mice of Group C consumed approximately 32% fewer calories, but similar amounts of essential dietary constituents as those of Group B. Calorie restriction of Group C provided modest (P = 0.05) or substantial survival advantage (P = 0.001) compared to the ad libitum feeding of Groups B or A, respectively. Progression to severe glomerular pathology was delayed among Group C mice, with more than a 5-week delay to heavy proteinuria (>100 mg/dl), a >4-week delay to hematuria, and a >5-week delay to median mortality, representing a 20% or 25% extension of median life span, compared to ad libitum-fed Group B and A mice, respectively. Mean glomerular histopathology scores were also lower in calorie-restricted mice compared to the ad libitum-ied cohorts (P = 0.001). Titers of anti-ss-DNA, ds-DNA, and ANCA autoantibodies developed in weanlings prior to the full imposition of calorie restriction and were not reduced significantly by calorie restriction.

Transplantation of MHC-compatible, T-cell-depleted, bone marrow cells has successfully treated autoimmunities, immunodeficiencies, malignancies, and developmental deficiencies of the hematopoietic system. Recombinant inbred SCG/Kj mice develop spontaneous crescentic glomerulonephritis, systemic vasculitis, and a lymphoproliferative disorder early in life. To determine whether the precipitous autoimmune disease of SCG/Kj mice could be treated by bone marrow transplantation, 30 SCG/Kj mice were engrafted with T-cell-depleted, bone marrow (TCDM) from allogeneic, MHC-compatible, autoimmune-resistant C3H/He donors, and 30 SCG/Kj mice served as controls and received TCDM from syngeneic, SCG/Kj donors. A significant survival advantage was evident from SCG/Kj mice engrafted with C3H/He TCDM (p < 0.005), and an 89% extension of median survival compared to recipients of SCG/Kj TCDM. Within 28 weeks post-transplantation, 62% of mice engrafted with SCG/Kj TCDM had died with clinical signs of fatal crescentic glomerulonephritis. This result compared with only 10% of mice engrafted with C3H/He TCDM. Mice engrafted with SCG/Kj TCDM developed significantly greater titers of autoantibodies to ss-DNA, ds-DNA, and myeloperoxidase (ANCA) (p < 0.001), had shorter latencies to the development of, and a greater incidence of proteinuria, hematuria, and peripheral lymphade-nopathy, and a greater mean grade of glomerular lesion (p < 0.001), than mice engrafted with C3H/He TCDM. These findings indicate that the genetic defect of the SCG/Kj strain of mice resides within the hematopoietic stem cells and provokes the speculation that bone marrow transplantation might be a useful means of treating progressive crescentic glomerulonephritis in humans.

Urinary levels of collagen- and elastin-crosslink amino acids have been used as biologic markers for degradation of collagen and elastin in the body. Circadian variation of collagen-crosslink amino acids is well known. The current study was undertaken to determine whether there is also circadian variation in excretion of elastin-crosslink amino acids. We used an isotope dilution-HPLC assay to measure the elastin-crosslink amino acids, desmosine (DES) and isodesmosine (IDES), and the collagen-crosslink amino acids, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), in urine. Sixteen apparently healthy subjects collected urine from 5:00 to 7:00 am, and from 5:00 to 7:00 pm. Mean urinary excretion of DES and IDES in women was 56% and 41% higher (P < 0.001), respectively, in am versus pm specimens when normalized by the creatinine content of the urine specimen. For men, the corresponding values were 11% and 13% higher (not statistically significant). Mean urinary excretion of HP and LP in women was 61% and 71% higher (P < 0.001), respectively, in am versus pm specimens. For men, the corresponding values were 11% and 19% higher (not statistically significant). Differences were not found in the am versus pm rates of excretion of creatinine in men or women. These findings demonstrate the occurrence of circadian variation in HP, LP, DES and IDES in women but not in men. We conclude that the time of collection of urine specimens, especially from women, must be taken into consideration in using the urinary levels of these crosslink amino acids as biologic markers for collagen or elastin degradation.

In a previous study, we showed that dietary gamma-linolenic acid (GLA), an omega-6 polyunsaturated fatty acid found in borage oil (BOR), attenuates the development of hypertension in young spontaneously hypertensive rats (SHR). The purpose of this study was to determine the effects of dietary GLA on established hypertension in adult rats, as well as its effects on components of the renin-angiotensin-aldosterone axis. For 5 weeks, male SHR (14–15 weeks old) were fed a basal fat-free diet to which 11% by weight of sesame oil (SES) or BOR was added. Systolic blood pressure (SBP), determined by the tail cuff method, and weight were measured weekly. Plasma renin activity (PRA), aldosterone (PA), and corticosterone (PC) levels were measured at the end of the dietary treatments. The adrenal glands were homogenized, and angiotensin II (ANG II) binding was measured and plotted according to Scatchard. Systolic blood pressure was 12 mmHg lower at Week 5 in SHR fed the BOR diet compared to SES-fed rats (P < 0.005). Weight gains were similar in both dietary groups. Plasma aldosterone was lower, PRA was higher, and the PA/PRA ratio was significantly lower (P < 0.05) in BOR-fed rats. Levels of PC were the same in both groups. The BOR-enriched diet reduced adrenal ANG II receptor density and affinity compared to the SES diet. Results suggest that BOR inhibits adrenal responsiveness to ANG II by an action on adrenal receptors. Our findings demonstrated that dietary GLA lowers SBP in adult SHR. This effect may be mediated, at least in part, by interference with the renin-angiotensin-aldosterone system at the level of adrenal ANG II receptors.

Glucocorticoids are potent immunosuppressants shown to be effective in the treatment of inflammatory diseases. Reportedly, they work, in part, by inhibiting cytokines and other transcription factors including AP-1. In this study we investigated the mechanisms of efficient repression of collagenase gene expression by dexamethasone in the human gingival fibroblast. Northern analyses showed that IL-1-dependent collagenase mRNA production was significantly decreased in the presence of dexamethasone. The influence of dexamethasone on the transcription factor NF-kB, STAT3, and AP-1 was investigated by using the gel mobility shift assay with nuclear extracts prepared from the cells grown in the presence of dexamethasone. We observed that in addition to AP-1, binding of NF-kB and STAT3 to DNA was also decreased significantly. Additionally, dexamethasone induced the transcription of the IkB-α gene suggesting that in the presence of dexamethasone, NF-kB quickly reassociates with newly synthesized IkB-α and markedly reduces the amount of NF-kB. CAT transfection studies utilizing collagenase promoter demonstrated a dose-dependent transcriptional inhibition of IL-1-induced gingival collagenase gene expression by dexamethasone. These data reveal that collagenase gene expression can be regulated by the impairment of IL-1-stimulated NF-kB, STAT3, and AP-1 activities, and can highlight a possible molecular mechanism for the anti-inflammatory effects of glucocorticoids.

In muscular dystrophy (MD) the imbalance between muscle protein synthesis and degradation may be an important factor leading to muscle wasting. The three major pathways of muscle proteolysis identified in skeletal muscle are: the lysosomal cathepsin pathway, the calcium-dependent calpain pathway, and the ATP-dependent ubiquitin pathway. Insulin-like growth factor I (IGF-I) and a high-protein diet (HPD) have been shown to reduce proteolysis in skeletal muscle. We examined the effect of 6 weeks of recombinant human IGF-I (rhIGF-l) alone or in combination with HPD treatment on the proteolytic pathways in skeletal muscle of 129 ReJ dystrophic (dy) mice. (A group of normal (Norm) nondystrophic (129 J) mice were included as controls). Untreated dy mice exhibited increased net proteolysis (P < 0.05), elevated net calpain activity (P < 0.01), and increased ubiquitin levels when compared to control mice (P < 0.05). Our evidence suggests that HPD and rhIGF-l decrease proteolysis in the 129 ReJ dy mouse. This effect appears attributable, at least in part, to reduced calpain-mediated myofibrillar breakdown (P < 0.05) due to decreased calpain autolysis or increased calpastatin levels. In contrast to calpain, cathepsin B activity was increased in HPD and rhIGF-l + HPD-treated dy muscle (P < 0.05) and unaltered in the rhIGF-l treated animals. Levels of free and protein-conjugated ubiquitin were also increased in rhIGF-l, and rhIGF-l + HPD treated dy animals (P < 0.05). The amelioration of muscle wasting in the 129 ReJ dy model by HPD and/or rhIGF-l may have potential implications in the treatment of human MD.