Cross-talk between receptor-mediated phospholipase C-beta and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes

Phospholipase C-beta (PLC-beta) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such as alpha1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10(-8)M), phenylephrine (PHE, 10(-5)M) or Ang II (10(-7)M) resulted in a rapid (0-10 min) activation of PLC-beta to an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10(-6)M) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0-5 min) translocation of PKC- immunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-alpha, -delta and -xi immunoreactivities was observed. PMA caused translocation of PKC-alpha, PKC-epsilon as well as PKC-delta. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10(-4)M) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10(-6)M) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-beta and after a lag-phase PLD, whereas PHE and Ang II activate only PLC-beta. PLC-beta stimulated by ET-1, may cross-talk with PLD via translocation of PKC-epsilon. These signals are possibly linked to the hypertrophic response.     

Role of the alpha 1-, and alpha 2- and beta-adrenoceptors of the median preoptic area on the water intake, renal excretion, and arterial pressure induced by ANG II

The present experiments were conducted to investigate the role of the alpha 1-, alpha 2- and beta-adrenergic receptors of the median preoptic area (MnPO) on the water intake and urinary electrolyte excretion, elicited by central injections of angiotensin II (ANG II). Prazosin (an alpha 1-adrenergic receptor antagonist) and yohimbine (an alpha 2-adrenergic receptor antagonist) antagonized the water ingestion, Na+, K+, and urine excretion induced by ANG II. Administration of propranolol, a beta-adrenergic receptor antagonist increased the Na+, K+, and urine excretion induced by ANG II. Previous treatment with prazosin and yohimbine reduced the pressor responses to ANG II. These results suggest that the adrenergic neurotransmission in the MnPO may actively participate in ANG II-induced dipsogenesis, natriuresis, kaliuresis, diuresis and pressor responses in a process that involves alpha 1-, alpha 2-, and beta-adrenoceptors.     

Effects of genistein and staurosporine on angiotensin II-induced DNA synthesis, protein synthesis and mitogen-activated protein kinase activation in vascular smooth muscle cells

This study examines the effects of protein kinase inhibitors and activator on angiotensin II-induced DNA synthesis and protein synthesis of rat aortic smooth muscle cells. In quiescent confluent cells, angiotensin II induced a concentration-dependent increase in thymidine incorporation and leucine incorporation. The tyrosine kinase inhibitor genistein caused an inhibition of the angiotensin II-induced DNA synthesis but not of the agent-induced protein synthesis. The protein kinase C inhibitors staurosporine and calphostin C caused an inhibition of the angiotensin II-induced protein synthesis but not of the agent-induced DNA synthesis. The protein kinase C activator phorbol 12-myristate 13-acetate stimulated protein synthesis. Angiotensin II stimulated mitogen-activated protein (MAP) kinases and the angiotensin II-induced MAP kinase activation was inhibited by genistein but not by staurosporine. These findings suggest that angiotensin II-induced DNA synthesis is at least partly mediated via protein-tyrosine phosphorylation and angiotensin II-induced protein synthesis is at least partly mediated by activation of protein kinase C. It seems likely that MAP kinase activation is involved in DNA synthesis but not in protein synthesis induced by angiotensin II.     

Germline mutations of the BRCA1 and BRCA2 genes in a breast and ovarian cancer patient

Nitric oxide (NO) has been implicated as a modulator of the vascular effects of angiotensin II (ANG II) in the kidney. We used a NO-sensitive microelectrode to study the effect of ANG II on NO release, and to determine the effect of selective inhibition of the ANG II subtype I receptor (AT1) with losartan (LOS) and candesartan (CAN). NO release from isolated and perfused renal resistance arteries was measured with a porphyrin-electroplated, carbon fiber. The vessels were microdissected from isolated perfused rat kidneys and perfused at constant flow and pressure in vitro. The NO-electrode was placed inside the glass collection cannula to measure vessel effluent NO concentration. ANG II stimulated NO release in a dose-dependent fashion: 0.1 nM, 10 nM and 1000 nM ANG II increased NO-oxidation current by 85+/-18 pA (n = 11), 148+/-22 pA (n = 11), and 193+/-29 pA (n = 11), respectively. These currents correspond to changes in effluent NO concentration of 3.4+/-0.5 nM, 6.1+/-1.1 nM, and 8.2+/-1.3 nM, respectively. Neither LOS (1 muM) nor CAN (1 nM) significantly affected basal NO production, but both AT1-receptor blockers markedly blunted NO release in response to ANG II (10 nM): 77+/-6% inhibition with LOS (n = 8) and 63+/-9% with CAN (n = 8). These results are the first to demonstrate that ANG II stimulates NO release in isolated renal resistance arteries, and that ANG II-induced NO release is blunted by simultaneous AT1-receptor blockade. Our findings suggest that endothelium-dependent modulation of ANG II-induced vasoconstriction in renal resistance arteries is mediated, at least in part, by AT1-receptor-dependent NO release.     

Control of vascular smooth muscle cell growth in fowl

In adult domestic fowl, angiotensin (ANG) receptors are present in the vascular smooth muscles (VSM) and in the endothelium, mediating vasorelaxation via endothelium-derived relaxing factor/cGMP. ANG II-induced relaxation is minor in chicks and becomes more marked as they mature but diminishes in adult birds, whereas ANG II neither relaxes nor contracts endothelium-denuded aortae from mature chickens. The present study examines in cultured fowl aortic SM cells whether (1) ANG II stimulates or inhibits VSM cell growth and, if so, whether this growth-stimulatory or -inhibitory effect changes with maturation/aging, and (2) S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, and cGMP attenuate the basal or stimulated VSM cell growth. [Asp1, Val5]ANG II (native fowl ANG II, 10(-6) M) markedly increased (increase from vehicle control, 226.5%; P < 0.01) [3H]thymidine (Thd) incorporation into DNA of quiescent VSM cells (first subculture) from 6-week-old chicks. This growth-stimulating effect was reduced with age (41.4, 29.6, and 3.2% at 9, 19, and 43 weeks of age, respectively). In contrast, platelet-derived growth factor (PDGF, 20 ng/ml) increased [3H]Thd incorporation similarly in chicks, pullets, and hens. Furthermore, ANG II significantly (45.9%, P < 0.01) attenuated the growth-promoting effect of fetal calf serum in cultured VSM cells from 6-week-old chicks. This inhibitory effect also decreased in older birds. ANG II showed neither a growth-stimulatory nor -inhibitory effect in cultured neointimal cells. SNAP attenuated dose dependently (20-60 microM) the basal and PDGF-induced VSM cell growth, whereas cGMP inhibited basal growth only at a high dose (100 microM). These results indicate that in fowl VSM cells, ANG II is mitogenic and antimitogenic in chicks but not in mature birds, suggesting that phenotypic modulation occurs in the ANG receptors/signaling mechanism with maturation/age or in neointimal cells, whereas the mitogenic mechanism via PDGF remains in both young and mature birds.     

Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1-34), PTHrP(1-34), PTHrP(1-141), PTHrP(100-114) and antisense oligonucleotides against PTHrP

Parathyroid hormone-related protein (PTHrP) is thought to be an important autocrine/paracrine factor for chondrocyte metabolism since mice lacking the PTHrP gene exhibit abnormal cartilage development. To determine the biological role of PTHrP in chondrocytes, we first compared the agonist potency of human (h) PTHrP(1-34) with hPTH(1-34) in cultured rat articular chondrocytes. Neither hPTHrP(1-34) nor hPTH(1-34) altered basal DNA synthesis, but attenuated the stimulatory effect of transforming growth factor beta (TGF-beta). Both agents suppressed the expression of alpha(1) type II collagen mRNA in a dose-response fashion with the same potency. In addition, the action of exogenously added hPTHrP(1-34) and hPTH(1-34) on intracellular cAMP and [Ca2+]i levels was similar. We next compared the effect of PTHrP within its entire amino acid sequence (1-141). With regard to thymidine incorporation, alpha(1) type II collagen gene expression and accumulation of cAMP and [Ca2+]i level, there was no significant difference between hPTHrP(1-34) and hPTHrP(1-141). PTHrP C-terminal (100-114) did not show any function. To further investigate PTHrP function, intracellular PTHrP translation was inhibited by a transgene of antisense oligonucleotides against PTHrP. Antisense oligonucleotides decreased PTHrP mRNA translation, specifically inhibited DNA synthesis in control as well as TGF-beta-treated chondrocytes and enhanced alpha(1) type II collagen mRNA expression in TGF-beta-treated chondrocytes. These results suggest that there is no significant difference between exogenously added hPTH(1-34), hPTHrP(1-34) and PTHrP(1-141) with regard to the biological action of these agents, including cell growth, differentiation and second messenger pathway. However, the result of DNA synthesis in the antisense PTHrP-inhibition study suggests that intracellular PTHrP may have an as yet unknown biological role, in addition to a classical PTH/PTHrP receptor-mediated function in the rat articular chondrocyte.     

Role of angiotensin II and vasopressin receptors within the supraoptic nucleus in water and sodium intake induced by the injection of angiotensin II into the medial septal area

In this study we investigated the effects of the injection into the supraoptic nucleus (SON) of non-peptide AT1- and AT2-angiotensin II (ANG II) receptor antagonists, DuP753 and PD123319, as well as of the arginine-vasopressin (AVP) receptor antagonist d(CH2)5-Tyr(Me)-AVP, on water and 3% NaCl intake induced by the injection of ANG II into the medial septal area (MSA). The effects on water or 3% NaCl intake were assessed in 30-h water-deprived or in 20-h water-deprived furosemide-treated adult male rats, respectively. The drugs were injected in 0.5 microliter over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. Antagonists were injected at doses of 20, 80 and 180 nmol. Water and sodium intake was measured over a 2-h period. Previous administration of the AT1 receptor antagonist DuP753 into the SON decreased water (65%, N = 10, P < 0.01) and sodium intake (81%, N = 8, P < 0.01) induced by the injection of ANG II (10 nmol) into the MSA. Neither of these responses was significantly changed by injection of the AT2-receptor antagonist PD123319 into the SON. On the other hand, while there was a decrease in water intake (45%, N = 9, P < 0.01), ANG II-induced sodium intake was significantly increased (70%, N = 8, P < 0.01) following injection of the V1-type vasopressin antagonist d(CH2)5-Tyr(Me)-AVP into the SON. These results suggest that both AT1 and V1 receptors within the SON may be involved in water and sodium intake induced by the activation of ANG II receptors within the MSA. Furthermore, they do not support the involvement of MSA AT2 receptors in the mediation of these responses.     

Dissociation of left ventricular hypertrophy, beta-myosin heavy chain gene expression, and myosin isoform switch in rats after ascending aortic stenosis

The regulation of angiotensin II (Ang II) receptors and Ang II-induced modulation of intracellular Ca2+ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)-salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca2+ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT1 subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II-specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca2+ responses induced by increasing Ang II concentrations (10[-12] to 10[-4] mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca2+ spike frequency were unaltered in cardiomyocytes from DOCA-salt-hypertensive rats. The density of AT1 subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca2+ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT1 receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT1 receptor status is unaltered. These findings report for the first time the cardiac cell-specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT1 receptor in cardiac hypertrophy in DOCA-salt-hypertensive rats.     

Endothelin expression and responsiveness in human ovarian carcinoma cell lines

To elucidate the potential role of endothelins (ETs) as growth regulators in ovarian carcinoma cells in culture, expression of endothelins and their receptors were measured in two ovarian cancer cell lines (PEO4 and PEO14), together with the effect of the exogenous addition of endothelins on the growth of these cell lines in vitro. RT-PCR analysis of mRNA prepared from PEO4 and PEO14 indicated the presence of ET-1 and ET-3 mRNA. Immunoreactive ET-1-like peptide was found in media from cultures of both PEO4 (1.7 +/- 0.4 fmol/10(6) cells/72 h) and PEO14 (20.2 +/- 6.8 fmol/10(6) cells/72 h) cell lines. Radioligand binding studies using 125I-ET-1 and membrane fractions were consistent with PEO4 cells having two receptor sites of either high affinity (Kd = 0.065 nM, Bmax = 0.047 pmol/mg protein) or lower affinity sites (Kd = 0.49 nM, Bmax = 0.23 pmol/mg protein). Studies using membrane fractions of PEO14 cells indicated that this cell line has only a single lower affinity binding site (Kd = 0.56 nM, Bmax = 0.31 pmol/mg protein). However, RT-PCR analysis indicated the presence of mRNA from both ETA and ETB receptors in PEO4 and PEO14 cell lines. Exogenous addition of ETs to PEO4 and PEO14 cells at concentrations of 10(-10)-10(-7)M resulted in specific dose-dependent increases in cell number for ET-1 (with maximum effects at 10(-10) and 10(-9)M for PEO4 and PEO14, respectively) and ET-2 (maximum effects at 10(-8) and 10(-9)M for PEO4 and PEO14, respectively) but not for ET-3. Experiments on the growth of PEO14 cells using BQ123 (ETA-R) antagonist and "antisense" oligonucleotide against the ETA-R, in the absence of exogenous ETs, suggested that immunoreactive ET-1-like material secreted by PEO14 cells can affect their growth in an autocrine manner. These results would be consistent with ET-1 acting as a possible autocrine growth regulator in human ovarian carcinoma cells.     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

The orally active nonpeptide endothelin A-receptor antagonist A-127722 prevents and reverses hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in Sprague-Dawley rats

Exposure to hypoxia is associated with increased pulmonary artery pressure and plasma endothelin (ET-1) levels and with selective enhancement of ET-1 peptide and messenger RNA (mRNA) and endothelin-A (ET-A) receptor mRNA in rat lung. Our study tested the hypothesis that A-127722, an orally active antagonist of the ET-A receptor, can prevent hypoxia-induced pulmonary hypertension and vascular remodeling in the rat. Pretreatment with A-127722 (3, 10, and 30 mg/kg/day in drinking water for 2 days) caused dose-dependent inhibition of the pulmonary vasoconstrictor response to short-term hypoxia (10% O2, 90 min). Long-term A-127722 treatment (10 mg/kg/day in drinking water for 2 weeks) instituted 48 h before hypoxic exposure attenuated the subsequent development of pulmonary hypertension, the associated right atrial hypertrophy, and pulmonary vascular remodeling. Institution of A-127722 treatment (10 mg/kg/day in drinking water for 4 weeks) after 2 weeks of hypoxia retarded the progression of established hypoxia-induced pulmonary hypertension and right atrial hypertrophy and reversed the pulmonary vascular remodeling despite continuing hypoxic exposure. These findings support the hypothesis that endogenous ET-1 plays a major role in hypoxic pulmonary vasoconstriction/hypertension, right heart hypertrophy, and pulmonary vascular remodeling and suggest that ET-A receptor blockers may be useful in the treatment and prevention of hypoxic pulmonary hypertension in humans.     

Alternative splicing of CYP2D mRNA in human breast tissue

Cerebrovascular arteriovenous malformations (AVMs) display abnormal vascular development and dysautoregulation of blood flow. Genetic mechanisms that contribute to the pathogenesis and phenotype of cerebral AVMs are unknown. As a first step in understanding the pathophysiology of AVMs, the authors investigated the hypothesis that endothelial dysfunction-specifically, deregulation of endothelin-1 (ET-1) secretion-contributes to the abnormal vascular phenotype and the lack of hemodynamic autoregulation elaborated by these lesions. Endothelin-1 peptide and preproendothelin-1 (ppET1) messenger RNA were not detected in the intranidal vasculature of all 17 patients with AVMs studied, but were prominently expressed in human control subjects with normal cerebrovasculature (p < 0.01). Although AVM vasculature lacked ET-1, its expression was prominent in vasculature distant from these lesions, suggesting local repression of the ppET-1 gene. Local repression of ET-1 was specific to AVMs; ET-1 in vascular malformations of patients with Sturge-Weber disease was actually elevated compared to normal controls (p < 0.01). Repression of the ppET-1 gene was an intrinsic phenotype of AVM endothelial cells and was not due to factors in the AVM microenvironment. The authors also showed that ETA receptor expression was low in AVM vasculature compared to normal controls. Together, these results demonstrate that the ppET-1 gene is locally repressed in AVM lesions and suggest a role for abnormal ppET-1 gene regulation in the pathogenesis and clinical sequelae of cerebral AVMs.     

Increased protein kinase C activity in myotrophin-induced myocyte growth

Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue. Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin. The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known. To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes. PKC activity was determined by measuring the incorporation of 32P into histone type III-S and PKCepsilon substrate peptide (epsilon(pep)) from [gamma-32P]ATP in neonatal myocytes. Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis. The effect of myotrophin on the stimulation of PKC activity and [3H]leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors. Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and beta-myosin heavy chain. To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth. Genistein attenuated the [3H]leucine incorporation induced by myotrophin. To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms. Results showed that both PKCalpha and PKCepsilon isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKCepsilon isoform was involved in myotrophin-induced adult myocyte hypertrophy. PKCdelta and PKCzeta do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin. Treatment with antisense oligonucleotides specific for PKCalpha and PKCepsilon isoforms further supported this result. PKCalpha is the major PKC isoform in neonatal myocytes and needs Ca2+ and phospholipids for its activation, and PKCepsilon (the Ca2+-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study. Treatment of neonatal myocytes with the PKCalpha and PKCepsilon antisense oligodeoxynucleotides for 5 days significantly reduced Ca2+-dependent and Ca2+-independent PKC activity, respectively, as well as the [3H]leucine incorporation induced by myotrophin. Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction. Myotrophin does not change PKC isoform expression (both Ca2+ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts. Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase-coupled pathway and translocation of PKC from the cytosol to the cell membrane.     

AT2-antagonist sensitive potentiation of angiotensin II-induced vasoconstrictions by blockade of nitric oxide synthesis in rat renal vasculature

1. Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II-induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT-R) antagonists on Ang II-induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single-pass circuit. 2. In the absence of NO blockade, the AT1-R antagonist L-158809 (500 nM) antagonized the Ang II-induced vasoconstrictions, while the AT2-R antagonist PD-123319 (500 nM) had no effect. 3. Perfusing kidneys in the presence of either NO synthase inhibitors, L-NAME (100 microM) or L-NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY-83583 (10 microM), significantly increased both molar pD2 (from 9.40+/-0.25 to 10.36+/-0.11) and Emax values (from 24.9+/-3.1 to 79.9+/-4.9 mmHg) of the concentration-response curve for Ang II-induced vasoconstriction. 4. In the presence of L-NAME, 500 nM L158809 abolished the Ang II-induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD-123319 reversed the left shift of the concentration-response curve for Ang II (molar pD2 value 9.72+/-0.13) leaving Emax value unaffected (91.3+/-7.6 mmHg). 5. In the presence of L-NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration-dependent manner by L-158809 (0.05-500 nM). By contrast, PD-123319 (0.5-500 nM) did not affect the 10 nM Ang II-induced vasoconstriction and concentration-dependently decreased the 0.1 nM Ang II-induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6. Similar to PD-123319, during NO blockade the AT2-R antagonist CGP-42112A at 5 nM decreased by 50% the 0.1 nM Ang II-induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II-induced vasoconstriction. 7. In conclusion, the renal Ang II-induced vasoconstriction, which is antagonized only by AT1-R antagonist in the presence of endogenous NO, becomes sensitive to both AT1- and AT2-R antagonists during NO synthesis inhibition. While AT1-R antagonist inhibited both L-NAME-potentiated and -augmented components of Ang II-induced vasoconstriction, AT2-R antagonists inhibited only the L-NAME-potentiated component.