René de Vries

Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor

Objective Mannose 6-phosphate receptors (M6PR) bind both renin and prorenin, and such binding contributes to renin/prorenin clearance but not to angiotensin generation. Here, we evaluated the kinetics of renin/prorenin binding to the recently discovered human (pro)renin receptor (h(P)RR), and the idea that such binding underlies tissue angiotensin generation. Methods and results Vascular smooth muscle cells from control rats and transgenic rats with smooth muscle h(P)RR overexpression were incubated at 4 or 37°C with human renin or prorenin. Incubation at 37°C greatly increased binding, suggesting that (pro)renin-binding receptors cycle between the intracellular compartment and the cell surface. Blockade of the M6PR reduced binding by approximately 50%. During M6PR blockade, h(P)RR cells bound twice as much prorenin as control cells, while renin binding was unaltered. Incubation of h(P)RR (but not control) cells with prorenin + angiotensinogen yielded more angiotensin than expected on the basis of the activity of soluble prorenin, whereas angiotensin generation during incubation of both cell types with renin + angiotensinogen was entirely due to soluble renin. The renin + angiotensinogen-induced vasoconstriction of isolated iliac arteries from control and transgenic rats was also due to soluble renin only. The recently proposed (P)RR antagonist ‘handle region peptide’, which resembles part of the prosegment, blocked neither prorenin binding nor angiotensin generation. Conclusions H(P)RRs preferentially bind prorenin, and such binding results in angiotensin generation, most likely because binding results in prorenin activation.

Vasoconstriction by in situ formed angiotensin II: role of ACE and chymase

OBJECTIVE To assess the importance, for vasoconstriction, of in situ angiotensin (Ang) II generation, as opposed to ang II delivery to AT receptors via the organ bath fluid. METHODS Ang I and II concentration-response curves in human and porcine coronary arteries (HCAs, PCAs) were constructed in relation to estimates of the clearances of Ang I and II (ClAngI, ClAngII) from the organ bath and the release of newly formed Ang II (RAngII) into the bath fluid. HCAs were from 25 heart valve donors (age 5-54 years), and PCAs from 14 pigs (age 3 months). RESULTS Ang I- and II-evoked constrictions were inhibited by the AT1 receptor antagonist, irbesartan, and were not influenced by the AT2 receptor antagonist, PD123319. In HCAs Ang II was only three times more potent than Ang I, wheres, in the experiments with Ang I, comparison of ClAngI with ClAngII and RAngII indicated that most of the arterially produced Ang II did not reach the bath fluid. Also in PCAs Ang I and II showed similar potency. In HCAs both the ACE inhibitor, captopril, and the chymase inhibitor, chymostatin, inhibited Ang I-evoked vasoconstriction, while only chymostatin had a significant effect on ClAngI. In PCAs Ang I-evoked vasoconstriction was almost completely ACE-dependent. CONCLUSIONS This study points towards the functional importance of in situ ACE- and chymase-dependent Ang II generation, as opposed to Ang II delivery via the circulation. It also indicates that functionally relevant changes in local Ang I-II conversion are not necessarily reflected by detectable changes in circulating Ang II.

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.

Nongenomic Effects of Aldosterone in the Human Heart. Interaction With Angiotensin II

Aldosterone exerts rapid “nongenomic” effects in various nonrenal tissues. Here, we investigated whether such effects occur in the human heart. Trabeculae and coronary arteries obtained from 57 heart valve donors (25 males; 32 females; 17 to 66 years of age) were mounted in organ baths. Aldosterone decreased contractility in atrial and ventricular trabeculae by maximally 34±3% and 15±4%, respectively, within 5 to 15 minutes after its application. The protein kinase C (PKC) inhibitor chelerythrine chloride, but not the mineralocorticoid receptor antagonists spironolactone and eplerenone, blocked this effect. Aldosterone also relaxed trabeculae that were prestimulated with angiotensin II (Ang II), and its negative inotropic effects were mimicked by hydrocortisone (at 10-fold lower potency) but not 17β-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 &mgr;mol/L aldosterone or 17β-estradiol (but not hydrocortisone) doubled the maximum contractile response (Emax) to Ang II. &Dgr;Emax correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P<0.01). Spironolactone and eplerenone did not block the potentiating effect of aldosterone. Studies in porcine renal arteries showed that potentiation also occurred at pmol/L aldosterone levels but not at 17β-estradiol levels <1 &mgr;mol/L. Aldosterone did not potentiate the α1-adrenoceptor agonist phenylephrine. In conclusion, aldosterone induces a negative inotropic response in human trabeculae (thereby antagonizing the positive inotropic actions of Ang II) and potentiates the vasoconstrictor effect of Ang II in coronary arteries. These effects are specific and involve PKC and ERK 1/2, respectively. Furthermore, they occur in a nongenomic manner, and require pathological aldosterone concentrations.

Bradykinin‐induced relaxation of coronary microarteries: S‐nitrosothiols as EDHF?

To investigate whether S‐nitrosothiols, in addition to NO, mediate bradykinin‐induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. Following preconstriction, concentration–response curves (CRCs) were constructed to bradykinin, the NO donors S‐nitroso‐N‐penicillamine (SNAP) and diethylamine NONOate (DEA‐NONOate) and the S‐nitrosothiols L‐S‐nitrosocysteine (L‐SNC) and D‐SNC. All agonists relaxed PCMAs. L‐SNC was ∼5‐fold more potent than D‐SNC. The guanylyl cyclase inhibitor ODQ and the NO scavenger hydroxocobalamin induced a larger shift of the bradykinin CRC than the NO synthase inhibitor L‐NAME, although all three inhibitors equally suppressed bradykinin‐induced cGMP responses. Complete blockade of bradykinin‐induced relaxation was obtained with L‐NAME in the presence of the large‐ and intermediate‐conductance Ca2+‐activated K+‐channel (BKCa, IKCa) blocker charybdotoxin and the small‐conductance Ca2+‐activated K+‐channel (SKCa) channel blocker apamin, but not in the presence of L‐NAME, apamin and the BKCa channel blocker iberiotoxin. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage‐dependent K+ channels and ATP‐sensitive K+ channels did not affect bradykinin‐induced relaxation. SNAP‐, DEA‐NONOate‐ and D‐SNC‐induced relaxations were mediated entirely by the NO‐guanylyl cyclase pathway. L‐SNC‐induced relaxations were partially blocked by charybdotoxin+apamin, but not by iberiotoxin+apamin, and this blockade was abolished following endothelium removal. ODQ, but not hydroxocobalamin, prevented L‐SNC‐induced increases in cGMP, and both drugs shifted the L‐SNC CRC 5–10‐fold to the right. L‐SNC hyperpolarized intact and endothelium‐denuded coronary arteries. Our results support the concept that bradykinin‐induced relaxation is mediated via de novo synthesized NO and a non‐NO, endothelium‐derived hyperpolarizing factor (EDHF). S‐nitrosothiols, via stereoselective activation of endothelial IKCa and SKCa channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries.

Nucleotide Excision DNA Repair Is Associated With Age-Related Vascular Dysfunction

Background Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Inhibitory effect of nitric oxide (NO) synthase inhibitors on naloxone-precipitated withdrawal syndrome in morphine-dependent mice.

The effect of intraperitoneally administered nitric oxide (NO) synthase inhibitors has been examined on the naloxone-precipitated withdrawal syndrome in morphine-dependent mice. L-NAME (30-200 mg/kg) and L-NOARG (7.5-50 mg/kg) induced a significant decrease of naloxone-precipitated withdrawal jumping and diarrhoea. However, L-NMMA (3.5-100 mg/kg), considered as a less potent NO synthase inhibitor, did not significantly affect the withdrawal signs in mice. Although a specificity of NO synthase inhibitors is not fully established, these results indicate that inhibition of NO synthesis in the central nervous system and periphery may significantly affect the morphine withdrawal phenomena. Accordingly, this study suggests an involvement of NO in morphine withdrawal syndrome.

7-Nitro indazole, an inhibitor of neuronal nitric oxide synthase, attenuates pilocarpine-induced seizures

7-Nitro indazole (25-100 mg/kg i.p.), an inhibitor of neuronal nitric oxide (NO) synthase, attenuated the severity of pilocarpine (300 mg/kg i.p.)-induced seizures in mice. This indicates that the decreased neuroexcitability of the central nervous system (CNS) following administration of 7-nitro indazole may be due to inhibition of neuronal NO synthase, implying that NO acts as an excitatory and proconvulsant factor in the CNS.

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.

Excitatory amino acid receptor antagonists and naloxone-precipitated withdrawal syndrome in morphine-dependent mice

The effects of the excitatory amino acid (EAA) receptor antagonists MK-801 (non-competitive NMDA receptor antagonist), DNQX (competitive non-NMDA receptor antagonist) and 5,7-DCKA (antagonist of glycine site of NMDA receptor) have been examined on the naloxone (4 mg/kg, i.p.)-precipitated withdrawal jumping behaviour in morphine-dependent mice. The results indicate that withdrawal jumping behaviour in morphine-dependent mice was attenuated by all three EAA receptor antagonists, MK-801, DNQX and 5,7-DCKA. However, MK-801, DNQX and 5,7-DCKA inhibited the jumping behaviour in a relatively narrow dose range.