Beril Tom

Bradykinin Potentiation by Angiotensin-(1-7) and ACE Inhibitors Correlates With ACE C- and N-Domain Blockade

Abstract—ACE inhibitors block B2 receptor desensitization, thereby potentiating bradykinin beyond blocking its hydrolysis. Angiotensin (Ang)-(1-7) also acts as an ACE inhibitor and, in addition, may stimulate bradykinin release via angiotensin II type 2 receptors. In this study we compared the bradykinin-potentiating effects of Ang-(1-7), quinaprilat, and captopril. Porcine coronary arteries, obtained from 32 pigs, were mounted in organ baths, preconstricted with prostaglandin F2&agr;, and exposed to quinaprilat, captopril, Ang-(1-7), and/or bradykinin. Bradykinin induced complete relaxation (pEC50=8.11±0.07, mean±SEM), whereas quinaprilat, captopril, and Ang-(1-7) alone were without effect. Quinaprilat shifted the bradykinin curve to the left in a biphasic manner: a 5-fold shift at concentrations that specifically block the C-domain (0.1 to 1 nmol/L) and a 10-fold shift at concentrations that block both domains. Captopril and Ang-(1-7) monophasically shifted the bradykinin curve to the left, by a factor of 10 and 5, respectively. A 5-fold shift was also observed when Ang-(1-7) was combined with 0.1 nmol/L quinaprilat. Repeated exposure of porcine coronary arteries to 0.1 &mgr;mol/L bradykinin induced B2 receptor desensitization. The addition of 10 &mgr;mol/L quinaprilat or Ang-(1-7) to the bath, at a time when bradykinin alone was no longer able to induce relaxation, fully restored the relaxant effects of bradykinin. Angiotensin II type 1 or 2 receptor blockade did not affect any of the observed effects of Ang-(1-7). In conclusion, Ang-(1-7), like quinaprilat and captopril, potentiates bradykinin by acting as an ACE inhibitor. Bradykinin potentiation is maximal when both the ACE C- and N-terminal domains are inhibited. The inhibitory effects of Ang-(1-7) are limited to the ACE C-domain, raising the possibility that Ang-(1-7) synergistically increases the blood pressure-lowering effects of N-domain-specific ACE inhibitors.

ACE- Versus Chymase-Dependent Angiotensin II Generation in Human Coronary Arteries A Matter of Efficiency?

Objective—The objective of this study was to investigate ACE- and chymase-dependent angiotensin I-to-II conversion in human coronary arteries (HCAs). Methods and Results—HCA rings were mounted in organ baths, and concentration-response curves to angiotensin II, angiotensin I, and the chymase-specific substrate Pro11-d-Ala12–angiotensin I (PA–angiotensin I) were constructed. All angiotensins displayed similar efficacy. For a given vasoconstriction, bath (but not interstitial) angiotensin II during angiotensin I and PA–angiotensin I was lower than during angiotensin II, indicating that interstitial (and not bath) angiotensin II determines vasoconstriction. PA–angiotensin I increased interstitial angiotensin II less efficiently than angiotensin I. Separate inhibition of ACE (with captopril) and chymase (with C41 or chymostatin) shifted the angiotensin I concentration-response curve ≈5-fold to the right, whereas a 10-fold shift occurred during combined ACE and chymase inhibition. Chymostatin, but not captopril and/or C41, reduced bath angiotensin II and abolished PA–Ang I–induced vasoconstriction. Perfused HCA segments, exposed luminally or adventitially to angiotensin I, released angiotensin II into the luminal and adventitial fluid, respectively, and this release was blocked by chymostatin. Conclusions—Both ACE and chymase contribute to the generation of functionally active angiotensin II in HCAs. However, because angiotensin II loss in the organ bath is chymase-dependent, ACE-mediated conversion occurs more efficiently (ie, closer to AT1 receptors) than chymase-mediated conversion.

Bradykinin potentiation by ACE inhibitors: a matter of metabolism

Studies in isolated cells overexpressing ACE and bradykinin type 2 (B2) receptors suggest that ACE inhibitors potentiate bradykinin by inhibiting B2 receptor desensitization, via a mechanism involving protein kinase C (PKC) and phosphatases. Here we investigated, in intact porcine coronary arteries, endothelial ACE/B2 receptor ‘crosstalk’ as well as bradykinin potentiation through neutral endopeptidase (NEP) inhibition. NEP inhibition with phosphoramidon did not affect the bradykinin concentration‐response curve (CRC), nor did combined NEP/ACE inhibition with omapatrilat exert a further leftward shift on top of the ≈10 fold leftward shift of the bradykinin CRC observed with ACE inhibition alone. In arteries that, following repeated exposure to 0.1 μM bradykinin, no longer responded to bradykinin (‘desensitized’ arteries), the ACE inhibitors quinaprilat and angiotensin‐(1‐7) both induced complete relaxation, without affecting the organ bath fluid levels of bradykinin. This phenomenon was unaffected by inhibition of PKC or phosphatases (with calphostin C and okadaic acid, respectively). When using bradykinin analogues that were either completely or largely ACE‐resistant ([Phe8Ψ(CH2‐NH)Arg9]‐bradykinin and [ΔPhe5]‐bradykinin, respectively), the ACE inhibitor‐induced shift of the bradykinin CRC was absent, and its ability to reverse desensitization was absent or significantly reduced, respectively. Caveolar disruption with filipin did not affect the quinaprilat‐induced effects. Filipin did however reduce the bradykinin‐induced relaxation by ≈25–30%, thereby confirming that B2 receptor‐endothelial NO synthase (eNOS) interaction occurs in caveolae. In conclusion, in porcine arteries, in contrast to transfected cells, bradykinin potentiation by ACE inhibitors is a metabolic process, that can only be explained on the basis of ACE‐B2 receptor co‐localization on the endothelial cell membrane. NEP does not appear to affect the bradykinin levels in close proximity to B2 receptors, and the ACE inhibitor‐induced bradykinin potentiation precedes B2 receptor coupling to eNOS in caveolae.

L-NAME-resistant bradykinin-induced relaxation in porcine coronary arteries is NO-dependent: effect of ACE inhibition

NO synthase (NOS) inhibitors partially block bradykinin (BK)‐mediated vasorelaxation. Here we investigated whether this is due to incomplete NOS inhibition and/or NO release from storage sites. We also studied the mechanism behind ACE inhibitor‐mediated BK potentiation. Porcine coronary arteries (PCAs) were mounted in organ baths, preconstricted, and exposed to BK or the ACE‐resistant BK analogue Hyp3‐Tyr(Me)8‐BK (HT‐BK) with or without the NOS inhibitor L‐NAME (100 μM), the NO scavenger hydroxocobalamin (200 μM), the Ca2+‐dependent K+‐channel blockers charybdotoxin+apamin (both 100 nM), or the ACE inhibitor quinaprilat (10 μM). BK and HT‐BK dose‐dependently relaxed preconstricted vessels (pEC50 8.0±0.1 and 8.5±0.2, respectively). pEC50s were &10 fold higher with quinaprilat, and &10 fold lower with L‐NAME or charybdotoxin+apamin. Complete blockade was obtained with hydroxocobalamin or L‐NAME+ charybdotoxin+apamin. Repeated exposure to 100 nM BK or HT‐BK, to deplete NO storage sites, produced progressively smaller vasorelaxant responses. With L‐NAME, the decrease in response occurred much more rapidly. L‐Arginine (10 mM) reversed the effect of L‐NAME. Adding quinaprilat to the bath following repeated exposure (with or without L‐NAME), at the time BK and HT‐BK no longer induced relaxation, fully restored vasorelaxation, while quinaprilat alone had no effect. Quinaprilat also relaxed vessels that, due to pretreatment with hydroxocobalamin or L‐NAME+charybdotoxin+apamin, previously had not responded to BK. In conclusion, L‐NAME‐resistant BK‐induced relaxation in PCAs depends on NO from storage sites, and is mediated via stimulation of guanylyl cyclase and/or Ca2+‐dependent K+‐channels. ACE inhibitors potentiate BK independent of their effect on BK metabolism.

Selective Angiotensin-Converting Enzyme C-Domain Inhibition Is Sufficient to Prevent Angiotensin I–Induced Vasoconstriction

Somatic angiotensin-converting enzyme (ACE) contains 2 domains (C-domain and N-domain) capable of hydrolyzing angiotensin I (Ang I) and bradykinin. Here we investigated the effect of the selective C-domain and N-domain inhibitors RXPA380 and RXP407 on Ang I–induced vasoconstriction of porcine femoral arteries (PFAs) and bradykinin-induced vasodilation of preconstricted porcine coronary microarteries (PCMAs). Ang I concentration-dependently constricted PFAs. RXPA380, at concentrations >1 &mgr;mol/L, shifted the Ang I concentration-response curve (CRC) 10-fold to the right. This was comparable to the maximal shift observed with the ACE inhibitors (ACEi) quinaprilat and captopril. RXP407 did not affect Ang I at concentrations ≤0.1 mmol/L. Bradykinin concentration-dependently relaxed PCMAs. RXPA380 (10 &mgr;mol/L) and RXP407 (0.1 mmol/L) potentiated bradykinin, both inducing a leftward shift of the bradykinin CRC that equaled ≈50% of the maximal shift observed with quinaprilat. Ang I added to blood plasma disappeared with a half life (t1/2) of 42±3 minutes. Quinaprilat increased the t1/2 ≈4-fold, indicating that 71±6% of Ang I metabolism was attributable to ACE. RXPA380 (10 &mgr;mol/L) and RXP407 (0.1 mmol/L) increased the t1/2 ≈2-fold, thereby suggesting that both domains contribute to conversion in plasma. In conclusion, tissue Ang I–II conversion depends exclusively on the ACE C-domain, whereas both domains contribute to conversion by soluble ACE and to bradykinin degradation at tissue sites. Because tissue ACE (and not plasma ACE) determines the hypertensive effects of Ang I, these data not only explain why N-domain inhibition does not affect Ang I–induced vasoconstriction in vivo but also why ACEi exert blood pressure–independent effects at low (C-domain–blocking) doses.

Positive inotropy of calcitonin gene-related peptide and amylin on porcine isolated myocardium

Isolated porcine myocardial trabeculae from right atria and left ventricles were paced at 1.5 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. Alpha calcitonin gene-related peptide (alpha-CGRP) increased contractile force in nearly half of the trabeculae, whereas the selective CGRP(2) receptor agonist [Cys(acetylmethoxy)(2,7)]-CGRP had effect in only a few. Preincubation with the CGRP(1) receptor antagonist alpha-CGRP-(8-37) (10(-6) M) almost completely blocked positive inotropic responses to alpha-CGRP. Amylin had weak positive inotropic effects in some atrial, but not in ventricular trabeculae. Adrenomedullin did not affect contractility in either atrial or ventricular trabeculae. In conclusion, these results suggest that alpha-CGRP has a positive inotropic effect that can be mediated by both CGRP(1) and CGRP(2) receptors. Amylin seems to have a potential positive inotropic effect on atrial tissue, whereas no direct effect of adrenomedullin could be measured.

Vasoactive intestinal peptide has a direct positive inotropic effect on isolated human myocardial trabeculae

The aim of the present study was to assess the inotropic effects of vasoactive intestinal peptide (VIP) on isolated myocardial trabeculae from the right atrium and the left ventricle of human hearts. Furthermore, using reverse transcriptase-PCR, we wanted to determine the presence of mRNAs encoding the three cloned human VIP receptors, VPAC(1), VPAC(2) and PAC(1). The trabeculae were paced at 1.0 Hz in tissue baths, and changes in isometric contractile force upon exposure to agonist were studied. VIP had a potent positive inotropic effect in some of the atrial and ventricular trabeculae tested. This effect was almost completely blocked by the VIP-receptor antagonist VIP-(6-28). mRNAs encoding the human VPAC(1), VPAC(2) and PAC(1) receptors were detected in human myocardial trabeculae from both the right atrium and the left ventricle. In conclusion, VIP has a direct positive inotropic effect in both the atria and the ventricles of the human heart. The presence of mRNAs for the VPAC(1), VPAC(2) and PAC(1) receptors suggest that VIP may mediate its effect via these receptors.

Effects of donitriptan on carotid haemodynamics and cardiac output distribution in anaesthetized pigs.

We investigated the effects of donitriptan, which possesses a uniquely high affinity and efficacy at 5-HT1B/1D receptors, on carotid and systemic haemodynamics in anaesthetized pigs. Donitriptan (0.16-100 μg kg-1, i.v.) dose-dependently decreased total carotid blood flow and vascular conductance (maximum response: -25 ± 3%). This effect was entirely due to a selective reduction in the cephalic arteriovenous anastomotic fraction (maximum response: -63 ± 3%; ED50%: 92 ± 31 nmol/kg); the nutrient vascular conductance increased. Donitriptan did not decrease vascular conductances in or blood flow to a number of organs, including the heart and kidneys; in fact, vascular conductances in the skin, brain and skeletal muscles increased. Cardiac output was slightly decreased by donitriptan, but this effect was confined to peripheral arteriovenous anastomoses. The haemodynamic effects of donitriptan were substantially reduced by the 5-HT1B/1D receptor antagonist GR127935. These results show that donitriptan selectively constricts arteriovenous anastomoses via 5-HT1B receptor activation. The drug should be able to abort migraine headaches and it is unlikely to compromize blood flow to vital organs.

Negative inotropic effect of bradykinin in porcine isolated atrial trabeculae: Role of nitric oxide

Objectives To investigate whether bradykinin affects cardiac contractility independently of its effects on coronary flow and noradrenaline release, and whether such inotropic effects, if present, are mediated via nitric oxide (NO). Methods Right atrial trabeculae were obtained from 35 pigs, suspended in organ baths and attached to isometric transducers. Resting tension was set at approximately 750 mg and tissues were paced at 1.5 Hz. Tissue viability was checked by constructing a concentration response curve (CRC) to noradrenaline. Next, CRCs were constructed to bradykinin, either under baseline conditions or after pre-stimulation with the positive inotropic agent forskolin (1 or 10 μmol/l), in the absence or presence of the bradykinin type 2 (B2) receptor antagonist d-Arg [Hyp3-Thi5, d-Tic7, Oic8]-bradykinin (Hoe 140) (1 μmol/l), the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) (100 μmol/l) and/or the NO scavenger hydroxocobalamin (200 μmol/l). Results Bradykinin exerted a negative inotropic effect, both with and without forskolin pre-stimulation, reducing contractility by maximally 22 ± 3.6% (mean ± SEM) and 23 ± 3.6%, respectively (pEC50 8.37 ± 0.23 and 8.62 ± 0.22, respectively). l-NAME reduced this effect in pre-stimulated, but not in unstimulated, trabeculae. Hoe 140 and hydroxocobalamin fully blocked the inotropic effect of bradykinin. Conclusions Bradykinin induces a modest negative inotropic effect in porcine atrial trabeculae that is mediated via B2 receptors and NO. The inconsistent results obtained with l-NAME suggest that it depends on NO synthesized de novo and/or NO from storage sites.

α1-adrenoceptor subtypes mediating vasoconstriction in the carotid circulation of anaesthetized pigs: possible avenues for antimigraine drug development

It has recently been shown that the α-adrenoceptors mediating vasoconstriction of porcine carotid arteriovenous anastomoses resemble both α1- and α2-adrenoceptors, but no attempt was made to identify the specific subtypes (α1A, α1B and α1D) involved. Therefore, the present study was designed to elucidate the specific subtype(s) of α1-adrenoceptors involved in the above response, using the α1-adrenoceptor agonist phenylephrine and α1-adrenoceptor antagonists 5-methylurapidil (α1A), L-765 314 (α1B) and BMY 7378 (α1D). Ten-minute intracarotid infusions of phenylephrine (1, 3 and 10 μgkg−1.min−1) induced a dose-dependent decrease in total carotid and arteriovenous anastomotic conductance, accompanied by a small tachycardia. These carotid vascular effects were abolished by L-765 314 (1000 μgkg−1; i.v.), while these responses were only attenuated by 5-methylurapidil (1000 μgkg−1; i.v.), and BMY 7378 (1000 μgkg−1; i.v.). Furthermore, intravenous bolus injections of phenylephrine (3 and 10 μgkg−1) produced a dose-dependent vasopressor response, which was only affected by 1000 μgkg−1 of 5-methylurapidil, while the other antagonists were ineffective. These results, coupled to the binding affinities of the above antagonists at the different α1-adrenoceptors, suggest that both α1A- and α1B-adrenoceptors mediate constriction of carotid arteriovenous anastomoses in anaesthetized pigs. In view of the less ubiquitous nature of α1B- compared to α1A-adrenoceptors, the development of potent and selective α1B-adrenoceptor agonists may prove to be important for the treatment of migraine.