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Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho. Accumulating evidence has demonstrated that the Rho/Rho-kinase-mediated pathway plays an important role in various cellular functions, not only in vascular smooth muscle contraction but also in actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions, all of which may be involved in the pathogenesis of arteriosclerosis/atherosclerosis. Indeed, animal experiments have demonstrated that Rho-kinase inhibitors effectively suppress coronary artery spasm and that long-term inhibition of Rho-kinase inhibits the development of coronary arteriosclerotic lesions and even causes regression of coronary vascular lesions in vivo. Recent clinical studies also have demonstrated the inhibitory effect of a Rho-kinase inhibitor on coronary artery spasm in patients with vasospastic angina and on exercise-induced myocardial ischemia in patients with stable effort angina with adequate safety. It is possible that Rho-kinase is also involved in the pathogenesis of other forms of cardiovascular diseases. Thus, Rho-kinase could be regarded as a novel therapeutic target in treatment of cardiovascular diseases.

Previous studies indicate that anoxia results in vascular smooth muscle contraction, and this phenomenon is mediated in part by a decrease in release of endothelium-derived relaxing factor (EDRF). The role of platelets that relax blood vessels by eliciting EDRF release on anoxic contraction is not known. To examine anoxic contraction in the presence of platelets, we exposed norepinephrine (NE)-precontracted rat aortic rings to washed platelets and then induced anoxia (switch from 95% O2 to 95% N2). Platelets transiently relaxed the precontracted aortic rings (decrease in tension 49 +/- 4%, n = 18). The subsequent anoxia-induced contraction was augmented, however (magnitude of contraction 71 +/- 12 vs. 49 +/- 6% in parallel buffer-treated rings, n = 18, p < 0.01). To determine the mechanism of platelet-mediated augmentation of anoxic contraction, vascular rings were treated with indomethacin, the thromboxane A2 (TxA2)/endoperoxide receptor antagonist SQ29,548, lipoxygenase inhibitor U60,257B, free oxygen radical scavengers superoxide dismutase (SOD) plus catalase, serotonin (S2) receptor blocker LY53,857, or the ADP scavenger apyrase. Although apyrase, U60,257B, SQ29,548, LY53,857, and SOD plus catalase had no effect on augmentation of anoxic contraction in the presence of platelets, treatment with indomethacin markedly decreased (p < 0.02) the augmented anoxic vasoconstriction. Because indomethacin decreases the activity of platelet-activating factor (PAF), vascular rings were treated with two different specific PAF receptor antagonists: L-659,989 and WEB 2170 BS. Both these agents blocked the platelet-augmented anoxic vasoconstriction. In addition, synthetic PAF (10(-7) and 10(-6) M) in itself potentiated anoxic contraction, mimicking the activity of platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained exposure of vascular smooth muscle to catecholamines results in desensitization of alpha 1-adrenoreceptor-mediated vascular smooth muscle contraction. The present study was designed to determine the effects of prolonged exposure of blood vessels to catecholamines on protein kinase C (PKC) activity. Incubation of rat aortic smooth muscle with 10 microM norepinephrine (NE) for 4 h resulted in a threefold decrease in sensitivity of the contractile response of rat aortic smooth muscle to the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDBu); this loss in sensitivity was dependent on the presence of endothelium. NE induced a 45% decrease in enzymatic activity of the soluble and particulate forms of PKC. With [3H]PDBu used to label phorbol ester receptor binding sites in the aorta, there was a 34% decrease in [3H]PDBu binding sites in NE-treated blood vessels without change in binding affinity for the ligand. To determine whether this loss in enzymatic activity and [3H]PDBu binding resulted from a decrease in the quantity of the enzyme, Western blot analyses were performed using a monoclonal antibody (MoAb) against PKC. This approach confirmed the presence of an 80-Kd immunoreactive PKC in the soluble fraction of rat aortic smooth muscle and demonstrated a 44% decrease in the amount of PKC in blood vessels after sustained exposure to catecholamines. Our results demonstrate that prolonged activation of alpha-adrenoceptors in blood vessels leads to down-regulation of PKC which may contribute to desensitization of contraction mediated by vasoconstrictors.

The effects of the nifedipine gastrointestinal therapeutic system (GITS) on blood pressure (BP), systemic vascular resistance (SVR), and left ventricular (LV) performance were determined in eight patients with essential hypertension. LV systolic and diastolic performance were assessed by first-pass radionuclide cineangiography at rest and during upright bicycle exercise after initial and long-term BP reduction. After initial treatment, end-diastolic volume (LVEDV) increased in association with an increase in stroke volume (SV), cardiac output (CO), and peak ejection rate. After long-term treatment, LVEDV decreased, SV and CO returned to pretreatment values, early diastolic filling fraction increased, and time to peak filling rate decreased. These hemodynamic changes are consistent with an initial predominant effect of vasodilation on LV function. With long-term treatment, the effects on LV diastolic performance are consistent with a positive lusitropic effect of nifedipine GITS. Nifedipine GITS is an effective agent for control of hypertension; its hemodynamic effects are consistent with both an effect on SVR due to decreased vascular smooth muscle contraction and a direct lusitropic effect on myocardial function.

Abdominal aortic aneurysm (AAA) is a common vascular disease with, as of yet, unclear mechanism. Increased elastase activity and elastin degradation in the aorta are consistent findings in human AAA. Also, elastase perfusion of the aorta promotes aortic dilation in animal models of AAA. Although elastase-induced degradation of extracellular matrix proteins and the ensuing inflammation of the aortic wall have been implicated as possible causes of the aortic dilation in AAA, little is known regarding the effects of elastase on the mechanisms of aortic smooth muscle contraction. The purpose of this study was to test the hypothesis that elastase promotes aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats non-treated or treated with elastase. Initial experiments suggested that elastase alone caused matrix degradation. To avoid potential degradation of the extracellular matrix proteins by elastase, the same experiments were repeated in the presence of saturating concentrations of elastin (10 mg/ml). In normal Krebs (2.5 mM Ca2+), phenylephrine (Phe, 10(-5) M) caused contraction of the aortic strips that was significantly inhibited by elastase. The elastase-induced inhibition of Phe contraction was concentration- and time-dependent. At 5 U/ml elastase, the inhibition of Phe contraction was rapid in onset (2.4 +/- 0.3 minutes) and complete in 32 +/- 4 minutes. The inhibitory effects of elastase on Phe contraction were partially reversible. In Ca2+-free (2 mM EGTA) Krebs, Phe caused a small contraction that was not inhibited by elastase, suggesting that elastase does not inhibit Ca2+ release from the intracellular stores. Membrane depolarization by 96 mM KCl, which stimulates Ca2+ entry from the extracellular space, caused a contraction that was inhibited by elastase in a time-dependent and reversible fashion. The reversible inhibitory effects of elastase, particularly in the presence of saturating concentrations of elastin, suggest that they are not due to dissolution of the extracellular matrix or permanent damage to the smooth muscle contractile proteins. Elastase also caused significant inhibition of Phe- and KCl-induced 45Ca2+ influx. These data suggest that elastase promotes aortic relaxation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction, and thus further explain the role of increased elastase activity during the early development of AAA.

Serotonin is one of the most important vasoactive substances and has been implicated in the pathogenesis of coronary artery spasm and of acute coronary syndrome. We have recently demonstrated that local and long-term treatment with interleukin-1beta(IL-1beta) causes coronary arteriosclerotic changes and hyperconstrictive responses to serotonin in pigs in vivo. However, it remains to be examined which serotonergic (5-HT) receptor subtype mediates coronary spasm and whether alterations in serotonergic receptors are involved in the abnormality. In this study, we examined the inhibitory effect of sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, on the serotonin-induced coronary spasm as well as the possible alterations of serotonergic receptors in our porcine model. A segment of the porcine coronary artery was carefully dissected and aseptically wrapped with cotton mesh absorbing IL-1beta-bound microbeads from the adventitia. Two weeks after the procedure, angiographic study was performed, followed by binding assay for 5-HT1B and 5-HT2A serotonergic receptors and reverse transcription-polymerase chain reaction (RT-PCR) analysis for mRNA of those receptors. Angiographic study showed coronary vasospastic responses to serotonin at the IL-1beta-treated site. Sarpogrelate dose-dependently inhibited the serotonin-induced coronary spasm, but it did not affect the prostaglandin F2alpha-induced vasoconstriction. Radiolabeled receptor-binding assay showed that receptor affinity or receptor number of the 5-HT1B, or 5-HT2A receptors did not differ significantly between the spastic and the control sites. Furthermore, RT-PCR analysis showed that the expression of neither 5-HT2A nor 5-HT1B receptor mRNA was significantly altered at the spastic site. These results indicate that serotonin-induced coronary spasm is mediated primarily by 5-HT2A receptor in our porcine model, although the 5-HT2A receptor was not up-regulated, suggesting that alteration in the signal-transduction pathway for vascular smooth muscle contraction beyond the 5-HT2A receptor plays a primary role in the pathogenesis of coronary spasm in our porcine model.

We wished to characterize the altered reactivity of vascular smooth muscle and endothelial cells in rat aorta during chronic treatment with cyclosporin. Male adult rats were treated orally for 6 weeks with either cyclosporin A (30 mg/kg/day in 1 ml olive oil, n = 9) or with vehicle alone (n = 10). Rings of isolated thoracic aorta were mounted in organ chambers to measure the change in isometric force in response to smooth muscle-contracting drugs and endothelium-dependent and independent vasodilators. Contractions to potassium chloride (20-80 mM) were markedly reduced in cyclosporin-treated rats. Contractions to phenylephrine (10(-10)-10(-6) M) were reduced at high concentrations (> or = 10(-7) M); those to endothelin-1 (10(-10)-10(-7) M) were not significantly altered. In contrast, contractions to angiotensin II (AII 10(-9)-10(-6) M) were significantly augmented. Endothelium-dependent relaxations to acetylcholine (ACh 10(-8)-10(-5) M) or ADP (10(-7)-10(-5) M) were reduced in cyclosporin-treated rats; endothelium-independent relaxations to SIN-1 (10(-7)-10(-5) M) and atrial natriuretic peptide (ANP 10(-10)-10(-7) M) remained unaffected. Thus, chronic treatment with cyclosporin affects both endothelium-dependent vasodilation and vascular smooth muscle contraction in rat aorta depending on the stimulus applied. The enhanced response to AII and the reduced release of endothelium-derived relaxing factor (EDRF) may contribute to augmented vascular tone and further damage to the arterial wall.

Endothelin (ET) ETA receptors on vascular smooth muscle are believed to mediate the vasoconstrictor effects of ET isopeptides, and ETB receptors on the endothelium are thought to mediate the vasodilator effects. This study has investigated the receptors mediating endothelin-induced contraction of isolated ring preparations of rat thoracic aorta (RTA) and rabbit carotid artery (RCA), pulmonary artery (RPA), and jugular vein (RJV). In RTA and RCA, ET-1 (EC50 4.5 and 5.2 nM, respectively) was 82- and 108-fold, respectively, more potent than ET-3, whereas the ETB receptor-selective agonists sarafotoxin S6c (S6c) and Ala1,3,11,15-ET-1 (4-Ala-ET-1) were without effect up toor = 1 microM. In contrast, in RPA and RJV, ET-1 (EC50 3.1 and 0.7 nM, respectively) and ET-3 (EC50 4.4 and 0.9 nM, respectively) were equipotent, and 4-Ala-ET-1 (EC50 10.7 and 2.1, respectively) and S6c (EC50 0.4 and 0.1 nM, respectively) were potent contractile agonists. The ETA receptor antagonist BQ123 (D-Val-Leu-D-Trp-D-Asp-Pro) competitively antagonized the effects of ET-1 in RTA and RCA (pA2 values 6.9 +/- 0.1 and 6.8 +/- 0.2, respectively) but did not antagonize (at 10 microM) contractions to ET-1, ET-3, or 4-Ala-ET-1 in RPA and RJV. In conclusion, contraction of vascular smooth muscle by endothelins can be mediated by both ETA and ETB receptors.

Amiloride (N-amidino-3,5-diamino-6-chloropyarzine carboxamide), a potassium sparing diuretic, decreases blood pressure and inhibits vascular smooth muscle contractility in rats. The purpose of these experiments was to investigate the mechanisms by which amiloride inhibits contraction of rat caudal artery. Caudal artery rings were mounted in a muscle chamber that was continuously flushed with circulated bathing medium and the contractions were recorded through a force transducer. The results demonstrate that amiloride competitively inhibited the norepinephrine-stimulated contraction of arterial rings (pA2 = 5.2). This suggests that amiloride and norepinephrine (NE) interact at the same site. The concentration required to produce half-maximum inhibition (IC50) of contraction produced by 10(-6) M norepinephrine was 48 +/- 6 microM. Amiloride (100 microM) shifted the concentration-response curves of norepinephrine and methoxamine to the right without significantly affecting the maximum contraction produced by these agonists. The EC50 value and the threshold concentration of both norepinephrine and methoxamine were increased by about 10-fold in the presence of 100 microM amiloride. Amiloride-induced inhibition of norepinephrine-stimulated contraction was reversible following removal of the drug. Amiloride-induced inhibition of vascular smooth muscle contraction was not blocked by ouabain or monensin. Amiloride at concentrations up to 100 microM did not inhibit K+ stimulated contractions. From these results we conclude that amiloride reversibly inhibits vascular smooth muscle contraction by competing with alpha-adrenoceptor agonists for binding to alpha 1-adrenoceptors.

Hypertension, both essential and experimental is characterised by increased vascular resistance. This is in part structural, due to increases in the wall-to-lumen ratio secondary to vascular hypertrophy; however, functional increases in responses to neurohumoral stimuli also occur. The latter may be a consequence of hypertrophy or if a selective increase is observed, an indication of a primary underlying hypertensive mechanism. In essential hypertension, enhanced responsiveness to both angiotensin II and alpha-adrenoceptor agonists are seen. However, in some experimental models, more selective increased responses have been reported. The mechanism of alpha-adrenoceptor-mediated vascular smooth muscle contraction is reviewed and the role of inositol phosphates and diacylglycerol as second messengers is discussed. The precise mechanism of increased vascular responses in hypertension is still uncertain.

The alpha 2-agonist, guanabenz, increases Na and water excretion with little change in renal blood flow, while clonidine has been reported to cause antinatriuresis proportional to renal vasoconstriction. We hypothesized that clonidine-mediated renal vasoconstriction involves alpha 1-receptors. Dose-response curves were constructed correlating decreases in renal blood flow with doses of phenylephrine, clonidine, and guanabenz injected as boluses into renal arteries of anesthetized dogs. Changes in renal blood flow before and after prazosin, yohimbine or verapamil were recorded. Guanabenz was a 10-fold weaker vasoconstrictor than clonidine, but was still effective when alpha 1-receptors were blocked. Tenfold differences were found between phenylephrine, clonidine, and guanabenz for dependence on alpha 1-receptors. Denervation did not significantly shift the curves. Verapamil markedly attenuated only clonidine and guanabenz, which supported their dependence on calcium channels for vascular smooth muscle contraction. Thus, postsynaptic alpha 2-adrenoceptors can contribute to renal vasoconstriction in the dog but the receptors are either less numerous on the vasculature or are less efficiently coupled to contractile elements than are alpha 1-receptors. Clonidine constricts the renal vasculature through both adrenoceptor subtypes.