Angiotensin II stimulates PGF(2 alpha)release in cultured neonatal rat ventricular myocytes via L-type calcium channels.

Angiotensin II (Ang II) has been shown to cause Prostaglandin F(2 alpha)(PGF(2 alpha)) release in neonatal rat ventricular myocytes and smooth muscle cells. In these cells, Ang II has also been shown to regulate growth. We used neonatal rat ventricular myocytes to investigate the role of calcium in maintenance of Ang II-induced PGF(2 alpha)release. The amount of PGF(2 alpha)produced was determined by radioimmunoassay. Ang II-induced PGF(2 alpha)release. Pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of diltiazm (voltage-sensitive L-type calcium channel blocker) produced significant inhibition in Ang II-induced PGF(2 alpha)release. Inhibition was first noted at 10(-8)M and was complete at 10(-6)M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of calcium channel blockers (conotoxin; voltage-sensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced no changes in Ang II-induced PGF(2 alpha)release. These results strongly suggest that Ang II-induced PGF(2 alpha)release in neonatal rat ventricular myocytes is maintained, at least in part, via increase in extracellular calcium influx.

Nitric oxide in CsA-induced hypertension: role of beta-adrenoceptor antagonist and thromboxane A2.

Cyclosporine A (CsA) is an immunosuppressive agent, which also causes hypertension. The effect of CsA on vascular responses was determined in Sprague-Dawley rats and rat aortic rings. Male rats weighing 250-300 g were given either CsA (25 mg/kg/day) in olive oil or vehicle by intraperitoneal (ip) injection for 7 days. CsA administration produced a 42% increase (P < 0.001) in mean arterial pressure (MAP) which reached a plateau after 3 days. The level of both nitrate/nitrite (NO2/NO3), metabolites of nitric oxide (NO), decreased by 50% (P < 0.001), but the level of thromboxane A2 (TBXA2) increased by 75% (P < 0.001), in the urine. When 10(-9) M of CsAwas added acutely to intact aortic rings from untreated rats, NO2/NO3 production decreased by 83% (P < 0.011), but TBXA2 production increased by 86% (P < 0.001). The effects of CsA were reversed both in vivo and in vitro by pretreatment with propranolol (15 mg/kg/day ip), beta-adrenoceptor antagonist. There were no changes in MAP and tension in rats treated with prop alone. In addition, in aorta of rats that were treated with CsA ip for 7 days, CsA significantly activated protein kinase C (PKC) translocation. This suggests that PKC mediate, in part, CsA-induced hypertension. In summary, CsA inhibits endothelial NO formation, activate PKC, and increaseTBXA2 production, with resulting increase in MAP, and this changes can be overcome by pretreatment with propranolol.

Angiotensin II stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes: roles of PKC and PGF2alpha.

Angiotensin II (Ang II) has been shown to regulate growth in smooth muscle cells. Protein kinase C (PKC), which mediates Ang II action, has been implicated in myocardial cell hypertrophy. Acute pressure overload in the left ventricles has been demonstrated to produce prostaglandin F2 alpha (PGF2alpha) release. Therefore, we used cultured neonatal rat ventricular myocytes to study Ang II, PKC and PGF2alpha and their relationship to hypertrophy. The amount of PGF2alpha produced was determined by radioimmunoassay, Ang II-induced hypertrophy and PGF2alpha release. Pretreatment with 10(-6) M of PKC inhibitor, 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7), blocked Ang II-induced hypertrophy and PGF2alpha release. In neonatal rat ventricular myocytes that were treated with either Ang II or PKC activator (Phorbol 12, 13, dibutyrate; PDBu), PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates, in part, Ang II-induced PGF2alpha release and hypertrophy. In summary, Ang II activates PKC, which causes PGF2alpha release and hypertrophy, and this PGF2alpha release and hypertrophy can be overcome by pretreatment with PKC inhibitor.

Adenosine induced direct negative inotropic effect is abolished during global ischemia: role of protein kinase C and prostacyclin.

Adenosine acts as a cardioprotective agent by producing coronary vasodilation, decreasing heart rate and by antagonizing the cardiostimulatory effect of catecholamines; adenosine also exerts a direct negative inotropic effect. Myocardial ischemia is known to be associated with enhanced levels of adenosine, increased protein kinase C (PKC) activity and prostacyclin (PGI2) release. The present study was conducted to determine if myocardial ischemia alters the cardioprotective effect of adenosine by increasing PKC activity and PGI2 release in the isolated rat heart perfused at 10 ml/min with Krebs-Henseleit buffer (KHB; 95% O2+5% CO2). Adenosine (10 mmol/min) reduced myocardial contractility as indicated by a decrease in contractility (dp/dtmax), heart rate (HR) and coronary perfusion pressure (PP). In hearts subjected to 30 min of ischemia (without perfusion) and then reperfused with KHB, adenosine failed to decrease dp/dtmax, HR or PP. However, during infusion of PKC inhibitor H-7 (1-(5-Isoquinolinesulfonyl)-2-methylpiperazine hydrochloride) (H-7; 6 mmol/min), which commenced 10 min before ischemia and continued throughout reperfusion, adenosine produced a decrease in dp/dtmax, HR and PP, similar to that before ischemia. Infusion of the PKC activator phorbol 12,13-dibutyrate (PDBu; 2 nmol/min) but not an inactive analogue in non-ischemic hearts prevented the adenosine induced decrease in dp/dtmax. During infusion of H-7, PDBu failed to block the direct negative inotropic effect of adenosine in non-ischemic hearts. In addition, pretreatment with H-7 or indomethacin (cyclooxygenase inhibitor) significantly reduced the PGI2 release following ischemia. This data suggest that PKC and PGI2 regulate the direct negative inotropic effect of adenosine, which is abolished during ischemia.

Nitric oxide in cyclosporine A-induced hypertension: role of protein kinase C.

Chronic treatment with cyclosporine A (CsA), an immunosuppressive agent, causes hypertension. The effect of CsA on vascular responses was determined in Sprague-Dawley rats and isolated rat aortic rings. Male rats weighing 250 to 300 g were given either CsA (25 mg/kg/day) in olive oil or vehicle by intraperitoneal injection for 7 days. Cyclosporine A administration produced a 42% increase (P<.001) in mean arterial pressure (MAP), which reached a plateau after 3 days. Conversely, the level of both nitrate/nitrite (NO2/NO3), metabolites of nitric oxide (NO), and 3', 5' cyclic guanosine monophosphate (cGMP), which mediates NO action, decreased by 50% (P<.001) and 35% (P<.001), respectively, in the urine. Thoracic aortic rings from rats treated with CsA, and precontracted with endothelin (10(-9) mol/L), showed a 35% increase (P<.001) in tension, whereas acetylcholine-induced (Ach; 10(-9) mol/L) endothelium-dependent relaxation was inhibited 65% (P<.001) compared with untreated rats. This response was similar to that of aortic rings, denuded of endothelium, from untreated rats in which Ach-induced relaxation was completely abolished (P<.001). Ach-induced formation of both NO2/NO3 and cGMP by both denuded and CsA-treated aortic rings was inhibited 95% (P<.001) and 65% P<.001), respectively, compared with intact aortic rings. The effects of CsA were reversed both in vivo and in vitro by pretreatment with L-arginine (L-Arg; 10 mg/kg/day intraperitoneally), the precursor of NO. There were no changes in MAP and tension in rats treated with L-Arg alone. In addition, in the aorta of rats that were treated intraperitoneally with CsA for 7 days, CsA significantly activated protein kinase C (PKC) translocation and decreased NO2/NO3 production. This suggest that PKC mediates, in part, CsA-induced hypertension. In summary, CsA activates PKC, which inhibits endothelial NO formation, with resulting increases in MAP and tension, and this inhibition can be overcome by L-Arg administration.

Angiotensin II-induced ET and PGI2 release in rat aortic endothelial cells is mediated by PKC.

Angiotensin II (Ang II) has been shown to stimulate the release of immunoreactive endothelin (ET) from cultured bovine ECs. Also, Ang II activates phospholipase A2 (PLA2) in various tissues, resulting in the release of arachidonic acid and formation of prostaglandins. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in Ang II-induced release of both ET and prostacyclin (PGI2). The amount of ET and PGI2 produced were determined by radioimmunoassay. Ang II-induced the release of both ET and PGI2. Pretreatment with 10(-6) M of any one of the PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine(CL), staurosporine, 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), and calphostin C blocked AII-induced release of both ET and PGI2. In rat aortic endothelial cells that were treated with either AII or PDBu, PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates AII-induced ET and PGI2 release. In summary, AII activates PKC which inhibits rat aortic endothelial cells ET and PGI2 formation, and this inhibition can be overcome by pretreatment with PKC inhibitors.

Cyclosporine A-induced prostacyclin release is maintained by extracellular calcium in rat aortic endothelial cells: role of protein kinase C.

Chronic treatment with the immunosuppressive drug Cyclosporine A (CsA) is associated with increased intracellular calcium in vascular smooth muscle cells, which may activate phospholipase A2. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in CsA-induced prostacyclin (PGI2) release. CsA (10(-9) M) produced a significant increase in PGI2 release. CsA-induced PGI2 release were inhibited 80-85% by 10(-9) M, and 99-100% by 10(-6) M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), staurosporine or 1-(5-isoquinolinesulfonyl)piperazine. Pretreatment with (10(-9) M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced PGI2 release. Conversely, pretreatment with (10(-9) M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium, mediates CsA-induced PGI2 release in rat aortic endothelial cells.

Nitric oxide in cyclosporine A-induced hypertension: endothelin receptors gene expression.

Cyclosporine A (CsA) is an immunosuppressive agent, which also causes hypertension. The effect of CsA on vascular responses was determined in spontaneously hypertensive rats and isolated rat aortic rings. Male rats weighing 250-300 g were given either CsA (25 mg/kg/day) in olive oil or vehicle by i.p. injection for 7 days. CsA administration produced a 27% increase (P < 0.001) in mean arterial pressure (MAP) which reached a plateau after 3 days. Conversely, the level of nitrate/nitrite, metabolites of nitric oxide (NO), decreased by 44% (P < 0.001) in the urine. In the presence of endothelin (ET) 10(-9) M, thoracic aortic rings from rats treated with olive oil, L-Arginine (L-Arg) or L-Arg+CsA showed a 100% increase (P < 0.001) in tension compared to the aortic rings from rats treated with CsA alone; aortic rings from rats treated with CsA alone did not respond to ET. The effects of CsA were reversed in both in vivo and in vitro by pretreatment with L-Arg (10 mg/kg/day ip), the precursor of NO. There were no changes in MAP and tension in rats treated with L-Arg alone. Possible explanation for lack of response to ET of aortic rings from CsA treated rats may be that CsA affected ET signalling pathway; ET receptors mRNA (messenger ribonucleic acid) gene expression was inhibited in aortic rings of rats treated with CsA. In summary, CsA inhibits endothelial NO formation, with resulting increases in MAP, and this inhibition can be overcome by parenteral administration of L-Arg.

Endothelin-induced prostacyclin production in rat aortic endothelial cells: role of calcium.

Endothelin (ET) is a potent vasoconstrictor peptide, released from endothelial cells, which is associated with prostaglandin (PG) release. The mechanism by which ET causes the release of PG is not clearly understood. We used rat aortic endothelial cells to investigate the role of calcium (Ca2+) in ET-1-induced prostacyclin (PGI2) release. ET-1 (10(-9) M) produced a significant increase in PGI2 release. Pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced PGI2 release. Inhibition was first noted at 10(-9) M and was complete at 10(-6) M. Conversely, pretreatment of rat aortic endothelial cells with different doses (10(-9) M and 10(-6) M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1- or PDBu-induced PGI2 release. These results provide further support for the concept that PKC mediates ET-induced PGI2 release in rat aortic endothelial cells via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.

Role of nitric oxide in cyclosporine A-induced hypertension.

Cyclosporine A (CsA) is an immunosuppressive agent that also causes hypertension. The effect of CsA on vascular responses was determined in Sprague-Dawley rats and isolated rat aortic rings. Male rats weighing 250 to 300 g were given either CsA (25 mg. kg-1. d-1) in olive oil or vehicle by intraperitoneal injection for 7 days. CsA administration produced a 42% increase (P<0.001) in mean arterial pressure (MAP) that reached a plateau after 3 days. Conversely, the levels of both nitrate/nitrite, metabolites of nitric oxide (NO), and cGMP, which mediates NO action, decreased by 50% (P<0.001) and 35% (P<0.001), respectively, in the urine. Thoracic aortic rings from rats treated with CsA and precontracted with endothelin (10(-9) mol/L) showed a 35% increase (P<0.001) in tension, whereas endothelium-dependent relaxation induced by acetylcholine (ACh, 10(-9) mol/L) was inhibited 65% (P<0.001) compared with that in untreated rats. This response was similar to that of endothelium-denuded aortic rings from untreated rats in which ACh-induced relaxation was completely abolished (P<0.001), but relaxation induced by S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) mol/L) was unaffected (P<0.001). ACh-induced formation of both nitrate/nitrite and cGMP by both denuded and CsA-treated aortic rings was inhibited 95% (P<0.001) and 65% (P<0.001), respectively, compared with intact aortic rings. The effects of CsA were reversed both in vivo and in vitro by pretreatment with L-arginine (10 mg. kg-1. d-1 IP), the precursor of NO. There were no changes in MAP and tension in rats treated with L-arginine alone. In summary, CsA inhibits endothelial NO activity, with resulting increases in MAP and tension, and this inhibition can be overcome by parenteral administration of L-arginine.

Protein kinase C mediates angiotensin II-induced contractions and the release of endothelin and prostacyclin in rat aortic rings.

Angiotensin II (Ang II) stimulation of vascular smooth muscle results in a myriad of intracellular signals that interact to produce the final physiologic response of the cell. We used rat aortic rings to investigate the role of protein kinase C (PKC) in Ang II-induced contractions and in the concomitant release of endothelin (ET) and prostacyclin (PGI2). Ang II (10(-9) M) produced a rapid contraction which was sustained for 10 min. When aortic rings were pretreated with graded concentrations of each of the four different inhibitors of PKC, that is, (i) 1-(5-isoquinolinesulfonylmethyl) piperazine (H7); (ii) 1-(5-isoquinolinesulfonyl) piperazine(CL); (iii) staurosporine; or (iv) calphostin C, inhibition of Ang II-induced contractions began at 10(-9) M, and was nearly complete at 10(-6) M. Ang II-induced contractions were associated with a 10-fold increase in the release of both ET and PGI2. Pretreatment with 10(-6) M of any one of the same four PKC inhibitors blocked Ang II-induced release of both ET and PGI2. Pretreatment with a blocker of the endothelin-A receptor, BQ123 (10(-6) M), inhibited, by approximately 50%, Ang II-induced contractions, and the release of both ET and PGI2. In aortic rings denuded of endothelium, Ang II-induced contractions, and the release of both ET and PGI2 were significantly reduced, compared to intact rings. We conclude that PKC mediates Ang II-induced contractions in rat aortic rings and that the secondary release of both ET and PGI2 during Ang II-induced contractions is mediated, at least in part, by PKC. In addition, approximately half of Ang II-induced contractile force and of PGI2 release is dependent upon the ET released from endothelial cells.

Cyclosporine A-induced contractions and prostacyclin release are maintained by extracellular calcium in rat aortic rings: role of protein kinase C.

Chronic treatment with the immunosuppressive drug, Cyclosporine A (CsA), is associated with increased intracellular calcium in vascular smooth muscle cells, which may cause vasoconstriction and/or activate phospholipase A2. We used rat aortic rings to investigate the role of protein kinase C (PKC) in CsA-induced contractions and secondary prostacyclin (PGI2) release. CsA (10(-9) M) produced a sustained contraction in rat aortic rings. Both CsA-induced contractions and PGI2 release were inhibited 84 to 89% by 10(-9) M, and 99 to 100% by 10(-6) M pretreatment doses of any of three different PKC inhibitors, i.e. 1-(5-isoquinolinesulfonylmethyl) piperazine (H7), staurosporine or 1-(5-isoquinolinesulfonyl) piperazine. Pretreatment with (10(-9) M) of diltiazem (a voltage-sensitive L-type calcium channel blocker) completely inhibited both CsA-induced contractions and PGI2 release. Conversely, pretreatment with (10(-9) M) of thapsigargin (an intracellular calcium channel blocker) did not alter the action of CsA. These results strongly suggest that PKC, in association with an influx of extracellular calcium mediates CsA-induced contractions and secondary PGI2 release in rat aortic rings.

Role of calcium in endothelin-induced contractions and prostacyclin release.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that induces characteristically long-lasting contractions. We used rat aortic rings to investigate the role of protein kinase C (PKC) in ET-1-induced contractions and prostacyclin (PGI2) release. ET-1 (10(-9) M) produced a gradual and sustained contraction in rat aortic rings. Pretreatment of aortic rings with different doses (10(-9) M and 10(-6) M) of diltiazem (voltage-sensitive L-type calcium channel blocker) produced significant inhibition of ET-1- and PDBu-induced contractions and PGI2 release. Inhibition was first noted at 10(-9) M and was complete at 10(-6) M. Conversely, pretreatment of aortic rings with different doses (10(-9) M and 10(-6) M) of calcium channel blockers (thapsigargin, an intracellular calcium channel blocker, or conotoxin, a voltage-sensitive N-type calcium channel blocker) produced no changes on ET-1-or PDBu-induced contraction or PGI2 release. These results provide further support for the concept that PKC mediates ET-induced contractions and PGI2 release in rat aortic rings via an increase in intracellular calcium and this increase is due to the influx of extracellular calcium and not to the release of calcium from the sarcoplasmic reticulum.

Endothelin-induced prostacyclin production in rat aortic rings is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic rings with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors, 1-(5-isoquinolinesulfonyl)piperazine(CL), staurosporine, and 1-(5-isoquinolinesulfonyltmethyl)piperazine (H7), blocked ET-induced PGI2 release. ET-induced PGI2 release was also blocked by pretreatment with inhibitors of either phospholipase A2 7,7-dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC, which activates phospholipase A2, which liberates arachidonic acid, which increases PGI2 production and release.

Action of protein kinase C in endothelin-induced contractions in rat aortic rings.

Endothelin (ET) is a potent vasoconstrictor peptide that induces characteristically long-lasting contractions. We used both intact and endothelium-denuded rat aortic rings to investigate the role of protein kinase C (PKC) in ET-induced contractions. ET (10(-9) M) and phorbol 12,13-dibutyrate (PDBu), a PKC activator, produced a gradual and sustained contraction of greater magnitude in denuded aortic rings than in intact rings. When aortic rings were pretreated with graded concentrations of different PKC inhibitors, inhibition of ET-induced contractions began at 10(-9)M and was nearly complete at 10(-3)M, and the reduction was greater in intact than in denuded rings. Pretreatment of aortic rings with PDBu or NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, potentiated ET-induced contractions. PKC enzyme assay showed activation of PKC in aortic rings that were treated with either ET or PDBu, inhibition after pretreatment with PKC inhibitors, and no change with 4 alpha-phorbol 12,13-didecanoate (PDD), an inactive phorbol ester. ET significantly increased nitrate and nitrite production, which was further increased by pretreatment with PKC inhibitors. PDBu prevented ET-induced nitrate/nitrite production, and PDD had no effect. These results strongly suggest that PKC mediates, in part, ET-induced contractions in rat aortic rings and that an intact endothelium is required for maximum inhibition by PKC inhibitors because PKC stimulated by ET inhibits nitric oxide release.