Vasodilator effect of urotensin II, one of the most potent vasoconstricting factors, on rat coronary arteries.

The effects of human urotensin II on coronary flow were studied in the perfused rat heart. Urotensin II transiently decreased coronary flow, then induced sustained vasodilatation. In the presence of a cyclooxygenase inhibitor, diclofenac, coronary vasodilatation was significantly inhibited. A nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (L-NNA), attenuated the urotensin-induced vasodilatation. These data suggest that urotensin II modulates coronary flow through factors such as cyclooxygenase products and nitric oxide to elicit coronary vasodilatation.

Differential modulation of nitric oxide and prostacyclin release in senescent rat heart stimulated by angiotensin II.

To elucidate the mechanism of age-related changes in the cardiovascular function stimulated with angiotensin II, we examined the effects of angiotensin II on the coronary flow, production of nitric oxide (NO) and prostacyclin, and on the cardiac function in the Langendorff-perfused young and aged rats' hearts. Angiotensin II decreased coronary flow, left ventricular dP/dt and heart rate. These effects were more pronounced in aged rats. Pretreatment with a NO synthase inhibitor, N(G)-nitro-L-arginine, significantly increased the angiotensin II-induced vasoconstriction in young rats. Angiotensin II increased the concentration of NO in the coronary effluent in young but not in aged rats. In contrast, angiotensin II stimulated the release of prostacyclin to a much greater extent in aged rats than in young rats. These results suggest that impaired production of NO may contribute to the greater constrictor effect of angiotensin II in the aged rat, although aging modulated the production of prostacyclin in a different manner. This age-related endothelial dysfunction may alter the physiological regulation of coronary flow and cardiac function stimulated with angiotensin II.

Inhibition of the expression of the gene for the angiotensin AT1 receptor by angiotensin II in the rat adrenal gland.

The expression of angiotensin AT1A and AT1B receptor mRNA after continuous angiotensin II administration was investigated in the rat adrenal gland. Angiotensin AT1 receptor mRNA detected by Northern blot analysis decreased to 52.7+/-16.1% of control after the administration of angiotensin II (20 microg/h) for 24 h, and to 70.8+/-8.0% after 1 week. A low dose of angiotensin II (0.2 microg/h) also decreased angiotensin AT1 receptor mRNA to 73.0+/-5.5% after 1 week. Competitive reverse transcription and polymerase chain reaction (RT-PCR) experiments revealed that both angiotensin AT1A and AT1B receptor mRNAs decreased after administration of angiotensin II (20 or 0.2 microg/h) for 1 week. Analysis of the angiotensin AT1A promoter by using luciferase-reporter system showed that angiotensin II (up to 1 microM) did not have any effects on the promoter activity (106+/-5.7% after 0.1 microM angiotensin II stimulation) in Y1 cells and cultured vascular smooth muscle cells, although phorbol myristate acetate (PMA) decreased the promoter activity by about 40% compared with control. These results suggest that angiotensin AT1 receptor gene expression in the rat adrenal gland is inhibited by angiotensin II and it may not be due to suppression of promoter activity. Other mechanisms such as destabilization of angiotensin AT1 receptor mRNA or angiotensin II-induced increased blood pressure may be involved in the inhibition.

Effect of ischemic preconditioning on myocardial oxygen consumption during ischemia.

Ischemic preconditioning (IPC) in the heart may reduce myocardial energy demand. The present study was undertaken to examine changes in myocardial oxygen consumption (MVO2) during ischemia by IPC in Langendorff perfused rat hearts. We assessed MVO2 during ischemia from the measurement of mitochondrial cyt. aa3 redox state by a two-wavelength reflectance spectrophotometry where T(1/2), the time from the onset of ischemia to the point for half reduction of cyt. aa3, was assumed to represent MVO2. The heart was preconditioned by three cycles of 5 min ischemia plus 5 min reperfusion and then subjected to 30 min global ischemia followed by reperfusion for 30 min. The T(1/2) was significantly longer in the preconditioned heart (30 +/- 6 s, n = 10) than the control heart (14 +/- 5 s, n = 9, P<0.001), indicating a reduction of MVO2 during ischemic period by IPC. The prolongation of T(1/2) was evident after only one IPC episode. When the heart was perfused with high K+ solution to abolish MVO2 for contractions, we still found the prolongation of T1(1/2) in the preconditioned heart (116 +/- 12 s, n = 6) compared to the control heart (86 +/- 10 s, n = 6, P<0.01), suggesting that decrease in contractile activity may be, in part but not completely, responsible for the reduction of MVO2. In contrast, the prolongation of T(1/2) was completely abolished by administration of a NO synthase inhibitor N omega-nitro-L-arginine in the high K+ arrested heart, demonstrating involvement of NO in the reduction of MVO2, presumably by suppression of mitochondrial respiratory chain. In conclusion, IPC reduces MVO2 during ischemia. The reduction of MVO2 in the preconditioned heart may be accounted for by decreased contractile activity and by depression of respiratory chain by NO.

Identification of a cis-acting glucocorticoid responsive element in the rat angiotensin II type 1A promoter.

Enhanced vascular responsiveness to angiotensin II at the AT1 receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells (< 12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 mumol/L) and was located at positions -756 to -770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (> 8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro-translated rat glucocorticoid receptors in a specific manner. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs.(ABSTRACT TRUNCATED AT 250 WORDS)

Species-related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis.

1. Experiments were carried out to clarify the mechanisms responsible for variations in the positive inotropic effect (PIE) of angiotensin II (AII) on ventricular muscles from various mammals. We examined the density of AII receptors, the relative proportions of receptor subtypes and the acceleration of the hydrolysis of phosphoinositide that was induced by AII, as well as the PIE of AII in ventricular muscles from the rabbit, dog, rat and ferret. 2. In the rabbit, AII (1 microM) in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM) elicited a concentration-dependent PIE, which was antagonized by a selective AT1 subtype antagonist, losartan, but not by an AT2 antagonist, PD123319. AII did not have any inotropic effects in ventricular muscles from the dog, rat and ferret. 3. Specific high-affinity binding of [125I]-AII, with a similar Kd value in each case (1-2 nM), was observed with membrane fractions derived from ventricular muscle of all four species tested. 4. In the rabbit, losartan and PD123319 each displaced approximately 50% of [125I]-AII specific binding having high affinity for the receptors, and indicating that AT1 and AT2 subtypes were present in equal numbers. In the other species the AT1 subtype of receptors was predominant. 5. In all four species AII caused a concentration-dependent acceleration of the hydrolysis of phosphoinositide in ventricular slices that had been prelabelled with myo-[3H]-inositol. 6. The results indicate that the signal-transduction process distal to acceleration of the hydrolysis of phosphoinositide may be responsible for the wide range of species variations in the inotropic action of AII on mammalian ventricular myocardium.

Modulation by aging of the coronary vascular response to endothelin-1 in the rat isolated perfused heart.

Changes with age in the coronary vascular response to endothelin-1 were investigated in perfused hearts isolated from 2-, 6- and 24-month-old (mo) male Fisher-344 rats. Endothelin-1 injected as a single bolus (0.3, 3 and 30 nmol) into the coronary artery supply caused dose-dependent vasoconstriction in all three age groups. While there was no age-related change in the vasoconstriction induced by the lower doses (0.3 and 3 nmol), the higher dose (30 nmol) elicited a more pronounced vasoconstriction in 6- and 24-mo rats than that in 2-mo rats. NG-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide formation, markedly enhanced the vasoconstriction induced by 30 nmol endothelin-1 in 2- and 6-mo rats but only slightly and non-significantly enhanced that vasoconstriction in 24-mo rats. Haemoglobin, which inhibits activation of guanylate cyclase by nitric oxide, enhanced the endothelin-1-induced vasoconstriction in 2-mo rats, but not in 6- and 24-mo rats. The acetylcholine-induced coronary vasodilation was more pronounced in 2- and 6-mo rats than in 24-mo rats and was attenuated by L-NNA in 2- and 6-mo rats. The coronary vasodilation induced by nitroprusside (0.1 mmol), a pharmacological precursor of nitric oxide, did not change with age. Endothelin-1 (30 nmol) markedly increased the release of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in all three age groups. The prostaglandin synthesis inhibitor indomethacin enhanced the endothelin-1-induced vasoconstriction in 2- and 6-mo rats to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characteristics of the positive inotropic effect of angiotensin II in the rabbit ventricular myocardium.

1. In order to elucidate the mechanism underlying the positive inotropic effect (PIE) of angiotensin II (AII), we measured changes in phosphoinositide hydrolysis and contractile force induced by AII in the rabbit ventricular myocardium. 2. AII (1.0 nM-3 microM) produced a PIE in a concentration-dependent manner in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM), the maximal response being about 40% of that to isoprenaline and the EC50 30 nM. 3. The PIE of AII was associated with a concentration-dependent increase in the total duration of contraction; the time to peak force and the relaxation time were prolonged. 4. AII (10 nM-30 microM) elicited an accumulation of [3H]-inositol monophosphate in a concentration-dependent manner in rabbit ventricular slices prelabelled with myo-[3H]-inositol. 5. The PIE and the accumulation of [3H]-inositol monophosphate induced by AII were inhibited by a non-selective AII receptor antagonist, saralasin (10 nM-1 microM) and by a selective AT1 receptor antagonist, losartan (10 nM-1 microM), but not a selective AT2 receptor antagonist, PD 123319 (1 microM). 6. A tumour-promoting phorbol ester, phorbol 12,13-dibutyrate (PDBu, 10-100 nM), inhibited the AII-induced PIE and [3H]-inositol monophosphate accumulation in a concentration-dependent manner. 7. These results suggest that AII exerts a PIE through activation of AT1 receptors and subsequent acceleration of phosphoinositide hydrolysis. Activation of protein kinase C by PDBu may inhibit the AII-induced stimulation of phosphoinositide hydrolysis and thereby the PIE of AII in the rabbit ventricular myocardium.

Influence of aging on the contractile response to endothelin of rat thoracic aorta.

Age-related changes in the contractile response to endothelin-1 and ACh were assessed in thoracic aortas isolated from 2-, 6- and 24-month-old male Fischer 344 rats. In aortic strips with an intact endothelium, the maximal contractile response to endothelin-1 decreased with development to maturity. Removal of the endothelium did not affect the contractile response to endothelin-1. Endothelin-1 did not elicit a relaxant response in phenylephrine-precontracted strips. The ACh-induced relaxation decreased in senescent rats. These results indicate that the contractile response of aortic smooth muscle to endothelin-1 decreases with age, and that the endothelial vasorelaxant factors do not contribute to this age-induced modulation.

Myocardial alpha 1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolism. Species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium.

Species-dependent variations of myocardial alpha 1-adrenoceptor-mediated positive inotropic effects of epinephrine were assessed in relation to characteristics of alpha 1-receptor bindings and acceleration of phosphatidylinositol metabolism in the isolated rat, rabbit, and dog ventricular myocardium. Epinephrine in the presence of the beta-adrenoceptor antagonist bupranolol (10(-6) M) elicited a positive inotropic effect through activation of alpha 1-adrenoceptors in rat and rabbit, whereas in dog ventricular myocardium, bupranolol abolished the positive inotropic effect of epinephrine. [3H]Prazosin bound to membrane fractions derived from rat, rabbit, and dog ventricular muscle with high affinities in a saturable and reversible manner. In dog, Bmax and Kd values of alpha 1-adrenoceptor binding sites were identical to those in rabbit ventricular muscle. The Bmax value of alpha 1-adrenoceptors in rat ventricle was the highest, amounting to two to four times those in rabbit and dog. Epinephrine displacement curves for the specific binding of [3H]prazosin in the membrane fraction of these species showed high and low affinity sites with slope factors significantly less than unity, which were shifted to single low affinity sites with slope factors close to unity by addition of 5'-guanylylimidodiphosphate. Accumulation of [3H]inositol 1-phosphate [( 3H]IP1) in ventricular slices prelabeled with [3H]myo-inositol was increased by epinephrine in a time- and concentration-dependent manner in rat ventricular slices. [3H]IP1 accumulation likewise was facilitated by alpha 1-adrenoceptor stimulation in rabbit ventricular slices, whereas the extent of [3H]IP1 accumulation was much less than that in rat. In dog ventricular slices, [3H]IP1 was not accumulated by epinephrine. In rabbit papillary muscle, the time course of increase in contractile force induced by alpha-adrenoceptors coincided with the prolongation of the action potential duration with a similar time course, which is in strong contrast to previous findings in rat that the contractile response was dissociated from the electrophysiological response to alpha-adrenoceptor stimulation. The present results indicate that a wide range of variation of alpha 1-adrenoceptor-mediated regulation of myocardial contractility may be ascribed to different contributions of facilitatory as well as inhibitory regulatory processes that lead to intracellular Ca2+ mobilization subsequent to myocardial alpha 1-adrenoceptor activation among mammalian species.

Different mechanisms involved in the positive inotropic effects of benzimidazole derivative UD-CG 115 BS (pimobendan) and its demethylated metabolite UD-CG 212 Cl in canine ventricular myocardium.

UD-CG 115 BS produced a positive inotropic effect in a concentration-dependent manner (EC50 = 9.2 x 10(-5) M, efficacy = 0.65) in isolated canine ventricular muscle. UD-CG 212 Cl also elicited a positive inotropic effect (EC50 = 1.9 x 10(-7) M, efficacy = 0.23); its potency was higher, but its efficacy was much less than that of UD-CG 115 BS. Although the effect of UD-CG 115 BS was not altered by a beta-adrenoceptor antagonist, bupranolol (3 x 10(-7) M), the response to UD-CG 212 Cl in high concentrations became transient in the presence of bupranolol: After reaching a peak, the force decreased gradually to the control level at greater than or equal to 10(-4) M. Both UD-CG 115 BS and UD-CG 212 Cl elevated the cyclic AMP level, but to a much smaller extent than other newly developed cardiotonic agents such as amrinone, milrinone, enoximone, and piroximone. Carbachol (3 x 10(-6) M) abolished the accumulation of cyclic AMP produced by these agents while it suppressed the maximum contractile response to UD-CG 115 BS by only 30%. The positive inotropic effect of UD-CG 212 Cl was converted to a negative effect by carbachol. Both UD-CG 115 BS and UD-CG 212 Cl produced a leftward shift in the concentration-response curve for the positive inotropic effect of isoproterenol. These results suggest that an elevation of cyclic AMP levels owing to cyclic AMP phosphodiesterase inhibition may be predominantly responsible for the positive inotropic effect of UD-CG 212 Cl but that a cyclic AMP-independent mechanism may contribute significantly to the positive inotropic effect of UD-CG 115 BS. UD-CG 212 Cl (greater than 3 x 10(-6) M) elicits a negative inotropic effect that is unmasked by beta-adrenoceptor blockade.

Subcellular mechanism of desensitization of the ATP-induced Ca2+ mobilization in human umbilical vein endothelial cells: role of intracellular Ca2+ stores.

Confluent monolayers of human umbilical vein endothelial cells subcultured on glass coverslips were loaded with the fluorescent Ca2+ indicator, fura-2. Changes in fura-2 fluorescence were detected by means of a fluorescence spectrophotometer. Both ATP and ADP (0.3-100 microM) caused a concentration-dependent transient peak response of the intracellular free calcium concentration ([Ca2+]i), followed by a lower sustained response. AMP and adenosine did not induce detectable changes in [Ca2+]i. The sustained response to ATP was abolished by superfusion with the Ca2(+)-free solution (with 1 mM EGTA), while the transient peak response was uninfluenced. The transient peak response to ATP (30 microM) was inhibited by pre-exposure to ATP in a graded manner depending on the concentration of ATP. The response to ATP recovered after washout for 20 min with the solution containing Ca2+, but not with the Ca2(+)-free solution. The transient peak response to ATP was markedly reduced by preceding exposure to histamine, while the response to histamine was not influenced by pre-exposure to ATP. These findings indicate that depletion and refilling of the ATP-sensitive intracellular Ca2+ store may be responsible for the desensitization and recovery of the ATP-induced [Ca2+]i response. The pharmacological characteristics of the ATP-sensitive intracellular Ca2+ store seem different from those of the histamine-sensitive store.

Enantiomers of dobutamine increase the force of contraction via beta adrenoceptors, but antagonize competitively the positive inotropic effect mediated by alpha-1 adrenoceptors in the rabbit ventricular myocardium.

Experiments were carried out to characterize the pharmacological properties of enantiomers and racemic mixture of dobutamine to modulate the myocardial contractility through alpha and beta adrenoceptors in the rabbit papillary muscle. Dobutamine caused the concentration-dependent positive inotropic effect: the rank order of potency was R-(+)- greater than (+/-) - greater than S-(-)-dobutamine. The positive inotropic effect of (+)-, (-)- and (+/-)-dobutamine was antagonized by a beta adrenoceptor antagonist, (+/-)-bupranolol in a competitive manner, but was not affected by an alpha-1 adrenoceptor antagonist, prazosin. The concentration-response curve for (-)-phenylephrine mediated by alpha adrenoceptors in the presence of 10(-6) M (+/-)-bupranolol was shifted by enantiomers of dobutamine to the right in a concentration-dependent manner. Thus, enantiomers of dobutamine antagonized the positive inotropic effect of (-)-phenylephrine in a competitive manner, and pA2 values [negative logarithm of the dissociation constant (KB)] for (+)- and (-)-dobutamine were 6.67 and 5.99, respectively. The specific binding of [3H]prazosin to membrane fractions of rabbit ventricular myocardium was displaced by dobutamine with a high potency: the -log Ki values for (+)- and (-)-dobutamine were 6.43 and 5.97, respectively, which correspond well with pA2 values of these compounds for functional modification. These findings indicate that enantiomers of dobutamine elicit the positive inotropic effect through activation of beta adrenoceptors, whereas both enantiomers behave as the competitive antagonist of myocardial alpha adrenoceptors mediating the positive inotropic effect in the isolated rabbit papillary muscle.