Activation of angiotensin II subtype 2 receptor induces catecholamine release in an extracellular Ca(2+)-dependent manner through a decrease of cyclic guanosine 3',5'-monophosphate production in cultured porcine adrenal medullary chromaffin Cells.

We have previously demonstrated that CGP 42112 (AT(2) agonist > or =1 nM) markedly reduces catecholamine biosynthesis through AT(2), which is the major angiotensin II (AngII) receptor subtype in cultured porcine chromaffin cells. Also, we have shown that CGP 42112 (> or =1 nM) induces a reduction in cGMP production in these cells. The present study showed that AngII reduced cGMP production via AT(2) in a manner similar to that found with CGP 42112. AngII (1 nM) significantly increased catecholamine secretion from cultured porcine adrenal medullary chromaffin cells. The stimulation was significantly inhibited by PD 123319 (AT(2) antagonist). The stimulation was moderately, but significantly, attenuated by CV-11974 (AT(1) antagonist, > or =10 nM), suggesting an involvement of AT(1). Moreover, CGP 42112 (> or =10 nM) markedly increased catecholamine release from these cells. The stimulation by CGP 42112 was abolished by PD 123319, whereas CV-11974 had no effect, indicating that this response is also mediated by AT(2). We further examined whether extracellular Ca(2+) is involved in the stimulatory effect of AT(2) on catecholamine secretion. Removal of external Ca(2+) significantly suppressed either AngII plus CV-11974 (100 nM; which simulates specific AT(2) stimulation) or CGP 42112- induced catecholamine secretion. AngII plus CV-11974 or CGP 42112 caused a sustained increase in intracellular Ca(2+) ([Ca(2+)](i)), as determined in fura-2-loaded chromaffin cells in an extracellular Ca(2+)-dependent manner. In the presence of EGTA, the subsequent addition of AngII with CV-11974 and CGP 42112 did not cause any increase in [Ca(2+)](i) levels. Consistent with this finding, CGP 42112 (10 nM to 1 microM) did not alter inositol triphosphate (IP(3)) production, a messenger for mobilization of Ca(2+) from intracellular storage sites. In addition, the intracellular Ca(2+) chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethylester (BAPTA) did not affect CGP 42112-induced catecholamine release. We tested whether a decrease in cGMP was the cause of the stimulatory effect of AT(2) on catecholamine secretion. Pretreatment with 8-bromo-cGMP (1 mM) prevented the stimulatory effect of AngII plus CV-11974 and CGP 42112 on both catecholamine secretion and [Ca(2+)](i). When 8-bromo-cGMP was added after application of AngII plus CV-11974 or CGP 42112, [Ca(2+)](i) induced by these agents was gradually reduced toward the baseline values. Similarly, guanylin completely abolished the AngII- plus CV-11974-induced increase in both NE secretion and [Ca(2+)](i). The Ca(2+) channel blockers, nicardipine and omega-conotoxin G VIA, at 1 microM in both cases, were also effective in inhibiting AT(2) stimulation-induced secretion. On the other hand, neither T-type voltage-dependent Ca(2+) channel blockers, flunarizine, nor Ni(2+) affected catecholamine release caused by AT(2) stimulation. These findings demonstrate that AT(2) stimulation induces catecholamine secretion by mobilizing Ca(2+) through voltage-dependent Ca(2+) channels without affecting intracellular pools and that these effects could be mediated by a decrease in cGMP production.     

Enhanced adrenomedullin production by mechanical stretching in cultured rat cardiomyocytes

Adrenomedullin (AM) is secreted from cultured cardiac myocytes. In this study, we examined whether mechanical stretching stimulates AM production in cardiac myocytes, and if so, whether angiotensin II (Ang II) is involved in that mechanism. Neonatal rat cardiac myocytes cultured in serum-free medium were stretched 10% or 20% on flexible silicone rubber culture dishes, and AM mRNA expression was examined by quantitative polymerase chain reaction. The AM mRNA levels in the myocytes stretched 10% and 20% for 24 hours significantly increased by 56% (P<0.05) and 88% (P<0.01), respectively, when compared with the levels in nonstretched cells. AM secretion into the medium after the myocytes were stretched 10% and 20% increased by 22% (P<0.05) and 45% (P<0.01), respectively. In nonstretched myocytes incubated with 10(-6) mol/L Ang II for 24 hours, AM mRNA and secretion increased by 86% (P<0.05) and 36% (P<0. 01), respectively. These effects of Ang II were abolished by 10(-6) mol/L CV-11974, an Ang II type I (AT(1)) receptor antagonist, but not by 10(-6) mol/L PD-123319, an Ang II type II antagonist. Stretch-induced increases of AM gene expression and secretion were significantly inhibited (P<0.05) in the presence of 10(-6) mol/L CV-11974 by 46% and 52%, respectively; however, they were not affected by 10(-6) mol/L PD-123319. These findings indicate that AM production from cardiac myocytes is augmented by mechanical stretching, partially through the AT(1) receptors, which suggests a local interaction between AM and the renin-angiotensin system in stretched cardiac myocytes.     

Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats

OBJECTIVE: To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT2R), downregulates RhoA/Rho kinase, which plays a role in AT1 receptor (AT1R)-mediated function. METHODS: In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10 mol/l), CGP42112A (10 mol/l, a selective AT2R agonist) +/- valsartan (10 mol/l, an AT1R antagonist), or the Rho kinase inhibitor fasudil (10 mol/l). AT1R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT2R, RhoA/Rho kinase and vasodilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasodilatory responses to Ang II (10-10 mol/l) were performed in norepinephrine pre-contracted vessels +/- valsartan(10 mol/l), PD123319 (10 mol/l, an AT2R antagonist) or fasudil (10 mol/l). RESULTS: A10 VSMC expressed AT1R and AT2R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 +/- 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 +/- 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 +/- 7%). PD123319 blocked these effects. Fasudil increased AngII-induced relaxation in SHRSP vessels. AT2R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased two-fold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation. CONCLUSIONS: Ang II/AT2R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasodilation in conditions associated with vascular AT2R upregulation, such as in SHRSP chronically treated with AT1R blockers, which may contribute to blood pressure lowering by these antihypertensive agents.     

Involvement of cytochrome P450 metabolites in the vascular action of angiotensin II on the afferent arterioles.

Recent studies have demonstrated that cytochrome P450-dependent metabolites of arachidonic acid (CYP450-AA) play important roles in the control of renal vascular resistance (RVR). In the present study, we examined the possible involvement of CYP450-AA in the vasoconstrictor action of angiotensin II (Ang II) on the afferent arterioles (Af-Arts), a vascular segment crucial to the control of RVR. Rabbit Af-Arts were microperfused at 60 mmHg in vitro, and the vasoconstrictor action of Ang II (10(-11)-10(-8) M, added to both the bath and lumen) was examined with or without blocking the activity of CYP450 epoxygenase or hydroxylase. Ang II decreased the luminal diameter of Af-Arts in a dose-dependent manner (34+/-2% of control diameter at 10(-8) M, n=9, p<0.0001). Pretreatment with miconazole, an inhibitor of CYP450 epoxygenase, at 10(-6) M decreased the basal diameter by 14+/-1% (n=6, p<0.01) and augmented the vasoconstrictor action of Ang II (7+/-3% of control diameter at 10(-8) M, p<0.001 vs. without miconazole). This augmentation was abolished by blocking the Ang II type 2 (AT2) receptor with PD 123319 at 10(-7) M. In contrast, pretreatment with 17-octadecynoic acid (17-ODYA, 10(-6) M), which inhibits both epoxygenase and hydroxylase activity, had no effect on the basal diameter but attenuated the vasoconstrictor action of Ang 11(46+/-2% of control diameter at 10(-8) M, p<0.01 vs. without 17-ODYA). Our results demonstrate that in the Af-Art, endogenous CYP450-AA are involved not only in the control of basal tone but also in the action of Ang II. Further, it appears that the CYP450 epoxygenase pathway attenuates Ang II action via AT2 receptors.     

Angiotensin II type 2 receptor inhibits vascular endothelial growth factor-induced migration and in vitro tube formation of human endothelial cells

Endothelial cell migration and tube formation in response to vascular endothelial growth factor (VEGF) play an important role in the process of angiogenesis. Recent data indicate that angiotensin type 2 (AT2) receptor stimulation is antiangiogenic. Therefore, we studied the effect of angiotensin II (Ang II) on VEGF-induced migration and in vitro tube formation of human endothelial cells. Ang II inhibited VEGF-induced migration of EA.hy926 cells, human coronary artery (HCA) and human dermal microvascular (HDM) endothelial cells (ECs) as well as tube formation by HDMECs. The AT2 receptor antagonist PD123,319 but not the AT1 receptor antagonist losartan blocked the inhibitory effect of Ang II. The inhibitory effect of Ang II on VEGF-induced migration of endothelial cells was mimicked by the specific AT2 receptor agonist CGP-42112A. The phosphorylation of Akt and its downstream effector endothelial NO synthase (eNOS) is pivotal to VEGF-induced angiogenesis. We therefore investigated the effect of Ang II on VEGF-induced Akt and eNOS phosphorylation. Ang II diminished the VEGF-induced phosphorylation of Akt and eNOS in endothelial cells, whereas the autophosphorylation of VEGF receptors was unaffected. CGP-42112A again mimicked and PD123,319 but not losartan blocked the inhibitory effect of Ang II. Treatment of endothelial cells with pertussis toxin (PTX) totally abolished the AT2 receptor-mediated inhibition of VEGF-induced endothelial cell migration and blocked the inhibition of Akt and eNOS phosphorylation. In conclusion, this study indicates that AT2 receptor stimulation inhibits VEGF-induced endothelial cell migration and tube formation via activation of a PTX-sensitive G protein. These findings may explain the reported antiangiogenic properties of the AT2 receptor.     

Angiotensin III stimulates aldosterone secretion from adrenal gland partially via angiotensin II type 2 receptor but not angiotensin II type 1 receptor

Angiotensin II (Ang II) and Ang III stimulate aldosterone secretion by adrenal glomerulosa, but the angiotensin receptor subtypes involved and the effects of Ang IV and Ang (1-7) are not clear. In vitro, different angiotensins were added to rat adrenal glomerulosa, and aldosterone concentration in the medium was measured. Ang II-induced aldosterone release was blocked (30.3 ± 7.1%) by an Ang II type 2 receptor (AT2R) antagonist, PD123319. Candesartan, an Ang II type 1 receptor (AT1R) antagonist, also blocked Ang II-induced aldosterone release (42.9 ± 4.8%). Coadministration of candesartan and PD123319 almost abolished the Ang II-induced aldosterone release. A selective AT2R agonist, CGP42112, was used to confirm the effects of AT2R. CGP42112 increased aldosterone secretion, which was almost completely inhibited by PD123319. In addition to Ang II, Ang III also induced aldosterone release, which was not blocked by candesartan. However, PD123319 blocked 22.4 ± 10.5% of the Ang III-induced aldosterone secretion. Ang IV and Ang (1-7) did not induce adrenal aldosterone secretion. In vivo, both Ang II and Ang III infusion increased plasma aldosterone concentration, but only Ang II elevated blood pressure. Ang IV and Ang (1-7) infusion did not affect blood pressure or aldosterone concentration. In conclusion, this report showed for the first time that AT2R partially mediates Ang III-induced aldosterone release, but not AT1R. Also, over 60% of Ang III-induced aldosterone release may be independent of both AT1R and AT2R. Ang III and AT2R signaling may have a role in the pathophysiology of aldosterone breakthrough.     

Angiotensin II Type 2 Receptor-Mediated Inhibition of NaCl Absorption Is Blunted in Thick Ascending Limbs From Dahl Salt-Sensitive Rats

NO reduces NaCl absorption by thick ascending limbs (TALs) by inhibiting the Na/K/2Cl cotransporter (NKCC2). We have shown that NO-induced inhibition of Na transport is reduced in Dahl salt-sensitive rat (SS) TALs. Angiotensin II increases NO production in TALs via angiotensin II type 2 receptor (AT(2)R). It is unknown whether AT(2)Rs regulate TAL NaCl absorption and whether this effect is reduced in SS rats. We hypothesized that AT(2)R activation decreases TAL Na transport via NO, and this effect is blunted in SS rats. In the presence of angiotensin II type 1 receptor antagonist losartan, AT(2)R activation with angiotensin II inhibited NKCC2 activity by 32±7% (P<0.03). AT(2)R antagonist PD-123319 abolished the effect of angiotensin II. Activation with the AT(2)R-selective agonist CGP42112A (10 nmol/L) decreased NKCC2 activity by 29±6% (P<0.03). The effect of CGP42112A on NKCC2 activity was blocked by PD-123319 and by NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. In Dahl salt-resistant rat TALs, 1 nmol/L of CGP42112A decreased NKCC2 activity by 23±4% (P<0.01). In SS TALs, it had no effect. TAL AT(2)R mRNA did not differ in SS versus salt-resistant rats. We conclude the following: (1) TAL AT(2)R activation decreases Na absorption; (2) this effect is mediated by AT(2)R-induced stimulation of NO; (3) AT(2)R-induced reduction of NKCC2 activity is blunted in SS rats; and (4) defects in AT(2)R/NO signaling rather than decreased AT(2)R expression likely account for the blunted effect in SS TALs. Impaired AT(2)R-mediated signaling in TALs could contribute to the Na retention associated with salt-sensitive hypertension.     

Effects of angiotensin II on nitric oxide generation in growing and resting rat aortic endothelial cells

OBJECTIVES: To assess the effects of angiotensin II (ang II) and its receptors on nitric oxide (NO) production and endothelial NO synthase (eNOS) activity and expression with respect to rat aortic endothelial cell (RAEC) growth. To also assess whether an intact endothelium is required for ang II activity. METHODS: RAEC were treated with different doses of ang II, Ca(2+) ionophore A23187, valsartan (an AT(1) receptor inhibitor) or PD-123319 (an AT(2) receptor inhibitor) alone or in combination for 24 h before measuring nitrite levels by Griess reaction as an index of NO production and eNOS activity by L-[3H]-arginine to L-[3H]-citrulline conversion assay. eNOS mRNA and protein expressions were determined by Northern and Western analyses, respectively. The requirement of endothelium for ang II-mediated relaxant/contractile effects was investigated by isometric tension studies. RESULTS: NO production and eNOS activity/expression were almost two-fold greater in proliferating RAEC. Ang II or Ca(2+) ionophore A23187 enhanced NO production in proliferating and confluent RAEC without altering the fold-difference in basal NO release. Both valsartan and PD-123319 significantly diminished NO production in RAEC treated with ang II but not Ca(2+) ionophore A23187 while NG-nitro-L-arginine (L-NNA, 10 micromol/l) equally decreased NO generation in response to both stimulators. L-NNA, valsartan and PD-123319 also abolished endothelium-dependent vasorelaxant responses to ACh and Ca(2+) ionophore A23187 in the presence of ang II. Sodium nitroprusside (SNP), a NO donor, increased endothelium-independent vasorelaxant responses that were augmented by valsartan but not L-NNA or PD-123319 in the presence of ang II. CONCLUSIONS: Ang II induces vascular NO production through endothelial AT(1) and AT(2)-receptors. This may be beneficial in counterbalancing its vasoconstrictor effect on vascular smooth muscle cells.     

Role of cardiac ATP-sensitive K+ channels induced by angiotensin II type 1 receptor antagonist on metabolism, contraction and relaxation in ischemia-reperfused rabbit heart

The role of cardiac adenosine triphosphate-sensitive K+ (K(ATP)) channels induced by angiotensin II type 1 (AT1) receptor antagonist, CV-11974, on myocardial metabolism and contraction during ischemia, and reperfusion by the phosphorus 31-nuclear magnetic resonance in Langendorff-perfused rabbit hearts was investigated. After 20 min of continuous normothermic global ischemia, 30 min of postischemic reperfusion was carried out. CV-11974 with or without the K(ATP) channel blocker, glibenclamide, or the bradykinin B2 receptor antagonist, D-Arg-[Hyp3,D-Phe7]bradykinin, was administered 40 min prior to the global ischemia. Adenosine triphosphate (ATP), creatine phosphate (PCr), inorganic phosphate (Pi), intracellular pH (pHi), left ventricular systolic developed pressure, left ventricular end-diastolic pressure (LVEDP), and coronary flow were measured. Twenty-eight hearts were divided into 4 experimental groups consisting of 7 hearts each. Group I consisted of controls, Group II perfused with CV-11974 (10(-6) mol/L), Group III perfused with CV-11974 (10(-6) mol/L) in combination with glibenclamide (10(-6) mol/L), and Group IV perfused with CV-11974 (10(-6) mol/L) in combination with D-Arg-[Hyp3,D-Phe7]bradykinin (10(-6) mol/L). Group II showed a significant inhibition of the decrease in ATP during ischemia and reperfusion compared with Group I (p<0.01), being 42+/-3% and 19+/-4% at ischemia, 69+/-3% and 47+/-4% at reperfusion in Group II and Group I, respectively. Group II also showed a significant inhibition of the increase in LVEDP during ischemia and reperfusion compared with Group I (p<0.01), being 13+/-4 mmHg and 52+/-8 mmHg at ischemia, 8+/-2 mmHg and 26+/-5 mmHg at reperfusion in Group II and Group I, respectively. However, Group II did not inhibit the decrease in ATP and the increase in LVEDP during ischemia and reperfusion. Group IV also showed no inhibition of the aforementioned parameters during the same period. These results suggest that CV-11974 has a significant beneficial effect for improving myocardial energy metabolism and relaxation during both myocardial ischemia and reperfusion, which is provided by K(ATP) channels and bradykinin B2 receptor. The cardioprotective quality of the AT1 receptor antagonist is caused by the K(ATP) channels that are mediated by the bradykinin B2 receptor.     

Angiotensin II type 2 receptor inhibits prorenin processing in juxtaglomerular cells.

Long-term treatment with an angiotensin II type 1 receptor blocker (ARB) has been shown to decrease the plasma renin activity (PRA) of hypertensive patients, whereas PRA remains elevated during angiotensin-converting enzyme inhibitor (ACEI) treatment. In the present study, we used rat juxtaglomerular (JG) cells to elucidate the mechanism(s) involved in the differential regulation of PRA between ARB and ACEI treatment. Addition of 100 nmol/l angiotensinogen (Aogen) to JG cells (n=6 primary cultures) significantly increased the medium angiotensin (Ang) II levels from 14 +/- 2 to 440 +/- 9 pg/ml and suppressed the renin secretion rate (RSR) from 39.6 +/- 5.4% to 6.3 +/- 1.8% without affecting active renin content (ARC) or total renin content (TRC). In the Aogen-treated cells, the ACEI, delapril hydrochloride (CV3317, 10 micromol/l), significantly decreased the medium Ang II levels to 58 +/- 14 pg/ml and increased RSR to 39.8 +/- 4.1% without affecting ARC or TRC. The ARB, an active metabolite of candesartan cilexetil (CV11974, 10 micromol/l), however, significantly increased the medium Ang II levels and RSR to 486 +/- 15 pg/ml and 40.9 +/- 9.8%, respectively, and decreased ARC from 63.2 +/- 6.8 to 21.6 +/- 3.6 ng of Ang l x h(-1) x million cells(-1) without affecting TRC. The decreases in ARC of the Aogen+CV11974-treated cells (n=6 primary cultures) were inhibited by an Ang II type 2 receptor blocker, PD123319 (10 micromol/l). JG cells (n=6 primary cultures) were also treated with an Ang II type 2 receptor agonist, CGP42212A (0.1 micromol/l). CGP42212A significantly increased RSR from 38.2 +/- 1.6% to 49.7 +/- 4.7% and decreased ARC from 60.8 +/- 3.0 to 25.3 +/- 2.8 ng of Ang l x h(-1) million cells(-1) without affecting TRC. Addition of CV11974 did not alter the RSR, ARC, or TRC of the CGP42212A-treated cells; however, PD123319 abolished the effects of CGP42212A. These results indicate that, distinct from ACEIs, ARBs inhibit prorenin processing of JG cells through Ang II type 2 receptors. Long-term treatment with an ARB may decrease PRA in part by diminishing the storage of active renin in JG cells.     

Effect of luminal angiotensin II receptor antagonists on proximal tubule transport.

The proximal tubule can endogenously synthesize and secrete luminal angiotensin II at a concentration approximately 100- to 1000-fold higher than that in the systemic circulation. We have recently shown that this endogenously produced and luminally secreted angiotensin II regulates proximal tubule volume reabsorption, which is a reflection of sodium transport within this segment. In this study, we use in vivo microperfusion of angiotensin II receptor antagonists into the lumen of the proximal tubule to examine the role of the luminal AT1 and AT2 receptor in the regulation of volume reabsorption. Systemically administered (intravenous) AT1 and AT2 receptor antagonists, acting through basolateral angiotensin II receptors, have previously been shown to inhibit proximal tubule transport. Luminal perfusion of 10(-6) mol/L Dup 753 (AT1 antagonist) and 10(-6) mol/L PD 123319 (AT2 antagonist) decreased proximal tubule volume reabsorption from 2.94 +/- 0.18 to 1.65 +/- 0.18 and 1.64 +/- 0.19 nL/mm x min, respectively, P < .01. Luminal perfusion of 10(-4) mol/L CGP 42112A, another AT2 antagonist, similarly decreased volume reabsorption to 1.32 +/- 0.36 nL/nm x min, P < .01. The inhibition of transport with AT1 and AT2 antagonist was additive, as luminal perfusion of 10(-6) mol/L Dup 753 plus 10(-6) mol/L 123319 resulted in a decrease in volume reabsorption to 0.41 +/- 0.31 nL/mm x min, P < .001 v control, P < .05 v Dup 753, and P < .01 v PD 123319. These results show that endogenously produced angiotensin II regulates proximal tubule volume transport via both luminal AT1 and AT2 receptors.     

Effect of angiotensin converting enzyme inhibitor and angiotensin II type 1 receptor antagonist on metabolism and contraction in ischemia-reperfused rabbit heart

The effect of angiotensin converting enzyme (ACE) inhibitor, temocaprilat and/or angiotensin II type 1 (AT1) receptor antagonist, CV-11974 on myocardial metabolism and contraction during ischemia and reperfusion was examined by phosphorus 31-nuclear magnetic resonance (31P-NMR) in Langendorff rabbit hearts. After normothermic 15 min global ischemia, postischemic reperfusion of 60min was carried out. Temocaprilat and/or CV-11974 were administered from 40 min prior to the global ischemia. Adenosine triphosphate (ATP), creatine phosphate (PCr), inorganic phosphate (Pi), intracellular pH (pHi), left ventricular developed pressure (LVDevP), left ventricular end-diastolic pressure (LVEDP) and coronary flow were measured. Twenty-eight hearts were divided into 4 experimental groups consisting of 7 hearts each: group I consisted of controls, group II was perfused with temocaprilat (10(-6)mol/L), group III was perfused with CV-11974 (10(-6)mol/L), and group IV was perfused with temocaprilat (10(-6)mol/L) in combination with CV-11974 (10(-6) mol/L). Groups II and III showed a significant (p<0.05) inhibition of an overshoot phenomenon of PCr during postischemic reperfusion compared with group I. Group IV also showed a more pronounced significant (p<0.01) inhibition of the overshoot of PCr during reperfusion compared with group I. Groups II, III and IV showed a significant (p<0.05) inhibition of the decrease in ATP during global ischemia (59+/-2, 54+/-3 and 54+/-7%, respectively) compared with group I (45+/-3%). Groups II and IV showed a significant (p<0.05) early recovery of ATP during reperfusion (81+/-2, 80+/-6%) compared with group I (71+/-3%) and group II (73+/-2%). Group IV showed no more significant recovery in ATP than group III. There were no differences in LVDevP, LVEDP and coronary flow among these groups. In conclusion, temocaprilat in combination with CV-11974 has significant potential for improving myocardial energy metabolism during both myocardial ischemia and reperfusion.     

Torasemide inhibits angiotensin II-induced vasoconstriction and intracellular calcium increase in the aorta of spontaneously hypertensive rats.

Torasemide is a loop diuretic that is effective at low once-daily doses in the treatment of arterial hypertension. Because its antihypertensive mechanism of action may not be based entirely on the elimination of salt and water from the body, a vasodilator effect of this drug can be considered. In the present study, the ability of different concentrations of torasemide to modify angiotensin II (Ang II)-induced vascular responses was examined, with the use of an organ bath system, in endothelium-denuded aortic rings from spontaneously hypertensive rats. Ang II-induced increases of intracellular free calcium concentration ([Ca(2+)](i)) were also examined by image analysis in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A dose-response curve to Ang II was plotted for cumulative concentrations (from 10(-9) to 10(-6) mol/L) in endothelium-denuded aortic rings (pD(2)=7.5+/-0.3). Isometric contraction induced by a submaximal concentration of Ang II (10(-7) mol/L) was reduced in a dose-dependent way by torasemide (IC(50)=0.5+/-0.04 micromol/L). Incubation of VSMCs with different concentrations of Ang II (from 10(-10) to 10(-6) mol/L) resulted in a dose-dependent rise of [Ca(2+)](i) (pD(2)=7.5+/-0.3). The stimulatory effect of [Ca(2+)](i) induced by a submaximal concentration of Ang II (10(-7) mol/L) was blocked by torasemide (IC(50)=0.5+/-0.3 nmol/L). Our findings suggest that torasemide blocks the vasoconstrictor action of Ang II in vitro. This action can be related to the ability of torasemide to block the increase of [Ca(2+)](i) induced by Ang II in VSMCs. It is proposed that these actions might be involved in the antihypertensive effect of torasemide observed in vivo.     

Angiotensin II enhances noradrenaline release from sympathetic nerves of the rat prostate via a novel angiotensin receptor: implications for the pathophysiology of benign prostatic hyperplasia

The renin-angiotensin system (RAS) is present in the human prostate and may be activated in benign prostatic hyperplasia (BPH), possibly contributing to the pathophysiology of this disorder by enhancing local sympathetic tone and cell growth. The functional role of the RAS in the prostate, however, is unknown. The present study was undertaken to determine whether angiotensin (Ang) II enhances sympathetic transmission in the prostate. The neuronal stores of the rat prostate were labelled with [(3)H]noradrenaline (NA). Ang II and Ang I enhanced [(3)H]NA release in a concentration-dependent manner. The Ang II receptor subtype 1 (AT(1) receptor) antagonist losartan and the AT(2) receptor antagonist PD123319 inhibited this facilitatory effect of Ang II and Ang I, whereas the other AT(2) receptor antagonist, CGP42112, was without effect. Bradykinin also increased [(3)H]NA release, which was inhibited by the B(2) receptor antagonist Hoe140. The angiotensin-converting enzyme inhibitor captopril inhibited the effect of Ang I, but potentiated that of bradykinin. Interestingly, captopril alone produced an increase in [(3)H]NA release which was inhibited by Hoe140. Losartan, but not PD123319 or CGP42112, inhibited [(125)I]-Ang II binding in Chinese hamster ovary cells transfected with the AT(1a) or AT(1b) receptor. In contrast, in cells expressing the AT(2) receptor, PD123319 and CGP42112, but not losartan, inhibited [(125)I]-Ang II binding. In conclusion, Ang II enhances the release of NA from sympathetic nerves of the rat prostate via a novel functional receptor distinct from the cloned AT(1a), AT(1b) or AT(2). These data provide direct evidence in support of a functional role for the local RAS in modulating sympathetic transmission in the prostate, which may have important implications for the pathophysiology of BPH.     

Effects of angiotensin II on nitric oxide generation in proliferating and quiescent rat coronary microvascular endothelial cells.

Nitric oxide (NO) and the mitogenic peptide angiotensin II (Ang II) have been implicated in endothelial cell growth. However, the putative relationship between these two opposing agents with respect to endothelial cell growth remains unknown. In this study, proliferating and confluent rat coronary microvascular endothelial cells (CMEC) were treated with different doses of Ang II, Ca2+ ionophore A23187, or valsartan (an Ang II type 1 (AT1) receptor inhibitor) alone or in combination for 24 h before measuring the nitrite levels as an index of NO generation. NO production and endothelial NO synthase (eNOS) mRNA/protein expression were found to be 3-fold greater in proliferating vs. quiescent CMEC. Treatments of CMEC with Ang II or Ca2+ ionophore A23187 equally increased NO production without altering the fold-difference in the basal release of NO from proliferating vs. confluent CMEC. Valsartan abolished NO production in CMEC treated with Ang II but not Ca2+ ionophore A23187. Treatments of endothelium-intact vascular rings with Ang II (1 nmol/l to 10 micromol/l) plus valsartan or PD-123319, an Ang II type 2 (AT2) receptor inhibitor, attenuated vascular responses to acetylcholine in an Ang II dose-dependent manner. In these rings, phenylephrine produced significant increases in contractile responses only at nmol/l concentrations of Ang II. In contrast, pharmacological and mechanical inactivation of endothelium enhanced contractile responses to phenylephrine at micromol/I concentrations of Ang II. These data demonstrate that Ang II stimulates NO production in CMEC in both an AT1- and an AT2 receptor-regulated manner, and that this stimulation of NO may be beneficial in counterbalancing the direct vasoconstrictor effect of Ang II on underlying smooth muscle cells.     

AT2 receptor-mediated relaxation is preserved after long-term AT1 receptor blockade

Angiotensin II type 2 receptor (AT2R) stimulation may cause vasodilation per se and may contribute to the antihypertensive effect produced by Angiotensin II type 1 receptor (AT1R) antagonists, given that AT1R blockade increases endogenous levels of Ang II, suggesting a physiological role for the unblocked AT2R. Thus, we first directly assessed whether or not there is desensitization to AT2R-mediated vasorelaxation because this is an important consideration, given the raised Ang II levels and the marked desensitization that is known to occur after AT1R stimulation. Second, we examined if AT2R-mediated vasorelaxation is preserved after long-term treatment with the AT1R antagonist candesartan cilexetil. Consecutive concentration-response curves to AT2R stimulation, with either Ang II (with AT1R blockade) or the selective agonist CGP42112, were studied in rat isolated mesenteric resistance arteries mounted in an arteriograph. AT2R stimulation with Ang II induced a concentration-dependent relaxation without desensitization. Similarly, CGP42112 evoked highly reproducible relaxation, which, like Ang II, was abolished by the AT2R antagonist PD123319. By contrast, AT1R-mediated contraction exhibited marked desensitization. In rats treated with candesartan cilexetil (2 mg/kg per day for 2 weeks), AT1R-mediated contraction was abolished, whereas AT2R-mediated relaxation evoked by either Ang II or CGP42112 was highly reproducible, PD123319-sensitive, and of a magnitude similar to that observed in na?ve animals. Therefore, this study has provided unequivocal evidence for the reproducible nature of AT2R-mediated vasorelaxation during short-term and long-term AT1R blockade. Such preservation of AT2R function is a prerequisite for the consideration of physiological role(s) of AT2R during AT1R blockade.     

Angiotensin II stimulates DNA and protein synthesis in vascular smooth muscle cells from human arteries: role of extracellular signal-regulated kinases.

OBJECTIVE: This study investigates the growth effects and associated signaling pathways of angiotensin II (Ang II) in human vascular smooth muscle cells. METHODS: Cultured vascular smooth muscle cells derived from resistance arteries (< 300 microm diameter) from subcutaneous gluteal biopsies of healthy subjects (n = 6) and human aortic vascular smooth muscle cells were used. Cells were studied between passages 3 and 6. Both 3H-thymidine and 3H-leucine incorporation were measured as indices of vascular smooth muscle cell hyperplasia (DNA synthesis) and cell hypertrophy (protein synthesis), respectively. Growth effects of Ang II (10(-12) - 10(-6) mol/l), in the absence and presence of 10(-5) mol/l losartan (AT1 antagonist) and PD123319 (AT2 antagonist), were determined. Ang II-induced effects were compared to those of endothelin-1. To determine whether extracellular signal-regulated kinase (ERK)-dependent pathways play a role in Ang II-mediated growth, cells were pretreated with the selective ERK kinase (MEK) inhibitor, PD98059 (10(-5) mol/l). ERK activation was determined by Western blot in the absence and presence of PD98059. RESULTS: Ang II dose-dependently increased 3H-thymidine incorporation in cells from aorta (Emax = 276 +/- 10.4% of control) and resistance arteries (Emax = 284 +/- 5.1% of control). Ang II also stimulated 3H-leucine incorporation in cells from aorta (Emax = 162 +/- 11.6 of control) and resistance arteries (Emax 175 +/- 10% of control). Unlike Ang II, endothelin-1 failed to significantly alter cellular growth, except at high concentrations (> 10(-7) mol/l), where it had a weak stimulatory effect Losartan, but not PD123319, blocked Ang II-stimulated growth responses. Ang II significantly increased phosphorylation of ERK-1 and ERK-2, with maximum responses obtained at 5 min. PD98059 inhibited Ang II-stimulated ERK activity and abrogated agonist-induced DNA and protein synthesis. Losartan, but not PD123319 inhibited Ang II-induced phosphorylation of ERK-1 and ERK-2. CONCLUSIONS: Ang II stimulates both hyperplasia and hypertrophy in vascular smooth muscle cells from human arteries. These growth effects are mediated via Ang II receptors of the AT1 subtype that are linked to ERK-dependent signaling pathways.     

Loss of biphasic effect on Na/K-ATPase activity by angiotensin II involves defective angiotensin type 1 receptor-nitric oxide signaling

Oxidative stress causes changes in angiotensin (Ang) type 1 receptor (AT1R) function, which contributes to hypertension. Ang II affects blood pressure via maintenance of sodium homeostasis by regulating renal Na(+) absorption through its effects on Na/K-ATPase (NKA). At low concentrations, Ang II stimulates NKA; higher concentrations inhibit the enzyme. We examined the effect of oxidative stress on renal AT1R function involved in biphasic regulation of NKA. Male Sprague-Dawley rats received tap water (control) and 30 mmol/L of L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mmol/L of Tempol (antioxidant) for 2 weeks. BSO-treated rats exhibited increased oxidative stress, AT1R upregulation, and hypertension. In proximal tubules from control rats, Ang II exerted a biphasic effect on NKA activity, causing stimulation of the enzyme at picomolar and inhibition at micromolar concentrations. However, in BSO-treated rats, Ang II caused stimulation of NKA at both of the concentrations. The effect of Ang II was abolished by the AT1R antagonist candesartan and the mitogen-activated protein kinase inhibitor UO126, whereas the Ang type 2 receptor antagonist PD-123319 and NO synthase inhibitor N(G)-nitro-L-arginine methyl ester had no effect. The inhibitory effect of Ang II was sensitive to candesartan and N(G)-nitro-L-arginine methyl ester, whereas PD-123319 and UO126 had no effect. In BSO-treated rats, Ang II showed exaggerated stimulation of NKA, mitogen-activated protein kinase, proline-rich-tyrosine kinase 2, and NADPH oxidase but failed to activate NO signaling. Tempol reduced oxidative stress, normalized AT1R signaling, unmasked the biphasic effect on NKA, and reduced blood pressure in BSO-treated rats. In conclusion, oxidative stress-mediated AT1R upregulation caused a loss of NKA biphasic response and hypertension. Tempol normalized AT1R signaling and blood pressure.     

Effect of atrial natriuretic factor on calcium fluxes in adrenal glomerulosa cells

We studied the effect of atrial natriuretic factor (ANF) on calcium influx and efflux in rat adrenal glomerulosa cells stimulated by angiotensin II (Ang II) or potassium ion, and observed how ANF inhibits the initial and sustained phases of the aldosterone response to Ang II or K+ using a superfusion system of dispersed adrenal glomerulosa cells. K+ (8 mM) significantly increased Ca2+ influx rate compared with basal rate (0.91 +/- 0.10 vs 0.42 +/- 0.04 nmol/min/10(6) cells; p less than 0.01). ANF (10(-8) M) did not inhibit the K+-induced increase in Ca2+ influx rate (0.99 +/- 0.18 nmol/min/10(6) cells). Ang II (10(-9) and 10(-8) M) stimulated Ca2+ influx rate (10(-9) M Ang II, 0.62 +/- 0.02; 10(-8) M Ang II, 0.71 +/- 0.09 vs basal, 0.44 +/- 0.03 nmol/min/10(6) cells; p less than 0.05), while ANF (10(-8) M) did not change the Ca2+ influx rate increased by Ang II (ANF + 10(-9) M Ang II, 0.62 +/- 0.06; ANF + 10(-8) M Ang II, 0.69 +/- 0.14 nmol/min/10(6) cells). In the Ca2+ efflux study ANF (10(-8) M) was perfused through the cells 10 minutes before the start of perfusion with Ang II (10(-9) M) or K+ (12 mM).(ABSTRACT TRUNCATED AT 250 WORDS)