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BASIC BIOLOGY

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

Rosa A. M. B. Borgheresi^*,1, Janine M. G. Leroy^*, Alvaro Yogi, Rosangela A. DosSantos, Maria C. Breno^* and Rita C. Tostes

^* Laboratory of Pharmacology, Butantan Institute, 05503-900 Sao Paulo, Brazil; and Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, 05508-900 Sao Paulo, Brazil

To whom requests for reprints should be addressed at ¹ Laboratory of Pharmacology, Butantan Institute, Av. Vital 1500, 05503-900 Sao Paulo, Brazil. E-mail: rosabbor-gheresi{at}butantan.gov.br

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-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.

Key Words: reptile • endothelin • sarafotoxin • endothelin receptors • vascular reactivity • cloning

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