Angiotensin receptor in the heart of Bothrops jararaca snake.

Angiotensin II interacts with specific cell surface angiotensin AT1 and AT2 receptors and, in some vertebrates, with an atypical angiotensin AT receptor. This study was designed to characterize the angiotensin receptor in the heart of Bothrops jararaca snake. A specific and saturable angiotensin II binding site was detected in cardiac membranes and yielded Kd=7.34+/-1.41 nM and B(max)=72.49+/-18 fmol/mg protein. Competition-binding studies showed an angiotensin receptor with low affinity to both angiotensin receptor antagonists, losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole) and PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate). Studies on the intracellular signaling pathways showed that phospholipase C/inositol phosphate breakdown and adenylylcyclase/cyclic AMP generation were not coupled with this angiotensin receptor. An adenylylcyclase enzyme sensitive to forskolin was detected. The results indicate the presence of an angiotensin receptor in the heart of B. jararaca snake pharmacologically distinct from angiotensin AT1 and AT2 receptors. It seems to belong to a new class of angiotensin receptors, like some other atypical angiotensin AT receptors that have already been described.

Receptor-mediated endocytosis of angiotensin II in rat myometrial cells.

The events involved in the processing of the angiotensin II (Ang II)-receptor complex were studied in primary cultures of rat myometrial cells. Ang II bound to rat myometrial cells in a specific, time- and temperature-dependent fashion. Pretreatment with cycloheximide did not interfere with binding up to 3 hr, but inhibited increases in binding observed over longer periods. The [3H]Ang II binding to intact cells was inhibited by dithiothreitol (DTT), and the rank order of potency of Ang II and nonpeptide antagonists to inhibit the [3H]Ang II binding was Ang II > Losartan >> PD 123319 or CGP 42112B, indicating the presence of the AT1 receptor type. Whereas most of the [3H]Ang II binding at 4 degrees was susceptible to acid or pronase treatment, binding at 35 degrees was resistant to both treatments, suggesting an internalization of the Ang II-receptor complex. Phenylarsine oxide (PAO) and N-ethylmaleimide (NEM) caused a concentration-dependent inhibition when the binding assay was performed at 35 degrees, but no effect was observed at 4 degrees, indicating that these agents did not alter cell-surface binding but actually prevented the internalization process. Simultaneous treatment with 1 mM DTT or beta-mercaptoethanol prevented the inhibitory effect of NEM, but only DTT could prevent the inhibition caused by PAO, indicating that two closely located sulfhydryl groups must be involved in the internalization process. Chloroquine (100 microM) inhibited the [3H]Ang II dissociation from cells, and monensin (25 microM) induced a 30% inhibition of [3H]Ang II binding (35 degrees, 3 hr), suggesting endosomal processing of the Ang II-receptor complex with receptor recycling to the cell surface. These results indicate that Ang II binding to AT1 receptors in rat myometrial cells is followed by internalization of the Ang II-receptor complex and recycling of the receptor to the cell surface.

Isolation and identification of angiotensin-like peptides from the plasma of the snake Bothrops jararaca.

Two distinct hypertensive peptides were purified and characterized from Bothrops jararaca (Bj) plasma incubated at pH 4, 37 degrees C, 24 hr. These peptides were active on rat and Bj blood pressure, on rat isolated uterus, on guinea pig isolated ileum and on Bj isolated duodenum. At the releasing conditions no further activities were found for kininases, angiotensinases or angiotensin converting enzymes. The peptides were purified by ethanol/ether extraction, Sephadex G-25 gel filtration, semipreparative reverse-phase (C-18) HPLC and analytical (C-18) HPLC. The amino-acid sequences of the purified peptides corresponded to (Ile5)AII and (Val5-Tyr9)AI and their molecular masses were confirmed by mass spectrometry as 1046.6 and 1348.0 respectively. The presence of those two angiotensins on Bj plasma may have some evolutionary significance since (Ile5)AII is known as a mammalian angiotensin and (Val5)AII as a non-mammalian one.

Alpha-1 adrenoceptor subtypes involved in noradrenaline-induced contraction of snake (Bothrops jararaca) isolated aorta

The effects of chloroethylclonidine (CEC) and calcium channel antagonists on noradrenaline-induced contractions of Bothrops jararaca aorta were investigated, in order to characterize the alpha-1 adrenoceptor subtypes present in this preparation. CEC (5 × 10−5or 10−4M) displaced to the right, in the same order of magnitude, the concentration-response curve of noradrenaline without affecting its maximum response. The residual response was blocked by prazosin (10−8 M) but not by idazoxan (10−5 M). Nifedipine (10−5 M) or diltiazem (10−5 M), in the absence of cocaine, did not have any effect on the concentration-response curve of noradrenaline; however, in the presence of cocaine, both the drugs were equipotent in displacing the curve, though diltiazem produced a non-parallel displacement. These results suggest the presence of two distinct alpha-1 adrenoceptor subtypes in Bothrops jararaca aorta: one CEC-sensitive (alpha-1B-like) and another CEC-insensitive, though the possibility of the presence of a single but different subtype cannot be excluded.

Experimental cryptorchidism in prepubertal rats: some effects on reactivity of the seminal vesicle to catecholamines.

The development of testes and genital accessory organs, the plasma testosterone concentration and the reactivity of seminal vesicles to catecholamines were studied in rats rendered cryptorchid at the time of prepubertal growth. The wet weights of the accessory organs and the testosterone concentrations were reduced and corresponded to those observed after castration. The response of the seminal vesicle to adrenaline and noradrenaline, but not to methoxamine, showed a reduction in sensitivity. It is suggested that an enhancement of neuronal catecholamine uptake may have occurred. The results also reinforce the idea that development, plasma testosterone concentration and reactivity of seminal vesicles are independent phenomena. In cryptorchidism, despite the consequences arising from the maintenance of the testes at body temperature rather than at the lower temperature of the scrotum, the age that the rat is rendered cryptorchid is also of fundamental importance.

Circulating vasotocin in the snake Bothrops jararaca.

1. There is biochemical and pharmacological evidence to suggest the presence of vasotocin in the blood and plasma of the snake Bothrops jararaca (Bj). 2. XE-64 extracts from Bj blood showed antidiuretic and hypotensive activities in rats and a contractile effect on rat isolated uterus, which was totally dialysable and inhibited by thioglycollate. 3. Extracts from Bj whole plasma presented an antidiuretic activity which was only partially dialysable. 4. The plasma extracts also showed oxytocic properties. 5. When EDTA and Sep-Pak C18 extraction were used, a better recovery and characterization of vasotocin by HPLC was obtained. 6. These results indicate the occurrence of free and bound circulating vasotocin in Bj, in an equilibrium dependent of its enzymatic hydrolysis.

Effect of chymotrypsin on rat blood pressure.

1. Chymotrypsin (Cht) administration (14 mg/kg, i.v.) to rats always caused hypertension; hypotension preceded this effect in 64% of the observations (n = 11). 2. A 68% reduction of circulating kininogen but not of angiotensinogen was observed after Cht administration. 3. Cht effects were not affected by captopril, [Sar1-Leu8]-angiotensin II and alpha-adrenoceptor antagonists. In 70% of the observations (n = 10) hypotension was abolished by a mixture of histamine H1- and H2-antagonists. Therefore histamine release may explain hypotension. 4. Cht released in vitro from rat plasma, a substance producing hypertension in the rat and contraction of the guinea-pig ileum. Both effects were antagonized by [Sar1-Leu8]-angiotensin II. 5. In spite of this angiotensin release in vitro, the hypertensive component of the in vivo response to Cht seems to be due to some other substance.

Hydrolysis of L-cystine-di-beta-naphthylamide and neurohypophyseal peptides by the plasma of the snake Bothrops jararaca.

1. Bothrops jararaca plasma or serum hydrolysed L-cystine-di-beta-naphthylamide (CNAse activity) at a degree comparable to that of plasma or serum in pregnant women. 2. In adult snakes, activity was less in males. It was not altered in pregnancy but increased after delivery, being higher at pH 6.4 (unspecific enzymes) than at pH 7.9 (true pregnant woman plasma oxytocinase). 3. Its optimum pH was 5.9, different from that of other known enzymes that hydrolyse the same substrate. 4. Bothrops jararaca plasma also hydrolysed vasopressin, oxytocin and vasotocin. 5. These hydrolysing activities were unexpected for an ovoviviparous reptile.

Active and inactive renin in the plasma of the snake Bothrops jararaca.

1. Complementing the work of Gervitz R. K., Hiraichi E., Fichman M. and Lavras A. A. C. Comp. Biochem. Physiol. 86A, 503-507 (1987), conditions have been established for measuring plasma renin activity (PRA) of the venomous snake Bothrops jararaca (Bj). 2. It corresponded to 115.9 +/- 11.5 ng equivalents of angiotensin II (AII) per ml of plasma (N = 13). 3. PRA did not increase when Bj plasma was submitted to acid-cryo-trypsin Bitis arietans venom activation of inactive renin. 4. This may indicate either absence of inactive renin in this plasma or lack of its activation, due to the already demonstrated (Nahas L., Kamiguti A. S., Betti F., Martins I. S. S. and Rodrigues M. I., Comp. Biochem. Physiol. 69A, 739-743, 1981; Prezoto B. C., Hiraichi E., Abdalla F. M. F. and Picarelli Z. P., Comp. Biochem. Physiol. 99C, 135-139, 1991) absence of factor XII.

The vasopressor action of angiotensin in the snake Bothrops jararaca.

1. Carotid blood pressure from anesthetized B. jararaca snakes was recorded in order to study angiotensin action in this reptile. 2. Whereas [Asn1,Val5] AII and AIII were less potent than [Asp1,Ile5] AII and [Asp1,Val5] AII, [Sar1,Ile5] AII was slightly more potent. 3. Captopril abolished the responses to AI (0.01-3 micrograms/kg). 4. [Sar1,Ala8] AII was uneffective but [Sar1,Leu8] AII or phenoxybenzamine were able to reduce AII vasopressor responses. 5. These results led to the conclusion that the vasopressor response of AII in B. jararaca is due to an interaction with its own receptor but, part of the AII receptor population seems to be coupled to the sympatho-adrenal system. Moreover, structural requirements seem to be necessary for the AII response in B. jararaca.

Effects of catecholamines on the isolated aorta of the snake Bothrops jararaca.

1. Effects of catecholamines in snakes have been examined using an aorta preparation isolated from Bothrops jararaca. Adrenaline, noradrenaline and isoprenaline produced dose-dependent contractions on this preparation. The relative potency was adrenaline greater than noradrenaline greater than isoprenaline. 2. Phentolamine displaced, to the right, the concentration-response curve of the three catecholamines tested, showing the presence of alpha-adrenoceptors in this preparation. 3. Isoprenaline has never produced a relaxation, even when the aorta was first contracted by BaCl2 and pretreated with phentolamine, indicating that beta-adrenoceptors are absent in this preparation. 4. In this Bothrops jararaca preparation, exclusively neuronal uptake was found, thus demonstrating that its existence was preserved during evolution.

Kallikrein-kinin system in the plasma of snakes.

Using pharmacological preparations suitable for assay of mammalian kinins, it was shown that Bothrops jararaca (Bj) venom and other kininogenases were unable to release kinins from snake plasma. The kallikrein-kinin system presents species-specificity in birds. In order to detect such a specificity in snakes, the effects of Bj venom on snake blood pressure and the effect of incubates of snake plasma with trypsin, on snake blood pressure and snake uterus, were studied. The possibility of activating snake plasma kallikrein with ellagic acid, glass beads or kaolin was also investigated. Whereas plasma of the snakes Waglerophis merremii (Wm) and Crotalus durissus (Cd), were shown to contain factor XII, prekallikrein, kininogen, kininases and to present a low but definite activation rate of the kinin system, the plasmas of Bj, Bothrops mojeni (Bm) and Oxyrophus trigeminus (Ot), yielded only kininogen and kininases. Activation of the system was not even detected by the sensitive substrate Ac-Phe-Arg-Nan (acetyl-phenylalanyl-arginyl-4nitro-anilide), indicating that the plasma of these species does not possess either factor XII and/or prekallikrein. Snake plasma may constitute an interesting model for the study of blood clotting, fibrinolytic and complement systems.

Effect of the neonatal steroid hormone treatment of rats on adult hormone levels and the reactivity of seminal vesicles to parasympathomimetic drugs.

1. Seminal vesicle reactivity to cholinergic agents, plasma testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations and seminal vesicle testosterone concentrations were determined in adult male rats treated during the first 6 h of life with 1.0 ml peanut oil (oil-treated), 1.0 mg testosterone propionate (TP-treated) or 1.2 mg 19-nor-testosterone homofarnesate (19-NT-treated). 2. At 90-100 days of age, the neonatally treated animals presented atrophied accessory genital organs and increased (TP-treated, N = 10) or unchanged (19-NT-treated, N = 11) pD2 values for acetylcholine (vehicle: 5.18 +/- 0.06, N = 10; TP-treated: 5.26 +/- 0.06, N = 10; 19-NT-treated: 5.14 +/- 0.09, N = 11), and acetyl-beta-methylcholine (vehicle: 5.19 +/- 0.07; TP-treated: 5.43 +/- 0.06; 19-NT-treated: 5.25 +/- 0.07). The relative intrinsic activity, alpha, of acetyl-beta-methylcholine increased after both hormonal treatments (vehicle: 0.85 +/- 0.03; TP-treated: 0.95 +/- 0.02; 19-NT-treated: 0.92 +/- 0.03). 3. No variation in mean adult plasma testosterone concentration was observed after neonatal treatment with either TP or 19-NT (vehicle: 752.93 +/- 273.66, N = 8; TP-treated: 459.05 +/- 88.32, N = 8; 19-NT-treated: 836.86 +/- 113.08, N = 7). However, testosterone content of seminal vesicles of adult rats was decreased in the animals treated with TP (N = 5) and 19-NT (N = 6) compared to controls. 4. These results indicate a specific effect of neonatal hormone treatment on androgen metabolism which is demonstrable in the adult.

Effect of the neonatal steroid hormone treatment of rats on adult hormone levels and the reactivity of seminal vesicles to parasympathomimetic drugs

Seminal vesicle reactivity to cholinergic agents, plasma testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) conmcentrations and seminal vesicle testosterone concentrations were determined in adult male rats treated during the first 6 h of life with 1.0 ml peanut oil (oil-treated), 1.0 mg testosterone propionate (TP-treated) or 1.2 mg 19-nor-testosterone homofarnesate (19-NT-treated). At 90-100 days of age, the neonatally treated animals presented atrophied accessory genital organsand increased (TP-treated, N=10) or unchanged (19-NT-treated, N=11) pD, values for acetylcholine (vehicle; 5.18 ñ 0.06, N=10; TP-treated; 5.26 ñ 0.06, N+10; 19-NT-treated; 5.14 ñ 0.09, , N=11), and acetyyl-beta-methylcholine (vehicle: 5.19 ñ 0.07; TP-treated: 5.43 ñ 0.06; 19-NT-treated 5.25 ñ 0.07). The relative intrinsic activity, alpha, of acetyl-beta-methylcholine increased after both hormonal treatments (vehicle: 0.85 ñ 0.03; TP-treated:0.95 ñ 0.02; 19-NT-treated: 0.92 ñ 0.03). No variation in mean adult plasma testosterone concentration was observed after neonatal treatmentwith either TP or 19-NT (vehicle:752.93 ñ 273.66, N+8; TP-treated; 459.05 ñ 88.32, N=8; 19-NT-treated: 836.86 ñ 113.08, N=7). However, testosterone content of seminal vesicles of adult rats was decreased in the animalstreated with TP (N=5) and 19-NT (N=6) compared to controls. These results indicate a specific effect of neonatal hormone treatment on androgen metabolism which is demonstrable in the adult

Effects of catecholamines on the isolated aorta of the snake Bothrops jararaca

Effects of catecholamines in snakes have been examined using an aorta preparation isolated from Bothrops jararaca. Adrenaline, noradrenaline and isoprenaline produced dose-dependent contractions on this preparation. The relative potency was adrenaline > noradrenaline > isoprenaline. 2. Phentolamine displaced, to the right, the concentration-response curve of the three catecholamines tested, showing the presence of alpha-adrenoceptors in this preparation. 3. Isoprenaline has never produced a relaxation, even when the aorta was first contracted by BaCl2 and pretreated with phentolamine, indicating that beta-adrenoceptors are absent in this preparation. 4. In this Bothrops jararaca preparation, exclusively neuronal uptake was found, thus demonstrating that its existence was preserved during evolution.

Characterization of postjunctional alpha-adrenoceptors in the isolated aorta of the snake Bothrops jararaca

To characterize the alpha-adrenoceptors present in Bothrops jararaca aorta, selective alpha-adrenoceptor agonists and antagonists were used. The relative order of potency of the tested agonists was noradrenaline > phenylephrine > clonidine. Prazosin was more potent than phentolamine and yohimbine in antagonizing noradrenaline response, since the PA2 values were 9.91, 7.59 and 7.33, respectively. Yohimbine was also able to block the contraction produced by phenylephrine, an alpha-1 agonist. These results suggest the existence of an alpha-1 subtype of adrenoceptor in this preparation which, however, presents some different characteristics from those described for mammals.

Kallikrein-kinin system in the plasma of the snake Bothrops jararaca.

1. Bothrops jararaca venom (BJV) caused a fall in the carotid artery blood pressure of the anaesthetized snake. This effect was tachyphylactic and was potentiated by captopril, a kininase II inhibitor; it was partially antagonized by promethazine plus cimetidine and was not affected by atropine. 2. Similar hypotensive effects were obtained by administration of trypsin or a partially purified BJV kininogenase to the snake. 3. Incubation of Bothrops jararaca plasma (BJP) with trypsin released a substance (or substances) that produced hypotension in the snake but not in the rat; this hypotensive effect was also potentiated by captopril. 4. The trypsinised plasma contracted Bothrops jararaca isolated uterus, a pharmacological preparation weakly sensitive to bradykinin. Trypsinised plasma was inactive on pigeon oviduct and rat uterus and displayed a weak action on the guinea-pig ileum. Similar effects were observed with incubates of a fraction of BJP, containing globulins, with a partially purified BJV kininogenase. 5. Like mammalian kinins, the substance(s) was(were) dialysable, thermostable in acid but not in alkaline pH, and inactivated by chymotrypsin but not by trypsin. Its(their) inactivation by BJP or BJP kininase II was inhibited by captopril. 6. These findings strongly suggest that, besides releasing histamine, BJV or trypsin release a kininlike substance (or substances) from the snake plasma. 7. Since BJV and other kininogenases active on mammalian plasma were shown to be unable to release kinins from BJP, in experiments conducted on pharmacological preparations suitable for the assay of mammalian kinins, these data also suggest that the snake Bothrops jararaca, like birds, may have developed its own kallikrein-kinin system.