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.

Characteristics of neural and humoral systems involved in the regulation of blood pressure in snakes.

Cardiovascular function is affected by many mechanisms, including the autonomic system, the kallikrein-kinin system (KKS), the renin-angiotensin system (RAS) and the endothelin system. The function of these systems seems to be fairly well preserved throughout the vertebrate scale, but evolution required several adaptations. Snakes are particularly interesting for studies related to the cardiovascular function because of their elongated shape, their wide variation in size and length, and because they had to adapt to extremely different habitats and gravitational influences. To keep the normal cardiovascular control the snakes developed anatomical and functional adaptations and interesting structural peculiarities are found in their autonomic, KKS, RAS and endothelin systems. Our laboratory has characterized some biochemical, pharmacological and physiological properties of these systems in South American snakes. This review compares the components and function of these systems in snakes and other vertebrates, and focuses on differences found in snakes, related with receptor or ligand structure and/or function in autonomic system, RAS and KKS, absence of components in KKS and the intriguing identity between a venom and a plasma component in the endothelin system.

Identification of novel bradykinin-potentiating peptides (BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure–function relationship of BPPs

Cardiovascular function is affected by many mechanisms, including the autonomic system, the kallikrein-kinin system (KKS), the renin-angiotensin system (RAS) and the endothelin system. The function of these systems seems to be fairly well preserved throughout the vertebrate scale, but evolution required several adaptations. Snakes are particularly interesting for studies related to the cardiovascular function because of their elongated shape, their wide variation in size and length, and because they had to adapt to extremely different habitats and gravitational influences. To keep the normal cardiovascular control the snakes developed anatomical and functional adaptations and interesting structural peculiarities are found in their autonomic, KKS, RAS and endothelin systems. Our laboratory has characterized some biochemical, pharmacological and physiological properties of these systems in South American snakes. This review compares the components and function of these systems in snakes and other vertebrates, and focuses on differences found in snakes, related with receptor or ligand structure and/or function in autonomic system, RAS and KKS, absence of components in KKS and the intriguing identity between a venom and a plasma component in the endothelin system.

Absence of oxytocin in the central nervous system of the snake Bothrops jararaca.

We used four complementary techniques to investigate the presence of oxytocin peptide in the hypophysis and brain of the snake Bothrops jararaca. A high-pressure liquid chromatographic analysis failed to show oxytocin in extracts of hypophysial and brain tissues but provided estimative values of the amounts of vasotocin (12 ng/mg hypophysis and 0.5 ng/mg brain) and mesotocin (500 pg/mg hypophysis and 8 pg/mg brain). Western blots with a polyclonal anti-oxytocin antibody failed to detect oxytocin in both tissues but detected compounds with higher molecular weight than oxytocin, as well as a relatively weak cross-reactivity with mesotocin. The reverse transcription-polymerase chain reaction analysis failed to detect the expression of oxytocin gene transcript, but detected a transcript related to the mesotocin-neurophysin precursor in both tissues. Immunohistochemistry with the same anti-oxytocin antibody detected strong staining in the neurohypophysis and in few fibers in the inner zone of the median eminence, which was not abolished by pre-adsorption of this antibody with oxytocin, vasopressin, vasotocin or mesotocin and might not be attributed to oxytocin. In conclusion, our data demonstrate the absence of oxytocin in the central nervous system of the snake B. jararaca and underline the pitfalls that can result from the use of a single technique to investigate the presence of peptides in tissues.

Absence of oxytocin in the central nervous system of the snake Bothrops jararaca

We used four complementary techniques toinvestigate the presence of oxytocin peptide in thehypophysis and brain of the snake Bothrops jararaca.A high-pressure liquid chromatographic analysis failedto show oxytocin in extracts of hypophysial and braintissues but provided estimative values of the amountsof vasotocin (12 ng/mg hypophysis and 0.5 ng/mgbrain) and mesotocin (500 pg/mg hypophysis and 8 pg/mg brain). Western blots with a polyclonal anti-oxytocinantibody failed to detect oxytocin in both tissuesbut detected compounds with higher molecular weightthan oxytocin, as well as a relatively weak cross-reactivitywith mesotocin. The reverse transcription–polymerasechain reaction analysis failed to detect theexpression of oxytocin gene transcript, but detected atranscript related to the mesotocin–neurophysin precursorin both tissues. Immunohistochemistry with thesame anti-oxytocin antibody detected strong staining inthe neurohypophysis and in few Wbers in the inner zoneof the median eminence, which was not abolished bypre-adsorption of this antibody with oxytocin, vasopressin,vasotocin or mesotocin and might not beattributed to oxytocin. In conclusion, our data demonstratethe absence of oxytocin in the central nervous system of the snake B. jararaca and underline the pitfallsthat can result from the use of a single techniqueto investigate the presence of peptides in tissues.

Receptor de angiotensina em musculatura vascular e cardíaca da serpente Bothroips jararaca / Angiotensin receptor in the cardiovascular system of Bothrops jararaca snake

Com o objetivo de caracterizar o receptor de angiotensina presente n sistema cardiovascular da serpente Bothrops jararaca, foram realizados estudos funcionais e com radioligantes em anéis de aorta e na musculatura cardíaca dessa serpente. Os três octapeptídeos estudados produziram efeito contrátil em aorta, com valores de pD2 de: 7,52 ñ O,17 [Asp1 , lle]All; 8,08 ñ O,10 [Asp1, Val5 ]All; 5,99 ñ O,05 [Asn1, Val5 ]All. A ordem de potência dos análogos de angiotensina foi:[Asp1 , Val5 ]All > [Asp1, lle5]All >>> [Asn1 , Val5 ]All, diferente do observado para outros vertebrados. [Sar1, lle8]All, antagonista não seletivo, losartan, antagonista seletivo AT1, e PDl23319, antagonista seletivo AT2, causaram deslocamento para a direita da curva concentração-efeito de [Asp1, lle5 ]All com diminuição do efeito máximo. Os valores de pKB foram, respectivamente, 5,16 ñ O,09; S,67 ñ O,31; 5,18 ñ O,62, os quais estão acima dos valores de pA2 e pKi3 descritos na literatura para estes antagonistas, sugerindo que o receptor de angiotensina presente na aorta da serpente Bothrops jararaca não é o AT1 ou o AT2. O prévio tratamento dos anéis de aorta com fenoxibenzamina, antagonista não seletivo de adrenoceptores alfa, não alterou a resposta contrátil induzida por [Asp1, lle5]All. A remoção da camada endotelial do vaso também não modificou a resposta induzida por [Asp1, lle5]All. Esses resultados indicam o não envolvimento de catecolaminas ou de fatores liberados do endotélio na resposta contrátil induzida pela angiotensina em aorta isolada de Bothrops jararaca. Ensaios de ligação da [3 H]All foram realizados em preparação bruta de membrana de coração da Bothrops jararaca, na ausência e na presença de [Asp1, lle5]All 10-4 M a 4§ C por lh e mostraram a presença de sítio específico e saturável para angiotensina. O valor de KD obtido foi de 7,34 ñ 1,41 nM e a densidade de receptores de 72,49 ñ 15,18 fmol/mg. Este sítio de ligação apresentou afinidade semelhante à descrita para o receptor de angiotensina em outras preparações biológicas. Experimentos de competição com análogos de angiotensina e com antagonistas peptídicos indicaram também a presença de um único sítio de ligação. A ordem de potência para os análogos de angiotensina e i para todos os antagonistas utilizados foi: [Asp1 , lle5]All = [Asp1, Val5]All = [Sar1, lle8]All > [Asn1, Val5]All >> [Sar1, Ala8]All >>>> losartan = PD 123319. Esses resultados gerem a presença de um ...(au)