Pharmacologic and Molecular Characterization of the Vascular ET A Receptor in the Venomous Snake Bothrops jararaca

Abstract

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.

Introduction

Endothelins (ETs) are a family of vasoactive peptides comprised of four endogenous 21-amino acid isoforms (ET-1, ET-2, ET-3, and ET4 or vasoactive intestinal contractor), as well as the 31-amino acid ET isoforms (ET-1, ET-2, and ET-3[1–31]; Refs. 1–3). Peptides with a high degree of sequence similarity to the ETs are the sarafotoxins (SRTXs), S6a, S6b, S6c, and S6d, a group of venom toxins first isolated from the gland of the snake Atractaspis engadensis (4–6). Recently, a new family of SRTXs was isolated from the venom of A. microlepidota microlepidota. This new family displays three additional amino acid residues at the C-terminus and were, therefore, named long-SRTXs (l-SRTXs; Ref. 7).

ET-1, the predominant isoform of the ET peptide family, and the other family members exert their effects through two specific G-protein–coupled receptors, called ET_A and ET_B (8–11). ET receptors are widely distributed in tissues from all vertebrates and in some invertebrates (12). In the vascular system of mammals, ET_A receptors are detected mainly on vascular smooth-muscle cells and mediate contraction (10), whereas ET_B receptors can be found both on vascular endothelial and smooth-muscle cells (13–15). Little evidence exists on the presence of ET receptors in tissues from reptiles.

The class Reptilia, including arboreal and terrestrial snakes (order Squamata), are the first vertebrates adapted to live in a terrestrial habitat (16). Because of their elongated body shape with long fluid columns, they are highly susceptible to hydrostatic pressure disturbances by the influence of gravity (17). In an earlier study, by using SRTX peptides, we found evidence of the presence of highly sensitive ET receptors in the aorta of the Brazilian poisonous snake Bothrops jararaca (18). Because one of the main physiologic functions of the ET system is to modulate the vascular tonus, we thought it would be interesting to characterize the ET receptors in the vascular system of this terrestrial snake. Therefore, in the present study we evaluated the blood pressure effects of ET-1, and functionally and molecularly characterized the vascular ET receptors in the B. jararaca snake.

Materials and Methods

Animals.

Experimental protocols followed standards and policies of the Committee for Animal Care and Use of the Butantan Institute. Briefly, adult B. jararaca snakes of both sexes, weighing up to 150 g, were maintained under controlled conditions of temperature and humidity, as previously described (19). The snakes were anesthetized with sodium pentobarbital, 30 mg/kg ip, before blood pressure measurements or decapitation, as previously described (20, 21).

Vascular Reactivity Studies.

A segment of approximately 4 cm of the aorta was excised from the area near the aortic arch, and 0.50-cm rings were cut and suspended in 10-ml organ baths, as described previously (18, 22). Contractile force was recorded with a Biopac MP100WS system, using the AcqKnowledge BioPac software (BioPac Systems Inc., Santa Barbara, CA).

Cumulative concentration-response curves (CCRCs) to ET-1, SRTX-b, ET-3, and SRTX-c (10⁻¹¹–10⁻⁶ M) were obtained in the presence and absence of the antagonists BQ-123, IRL-1038, and PD-142893. The antagonists were added to the bath 30 mins before beginning of the agonist CCRCs. Vascular reactivity to ET-1 and SRTX-c in the presence of 10⁻⁴ M N_ω-nitro-l-arginine methyl ester, hydrochloride (L-NAME; a nitric oxide synthase inhibitor) plus 3 × 10⁻⁶ M indomethacin (Indo; a cyclooxygenase inhibitor) as well as to the selective ET_B agonist, IRL-1620 (10⁻⁸–3 × 10⁻⁶ M), was also evaluated. A single CCRC was performed in each aortic ring to avoid the possibility of time-dependent changes in vascular responsiveness and tachyphylaxis phenomena.

Cloning and Sequencing of Partial Complementary DNA (cDNA) of Snake ETA-Receptor.

Total cellular RNA was isolated from the thoracic aortae using TRizol reagent. First-strand cDNA was synthesized using 2 μg total RNA, Moloney murine leukemia virus (M-MLV)–reverse transcriptase (RT), RNAase inhibitor (RNAsin), and oligo(dT) primer, at 42°C for 50 mins. cDNA was amplified by polymerase chain reaction (PCR), using the set of primers (5′–3′): TAC GAG AAC AAG TGT ATG AGG (forward) and AAG ACA TGA CTG AAA ACA ATT (reverse), which were designed from the Gallus gallus ET_A receptor sequence (Genebank AF472618); and Taq Platinum polymerase; with denaturing cycles at 94°C for 30 secs and annealing at 62°C. PCR products were resolved in 1% agarose gel electrophoresis, stained with ethidium bromide, and band intensities were measured using Kodak Digital Science software (Eastman Kodak Co., Rochester, NY). A unique band (with the expected size of ~836 bp) was excised, purified, and cloned using the pGEM-T vector system. The vectors were sequenced using a Sequencing MegaBACE kit with a capillary sequencer MegaBACE 1000, and the sequence obtained was aligned (Clustal W, http://www.ebi.ac.uk/clustalw/) and compared with other ET_A sequences.

Solutions and Drugs.

Bovine serum albumin, norepinephrine, acetylcholine hydrochloride, dimethyl sulfoxide, L-NAME, and indo were obtained from Sigma Chemical Company (St. Louis, MO); ET-1, ET-3, SRTX-b, SRTX-c, BQ-123, IRL-1038, IRL-1620, and PD-142893 were from American Peptide Co. Inc. (Sunnyvale, CA); TRizol, M-MLV-RT, Taq Platinum polymerase, oligo(dT), and primers were from Invitrogen Corp. (Carlsbad, CA); DNase I, RNAase inhibitor, PCR purification kit, and pGEM-T vector system were from Promega Bioscience Inc. (Granada, CA); NcoI and PstI were from MBI Fermentas (Hanover, MD); Sequencing MegaBACE kit and sequencer MegaBACE 1000 were from Amersham Bioscience Corp. (Piscataway, NJ). Pentobarbital sodium was kindly donated by the Cristália laboratory (Campinas, São Paulo, Brazil).

Data Analysis and Statistics.

Data are expressed as mean ± SEM; n indicates the number of animals. The concentration of the agonist producing a half-maximal response (EC₅₀) was determined after logarithmic transformation of the normalized concentration-response curves, and is reported as the negative logarithm (−log EC₅₀ = rank order of potency [pD₂] values) of the mean of individual values for each tissue. The antagonist potencies were expressed as pK_B values, that is, the negative log of the dissociation constant, K_B, which is equal to the molar concentration of the antagonist divided by the concentration ratio minus one (23). The groups were compared by one-way analysis of variance (ANOVA) followed by the Bonferroni test for multiple comparisons, or by the two-tailed Student’s t test. P < 0.05 was considered statistically significant.

Results

In Vivo Effects of ET-1 on B. jararaca Blood Pressure.

Intravenous infusion of 1.2–40 pmol/kg ET-1 on anesthetized B. jararaca snakes induced a dose-dependent biphasic response: a small hypotension followed by a long-lasting hypertensive response (Fig. 1). Higher doses of ET-1 (~40 nmol/kg) initially produced greater changes in blood pressure, which were followed by irreversible cardiac arrest (data not shown). Similar results were observed with infusion of SRTX-b.

Pharmacologic Characterization of ET Receptors.

As shown in Figure 2 , the agonists ET-1, SRTX-b, ET-3, SRTX-c, and IRL-1620 each contracted B. jararaca aortic rings in a concentration-dependent manner. The rank order of potency (pD₂) of these peptides was ET-1 > SRTX-b ⋙ ET-3 > SRTX-c ≫ IRL-1620. Responses to ET-3 and SRTX-c were observed only at concentrations higher than 10⁻⁸ M, and maximal responses were not observed at the highest concentration tested (10⁻⁶ M). The selective ET_B agonist, IRL-1620, only induced contraction at concentrations greater than 10⁻⁷ M, and at the concentration of 3 × 10⁻⁶ M, the maximum effect was not achieved. The pharmacologic parameters are summarized in Table 1 .

BQ-123, a selective ET_A antagonist, right-shifted the CCRCs to ET-1 and SRTX-b in B. jararaca aortae in a concentration-dependent manner, with pK_B values of 5.57 ± 0.16 (n = 10) and 5.60 ± 0.26 (n = 11), respectively (Fig. 3A and B ). The nonselective ET_A/ET_B receptors antagonist, PD-142893, similarly inhibited the contractile response to ET-1 (Fig. 3C ), with a pK_B value of 5.45 ± 0.26. The effects of both BQ-123 and PD-142893 on the CCRCs to ET-3 and SRTX-c were not clearly demonstrated.

In the presence of IRL-1038, a selective ET_B antagonist, the CCRC to SRTX-c, but not to ET-1, was left-shifted (Fig. 4A ). Responses to ET-1 and SRTX-c were potentiated when B. jararaca aortae were pretreated with 10 ⁻⁴ M L-NAME plus 3 × 10⁻⁶ M Indo (Fig. 4B ).

Molecular Characterization of ET Receptors.

We used various sets of primers from different species (human, rat, chicken, and Xenopus), in attempt to identify the presence of ET_A and ET_B receptors in the B. jararaca aortae. The analysis of PCR products showed a single band (with the expected size, ~836 bp) for the set of primers designed from the Gallus gallus ET_A receptor sequence (GenBank AF472618). No bands were identified for the ET_B receptor with the sets of primers we have used so far. Purification, cloning and sequencing of the cDNA fragment was performed, and oligonucleotides as well as the predicted amino acid sequences are shown in Figure 5 . The B. jararaca ET_A sequence has been deposited in the GenBank Data Library under accession number AY849868. The alignment made by CLUSTAL W showed an identity of 74% with human (24), 73% with rat (25), 77% with chicken (26), and 71% with Xenopus (27) ET_A receptors. The respective accession numbers are L06622 (hET_A), NM012550 (rET_A), AF472618.1 (cET_A), and U06633 (xET_A). A very high sequence similarity with the chicken embryo ET_A receptor was observed (BLAST identities = 62/ 74 [83%]; 91/107 [85%]; 155/185 [83%]; and 172/197 [87%]).

Discussion

The sarafotoxin and ET peptide families have a common evolutionary ancestry, despite their evolution in very diverse systems (28). In vivo and in vitro experiments have shown that SRTXs and ETs resemble each other in their pharmacologic activity and binding properties in different mammalian tissues as well as in tissues from lower vertebrates, such as tilapia fish, torpedo, toads, and lizards (29). Similar to mammals, SRTXs/ETs also modulate cardiovascular responses in lower vertebrates.

To pharmacologically characterize vascular ET receptors in the B. jararaca, we evaluated functional responses to SRTXs and ETs in isolated aortic rings. Vascular responses to ET-1 were very similar to those produced by SRTX-b, whereas responses to ET-3 were similar to SRTX-c, in agreement with previous reports showing a clear relationship between the sequences of the peptides and their biologic activity (6). The presence of typical ET_A receptors in the B. jararaca aorta was indicated by the rank order of potency for the agonists, with ET-1 being significantly more potent than ET-3. The potencies of the agonists observed in the present study are similar to those described in mammalian veins and arteries (13, 30) and in the guinea pig ileum (9). However, although pharmacologic data provides indirect evidence for the possible presence of ET_B-receptors, conclusive proof will require additional studies.

Few studies have addressed the cardiovascular actions of ETs/SRTXs in lower vertebrates, but it seems that the biologic activities of these peptides are well conserved across evolutionary lines. ET-1 induces potent constriction in arteries and vein of the frog Rana pipiens (31) and in various vascular tissues of trout (32). In the Alligator mississipiensis, ET-1 produces hemodynamic responses similar to those in mammals, but with a hypotensive phase of greater magnitude and duration (33). Interestingly, although nitric oxide production seems to be important in tonic regulation of the A. mississipiensis vasculature, it is not involved in ET-1–induced hypotension in this reptile (33).

The high degree of sequence similarity between the ET_A receptors expressed by the chicken and by the reptile used in this study may be related to the fact that the B. jararaca snake holds a terrestrial habitat. Interestingly, only negligible cardiovascular effects are observed in the aquatic snake Natrix tessellata in response to SRTX-b and ET-1 stimulation. Furthermore, very few binding sites for ET-1 were found in hearts from this animal and they were nonspecific (29). One may suggest that differences in response to SRTXs/ETs between aquatic and terrestrial snakes may be related to cardiovascular physiologic adaptations undergone by the terrestrial animals. It is well documented that terrestrial snakes are characterized by a number of attributes that are deficient or lacking in species that do not experience the hydrostatic stress of gravity (17).

In summary, our results suggest that both ET_A and ET_B receptors mediate vascular responses to SRTXs/ETs in B. jararaca. Characterization of ET receptors in lower vertebrates may contribute to a better understanding of the evolution as well as mechanisms involved in the functioning of the cardiovascular system.

Table 1.
Comparison of Potencies and Maximum Contractile Responses for ET Receptors Agonists in Aortae from B. jararaca^a

ET-1 SRTX-b ET-3 SRTX-c IRL₁₆₂₀

^apD₂ negative lagarithm of EC₅₀ values; EC₅₀, concentration (M ) of the agonist producing a half-maximal response; E_max, maximum contractile response obtained; nd, not determined; n, number of animals.

^bEstimated values from CCRC without maximal saturation at the highest concentrations of agonist.

pD₂ 8.87 ± 0.09 8.14 ± 0.08 6.90 ± 0.25^b < 6.0 ± 0.47^b < 6.0 ± 0.5^b

EC₅₀ (M) 1.39 × 10⁻⁹ 6.83 × 10⁻⁹ 1.10 × 10^−7b >4.85 × 10^−7b >4.9 × 10^−7b

E_max 2.49 ± 0.24 2.61 ± 0.29 2.43 ± 0.41^b nd nd

n 35 18 13 15 3

Figure 1.
Representative trace of the effects of ET-1 on blood pressure of a 225 g female B. jararaca snake under 30 mg/kg pentobarbital anesthesia. ET-1 (1.2 pmol/kg) produced a biphasic response, a small hypotension followed by a long-lasting hypertensive response. The return to baseline occurred after approximately 1 hr. The effects of intravenous injections of a 0.6% saline solution, 97 nmol/kg angiotensin I (Ang I), and 34 nmol/kg acetylcholine (ACh) are also shown. (Color figure available in the on-line version.)

Figure 2.
CCRCs to ET receptor agonists in B. jararaca aortae. ▪, ET-1 (n = 35); ▴, SRTX-b (n = 18); ▾, ET-3 (n = 13); ♦, SRTX-c (n = 15); and •, IRL-1620 (n = 3). Each point represents the means ± SEM for agonist-induced contractile responses (in grams).

Figure 3.
Effects of BQ-123 and PD-142893 in the CCRCs to ET-1 and SRTX-b. Each agonist was tested in control conditions, i.e., in the absence of the antagonist (▪) or in the presence of BQ-123 or PD-142893 at 10⁻⁶ M (▵) and 3 ×10⁻⁶ M (○). ET-1 (n = 33, 10, 8 for control, 10⁻⁶ M, and 3 × 10⁻⁶ M BQ-123; and n = 35, 4, and 3 for control, 10⁻⁶, and 3 ×10⁻⁶ M PD-142893, respectively); SRTX-b (n = 18, 11, 10). Each point represents the mean ± SEM of agonist-induced contractile responses (in grams) for n experiments.

Figure 4.
Effects of IRL-1038 on the CCRCs to SRTX-c (A) and of L-NAME plus indo on the contractile responses to ET-1 (B) and SRTX-c (C) . Each agonist was tested in control conditions, i.e., in the absence of the antagonist (▪) or in the presence of 10⁻⁶ M IRL₁₀₃₈ (▵), 3 × 10⁻⁶ M IRL₁₀₃₈ (⋄), or 10⁻⁴ μM L-NAME plus 3 × 10⁻⁶ M indomethacin (). SRTX-c (n = 6, 5, and 4 for control, 10⁻⁶ M, and 3 × 10 ⁻⁶ M IRL₁₀₃₈, respectively). In the experiments with L-NAME plus indomethacin, ET-1, and SRTX-c, n is equal to 5. Each point represents the mean ± SEM of agonist-induced contractile responses (in grams) for n experiments.

Figure 5.
(A) Partial nucleotide sequence of the vascular ET_A receptor from the serpent B. jararaca.* , Identical bases in the sequence of ET_A receptors from B. jararaca* with ET_A receptors from chicken (Gallus gallus). (B) Predicted amino acid sequence of the vascular ET_A receptor from the serpent B. jararaca. Amino acids that differ between the B. jararaca and Gallus gallus sequences are underlined.

	ET-1	SRTX-b	ET-3	SRTX-c	IRL₁₆₂₀
^apD₂ negative lagarithm of EC₅₀ values; EC₅₀, concentration (M ) of the agonist producing a half-maximal response; E_max, maximum contractile response obtained; nd, not determined; n, number of animals.
^bEstimated values from CCRC without maximal saturation at the highest concentrations of agonist.
pD₂	8.87 ± 0.09	8.14 ± 0.08	6.90 ± 0.25^b	< 6.0 ± 0.47^b	< 6.0 ± 0.5^b
EC₅₀ (M)	1.39 × 10⁻⁹	6.83 × 10⁻⁹	1.10 × 10^−7b	>4.85 × 10^−7b	>4.9 × 10^−7b
E_max	2.49 ± 0.24	2.61 ± 0.29	2.43 ± 0.41^b	nd	nd
n	35	18	13	15	3

Footnotes

Financial support was provided by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) (00/12126-9) and the Butantan Foundation, Brazil.

Acknowledgements

We thank Dr. Inácio de Loyola M. Junqueira (Biotechnology Center, Butantan Institute) for donation of reagents, and Dr. Edna T. Kimura for the sequencing experiments.

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