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We have previously demonstrated that the phosphorylation of p38 MAPK, through spinal AT 1 receptor activation, is involved in formalin-induced nociception and follows accompanied by the increase in spinal angiotensin (Ang) II levels. We have also found that Ang (1-7), an N-terminal fragment of Ang II generated by ACE2, prevents the Ang II-induced nociceptive behavior via spinal MAS1 and the inhibition of p38 MAPK phosphorylation. Here, we examined whether the ACE2 activator diminazene aceturate (DIZE) can prevent the formalin-induced nociception in mice. The i.t. administration of DIZE attenuated the second, but not the first phase of formalin-induced nociceptive response. An increase in the activity of spinal ACE2 was measured following DIZE administration. The inhibitory effect of DIZE on nociception was abolished by the i.t. co-administration of the MAS1 antagonist A779. The i.t. administration of Ang (1-7) showed a similar effect on the second phase of the response which was also attenuated by A779. Furthermore, DIZE and Ang (1-7) each inhibited the formalin-induced phosphorylation of p38 MAPK on the dorsal lumbar spinal cord. This inhibition was again prevented by A779. ACE2 was expressed in neurons and microglia but absent from astrocytes in the superficial dorsal horn. Our data show that the i.t.-administered DIZE attenuates the second phase of the formalin-induced nociception which is accompanied by the inhibition of p38 MAPK phosphorylation. They also suggest the involvement of MAS1 activation on spinal neurons and microglia in response to the increase in Ang (1-7) following ACE2 activation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

DAA-I (des-aspartate-angiotensin I), an endogenous angiotensin, had been shown earlier to ameliorate animal models of cardiovascular diseases via the angiotensin AT1 receptor and prostaglandins. The present study investigated further the action of DAA-I on the release of PGE2, PGI2, PGF2α and TXA2 in HUVEC. 10(-11)-10(-8)M DAA-I and 15min incubation specifically released PGE2 and PGI2. The release was inhibited by losartan and indomethacin but not by PD123319 and NS398 indicating that the angiotensin AT1 receptor and COX-1 mediate the release. At concentrations higher than 10(-7)M, DAA-I mimics the action of angiotensin II by releasing TXA2 but had no effect on the production of PGF2α. At similar concentrations and 4h incubation, DAA-I increased the release of the 4 prostaglandins via the angiotensin AT1 receptor and COX-2, again mimicking the action of angiotensin II. HUVEC that were preincubated with DAA-I or angiotensin II, released similar profiles of prostaglandins when incubated with arachidonic acid after the angiotensin had been washed off. We postulate that the internalized DAA-I/receptor complex remains active and mediates the conversion of arachidonic acid to the respective prostaglandins. The release of PGE2 and PGI2 via the angiotensin AT1 receptor and COX-1 is a novel specific action of DAA-I and is likely responsible for its beneficial effects seen in earlier studies. This specific action is definable as a biased agonism of the angiotensin AT1 receptor, which identifies DAA-I as a novel biased agonist and potential therapeutic that is able to produce specific prostaglandins at nanomolar concentrations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The review describes DAA-I (des-aspartate-angiotensin-I) as a prototype of a novel class of drugs that acts as agonists on the angiotensin AT1 receptor or ARAs (angiotensin receptor agonists). DAA-I is a component of the renin angiotensin system. Earlier studies showed that it was rapidly metabolized to angiotensin III. However, when administered at doses below the Km of enzymes, DAA-I produces specific actions that antagonize the deleterious actions of angiotensin II. DAA-I exerts protective actions in animal models of eight human pathologies in which angiotensin II is implicated. The pathologies include cardiac hypertrophy, neointima growth and cardiovascular hypertrophy, myocardial-ischemia reperfusion injury, hyperglycemia and insulin resistance, chemical induced inflammation, and exercise-induced skeletal muscle inflammation. Binding of DAA-I to the angiotensin AT1 receptors releases prostaglandins, which could either function as autocrines/paracrines or second messengers and attenuate the deleterious actions of angiotensin II. It is possible that in in vivo DAA-I functions as a physiological antagonist to angiotensin II, and exogenous DAA-I is a novel class of angiotensin receptor drug that could rival the angiotensin receptor blockers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cerebrovascular disease (CVD) ranks as the top three health risks, specially cerebral ischemia characterized with the damage of blood-brain barrier (BBB). The angiotensin Ang-(1-7) was proven to have a protective effect on cerebrovascular diseases. However, its role on blood-brain barrier and the underlying molecular mechanism remains unclear. In this study, Ang-(1-7) significantly relieved damage of ischemia reperfusion injury on blood-brain barrier in cerebral ischemia reperfusion injury (IRI) rats. Furthermore, its treatment attenuated BBB permeability and brain edema. Similarly, Ang-(1-7) also decreased the barrier permeability of brain endothelial cell line RBE4. Further analysis showed that Ang-(1-7) could effectively restore tight junction protein (claudin-5 and zonula occludens ZO-1) expression levels both in IRI-rats and hypoxia-induced RBE4 cells. Furthermore, Ang-(1-7) stimulation down-regulated hypoxia-induced matrix metalloproteinase-9 (MMP-9) levels, whose silencing with (matrix metalloproteinase-9 hemopexin domain) MMP9-PEX inhibitor significantly increased the expression of claudin-5 and ZO-1. Further mechanism analysis demonstrated that Ang-(1-7) might junction protein levels by tissue inhibitor of metalloproteinase 1 (TIMP1)-MMP9 pathway, because Ang-(1-7) enhanced TIMP1 expression, whose silencing obviously attenuated the inhibitor effect of Ang-(1-7) on MMP-9 levels and decreased Ang-(1-7)-triggered increase in claudin-5 and ZO-1. Together, this study demonstrated a protective role of Ang-(1-7) in IRI-induced blood-brain barrier damage by TIMP1-MMP9-regulated tight junction protein expression. Accordingly, Ang-(1-7) may become a promising therapeutic agent against IRI and its complications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The high frequency of rhinovirus (RV) infection and the lack of an effective treatment, underline the importance of research on novel anti-rhinoviral agents. The present study investigated the effects of des-aspartate-angiotensin I (DAA-I) on the survival of RV14-infected H1HeLa cells; and the early inflammatory processes in RV14-infected A549 lung epithelial cells. The study rationale was based on earlier findings showing that DAA-I is an effective anti-inflammatory agent, and that symptoms and severity of rhinoviral infection are related to the underling inflammation. RV14 concentration dependently caused the death of H1HeLa cells and DAA-I, at concentrations of 10⁻¹⁰ to 10⁻¹² M, attenuated the lethal action of RV14 indicating that that DAA-I exerts antiviral action. Unlike its action on H1HeLa cells, RV14 did not cause apparent cytopathic effect on A549 cells, and these cells were used to study the antiviral action of DAA-I. RV14 induced overexpression of ICAM-1, E-selectin and overproduction of superoxide in A549 cells, and DAA-I attenuated the three increases to basal level at concentrations of 10⁻¹⁰ to 10⁻¹² M. Losartan, an angiotensin AT₁ receptor antagonist, blocked the inhibitory action of DAA-I on superoxide overproduction indicating that the AT₁ receptor mediates the action of DAA-I. The present data represent a novel demonstration of the antiviral action of an angiotensin peptide, and a possible involvement of the renin angiotensin system in viral infection. Indeed the angiotensin AT₁ receptor has been reported to be obligatory for the development of virus-induced myocardial injury through the proinflammatory action of angiotensin II via the NF-κB/cytokine pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The present study investigated the actions of des-aspartate-angiotensin I (DAA-I) on infarct size and three early inflammatory events in acute myocardial ischemia-reperfusion injury in rats. The rationale was based on earlier findings showing that chronic daily administration of DAA-I attenuated infarct size of ischemic-reperfused rat heart, and cardiac hypertrophy in pressure overload rats. Anesthetized rats were subjected to 45 min of ischemia and 5h of reperfusion. Infarcted area, serum creatine kinase, and tissue myeloperoxidase activity were determined. The expression of intercellular adhesion molecule-1 (ICAM-1) was also investigated by immunohistochemistry and Western blotting. Intravenous administration of DAA-I at 5 min post reperfusion reduced myocardial infarct size by 45.5%, lowered serum creatine kinase activity, decreased myeloperoxidase activity in cardiac tissue, and inhibited the expression of ICAM-1 in cardiac capillary endothelium. The maximum effective dose was 1013 pmol/kg, and the cardioprotective actions of DAA-I were blocked by indomethacin. The data showed that the cardioprotection accorded by DAA-I was the result of its anti-inflammatory actions on early inflammatory processes in myocardial ischemia-reperfusion injury. The anti-inflammatory processes were indomethacin sensitive and probably mediated by prostaglandins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although a number of studies have shown that perivascular adipose tissue (PVAT) attenuates arterial contraction through the release of perivascular-derived relaxation factors (PVRF), the role of PVAT in modulating venous function and its mechanism(s) remained unknown. Here we examined the role of PVAT in the modulation of vascular function in the inferior vena cava. Venous rings from male Wistar rats were prepared with both endothelium and PVAT intact, with either PVAT or endothelium removed, or with both endothelium and PVAT removed for functional studies. Contractile response to phenylephrine, U 46619, or 5-hydroxytryptamine was significantly attenuated in PVAT+ as compared with PVAT- veins. PVAT- vessels with intact endothelium (E+) pre-contracted with phenylephrine showed a concentration-dependent relaxation response to angiotensin 1-7 [Ang-(1-7)], and this response was abolished by the removal of endothelium, and by Ang-(1-7) (Mas) receptor antagonists D-Ala-Ang-(1-7) (A779) or D-Pro(7)-Ang-(1-7). Donor solution incubated with a PVAT+ ring induced a relaxation response in the E+ recipient vessel but not in E- recipient vessel. The use of specific channel blockers and enzyme inhibitors showed that Ang-(1-7) is a transferable PVRF that induces endothelium-dependent relaxation through NO release and activation of voltage-dependent potassium (K(+)) channels (K(v)) channels. We conclude that venous PVAT attenuates agonist-induced contraction by releasing Ang-(1-7), which causes relaxation of smooth muscle through endothelial NO release and activation of K(v) channels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The mechanisms by which angiotensin-(1-7) [Ang-(1-7)] exerts its beneficial effects on end-organ damage associated with diabetes and hypertension are not well understood. The purpose of this study was A) to compare the effects of apocynin with Ang-(1-7) on renal vascular dysfunction and NADPH oxidase activity in a combined model of diabetes and hypertension and B) to further determine whether chronic treatment with Ang-(1-7) can modulate renal catalase, and peroxisome proliferator activated receptor- gamma (PPAR-gamma) levels in streptozotocin-induced diabetes in both normotensive Wistar Kyoto rats (WKY) and in spontaneously hypertensive rats (SHR). Apocynin or Ang-(1-7) treatment for one month starting at the onset of diabetes similarly attenuated elevation of renal NADPH oxidase activity in the diabetic SHR kidney and reduced the degree of proteinuria and hyperglycemia, but had little or modest effect on reducing mean arterial pressure. Both drugs also attenuated the diabetes-induced increase in renal vascular responsiveness to endothelin-1. Induction of diabetes in WKY and SHR animals resulted in significantly reduced renal catalase activity and in PPAR-gamma mRNA and protein levels. Treatment with Ang-(1-7) significantly prevented diabetes-induced reduction in catalase activity and the reduction in PPAR-gamma mRNA and protein levels in both animal models. Taken together, these data suggest that activation of Ang-(1-7)-mediated signaling could be an effective way to prevent the elevation of NADPH oxidase activity and inhibition of PPAR-gamma and catalase activities in diabetes and/or hypertension., ((c) 2010 Elsevier B.V. All rights reserved.)

Therapeutic modulation of the renin-angiotensin system is not complete without taking into consideration the beneficial effects of angiotensin-(1-7) in cardiovascular pathology. Various pharmacological pathways are already exploited to involve this heptapeptide in therapy as both inhibitors of angiotensin-converting enzyme and angiotensin II type 1 receptor blockers increase its levels. These drugs and administered angiotensin-(1-7) elicit various common effects, and some effects of the drugs are partially mediated by angiotensin-(1-7). The pharmacodynamic profile of angiotensin-(1-7) is rather complex, and in vitro and in vivo studies demonstrated a wide palette of effects for angiotensin-(1-7), some of them potentially beneficial for cardiovascular disease. Using various animal models to study cardiovascular physiology and disease it was shown that angiotensin-(1-7) has antihypertensive, antihypertrophic, antifibrotic and antithrombotic properties, all properties that may prove beneficial in a clinical setting. We also observed a novel action of angiotensin-(1-7), namely its capacity to stimulate the proliferation of endothelial progenitor cells. Access of angiotensin-(1-7) to the clinic, however, is restricted due to its unfavorable pharmacokinetic properties. In order to benefit of the therapeutic potential of angiotensin-(1-7) it is crucial to increase its half-life, either by using more stable analogues, which are now under development, or specific delivery methods. We here review the pharmacological characteristics and therapeutic potential of angiotensin-(1-7), implementing the experimental strategies taken to exploit the pharmacological mechanism of this heptapeptide in a clinical setting, and present our contribution to this field of research.

Angiotensin 1-7, a heptapeptide derived from metabolism of either angiotensin I or angiotensin II, is a biologically active peptide of the renin-angiotensin system. The present study investigated the effect of angiotensin 1-7 on the vasopressor action of angiotensin II in the renal and mesenteric vasculature of Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and streptozotocin-induced diabetic rats. Angiotensin II-induced dose-dependent vasoconstrictions in the renal vasculature. The pressor response was enhanced in the SHR and reduced in the streptozotocin-diabetic rat compared to WKY rats. Angiotensin 1-7 attenuated the angiotensin II pressor responses in the renal vasculature of WKY and SHR rats. However, the ability to reduce angiotensin II response was diminished in diabetic-induced rat kidneys. The effect of angiotensin 1-7 was not inhibited by 1-[(4-(Dimethylamino)-3-methylphenyl] methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319), an angiotensin AT(2) receptor antagonist. (D-ALA(7))-Angiotensin I/II (1-7) (D-ALA) (an angiotensin 1-7 receptor antagonist), indomethacin (a cyclo-oxygenase inhibitor), and N(omega)-Nitro-L-Arginine Methyl Ester (L-NAME)(a nitric oxide synthetase inhibitor) abolished the attenuation by angiotensin 1-7 in both WKY rats and SHR, indicating that its action is mediated by angiotensin 1-7 receptor that is either coupled to the release of prostaglandins and/or nitric oxide. The vasopressor responses to angiotensin II in mesenteric vasculature bed was also dose-dependent but smaller in magnitude compared to the renal vasculature. The responses to angiotensin II were relatively smaller in SHR but no significant difference was observed between WKY and streptozotocin-induced diabetic rats. Angiotensin 1-7 attenuated the angiotensin II pressor responses in WKY, SHR and diabetic-induced mesenteric bed. The attenuation was observed at the lower concentrations of angiotensin II in WKY and diabetic-induced rats but at higher concentrations in SHR. Similar observation as in the renal vasculature was seen with PD123319, D-ALA, and L-NAME. Indomethacin reversed the attenuation by angiotensin 1-7 only in the SHR mesenteric vascular bed. The present findings support the regulatory role of angiotensin 1-7 in the renal and mesenteric vasculature, which is differentially altered in hypertension and diabetes.

This study analyzed the influence of two main metabolites of angiotensin II, angiotensin IV and angiotensin-(1-7), on basal and angiotensin II-dependent [Ca2+](i) in rat mesangial cells. Angiotensin IV behaved as a weak agonist. Its effects were abolished by angiotensin AT(1) receptor antagonists. Treatment with angiotensin II abolished the effect of a subsequent treatment with angiotensin IV whereas two successive angiotensin IV-dependent [Ca2+](i) peaks were obtained. Angiotensin II increased [Ca2+](i) in a Ca2+-free medium whereas angiotensin IV was inactive. Leucine-valine-valine-hemorphin 7, a hemorphin specific for the angiotensin AT(4) receptor, was devoid of any agonistic or antagonistic effect. In contrast, angiotensin-(1-7), if without influence on basal [Ca2+](i), inhibited angiotensin II- and angiotensin IV-dependent [Ca2+](i) increases. Total inhibition of the angiotensin IV effect was obtained whereas association of angiotensin-(1-7) to 8-(NN-diethylamino)-octyl-3,4,5-trimethoxybenzoate, an inhibitor of inositol phosphate-mediated Ca2+ release, was necessary to suppress the effect of angiotensin II. These results provide evidence that angiotensin II metabolites may participate in the control of [Ca2+](i) in mesangial cells at the initial stage of binding to the angiotensin AT(1) receptors.

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

We used a vasoreactivity assay to examine the functional significance of angiotensin I-converting enzyme overexpression in smooth muscle cells after vascular injury. Rat carotid arteries isolated at days 2 to 14 after in vivo endothelial denudation were compared with the contralateral freshly denuded (control) vessels. Arterial rings were constricted ex vivo with angiotensin I in the absence or presence of the angiotensin I-converting enzyme inhibitors captopril (300 nM and 3 microM) or perindoprilate (1 nM). Angiotensin I-converting enzyme activity was determined by cleavage of the chromogenic substrate Hip-His-Leu. Angiotensin I-converting enzyme activity in injured arteries was increased (2-fold) at day 7 only after vascular injury. Contractions to angiotensin I were unaffected after injury. Inhibition by captopril and perindoprilate of angiotensin I-induced contractions was significantly less potent in injured arteries at day 7 as compared to control vessels. Mechanical removal of neointimal smooth muscle cells normalized the inhibition by captopril in injured arteries at day 7. Captopril did not affect angiotensin II-induced contractions. Thus, upregulation of angiotensin I-converting enzyme after arterial injury confers resistance to angiotensin I-converting enzyme inhibitors.

The influence of the angiotensin-converting enzyme inhibitor captopril on bradykinin-and angiotensin I-induced responses with special regard to nitric oxide (NO) was studied. Auxometric tension and angiotensin-converting enzyme activity was studied in isolated porcine iliac arteries. Captopril potentiated bradykinin-induced contraction of preparations with intact endothelium; this potentiation was not seen with the kininase I inhibitor mergepta or a bradykinin B(1)-receptor antagonist. Captopril did not affect bradykinin-induced relaxation. The captopril-mediated increase of bradykinin-induced contraction was only seen in preparations with intact endothelium, while captopril did not affect arterial strips treated with Nomega-nitro-L-arginine. Angiotensin I-induced contractions was less reduced by captopril when the strips were pretreated with Nomega-nitro-L-arginine. Both captopril and the NO donor S-nitroso-N-acetyl-penicillamine inhibited angiotensin-converting enzyme activity. An additional reduction in angiotensin-converting enzyme activity was seen when S-nitroso-N-acetyl-penicillamine was added to captopril-treated preparations. In conclusion, captopril increased bradykinin-induced contraction in a NO-dependent manner. This potentiation is probably mediated by the increased metabolism of bradykinin by kininase I, and the additive angiotensin-converting enzyme inhibitory effect of captopril and NO.

We investigated the contribution of chymase-dependent conversion of angiotensin I to angiotensin II in hamster cheek pouch. To investigate the converting activities in intact tissues, angiotensin I or II was applied to microvessels of the intact cheek pouch, and the vascular contractile response was recorded. Angiotensin I or angiotensin II (20 nM) induced a rapid contraction of arterioles, irrespective of their diameter. In the presence of I mM captopril, there was no contraction in response to angiotensin I in arterioles < 25 microm in diameter, whereas contraction was still observed in larger arterioles. Chymostatin (100 microM) treatment also reduced the response to angiotensin I in arterioles > 40 microm in diameter. Treatment with 1 mM captopril and 100 microM chymostatin resulted in the loss of response to angiotensin I, but not to angiotensin II, in all arterioles. Treatment of microvessels with 100 microg/ml compound 48/80 enhanced angiotensin I-induced vascular contraction response, suggesting the significance of mast cells as a source of cheek pouch chymase.

We previously reported that endogenous angiotensin II is released to cause mitogen-activated protein (MAP) kinase stimulation in the media portion of the vasculature. In this study, we examined whether a functional renin-angiotensin system is indeed present within the media of the vasculature. In rat aortic strips, endothelium removal produced an increase of MAP kinase activity. The MAP kinase activation was inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of the MAP kinase activation by pepstatin A, captopril and the angiotensin receptor antagonist losartan was almost the same. Pepstatin A inhibited MAP kinase activation induced by renin but not by angiotensin I and angiotensin II. Captopril inhibited the MAP kinase activation induced by angiotensin I but not by angiotensin II. In nephrectomized rat aortic strips, endothelium removal also produced an increase in MAP kinase activity, but the MAP kinase activation was considerably small and minimally inhibited by losartan. Nephrectomy produced a marked decrease in plasma renin activity. These findings suggest that an apparently fully intact and functional renin-angiotensin system is present in the media of the rat vasculature and this system serves to increase MAP kinase activity. It appears that renin plays the determining role in the regulation of angiotensin generation also in the media and the major source of the renin is renin of kidney origin.

The hemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is degraded by ACE. This study was designed to examine the effect of Ac-SDKP on the contractions to angiotensin I. Experiments were performed on rat aortic rings with endothelium exposed to nitro-L-arginine. Ac-SDKP (10 and 100 microM) significantly augmented angiotensin I ED20 (from 2.0+/-0.4 to 4.2+/-1.0 and 5.0+/-0.9 nM) and ED50 (from 4.3+/-0.7 to 8.6+/-1.0 and 10.7+/-1.3 nM, respectively), but did not alter its maximal response. The contractions to angiotensin II were not affected by Ac-SDKP. No degradation of exogenous Ac-SDKP nor detectable release of endogenous Ac-SDKP were observed in the incubation medium. These results suggest that Ac-SDKP impairs angiotensin I response by inhibiting ACE and subsequent angiotensin II formation.

Using a number of agonist and antagonist compounds, we attempted to characterize the responses and receptors involved in the effects of 5-hydroxytryptamine (5-HT) in the in situ autoperfused rat kidney. An intra-arterial (i.a.) bolus injection of 5-HT (0.0125 to 0.1 microg/kg) increased renal perfusion pressure in a dose-dependent way but did not change the systemic blood pressure. The 5-HT2 receptor agonist, (1-(3-chlorophenyl) piperazine), m-CPP, caused a local vasoconstrictor effect in the autoperfused rat kidney. An intra-arterial injection of 5-carboxamidotryptamine, 5-CT and 1-(m-chlorophenyl)-biguanide (m-CPBG) did not modify the renal perfusion pressure. The vasoconstrictor effect elicited by 5-HT and m-CPP was significantly decreased by ritanserin, enalapril and losartan but was not modified by prazosin, propranolol or indomethacin pretreatment. Our data suggest that the vasoconstrictor serotonergic response induced in the in situ autoperfused rat kidney is mediated through angiotensin II activation by a local 5-HT2 receptor mechanism.

Responses to angiotensin I and antiogensin I-(3-10), the precursors for angiotensin II and IV, were investigated in the mesenteric vascular bed of the cat. Under constant-flow conditions, injections of precursors and the active peptides into the mesenteric arterial perfusion circuit caused dose-related increases in receptor antagonist that were attenuated by the angiotensin AT1 receptor antagonist DuP532 (2-propyl-4-pentafluorethyl-1-[2'-(2H-tetrazol-5-YL)-1,1'-bi phenyl-4-YL methyl]1H-imidazole-5-carboxylic acid), but not by the angiotensin AT2 receptor antagonist PD123,319 ((S)1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl )-4,5,6,7- tetrahydro-1H-imadazo[4,5-c]pyridine-6-carboxylic acid, ditriflouroacetate]). Responses to angiotensin I and II were similar as were responses to angiotensin I-(3-10) and angiotensin IV, and these responses were not altered by the presence of a time-delay coil in the perfusion circuit. Responses to angiotensin I and angiotensin I-(3-10) were decreased by the angiotensin converting enzyme inhibitor enalaprilat in a dose of the angiotensin converting enzyme inhibitor that had no effect on responses to angiotensin II and IV and that enhanced vasodilator responses to bradykinin. The putative angiotensin AT2 receptor agonist, p-aminophenylalanine6-angiotensin II, produced dose-related increases in mesenteric arterial perfusion pressure that were reduced by DUP532, suggesting that they are mediated by angiotensin AT1 receptors. These results suggest that angiotensin I and angiotensin I-(3-10) are rapidly and efficiently converted by an angiotensin converting enzyme-dependent pathway into active peptides that induce vasoconstriction by activating angiotensin AT1 receptors in the mesenteric vascular bed of the cat.

The ability of intracerebroventricularly (i.c.v.) administered D-amino acid-substituted analogues of des-Asp-angiotensin I to attenuate the central pressor action of angiotensin III in the rat was investigated. Of the 9 D-amino acid-substituted analogues, only D-tyrosine-des-Asp-angiotensin I was active. I.c.v. D-tyrosine-angiotensin I but not i.c.v. D-isoleucine-angiotensin I (when prevented from degradation by angiotensin converting enzyme with captopril) also attenuated the central pressor action of angiotensin III. In vitro incubation of angiotensin I, D-tyrosine-angiotensin I and D-isoleucine-angiotensin I with brain homogenate resulted in the formation of des-Asp-angiotensin I, D-tyrosine-des-Asp-angiotensin I and D-isoleucine-des-Asp-angiotensin I, respectively. This shows that i.c.v. angiotensin I and D-tyrosine-angiotensin I were converted by brain aminopeptidase to des-Asp-angiotensin I and D-tyrosine-des-Asp-angiotensin I, respectively, which then attenuated the pressor action of angiotensin III. When compared to the findings of similar D-substitution studies carried out with angiotensin II and [Sar1,Ile8]angiotensin II by other investigators, des-Asp-angiotensin I has a stringent structural-activity relationship. These findings suggest that, at the physiological level, des-Asp-angiotensin I is formed from angiotensin I and that the nonapeptide probably acts on a distinct subtype of angiotensin receptors.

The mechanism of the NG-nitro-L-arginine (L-NNA)-induced pressor response was examined in pentobarbital-anesthetized dogs. The pressor effect of L-NNA (50 mg/kg, i.v.) was significantly and equally diminished by pretreatment with either hexamethonium (25 mg/kg, i.v.) or phentolamine (5 mg/kg, i.v.). The intracisternal administration of L-NNA (1 mg/kg), which did not cause changes in cardiovascular parameters when administered systemically, produced a significant pressor response and tachycardia. Furthermore, significant suppression of L-NNA-induced pressor responses was observed after treatment of dogs with captopril (5 mg/kg, i.v.) or a non-peptide angiotensin II receptor antagonist, losartan (10 mg/kg, i.v.), or bilateral occlusion of renal veins. The inhibitory effects of hexamethonium and losartan were additive. These results suggest that, in addition to vasoconstriction due to the inhibition of endothelial nitric oxide production, increased activity of the sympathetic nervous and renin-angiotensin systems contributes significantly to the development of pressor responses produced by the intravenous injection of L-NNA in anesthetized dogs.

In the presence of 3 x 10(-6) M captopril, 5 x 10(-7) M des-Asp-Angiotensin I was found to inhibit the electrically (1 and 2 Hz) induced contraction of the rabbit pulmonary artery but had no significant effect on the noradrenaline-stimulated contraction. 2.8 x 10(-6) M indomethacin and 10(-6) M losartan but not 10(-6) M (S) 1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4, 5,6,7- tetrahydro-1H-imidazo(4,5-c]pyridine-6-carboxylic acid, ditrifluoroacetate, dihydrate (PD123319) attenuated the inhibition. The inhibition of the electrically stimulated contraction by 5 x 10(-7) M des-Asp-angiotensin I coincided with a significant drop in the accompanying evoked 3H overflow from re-uptaken [3H]noradrenaline. The results indicate that des-Asp-angiotensin I acts presynaptically on a subtype of angiotensin receptor that involves the release of prostaglandin(s). In addition, this receptor subtype is susceptible to blockade by angiotensin AT1- but not AT2-specific receptor antagonists. It was suggested that this receptor subtype is identifiable with the recently described angiotensin AT1B receptor subtype found in the brain, pituitary and adrenal glomerulosa. These findings demonstrated a direct action of sub-micromolar concentrations of des-Asp-angiotensin I on a blood vessel and indicate that the nonapeptide is an active angiotensin per se.

The present study was undertaken to characterize the direct chronotropic effect of bradykinin in isolated spontaneously beating atria of the guinea pig. Bradykinin caused concentration-dependent increases in the beating rate of atria. In contrast, the active metabolite of bradykinin and the typical bradykinin B1 receptor agonist, Des-Arg9-bradykinin, had no effect on the beating rate of atria. Inhibition of converting enzyme or neutral endopeptidase by captopril or SQ-28603, respectively, did not affect beating rate but potentiated bradykinin-induced increase in beating rate. The potent bradykinin B2 receptor antagonist, HOE 140, antagonized bradykinin-induced chronotropic effect. In contrast, the bradykinin B1 receptor antagonist, Lys-[Leu8]Des-Arg9-bradykinin, had no effect. The increase in beating rate caused by bradykinin was not affected by blockade of beta 1-adrenoceptors, cyclooxygenase, or nitric oxide synthesis using atenolol, indomethacin and N omega-nitro-L-arginine, respectively. Unlike bradykinin, angiotensin I and angiotensin II caused very small or no change in beating rate in the presence or absence of captopril and SQ-28603. These results indicate that bradykinin causes a direct positive chronotropic effect which is mediated by activation of bradykinin B2 receptors independently of prostaglandins and beta 1-adrenoceptors.

Nanomolar concentrations of des-Asp-angiotensin I potentiated the contractile action of angiotensin II on the rabbit aortic ring but attenuated the contractile action of angiotensin III in the same tissue. Indomethacin had no effect on the potentiation of angiotensin II but inhibited the attenuation of angiotensin III. The action of angiotensin II, angiotensin III and des-Asp-angiotensin I was not inhibited by (S)-1-}[4-(dimethylamino)-3-methylphenyl]methyl}-5-(diphenylacetyl )-4,5,6, 7-tetrahydro-1H-imidazo-[4,5-c]pyridine-6-carboxylic acid, ditrifluoroacetate, dihydrate (PD123319), an angiotensin AT2 receptor antagonist. The data show that angiotensin II and angiotensin III act on different subclasses of angiotensin receptors and that their actions are differentially modulated by des-Asp-angiotensin I. The data also indicate the possibility that des-Asp-angiotensin I is a functional peptide that modulates the contractile action of the two angiotensins at sub-nanomolar concentrations.

Responses to angiotensin IV were investigated and compared with responses to angiotensin II in the pulmonary vascular bed of the intact-chest cat under constant flow conditions. Under low resting tone conditions, intralobar injections of angiotensin IV caused dose-related increases in lobar arterial pressure. Angiotensin II also increased lobar arterial pressure and was 100 fold more potent than angiotensin IV. Dose-response curves for both peptides were parallel, and the time-to-peak increase in lobar arterial pressure in response to angiotensin IV and angiotensin II was similar whereas the duration of the response to angiotensin IV was significantly shorter. Following administration of the angiotensin receptor subtype 1 (AT1) antagonist, DuP 532 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4- yl)-methyl]imidazole), responses to angiotensin IV and angiotensin II were reduced in a similar manner, whereas pulmonary pressor responses to serotonin were not altered. In contrast to the inhibitory effects of the angiotensin AT1 receptor antagonist, administration of PD 123,319 ((S)-1-[[4-(dimethylamino)-3-methyl-phenyl]methyl-5-(diphenylacetyl++ +)- 4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxylic acid), an angiotensin AT2 receptor antagonist, did not change responses to angiotensin II and angiotensin IV. The results of the present study demonstrate that angiotensin IV has significant vasoconstrictor activity in the pulmonary vascular bed, and suggest that pressor responses to angiotensin IV are mediated by the activation of angiotensin AT1 receptors. These data indicate that angiotensin IV is 100-fold less potent than angiotensin II and suggest that the hexapeptide may have a lower apparent affinity for the angiotensin AT1 receptor than does angiotensin II in the pulmonary vascular bed of the cat.

Intracerebroventricularly administered des-Asp-angiotensin I, when prevented from degradation by prior administration of captopril, attenuated dose-dependently the central pressor actions of angiotensin II and angiotensin III in the spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. This finding is the first demonstration of an intrinsic action of des-Asp-angiotensin I and, together with earlier finding of its increased production in the hypothalamus of the spontaneously hypertensive rat, may support the suggestion that the nonapeptide is a functional angiotensin that regulates the pressor action of angiotensin II and angiotensin III in the brain.

Angiotensin II and angiotensin III stimulated prostacyclin release in a time- and dose-dependent manner in both the prostatic and the non-prostatic part of the rabbit vas deferens. Also, angiotensin I enhanced the production of prostacyclin and its effect was blocked by captopril. Losartan, a type 1 (angiotensin AT1)-selective receptor antagonist, prevented the angiotensin II-induced prostacyclin release. The agonist peptide, p-aminophenylalanine angiotensin II, and the type 2 (angiotensin AT2)-selective receptor antagonist, PD123319, were found active only in the prostatic portion, suggesting heterogeneity of the receptor population. In conclusion, an angiotensin AT1 receptor mostly mediates the angiotensin-induced release of prostacyclin in the rabbit vas deferens.

To investigate the role of local renin angiotensin systems in the functional responses to angiotensin I and II, the effects of angiotensin I and angiotensin II on resistance were measured in perfused hindlimbs of rats under normal conditions and during infusion of enalaprilate, lisinopril, zabiciprilate and captopril at two infusion rates. The angiotensin-converting enzyme (ACE) inhibitors significantly increased ED50 and decreased maximal resistance changes of angiotensin I dose dependently, without effects on angiotensin II responses. Captopril increased the ED50 of angiotensin I significantly more than did the other ACE inhibitors at a low infusion rate. The ACE inhibitors, except for lisinopril, increased the ED50 of angiotensin I pressor responses in vivo to the same extent, and were similarly potent to inhibit plasma ACE activity at 15 min after injection. The ratio of the doses of the ACE inhibitors used in vivo was the same as in perfused hindlimbs. These results suggest that, in the hindlimbs, angiotensin I causes ACE-dependent vasoconstriction. Captopril may be more effective to inhibit local ACE than the other ACE inhibitors investigated.

The responses to angiotensinogen, angiotensinogen-(1-14) (tetradecapeptide, TDP), angiotensin I, and angiotensin II and the effect of the renin inhibitor, CH-66, the angiotensin-converting enzyme (ACE) inhibitor, perindopril, and the receptor antagonist, saralasin, were investigated in isolated femoral resistance arteries of the rat. The response to angiotensinogen in the presence of kallikrein was also investigated. Angiotensin I and TDP elicited a contraction which was not reduced by CH-66 but was inhibited by perindopril and saralasin. The response to angiotensinogen was small and not blocked by saralasin but the response to angiotensinogen that was mixed with renin for a few seconds was saralasin-sensitive and perindopril and CH-66 showed a tendency to block this response. The response to angiotensinogen was enhanced in the presence of kallikrein. These results suggest (1) the presence of a partial renin-angiotensin system (RAS) in this preparation, (2) that TDP is not converted via renin while both angiotensin I and TDP are converted through ACE in this preparation and (3) that the responses to angiotensinogen and TDP are different.

Angiotensin I and II inhibited the nerve stimulation-induced pressure changes in isolated, perfused rabbit ear artery with an IC50 of 3.07 and 0.36 nM, respectively. Neither angiotensin I nor angiotensin II affected the basal pressure or the pressure changes elicited by exogenously administered norepinephrine (NE). The potency of angiotensin I was unaltered by 10(-5) M captopril, indicating that conversion by angiotensin converting enzyme (ACE) was not necessary and did not take place. [Sar1,Val5,Ala8]angiotensin II (3 x 10(-8) M) antagonized the effect of angiotensin I. These findings could have implications regarding ACE inhibitor therapy and the pathophysiology of migraine.

The effect of acute administration of SQ 14,225, a new angiotensin converting enzyme inhibitor, on the drinking response of female rats administered either isoprenaline, angiotensin I, or angiotensin II was studied during 2 h after treatment. Administration of isoprenaline (25 micrograms/kg body wt) was accompanied by a significant increase in water intake when compared with saline-treated controls. Acute administration of a constant dose of isoprenaline (25 micrograms/kg body wt) and increasing doses of SQ 14,225 (5--50 mg/kg) was accompanied by a dose-related, linear decrease in water intake. Acute administration of either angiotensin I or angiotensin II (200 micrograms/kg body wt) was accompanied by a significant increase in water intake. The dipsogenic response to angiotensin II was not affected by acute administration of 35 mg SQ 14,225/kg body wt. However, at the same dose of SQ 14,225, angiotensin I-induced thirst was attenuated. Since isoprenaline-induced and angiotensin I-induced, but not angiotensin II-induced, thirsts are blocked by SQ 14,225, the results suggest that isoprenaline-induced thirst is mediated by way of the renin--angiotensin system.

To evaluate the cardiac effect of an inhibitor of angiotensin-converting enzyme, the effect of intracoronary (i.c.) captopril on coronary blood flow and regional myocardial function was examined in the anesthetized open-chest dog. Blood flow of the left circumflex coronary artery (LCX), left ventricular pressure (LVP), aortic pressure (AoP) and regional myocardial segment length were measured continuously. Captopril i.v. (0.3 mg/kg) produced an immediate reduction in AoP and an increase in percent shortening of myocardial segments followed by a decrease in coronary vascular resistance and increases in heart rate and LVdP/dt. Reductions in LCX flow induced by i.c. angiotensin were attenuated and i.c. bradykinin-induced increases in LCX flow were augmented after captopril. On the contrary, i.c. infusion of captopril (0.01 mg/min) into the LCX caused no change in hemodynamic variables and myocardial shortening although responses to angiotensin I and bradykinin were markedly modified. These results suggest that captopril may have no direct cardiac effect.

The cardiovascular pharmacology of SQ 14,225 (D-3-mercapto-2-methylpropanoyl-L-proline), a new orally effective inhibitor of angiotensin-coverting enzyme (ACE) was investigated in conscious normotensive rabbits. Intravenous administration of SQ 14,225 (3.1-310.0 microgram/kg) resulted in a dose related inhibition of the pressor responses to 310 ng/kg, i.v. of angiotensin I (AI) without diminishing the pressor responses to 100 ng/kg, i.v. of angiotensin II (AII). In fact, the responsiveness of the rabbits to AII was significantly enhanced by higher doses of SQ 14,225. This enhancement of the pressor effects of AII was found to be related to the inhibition of ACE and the resulting decrease in the levels of endogenous AII. In addition, SQ 14,225 (1.0 mg/kg, i.v.) markedly potentiated the magnitude and duration of the vasodepressor responses elicited by bradykinin (1.0 microgram/kg. i.v.). At a dose of 1.0 mg/kg. i.v., SQ 14,225 had no effect on the vasodepressor effects of intravenously administered isoproterenol (0.4 microgram/kg), acetylcholine (1.0 microgram/kg) or prostaglandin E2 (3.0 microgram/kg, i.v.). The pressor responses to norepinephrine (3.0 microgram/kg, i.v.) were similarly unaffected by SQ 14,225 (1.0 mg/kg, i.v.). In normal rabbits SW 14,225 (1.0 mg/kg, i.v.) caused a small but significant decrease in arterial pressure; it had no such effect in anephric rabbits. The observation of this study indicate that SQ, 14,225 is a specific inhibitor of ACE in conscious rabbits.

In the present study, pretreatment of rats with captopril significantly stimulated prostacyclin (PGI2) synthesis by their isolated aorta. This effect was maximal at captopril doses of 1.0 mumol/kg body weight. When added directly into the incubation buffer, captopril at final concentrations of 50 and 500 nM also increased the synthesis of PGI2 by isolated rat aorta. Our results show that captopril stimulates PGI2 synthesis in vascular tissue and that this effect may be due at least in part to a direct action of this substance.

The effect of the selective kinin B1 receptor agonist des-Arg9-BK was studied on blood pressure and on the in vitro aorta of rabbits pretreated 18 h earlier with lipopolysaccharide from E. coli, an infusion of bradykinin or with one of three angiotensin converting enzyme inhibitors captopril, enalapril or teprotide. The hypotensive response in vivo and contractile response seen on the in vitro aorta was selectively increased to des-Arg9-BK in all pretreated groups compared to controls, effects which were blocked by the selective competitive kinin B1 receptor antagonist des-Arg9-[Leu8]BK. Dexamethasone given to lipopolysaccharide pretreated rabbits had no effect on the increased hypotensive response seen with des-Arg9-BK. The skin vascular permeability response to des-Arg9-BK, bradykinin and histamine remained unchanged in the groups pretreated with lipopolysaccharide or captopril compared to controls. The possible mechanism(s) whereby angiotensin converting enzyme inhibitors produce this effect and the possible relevance to the inflammatory side-effects seen with this group of drugs is discussed.

(4R)-3-[(2S)-3-Mercapto-2-methylpropanoyl]-4-thiazolidinecarboxylic acid (YS-980), a potent angiotensin I (AI) converting enzyme inhibitor, inhibited the contractile response of isolated guinea-pig ileum to AI but not to AII, while it potentiated the response to bradykinin. Other sulfhydryl compuonds also produced inhibition of AI action, and the effects were closely correlated with their potentiating activities against bradykinin action. These results suggest that it is mainly inhibition of the enzyme in the tissue which participates in the mechanism of potentiation of kinin action by YS-980.

Oral administration of SQ 14,225 (0.03--3 mg/kg) to conscious normotensive dogs caused inhibition of the pressor response to intravenously administered angiotensin I (AI), the duration of which was dose-dependent. All doses of 0.1 mg/kg or greater caused 85--95% inhibition 30 min after administration whereas 0.03 mg/kg produced only a 25% inhibition. Pressor responses to angiotensin II (AII) were not similarly inhibited. Blood pressure was moderately reduced in a dose-related manner and followed the same pattern as inhibition of the AI pressor responses. The maximum change occurred after 1.0 mg/kg with only a more rapid onset occurring after the 3.0 mg/kg dose. Heart rate was not appreciably changed. SQ 14,225 also increased plasma renin activity (PRA), the levels and duration of which were dose-related. These data indicate that SQ 14,225 is an orally effective, potent inhibitor of angiotensin I-converting enzyme (ACE) in dogs. It appears that in mongrel dogs, ACE inhibition results in a slight to moderate reduction in blood pressure and an increase in PRA.

The extent to which cardiac neuroeffector function may be modulated by angiotensin II (AII) generated locally from angiotensin I (AI) was investigated in guinea-pig spontaneously beating isolated atria radiolabelled with [3H]noradrenaline. AI and AII were equipotent in increasing the rate and force of atrial contractions with a threshold concentration between 0.1 and 1.0 nM. In contrast, AI was approximately twenty times less potent than AII in enhancing the stimulation-induced (S-I) efflux of radioactivity. The actions of AI and AII on rate and force of atrial contractions, as well as those on S-I efflux of radioactivity, were substantially blocked by the AII receptor antagonist saralasin. On the other hand, the converting enzyme inhibitors captopril and MK-422 selectively blocked the actions of AI without affecting those of AII. These results suggest that significant local conversion of AI to AII occurs in guinea-pig isolated atria. This locally generated AII can act on myocardial AII receptors to increase rate and force of atrial contractions, and on AII receptors located on sympathetic nerve terminals to facilitate sympathetic neurotransmission. The lower potency of AI compared to AII in modulating transmitter release, in contrast to the equal potency of the peptides in mediating chronotropic and inotropic responses, may be due to conversion of AI to AII at sites remote from the neuroeffector junction.

Intravenous Captopril (20 microgram) in rats produced similar inhibition of the pressor responses to intravenous ANG I (70%) and of plasma-converting enzyme activity (CEA) measured fluorometrically (72%). One week oral treatment with Captopril (50 mg/kg per day) inhibited ANG I pressor responses more (84%) than plasma CEA (23%). Four-month oral treatment of normotensive and spontaneously hypertensive rats with Captopril (50 mg/kg per day) led to a 68% and 71% inhibition of the ANG I pressor responses, but to a 123% and 94% increase of plasma CEA, respectively. Thus, plasma CEA measurements can be dissociated from the in vivo inhibition of converting enzyme (CE). Chronic oral Captopril treatment induces CE biosynthesis.

Trandolapril (RU 44570), a new non-sulfhydryl angiotensin-converting enzyme (ACE) inhibitor chemically related to enalapril, and its diacid (RU 44403), were investigated for their ability to inhibit angiotensin-converting enzyme. Trandolapril attenuated angiotensin I (Ang I)-induced pressor responses following i.v. administration to rats and dogs with ID50 values of 13.1 +/- 1.3 and 21.1 +/- 2.3 micrograms/kg. RU 44403 produced corresponding values of 9.9 +/- 0.7 and 7.2 +/- 2.3 micrograms/kg. Trandolapril (3-300 micrograms/kg) produced a dose-related attenuation of Ang I-induced pressor responses (ID50 30 micrograms/kg) following oral administration to rats. Oral administration of trandolapril (30-1000 micrograms/kg) to dogs inhibited Ang I pressor responses for over 6 h. The depressor action of bradykinin in the rat was potentiated by i.v. trandolapril and RU 44403 with ED50 values of 5.5 +/- 0.8 and 4.9 +/- 0.3 micrograms/kg respectively. Trandolapril was 2.3-10-fold more potent than enalapril in all experiments, depending on species or route of administration. RU 44403 and MK 422 were approximately equipotent, implying that trandolapril was more readily hydrolysed than enalapril. Trandolapril (0.3-30 mg/kg) produced dose-related, long-lasting (greater than 24 h) reductions in blood pressure (BP) in spontaneously hypertensive rats (SHR) following oral administration. The anti-hypertensive effect was potentiated significantly in hydrochlorothiazide-pretreated SHR when the plasma renin activity was increased. Enalapril was 10-fold less potent than trandolapril in reducing BP. The anti-hypertensive action of trandolapril (3 mg/kg) was abolished in SHR that were bilaterally nephrectomized 24 h beforehand, but was maintained in SHR pretreated by indomethacin (5 mg/kg p.o.). Trandolapril (1 mg/kg i.v.) produced a modest and transient reduction in BP in anesthetized dogs. Trandolapril produced dose-related (30-1000 micrograms/kg) reductions in BP, total peripheral resistance and heart work in dogs pretreated with hydrochlorothiazide to increase plasma renin activity.

Chronotropic responses to angiotensin I and angiotensin II, vasopressin and bradykinin were measured in guinea pig isolated right atria. Angiotensin II (100-30,000 pg/ml) was slightly more potent than angiotensin I and caused a maximum tachycardia of 30-40 b/min; only 20% of the maximum response to (--)-noradrenaline. Propranolol (1 micro M) or reserpine pretreatment (1 mg/kg i.p., 24 h) did not alter the response to angiotensin II or bradykinin. Converting enzyme inhibition by captopril (10 micrograms/ml) did not affect resting rate nor the response to angiotensin II but shifted the location of the angiotensin I curve by 40 fold to the right. Bradykinin (5-500 ng/ml) caused small increases in rate while vasopressin 1-100 ng/ml was completely without effect. These results suggest that angiotensin II has a small positive chronotropic effect that is not dependent on tissue noradrenaline release or beta-adrenoceptors and that tissue converting enzyme is active in right atria. Relatively high concentrations of angiotensin and bradykinin were required to directly stimulate the sino-atrial node compared with plasma levels measured during physiological stimuli. Therefore these effects on atria are probably of little physiological significance for peptide concentrations in plasma but may be important in relation to local tissue generation of angiotensin II.

The ability of captopril and one of its metabolites, the disulphide dimer have been studied for their ability to affect angiotensin I and bradykinin responses. Captopril disulphide dimer (i.v.) potentiated the vasodilatory effects of bradykinin in urethane-anaesthetized rats, at doses of 0.1 and 0.3 mg/kg but did not inhibit the angiotensin I-mediated pressor response at these same concentrations. This activity of captopril disulphide dimer in vivo was not seen with bradykinin responses in isolated guinea pig ileum except at bath concentrations much higher than for captopril (10(-5) M). However captopril and captopril disulphide dimer at oral doses of 10 mg/kg both lowered systolic and diastolic blood pressures in spontaneously hypertensive rats. These studies are consistent with an in vivo bradykinin-potentiating activity of captopril disulphide dimer and suggest a possible antihypertensive activity of disulphide metabolites of captopril.

A carboxypeptidase inhibitor (DL-2-mercaptomethyl-3-guanidoethylthiopropranoic acid) (mergetpa) was used to block the conversion of kinins and B2 receptor antagonists into metabolites devoid of the C-terminal Arg. Experiments were carried out on rabbit isolated aortae (a B1 receptor system) or rabbit jugular veins and dog carotid arteries (two B2 receptor systems). The contractile effect of bradykinin in the rabbit aorta was significantly reduced by mergetpa while that of desArg9-BK was not modified. pA2 values of B2 receptor antagonists, [Thi5,8,D-Phe7]bradykinin and [Thi6,9,D-Phe8]kallidin were markedly reduced by mergetpa. The apparent affinity (pA2) of a B1 receptor antagonist, [Leu9]desArg10-kallidin was not affected. Carboxypeptidases inhibition did not modify the activities of bradykinin or the affinities of B2 receptor antagonists in the rabbit jugular vein and the dog carotid artery. An inhibitor of kininase II (D-3-mercapto-2-methylpropranoyl-L-proline (S,S] (captopril) reduced the contractile effects of angiotensin I in the three preparations and potentiated the stimulatory or inhibitory effects of bradykinin: captopril did not have effect on the affinities of B2 receptor antagonists and did not modify the effects of angiotensin II. Comparative experiments performed in tissues with or without endothelium gave the same results with both mergetpa and captopril. The present findings suggest that bradykinin and B2 receptor antagonists are converted by carboxypeptidases into biologically active B1 receptor agonist or antagonists. This is the reason why B2 receptor antagonists are not selective.

Changes in the biological activities of a number of autacoids after a single passage through the human perfused fetoplacental lobule have been assessed by bioassay, whilst recording fetal vascular resistance. Bradykinin did not affect vascular resistance, but its biological activity on the superfused bioassay tissues fell by approximately 98%, whereas des-Asp1-angiotensin I activity increased at least 80-fold and the vascular resistance rose. All these effects were inhibited by captopril. Angiotensin II increased vascular resistance but its activity on the bioassay tissues was not changed. 5-Hydroxytryptamine activity was reduced by 67-90% and resistance to flow was not affected. The activities of prostaglandins D2, E2, and F2 alpha were slightly reduced. Prostaglandins D2 and F2 alpha caused vasoconstriction, their maximum effects being greater than those of either of the angiotensins. The TxA2-mimetic U46619 was approximately 90 times more potent than PGF2 alpha, as a vasoconstrictor, but the maximal effects were comparable. Thus, autacoid activity can be reduced, augmented or not affected during passage through the human perfused fetal placental vasculature.

An angiotensin antagonist, Sarcosyl1-Cysteinyl(S-Methyl)8-angiotensin I [Sar1-Cys(Me)8-ANG I] was synthesized and its pharmacological properties evaluated in vivo (rat blood pressure assay) and in vitro (rabbit aortic strips, guinea-pig ileum and rat uterus assays). It was found to be an extremely potent angiotensin II (ANG II) antagonist in the rat pressor assay (dose ratio for ANG II of 1300 during infusion of 5.0 micrograms/kg/min Sar1-Cys(Me)8-ANG I) and a moderately effective antagonist in guinea-pig ileum (pA2 congruent to 8.2). Moderate antagonism was also seen in the rabbit aortic strip preparation (pA2 congruent to 8.1) while the analog was inactive in the rat uterus assay. In each of the preparations where antagonist activity was observed there was evidence of non-competitive antagonism. Most striking was the inability of extremely high doses ( up to 125 micrograms ANG II/kg) of ANG II to overcome the Sar1-Cys(Me)8-ANG I blockade. In both the rat pressor and guinea-pig ileum assays the Sar1-Cys(Me)8-ANG I antagonism is completely abolished in the presence of the converting enzyme inhibitor SQ14225 (Captopril-Squibb). Organ selectivity of this analog is discussed in terms of the inherent activity of the active principle (i.e. the Sar1-Cys(Me)8-angiotensin II [Sar1-Cys(Me)8-ANG II] released by the action of converting enzyme) and the availability of converting enzyme in each bioassay.

The effect of angiotensin-converting enzyme inhibition by captopril on the release of a prostacyclin-like substance by bradykinin, angiotensin I and angiotensin II was studied by means of the blood-bathed bioassay technique of Vane. Administration of captopril abolished the release of prostacyclin-like substance induced by angiotensin I, potentiated the release provoked by bradykinin and did not alter that due to angiotensin II. Potentiation of the bradykinin-induced renal vasodilatation with captopril could be completely reversed by indomethacin, which also abolished the kinin-induced release of prostacyclin-like substance. Potentiation of the bradykinin-induced hypotension was markedly attenuated but not completely reversed by cyclo-oxygenase inhibition. It is suggested that following converting inhibition increased production of prostacyclin by elevated kinin levels may contribute to the antihypertensive action of angiotensin-converting enzyme inhibitors.

Angiotensin I (AI) and angiotensin II (AII) caused concentration-dependent increases in atrial rate in guinea pig isolated right atria. Converting enzyme inhibitors captopril and MK421-diacid did not alter the responses to AII but displaced the curves to AI to the right. The atrial response to generated AII from AI was used as a bioassay to estimate the dissociation constants of converting enzyme inhibitors (Kb) and test for kinetics of simple competition. MK421-diacid was 12-40 times more potent than captopril. However, estimations of Kb for captopril and MK421-diacid were unsatisfactory because at high concentrations of inhibitors the curves to AI were not displaced according to simple competition. We conclude that AI in high concentration can stimulate AII receptors accounting for the stationary displacement of curves to AI in the presence of converting enzyme inhibitors. MK421-diacid also potentiates responses to bradykinin in this assay.

The sensitivity of the guinea pig isolated ileum to bradykinin, but not to other agonists, was increased ca. 2-fold during the 3-4 h following mounting of the preparation under 1 g load. Concomitantly, a decrease was observed in the bradykinin potentiating effect of BPP9a, and of potentiator B but not potentiator C. This decrease was observed only with analogues of BPP9a or potentiator B which retained one or both of the basic amino acid residues in these peptides. Similar stretching of the rat isolated uterus did not affect bradykinin sensitivity or the potency of bradykinin potentiating peptides. Kininase activity of the ileum significantly increased during the 4 h period after mounting of the loaded preparation, but was not affected by treatment with BPP9a. It is proposed that bradykinin sensitivity is favoured by the changes in sodium and potassium transport in the cell membrane caused by stretching of the ileum, and that a similar mechanism may be partly responsible for the action of bradykinin potentiating peptides.