M.C. Breno

Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

Pharmacologic and molecular characterization of the vascular ETA receptor in the venomous snake Bothrops jararaca.

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, ETA and ETB. 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 ETA 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 ETA receptors. BQ-123, a selective ETA antagonist, inhibited contractions induced by ET-1 and SRTX-b with expected negative log of the dissociation constant, KB, (pKB) values for mixed ETA/ETB receptor populations. The nonselective ETA/ETB receptors antagonist, PD-142893, produced similar inhibition. The ETB antagonist, IRL-1038, potentiated contractile responses to SRTX-c. ET-1 and SRTX-c responses were also potentiated when aortic rings were pretreated with Nω-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 ETA receptor sequence, enabled isolation, purification, cloning, and sequencing of a single band. The partial sequence of the B. jararaca ETA receptor showed a very high sequence similarity with ETA 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 ETA receptors.

Presence of functional angiotensin II receptor and angiotensin converting enzyme in the aorta of the snake Bothrops jararaca

AIM Angiotensin II (Ang II) interacts with AT(1) and AT(2) receptors and, in some vertebrates, with an Ang II binding site showing low affinity for AT(1) and AT(2) receptor antagonists. This study was carried out to characterize the Ang II receptor, and the presence of an angiotensin-converting enzyme (ACE) in the aorta of the Bothrops jararaca snake. MAIN METHOD Contraction induced by Ang I or II in aortic ring from the snake was evaluated in the absence or in the presence of ACE-blocker or Ang II antagonists. KEY FINDINGS Ang II analogs, modified at positions 1 and 5, induced vasoconstriction with differences in their potencies. The relative rank order was: [Asp(1), Val(5)] Ang II=[Asp(1), Ile(5)] Ang II>>>[Asn(1), Val(5)] Ang II. ACE-like activity was detected, as well as an Ang II receptor with low affinity for AT(1) and AT(2) selective receptor antagonists (pK(B) values of 5.62±0.23 and 5.08±0.25). A disulfide reducing agent almost abolished the Ang II effect, while an alpha adrenoceptor antagonist, or removing the endothelium, did not modify the Ang II effect. These results indicate that the B. jararaca aorta has an Ang II receptor pharmacologically distinct from AT(1) and AT(2) receptors, and the vasoconstrictor effect observed is independent of catecholamine or endothelium modulation. ACE and the AT receptor in the aorta of B. jararaca may be part of a tissue renin-angiotensin system. SIGNIFICANCE The data contribute to the knowledge of the renin-angiotensin system in vertebrate species, and provide insight into the understanding of snake Ang II receptor characteristics and diversity.