Batenburg, W.W.

Nitrite- and nitroxyl-induced relaxation in porcine coronary (micro-) arteries: Underlying mechanisms and role as endothelium-derived hyperpolarizing factor(s) (Article)

2012-11-01T00:00:00Z

To investigate the vasorelaxant efficacy of nitrite and nitroxyl (HNO) in porcine coronary (micro)arteries (PC(M)As), evaluating their role as endothelium-derived hyperpolarizing factors (EDHFs), preconstricted PCAs and PCMAs were exposed to UV light (a well-known inductor of nitrite; wave-length: 350-370 nm), nitrite, the HNO donor Angeli's salt, or bradykinin. UV light-induced relaxation of PCAs increased identically after endothelium removal and endothelial nitric oxide (NO) synthase (eNOS) blockade. UV light-induced relaxation diminished during Na+-K+-ATPase inhibition and S-nitrosothiol-depletion, and disappeared during NO scavenging with hydroxocobalamin or soluble guanylyl cyclase (sGC) inhibition with ODQ. Nitrite-induced relaxation of PCAs required millimolar levels, i.e., >1000 times endogenous vascular nitrite. Angeli's salt relaxed PCMAs more potently than PCAs, and this was due to the fact that HNO directly activated sGC in PCMAs, whereas in PCAs this occurred following its conversion to NO only. sGC activation by NO/HNO resulted in Na+-K+-ATPase stimulation and Kvchannel activation. The HNO scavenger l-cysteine blocked bradykinin-induced relaxation in PCAs, and potentiated it in PCMAs. The latter did not occur in the presence of hydroxocobalamin, suggesting that it depended on l-cysteine-induced generation of vasorelaxant S-nitrosothiols. In all experimental setups, incubation with red wine extract mimicked the effects of ODQ. In conclusion, nitrite, via its conversion to NO and S-nitrosothiols, and HNO, either directly, or via its conversion to NO, mediate relaxant effects involving the sGC-cGMP pathway, Na+-K+-ATPase and/or Kvchannels. Red wine extract counteracts these beneficial effects. NO blocks nitrite activation, and HNO, but not nitrite, may act as EDHF in the coronary vascular bed.

Renin-and prorenin-induced effects in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor does (pro)renin-(pro)renin receptor interaction actually occur? (Article)

2011-12-01T00:00:00Z

Renin/prorenin binding to the (pro)renin receptor ([P]RR) results in direct (angiotensin-independent) secondmessenger activation in vitro, whereas in vivo studies in rodents overexpressing prorenin (≈400-fold) or the (P)RR do not support such activation. To solve this discrepancy, DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release were evaluated in wild-type and human (P)RR-overexpressing vascular smooth muscle cells after their incubation with 1 to 80 nmol/L of (pro)renin. Human prorenin (4 nmol/L, ie, ≈800-fold above normal) + angiotensinogen increased DNA synthesis in human (P)RR cells only in an angiotensin II type 1 receptor-dependent manner. Prorenin at this concentration also increased plasminogen-activator inhibitor 1 release via angiotensin. Prorenin alone at 4 nmol/L was without effect, but at 20 nmol/L (≈4000-fold above normal) it activated extracellular signal-regulated kinase 1/2 directly (ie, independent of angiotensin). Renin at concentrations of 1 nmol/L (≈2000-fold above normal) and higher directly stimulated DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release in wild-type and human (P)RR cells, and similar effects were seen for rat renin, indicating that they were mediated via the rat (P)RR. In conclusion, angiotensin generation depending on prorenin-(P)RR interaction may occur in transgenic rodents overexpressing prorenin several 100-fold. Direct (pro)renin-induced effects via the (P)RR require agonist concentrations that are far above the levels in wild-type and transgenic rats. Therefore, only prorenin (and not [P]RR) overexpression will result in an angiotensin-dependent phenotype, and direct renin-(P)RR interaction is unlikely to ever occur in nonrenin-synthesizing organs.

Urinary renin, but not angiotensinogen or aldosterone, reflects the renal renin-angiotensin-aldosterone system activity and the efficacy of renin-angiotensin-aldosterone system blockade in the kidney (Article)

2011-11-01T00:00:00Z

Objective: To study which renin-angiotensin-aldosterone system (RAAS) component best reflects renal RAAS activity. Methods and Results: We measured urinary and plasma renin, prorenin, angiotensinogen, aldosterone, albumin and creatinine in 101 diabetic and nondiabetic patients with or without hypertension. Plasma prorenin was elevated in diabetic patients. Urinary prorenin was undetectable. Urinary albumin and renin were higher in diabetic patients. Men had higher plasma renin/prorenin levels, and lower plasma angiotensinogen levels than women. Plasma creatinine and albumin were also higher in men. Urinary RAAS components showed no sexual dimorphism, whereas urinary creatinine and albumin were higher in men. Angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor blockers increased plasma renin and decreased plasma angiotensinogen, without altering plasma aldosterone. In contrast, in urine, these drugs decreased renin and aldosterone without affecting angiotensinogen. When analyzing all patients together, urinary angiotensinogen excretion closely mimicked that of albumin, whereas urinary angiotensinogen and albumin levels both were 0.05% or less of their concomitant plasma levels. This may reflect the identical glomerular filtration and tubular handling of both proteins, which have a comparable molecular weight. In contrast, urinary renin excretion did not correlate with urinary albumin excretion, and the urinary/plasma concentration ratio of renin was more than 200 times the ratio of albumin, despite its comparable molecular weight. Urinary aldosterone excretion closely followed urinary creatinine excretion. Conclusion: The increased urinary renin levels in diabetes and the decreased urinary renin levels following RAAS blockade, occurring independently of changes in plasma renin, reflect the activated renal RAAS in diabetes and the success of RAAS blockade in the kidney, respectively. Urinary renin, therefore, more closely reflects renal RAAS activity than urinary angiotensinogen or aldosterone.

Specific coronary drug-eluting stents interfere with distal microvascular function after single stent implantation in pigs (Article)

2010-07-01T00:00:00Z

Objectives The aim of this study was to compare the effects of single drug-eluting stents (DES) on porcine coronary function distal to the stent in vivo and in vitro. Background The mechanism of endothelial dysfunction occurring in human coronary conduit arteries up to 9 months after DES implantation is unknown. Methods A sirolimus-eluting stent (SES), paclitaxel-eluting stent (PES), and a bare-metal stent (BMS) were implanted in the 3 coronary arteries of 11 pigs. After 5 weeks, in vivo responses in distal coronary flow to different doses of bradykinin (BK) and nitrates were measured. In vitro, vasodilation to BK and nitrates, as well as vasoconstriction to endothelin (ET)-1 were assessed in both distal coronary conduit and small arteries. In addition, contributions of nitric oxide (NO) and endotheliumderived hyperpolarizing factors (EDHFs) and cyclic guanosine monophosphate (cGMP) responses to BK-stimulation were determined in vitro. Results Both DES did not alter in vivo distal vasomotion. In vitro distal conduit and small arterial responses to BK were also unaltered; DES did not alter the BK-induced increase in cGMP. However, after NO synthase blockade, PES showed a reduced BK-response in distal small arteries as compared with BMS and SES (p < 0.05). The ET-1-induced vasoconstriction and vascular smooth muscle cell function were unaltered. Conclusions In this study of single stenting in healthy porcine coronaries for 5 weeks, SES did not affect distal coronary vascular function, whereas PES altered distal endothelial function of small arteries under conditions of reduced NO bioavailability. Therefore, specifically the EDH-component of microvascular function seems affected by PES.

Light-induced vs. bradykinin-induced relaxation of coronary arteries: Do S-nitrosothiols act as endothelium-derived hyperpolarizing factors? (Article)

2009-08-01T00:00:00Z

BACKGROUND: Light-induced relaxation depends on S-nitrosothiols. S-Nitrosothiols may also serve as endothelium-derived hyperpolarizing factors, mediating the relaxant response of porcine coronary arteries (PCAs) to bradykinin. Here we compared the mechanism of light-induced and bradykinin-induced PCA relaxation. METHODS: PCAs were mounted in organ baths in the dark, preconstricted and exposed to polychromatic light (5 min) or 100 nmol/l bradykinin. RESULTS: Light relaxed PCAs by maximally 71 ± 1%. S-Nitrosothiol depletion abolished this relaxation. Relaxations diminished following repetitive light exposures, particularly if the dark periods between the light exposures were less than 10 min, and increased following endothelium removal or nitric oxide synthase blockade with N-nitro-L-arginine methyl ester (L-NAME), despite the prevention of guanosine-3′,5′-cyclic monophosphate generation by the latter two procedures. Thus, reloading of the storage pools occurs in the dark, endothelial nitric oxide inhibits this process and photorelaxation does not depend on guanosine-3′,5′-cyclic monophosphate. Bradykinin relaxed PCAs by 69 ± 3%. The nitric oxide scavenger hydroxocobalamin and the Na-K ATPase inhibitor ouabain abolished the responses to bradykinin and light. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one abolished the response to light, and, like L-NAME, blocked the response to bradykinin by more than 50%. On top of L-NAME, intermediate and small conductance Ca-dependent K channel (IKCa/SKCa) blockade further reduced the response to bradykinin and enhanced photorelaxation. CONCLUSION: Photorelaxation depends on stored S-nitrosothiols and their release/synthesis is negatively affected by endothelial nitric oxide and IKCa/SKCa. S-Nitrosothiols activate endothelial IKCa/SKCa and, via guanylyl cyclase, smooth muscle Na-K ATPase. Thus, they possess all properties of a bradykinin-induced endothelium-derived hyperpolarizing factor.

Diabetic complications: A role for the prorenin-(pro)renin receptor-TGF-β1 axis? (Article)

2009-04-29T00:00:00Z

Morbidity and mortality of diabetes mellitus are strongly associated with cardiovascular disease including nephropathy. A discordant tissue renin-angiotensin system (RAS) might be a mediator of the endothelial dysfunction leading to both micro- and macrovascular complications of diabetes. The elevated plasma levels of prorenin in diabetic subjects with microvascular complications might be part of this discordant RAS, especially since the plasma renin levels in diabetes are low. Prorenin, previously thought of as an inactive precursor of renin, is now known to bind to a (pro)renin receptor, thus activating locally angiotensin-dependent and -independent pathways. In particular, the stimulation of the transforming growth factor-β (TGF-β) system by prorenin could be an important contributor to diabetic disease complications. This review discusses the concept of the prorenin-(pro)renin receptor-TGF-β1axis, concluding that interference with this pathway might be a next logical step in the search for new therapeutic regimens to reduce diabetes-related morbidity and mortality.

Effects of angiotensin II and its metabolites in the rat coronary vascular bed: Is angiotensin III the preferred ligand of the angiotensin AT2 receptor? (Article)

2008-07-07T00:00:00Z

Aminopeptidases metabolize angiotensin II to angiotensin-(2-8) (= angiotensin III) and angiotensin-(3-8) (= angiotensin IV), and carboxypeptidases generate angiotensin-(1-7) from angiotensin I and II. Angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin II type 1 (AT1) receptor blockers affect the concentrations of these metabolites, and they may thus contribute to the beneficial effects of these drugs, possibly through stimulation of non-classical angiotensin AT receptors. Here, we investigated the effects of angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) in the rat coronary vascular bed, with or without angiotensin AT1- or angiotensin II type 2 (AT2) receptor blockade. Results were compared to those in rat iliac arteries and abdominal aortas. Angiotensin II, angiotensin III and angiotensin IV constricted coronary arteries via angiotensin AT1receptor stimulation, angiotensin III and angiotensin IV being ≈ 20- and ≈ 8000-fold less potent than angiotensin II. The angiotensin AT2receptor antagonist PD123319 greatly enhanced the constrictor effects of angiotensin III, starting at angiotensin III concentrations in the low nanomolar range. PD123319 enhanced the angiotensin II-induced constriction at submicromolar angiotensin II concentrations only. Angiotensin-(1-7) exerted no effects in the coronary circulation, although, at micromolar concentrations, it blocked angiotensin AT1receptor-induced constriction. Angiotensin AT2receptor-mediated relaxation did not occur in iliac arteries and abdominal aortas, and the constrictor effects of the angiotensin metabolites in these vessels were identical to those in the coronary vascular bed. In conclusion, angiotensin AT2receptor activation in the rat coronary vascular bed results in vasodilation, and angiotensin III rather than angiotensin II is the preferred endogenous agonist of these receptors. Angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) do not exert effects through non-classical angiotensin AT receptors in the rat coronary vascular bed, iliac artery or aorta.

Corticosteroid-dependent, aldosterone-independent mineralocorticoid- receptor activation in the heart (Article)

2008-07-01T00:00:00Z

Prorenin anno 2008 (Article)

2008-06-01T00:00:00Z

For many years, prorenin has been considered to be nothing more than the inactive precursor of renin. Yet, its elevated levels in diabetic subjects with microvascular complications and its extrarenal production at various sites in the body suggest otherwise. This review discusses the origin, regulation, and enzymatic activity of prorenin, its role during renin inhibition, and the angiotensin-dependent and angiotensin-independent consequences of its binding to the recently discovered (pro)renin receptor. The review ends with the concept that prorenin rather than renin determines tissue angiotensin generation.

Aliskiren-binding increases the half life of renin and prorenin in rat aortic vascular smooth muscle cells (Article)

2008-06-01T00:00:00Z

Renin inhibition with aliskiren has been reported to cause a greater rise in renin than other types of renin-angiotensin system blockade, thereby potentially leading to angiotensin generation or stimulation of the human (pro)renin receptor (h(P)RR). Here we studied whether this rise in renin is attributable to an aliskiren-induced change in the prorenin conformation, allowing its detection in renin assays, or a change in renin/prorenin clearance. We also investigated whether aliskiren affects (pro)renin binding to its receptors, using rat aortic vascular smooth muscle cells (VSMCs) overexpressing the h(P)RR. Methods and Results-A 48-hour incubation with aliskiren at 40C converted the prorenin conformation from "closed" to "open," thus allowing its recognition in active site-directed renin assays. VSMCs accumulated (pro)renin through binding to mannose 6-phosphate receptors (M6PRs) and h(P)RRs. Aliskiren did not affect binding at 40C. At 370C, aliskiren increased (pro)renin accumulation up to 40-fold, and M6PR blockade prevented this. Aliskiren increased the intracellular half life of prorenin 2 to 3 times. Conclusion-Aliskiren allows the detection of prorenin as renin, and decreases renin/prorenin clearance. Both phenomena may contribute to the "renin" surge during aliskiren treatment, but because they depend on aliskiren binding, they will not result in angiotensin generation. Aliskiren does not affect (pro)renin binding to its receptors.

The (pro)renin receptor: A new addition to the renin-angiotensin system? (Article)

2008-05-13T00:00:00Z

The renin-angiotensin system is still incompletely understood. In particular, the function of prorenin, the inactive precursor of renin, is unknown. Yet, prorenin levels are > 10-fold higher than renin levels, and prorenin increases even further in subjects with diabetes mellitus displaying microvascular complications. The recent discovery of a (pro)renin binding receptor may shed light on the role of prorenin. This review discusses the possibility that prorenin binding to this receptor results in prorenin activation, thereby allowing angiotensin generation, and that prorenin simultaneously acts as an agonist of this receptor, inducing angiotensin-independent effects. Transgenic animals overexpressing the receptor, as well as a receptor antagonist are now available, and future studies should reveal to what degree this concept is applicable to humans as well.

Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide (Article)

2008-03-01T00:00:00Z

The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of I-renin or I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate-labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling.

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 (Article)

2007-12-01T00:00:00Z

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.

Prorenin and the (pro)renin receptor - An update (Article)

2007-05-01T00:00:00Z

ACE phenotyping as a first step toward personalized medicine for ACE inhibitors. Why does ACE genotyping not predict the therapeutic efficacy of ACE inhibition? (Article)

2007-03-01T00:00:00Z

Angiotensin (Ang)-converting enzyme (ACE) inhibitors are widely used for the treatment of cardiovascular diseases. Not all patients respond to ACE inhibitors, and it has been suggested that genetic variation might be a useful marker to predict the therapeutic efficacy of these drugs. In particular, the ACE insertion (I)/deletion (D) polymorphism has been investigated in this regard. Despite a decade of intensive research involving the genotyping of thousands of patients, we still do not know whether ACE genotyping helps in predicting the success of ACE inhibition. This review critically addresses the concept that predictive information on therapeutic efficacy of ACE inhibitors might be obtained based on ACE genotyping. It answers the following questions: Do higher ACE levels really result in higher Ang II levels? Is ACE the only converting enzyme in humans? Does ACE inhibition affect ACE expression? Why does ACE have 2 catalytically active domains? What is the relevance of ACE inhibitor-induced signaling through membrane-bound ACE? The review ends with the proposal that ACE phenotyping may prove to be a better first step toward personalized medicine for ACE inhibitors than ACE genotyping.

Nongenomic effects of aldosterone in the human heart: interaction with angiotensin II. (Article)

2005-10-01T00:00:00Z

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 17beta-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 micromol/L aldosterone or 17beta-estradiol (but not hydrocortisone) doubled the maximum contractile response (Emax) to Ang II. DeltaEmax 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 17beta-estradiol levels <1 micromol/L. Aldosterone did not potentiate the alpha1-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.

Angiotensin II-induced vasodilation: role of bradykinin, NO and endothelium-derived hyperpolarizing factors (Doctoral Thesis)

2005-06-30T00:00:00Z

Interactie tussen AT1 en a1-adrenerge receptoren (Hoofdstuk 6) Carvedilol, de selectieve ß1-adrenoceptor antagonist metoprolol, de niet-selectieve ßadrenoceptor antagonist propranolol, en de a1-adrenoceptor antagonist prazosin beïnvloedden geen van allen de constrictoire respons van HCMA’s op Ang II. Ang II, na toevoeging aan het orgaan bad in een lage (non-constrictoire) concentratie, versterkte de respons op de a1-adrenoceptor agonist fenylefrine enorm. Zowel carvedilol als de AT1 receptor antagonist irbesartan remden deze door Ang II geïnduceerde potentiatie. Carvedilol remde eveneens de door Ang II versterkte ophoping van inositolfosfaten onder invloed van fenylefrine in hartspiercellen. Samenvattend kan gesteld worden dat AT1-a1-receptor ‘crosstalk’, mogelijk via inositolfosfaten, HCMA’s gevoeliger maakt voor a1-adrenoceptor agonisten. De a1-adrenoceptor blokkerende effecten van carvedilol zorgen er voor dat carvedilol dit potentiërende effect van Ang II tegen kan gaan. Dit verklaart waarom het bloeddrukverhogende effect van Ang II bij patiënten met hartfalen die behandeld worden met carvedilol kleiner is dan bij patiënten die behandeld worden met metoprolol.

Selective angiotensin-converting enzyme C-domain inhibition is sufficient to prevent angiotensin I-induced vasoconstriction. (Article)

2005-01-01T00:00:00Z

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 mumol/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 < or =0.1 mmol/L. Bradykinin concentration-dependently relaxed PCMAs. RXPA380 (10 micromol/L) and RXP407 (0.1 mmol/L) potentiated bradykinin, both inducing a leftward shift of the bradykinin CRC that equaled approximately 50% of the maximal shift observed with quinaprilat. Ang I added to blood plasma disappeared with a half life (t(1/2)) of 42+/-3 minutes. Quinaprilat increased the t(1/2) approximately 4-fold, indicating that 71+/-6% of Ang I metabolism was attributable to ACE. RXPA380 (10 micromol/L) and RXP407 (0.1 mmol/L) increased the t(1/2) approximately 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.

Angiotensin II type 2 receptor-mediated vasodilation in human coronary microarteries. (Article)

2004-05-18T00:00:00Z

BACKGROUND: Angiotensin (Ang) II type 2 (AT2) receptor stimulation results in coronary vasodilation in the rat heart. In contrast, AT2 receptor-mediated vasodilation could not be observed in large human coronary arteries. We studied Ang II-induced vasodilation of human coronary microarteries (HCMAs). METHODS AND RESULTS: HCMAs (diameter, 160 to 500 microm) were obtained from 49 heart valve donors (age, 3 to 65 years). Ang II constricted HCMAs, mounted in Mulvany myographs, in a concentration-dependent manner (pEC50, 8.6+/-0.2; maximal effect [E(max)], 79+/-13% of the contraction to 100 mmol/L K+). The Ang II type 1 receptor antagonist irbesartan prevented this vasoconstriction, whereas the AT2 receptor antagonist PD123319 increased E(max) to 97+/-14% (P<0.05). The increase in E(max) was larger in older donors (correlation DeltaE(max) versus age, r=0.47, P<0.05). The PD123319-induced potentiation was not observed in the presence of the NO synthase inhibitor L-NAME, the bradykinin type 2 (B2) receptor antagonist Hoe140, or after removal of the endothelium. Ang II relaxed U46619-preconstricted HCMAs in the presence of irbesartan by maximally 49+/-16%, and PD123319 prevented this relaxation. Finally, radioligand binding studies and reverse transcription-polymerase chain reaction confirmed the expression of AT2 receptors in HCMAs. CONCLUSIONS: AT2 receptor-mediated vasodilation in the human heart appears to be limited to coronary microarteries and is mediated by B2 receptors and NO. Most likely, AT2 receptors are located on endothelial cells, and their contribution increases with age.

Bradykinin-induced relaxation of coronary microarteries: S-nitrosothiols as EDHF? (Article)

2004-05-01T00:00:00Z

1. To investigate whether S-nitrosothiols, in addition to NO, mediate bradykinin-induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. 2. 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 approximately 5-fold more potent than D-SNC. 3. 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. 4. Complete blockade of bradykinin-induced relaxation was obtained with L-NAME in the presence of the large- and intermediate-conductance Ca(2+)-activated K(+)-channel (BK(Ca), IK(Ca)) blocker charybdotoxin and the small-conductance Ca(2+)-activated K(+)-channel (SK(Ca)) channel blocker apamin, but not in the presence of L-NAME, apamin and the BK(Ca) channel blocker iberiotoxin. 5. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage-dependent K(+) channels and ATP-sensitive K(+) channels did not affect bradykinin-induced relaxation. 6. 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. 7. L-SNC hyperpolarized intact and endothelium-denuded coronary arteries. 8. 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 IK(Ca) and SK(Ca) channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries.

Mediators of bradykinin-induced vasorelaxation in human coronary microarteries. (Article)

2004-02-01T00:00:00Z

To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter approximately 300 microm) obtained from 42 heart valve donors (20 men and 22 women; age range, 3 to 65 years; mean age, 46 years) were mounted in Mulvany myographs. In the presence of the cyclooxygenase inhibitor indomethacin, bradykinin relaxed preconstricted HCMAs in a concentration-dependent manner. N(G)-nitro-L-arginine methyl ester and ODQ (inhibitors of nitric oxide [NO] synthase and guanylyl cyclase, respectively) and the NO scavenger hydroxocobalamin, either alone or in combination, shifted the bradykinin concentration-response curve to the right. Removal of H2O2 (with catalase), inhibition of cytochrome P450 epoxygenase (with sulfaphenazole or clotrimazole) or gap junctions (with 18alpha-glycyrrhetinic acid or carbenoxolone), and blockade of large- (BK(Ca)) and small- (SK(Ca)) conductance Ca2+-dependent K+ channels (with iberiotoxin and apamin), either alone or in addition to hydroxocobalamin, did not affect bradykinin. In contrast, complete blockade of bradykinin-induced relaxation was obtained when we combined the nonselective BK(Ca) and intermediate-conductance (IK(Ca)) Ca2+-dependent K+ channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K+ channels (K(IR)) and Na+/K+-ATPase (with BaCl2 and ouabain, respectively) shifted the bradykinin concentration-response curve 10-fold to the right but did not exert an additional effect in the presence of hydroxocobalamin. In conclusion, bradykinin-induced relaxation in HCMAs depends on (1) the activation of guanylyl cyclase, K(IR), and Na(+)/K(+)-ATPase by NO and (2) IK(Ca) and SK(Ca) channels. The latter are activated by a factor other than NO. This factor is not a cytochrome P450 epoxygenase product or H2O2, nor does it depend on gap junctions or BK(Ca) channels.