Wendy W. Batenburg

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.

Prorenin and Renin-Induced Extracellular Signal-Regulated Kinase 1/2 Activation in Monocytes Is Not Blocked by Aliskiren or the Handle-Region Peptide

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 125I-renin or 125I-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.

Angiotensin II Type 2 Receptor–Mediated Vasodilation in Human Coronary Microarteries

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 μm) 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 [Emax], 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 Emax to 97±14% (P <0.05). The increase in Emax was larger in older donors (correlation ΔEmax 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.

Aliskiren-Binding Increases the Half Life of Renin and Prorenin in Rat Aortic Vascular Smooth Muscle Cells

Objective—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 4°C 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 4°C. At 37°C, 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.

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.

Angiotensin II-aldosterone interaction in human coronary microarteries involves GPR30, EGFR, and endothelial NO synthase.

AIMS The aim of this study was to investigate the aldosterone-angiotensin (Ang) II interaction in human coronary microarteries (HCMAs). METHODS AND RESULTS HCMAs, obtained from 75 heart-beating organ donors, were mounted in myographs and exposed to Ang II, either directly or following a 30-min pre-incubation with aldosterone, 17β-oestradiol, hydrocortisone, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the extracellular regulated kinase 1/2 (ERK1/2) inhibitor PD98059, the GPR30 antagonist G15, or the epidermal growth factor receptor (EGFR) antagonist AG1478. Ang II constricted HCMAs in a concentration-dependent manner. All steroids, at nanomolar levels, potentiated Ang II and G15 prevented this effect. The potentiation disappeared or was reversed into Ang II antagonism at micromolar steroid levels. NO synthase (NOS) inhibition prevented the latter antagonism in the case of 17β-oestradiol, whereas both aldosterone and 17β-oestradiol at micro- (but not nano-) molar levels induced endothelial NOS phosphorylation in human umbilical vein endothelial cells. AG1478, but not SB203580 or PD98059, abolished the Ang II-induced contraction in the presence of aldosterone or 17β-oestradiol, and none of these drugs affected Ang II alone. CONCLUSION Steroids including aldosterone affect Ang II-induced vasoconstriction in a biphasic manner. Potentiation occurs at nanomolar steroid levels and depends on GPR30 and EGFR transactivation. At micromolar steroid levels, this potentiation either disappears (aldosterone and hydrocortisone) or is reversed into an inhibition (17β-oestradiol), and this is due to the endothelial NOS activation that occurs at such concentrations.

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.

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.

Mediators of Bradykinin-Induced Vasorelaxation in Human Coronary Microarteries

Abstract—To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter ≈300 &mgr;m) 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. NG-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 18&agr;-glycyrrhetinic acid or carbenoxolone), and blockade of large- (BKCa) and small- (SKCa) 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 BKCa and intermediate-conductance (IKCa) Ca2+-dependent K+ channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K+ channels (KIR) 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, KIR, and Na+/K+-ATPase by NO and (2) IKCa and SKCa 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 BKCa channels.

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?

Renin/prorenin binding to the (pro)renin receptor ([P]RR) results in direct (angiotensin-independent) second-messenger 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.