Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity

The hypothesis that Rho kinase is involved in myogenic reactivity was investigated in pressurized rat tail small arteries using videomicroscopic diameter determination and calcium fluorimetry. The potent Rho kinase inhibitor Y-27632 reversibly increased vessel diameter at 80 mmHg without changing the intracellular calcium concentration ([Ca](i)) shifting the relationship between diameter change and [Ca](i) to higher calcium levels. Neither endothelium removal nor inhibition of neural transmission affected the Y-27632-induced effect. Y-27632 at 3 x 10(-6) mol/l attenuated the myogenic response in the pressure range from 10 to 120 mmHg, shifting the relationship between vessel tone and [Ca](i) to higher calcium levels. In addition, the Y-27632-induced shift of the relationship between vessel tone and [Ca](i) was larger at 80 than at 10 mmHg. These results suggest that smooth muscle cell Rho kinase in rat tail small arteries 1) is in an active state partly determining the level of the myogenic tone, and 2) alters the strength of the myogenic response by changing calcium sensitivity, probably caused by the pressure-induced activation of the kinase.     

Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats

Recent evidence suggests that Rho/Rho kinase signaling plays an important role in the sustained vasoconstriction induced by many agonists and is involved in the pathogenesis of systemic vascular diseases. However, little is known about its role in increased vascular tone in hypoxic pulmonary hypertension (PH). The purpose of this study was to examine whether Rho/Rho kinase-mediated Ca2+ sensitization contributed to sustained vasoconstriction and increased vasoreactivity in hypoxic PH in rats. Acute intravenous administration of Y-27632, a Rho kinase inhibitor, nearly normalized the high pulmonary arterial blood pressure and total pulmonary resistance in chronically hypoxic rats. In contrast to nifedipine, Y-27632 also markedly decreased elevated basal vascular tone in hypertensive blood-perfused lungs and isolated pulmonary arteries. Y-27632 and another Rho kinase inhibitor, HA-1077, completely reversed nitro-L-arginine-induced vasoconstriction in physiological salt solution-perfused hypertensive lungs, whereas inhibitors of myosin light chain kinase (ML-9), protein kinase C (GF-109203X), phosphatidylinositol 3-kinase (LY-294002), and tyrosine kinase (tyrphostin A23) caused only partial or no reversal of the vasoconstriction. Vasoconstrictor responses to KCl were augmented in hypertensive physiological salt solution-perfused lungs and pulmonary arteries, and the augmentation was eliminated by Y-27632. These results suggest that Rho/Rho kinase-mediated Ca2+ sensitization plays a central role in mediating sustained vasoconstriction and increased vasoreactivity in hypoxic PH.     

Characteristics of myogenic reactivity in isolated rat mesenteric veins

Mechanisms of mechanically induced venous tone and its interaction with the endothelium and key vasoactive neurohormones are not well established. We investigated the contribution of the endothelium, l-type voltage-operated calcium channels (L-VOCCs), and PKC and Rho kinase to myogenic reactivity in mesenteric vessels exposed to increasing transmural pressure. The interaction of myogenic reactivity with norepinephrine (NE) and endothelin-1 (ET-1) was also investigated. Pressure myography was used to study isolated, cannulated, third-order rat mesenteric small veins and arteries. NE and ET-1 concentration response curves were constructed at low, intermediate, and high transmural pressures. Myogenic reactivity was not altered by nitric oxide synthase inhibition with N(ω)-nitro-L-arginine (L-NNA; 100 μM) or endothelium removal in both vessels. L-VOCCs blockade (nifedipine, 1 μM) completely abolished arterial tone, while only partially reducing venous tone. PKC (chelerythrine, 2.5 μM) and Rho kinase (Y27632, 3 μM) inhibitors largely abolished venous and arterial myogenic reactivity. There was no significant difference in the sensitivity of NE or ET-1-induced contractions within vessels. However, veins were more sensitive to NE and ET-1 when compared with corresponding arteries at low, intermediate, and high transmural pressures, respectively. These results suggest that 1) myogenic factors are important contributors to net venous tone in mesenteric veins; 2) PKC and Rho activation are important in myogenic reactivity in both vessels, while l-VOCCs play a limited role in the veins vs. the arteries, and the endothelium does not appear to modulate myogenic reactivity in either vessel type; and 3) mesenteric veins maintain an enhanced sensitivity to NE and ET-1 compared with the arteries when studied under conditions of changing transmural distending pressure.     

Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity.

Several recent studies have implicated the RhoA-Rho kinase pathway in arterial myogenic behavior. The goal of this study was to determine the effects of Rho kinase inhibition (Y-27632) on cerebral artery calcium and diameter responses as a function of transmural pressure. Excised segments of rat posterior cerebral arteries (100-200 microm) were cannulated and pressurized in an arteriograph at 37 degrees C. Increasing pressure from 10 to 60 mmHg triggered an elevation of cytosolic calcium concentration ([Ca(2+)](i)) from 113 +/- 9 to 199 +/- 12 nM and development of myogenic tone. Further elevation of pressure to 120 mmHg induced only a minor additional increase in [Ca(2+)](i) and constriction. Y-27632 (0.3-10 microM) inhibited myogenic tone in a concentration-dependent manner at 60 and 120 mmHg with comparable efficacy; conversely, sensitivity was decreased at 120 vs. 60 mmHg (50% inhibitory concentration: 2.5 +/- 0.3 vs. 1.4 +/- 0.1 microM; P < 0.05). Dilation was accompanied by further increases in [Ca(2+)](i) and an enhancement of Ca(2+) oscillatory activity. Y-27632 also effectively dilated the vessels permeabilized with alpha-toxin in a concentration-dependent manner. However, dilator effects of Y-27632 at low concentrations were larger at 60 vs. 100 mmHg. In summary, the results support a significant role for RhoA-Rho kinase pathway in cerebral artery mechanotransduction of pressure into sustained vasoconstriction (myogenic tone and reactivity) via mechanisms that augment smooth muscle calcium sensitivity. Potential downstream events may involve inhibition of myosin phosphatase and/or stimulation of actin polymerization, both of which are associated with increased smooth muscle force production.     

Rho kinase is involved in Ca2+ entry of rat penile small arteries

Tonic physiological activity of RhoA/Rho kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca(2+) sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of Ca(2+) entry induced by alpha(1)-adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular Ca(2+) ([Ca(2+)](i)) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca(2+)](i) and tension elicited by the alpha(1)-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca(2+)](i). In the presence of nifedipine, Y-27632 still inhibited the non-L-type Ca(2+) signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca(2+) entry evoked by store depletion with cyclopiazonic acid but largely reduced the Ba(2+) influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KCl markedly reduced tension without changing [Ca(2+)](i). In alpha-toxin-permeabilized penile arteries stimulated with threshold Ca(2+) concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or Phe in the presence of GTPgammaS. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca(2+). These results suggest that Rho kinase, besides its contribution to the Ca(2+) sensitization of the contractile proteins, is also involved in the regulation of Ca(2+) entry through a nonselective cation channel activated by alpha(1)-adenoceptor stimulation in rat penile arteries.     

Rho-Rho kinase pathway is involved in the regulation of myogenic tone and pump activity in isolated lymph vessels

To evaluate whether or not Rho-Rho kinase pathway is involved in the regulation of mechanical activity of lymph vessels, effects of Y-27632 and okadaic acid on lymph pump activity and myogenic, pressure- and agonist-induced tone were examined in isolated rat lymph vessels. Y-27632 caused a significant dilation with a cessation of the lymph pump activity. Y-27632 also produced a dose-related dilation of the lymph vessels precontracted by norepinephrine (NE)-, U-46619- or 80 mM KCl. Okadaic acid significantly constricted the lymph vessels and reduced the frequency of the lymph pump activity. Okadaic acid also produced a dose-related constriction of the lymph vessels precontracted by NE or U-46619. The Y-27632-induced decrease of the frequency of lymph pump activity was significantly reversed by the pretreatment with okadaic acid. In the presence of Y-27632, the pressure-mediated tone of the lymph vessel was significantly decreased. On the other hand, okadaic acid significantly increased the pressure-mediated tone. These findings suggest that Rho kinase and myosin phosphatase activity in lymphatic smooth muscles may contribute to the regulation of lymph pump activity and may be also involved in the control of myogenic pressure- and agonist-induced tone.     

Characteristics of myogenic tone in the rat ophthalmic artery

The pressure-induced constriction in the rat ophthalmic artery was characterized. Ophthalmic arteries were isolated, cannulated in an arteriograph, and pressurized. Arteries developed 25% constriction at 70 mmHg of intraluminal pressure. Arteries maintained almost similar diameter over the range of pressures 50-210 mmHg, and forced dilatation was observed at pressures >210 mmHg. Denudation of endothelium increased the sensitivity of arteries to pressure-induced constriction, and significantly higher myogenic tone was observed in the pressure range of 10-100 mmHg. Indomethacin and cyclooxygenase-2 inhibition by SC-236 decreased myogenic tone, whereas cyclooxygenase-1 inhibition by SC-560 potentiated myogenic tone in a lower concentration range and decreased at a higher concentration. Pressure-induced constriction was completely blocked by 1 microM nifedipine. Phospholipase C inhibition by 6 microM U-73122 decreased myogenic tone by 39%, whereas PKC inhibitor GF-109203X (3 microM) had no effect. Constriction to phenylephrine was significantly decreased by U-73122 (1 microM) and GF-109203X (3 microM) at an intraluminal pressure of 10 mmHg. Rho-kinase inhibition by Y-27632 (30 microM) and HA-1077 (30 microM) decreased myogenic tone by 75% and 73%, respectively, and 1 microM Y-27632 significantly decreased myogenic tone developed in response to graded increases in pressure. These results suggest that rat ophthalmic artery has an efficient pressure-dependent autoregulatory function that is modulated by endothelium. Contribution of phospholipase C-activation to myogenic tone is minimal, whereas Rho-kinase activation plays a predominant role in the myogenic reactivity in this artery.     

Pyrimidine nucleotides suppress KDR currents and depolarize rat cerebral arteries by activating Rho kinase

This study examined whether, and by what signaling and ionic mechanisms, pyrimidine nucleotides constrict rat cerebral arteries. Cannulated cerebral arteries stripped of endothelium and pressurized to 15 mmHg constricted in a dose-dependent manner to UTP. This constriction was partly dependent on the depolarization of smooth muscle cells and the activation of voltage-operated Ca(2+) channels. The depolarization and constriction induced by UTP were unaffected by bisindolylmaleimide I, a PKC inhibitor that abolished phorbol ester (PMA)-induced constriction in cerebral arteries. In contrast, the Rhokinase inhibitor Y-27632 attenuated the ability of UTP to both constrict and depolarize cerebral arteries. With patch-clamp electrophysiology, a voltage-dependent delayed rectifying K(+) (K(DR)) current was isolated and shown to consist of a slowly inactivating 4-aminopyridine (4-AP)-sensitive and an -insensitive component. The 4-AP-sensitive K(DR) current was potently suppressed by UTP through a mechanism that was not dependent on PKC. This reflects observations that demonstrated that 1) a PKC activator (PMA) had no effect on K(DR) and 2) PKC inhibitors (calphostin C or bisindolylmaleimide I) could not prevent the suppression of K(DR) by UTP. The Rho kinase inhibitor Y-27632 abolished the ability of UTP to inhibit the K(DR) current, as did inhibition of RhoA with C3 exoenzyme. Cumulatively, these observations indicate that Rho kinase signaling plays an important role in eliciting the cerebral constriction induced by pyrimidine nucleotides. Moreover, they demonstrate for the first time that Rhokinase partly mediates this constriction by altering ion channels that control membrane potential and Ca(2+) influx through voltage-operated Ca(2+) channels.     

Differential participation of protein kinase C and Rho kinase in alpha 1-adrenoceptor mediated contraction in rat arteries

The major functional alpha1-adrenoceptor in the rat aorta is of the alpha1Dsubtype and that in the caudal artery is of the alpha1A subtype. In the present study, the participation of protein kinase C (PKC) and Rho kinase (RhoK) in contractile responses to stimulation of the alpha1-adrenoceptors in these two arteries was investigated. Both the PKC inhibitor Ro-318220 and the RhoK inhibitor Y-27632 significantly blocked contractile responses of the aorta to phenylephrine (PE) and the selective alpha1A-adrenoceptor agonist A61603. When used in combination, the inhibitors had an additive blocking effect. In the caudal artery, Y-27632 but not Ro-318220 inhibited contractile responses to PE and A61603, and, in combination, the antagonism produced was no greater than that by Y-27632 alone. Contractile responses to direct activation of PKC with phorbol 12,13-dibutyrate were much smaller and levels of CPI-17 (PKC-activated protein phosphatase inhibitor of 17 kDa) were much lower in the caudal artery than the aorta. The results suggest that both PKC and RhoK contribute independently to contractile responses to stimulation of alpha1D-adrenoceptors in the aorta. However, RhoK, but not PKC, participates in contractile responses to stimulation of alpha1A-adrenoceptors in the caudal artery. This difference may largely be due to differences between the two arteries in the extent to which PKC participates in contraction.     

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca(2+) sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 microm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 microM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.     

Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca(2+)](i)). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca(2+) desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca(2+)-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca(2+)](i). We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca(2+) sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca(2+) desensitization through inhibition of RhoA/ROK.     

Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery

During pregnancy, reduced vascular responses to constrictors contribute to decreased uterine and total vascular resistance. Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor that exerts its actions via diverse signaling pathways, and its biosynthesis increases in preeclampsia. In this study, we hypothesized that maternal vascular responses to TxA(2) will be attenuated via Rho kinase, PKC, p38 MAPK, and ERK1/2 signaling pathways. Isolated ring segments of uterine and small mesenteric arteries from late pregnant (19-21 days) and virgin rats were suspended in a myograph, and isometric force was measured. Pregnancy did not affect uterine and mesenteric artery responses to the TxA(2) analog U-46619 (10(-9)-10(-5) M), but transduction signals associated with these contractions were different between pregnant and nonpregnant rats. Inhibition of Rho kinase (10(-6) M Y-27632) reduced sensitivity to U-46619 in virgin uterine vessels but did not inhibit these contractions in pregnant uterine arteries and had no effect on mesenteric vessels. Treatment of arterial segments with a PKC inhibitor (10(-6) M bisindolylmaleimide I) reduced U-46619-induced contractions in virgin uterine and mesenteric arteries and in pregnant mesenteric arteries. Pregnant uterine arteries, however, were unresponsive to PKC inhibition. Inhibition of ERK1/2 (10(-5) M PD-98059) and p38 MAPK (10(-5) M SB-203580) reduced U46619-induced contractions in nonpregnant vessels and in pregnant uterine and mesenteric vessels. These data suggest that normal pregnancy does not affect uterine and mesenteric contractile responses to TxA(2) but reduces the contribution of Rho kinase and PKC signaling pathways to these contractions in the uterine vasculature. In contrast, the role of ERK1/2 and p38 MAPK in U-46619-induced uterine contractions remains unchanged with pregnancy. TxA(2)-associated transduction signals and its regulators might present potential targets for the development of new treatments for preeclampsia and other pregnancy-associated vascular diseases.     

Acute and chronic NOS inhibition enhances alpha(2)- adrenoreceptor-stimulated RhoA and Rho kinase in rat aorta

We demonstrated that arteries from rats made hypertensive with chronic nitric oxide (NO) synthase (NOS) inhibition (N(omega)-nitro-L-arginine in drinking water, LHR) have enhanced contractile sensitivity to alpha(2)-adrenergic receptors (alpha(2)-AR) agonist UK-14304 compared with arteries from normotensive rats (NR). NO may regulate vascular tone in part through suppression of RhoA and Rho kinase (ROK). We hypothesized that enhanced RhoA and ROK activity augments alpha(2)-AR contraction in LHR aortic rings. Y-27632 eliminated UK-14304 contraction in LHR and NR aortic rings. The order of increasing sensitivity to Y-27632 was the following: endothelium-intact NR, LHR, and endothelium-denuded NR. UK-14304 stimulated RhoA translocation to the membrane fraction in LHR and denuded NR but not in intact NR aorta. Basally, more RhoA was present in the membrane fraction in denuded NR than in intact NR or LHR aorta. Relaxation to S-nitroso-N-acetyl-penicillamine and Y-27632 in denuded ionomycin-permeabilized rings was greater in NR than in LHR. Together these studies indicate alpha(2)-AR contraction depends on ROK activity more in NR than LHR aorta. Additionally, endogenous NO may regulate RhoA activation, whereas chronic NOS inhibition appears to cause RhoA desensitization.     

Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries

Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca(2+)](i)) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca(2+)](i) was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca(2+)](i). However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca(2+)](i), rather than enhanced augmentation of myofilament calcium sensitivity.     

Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.     

Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle

Gender differences in vascular reactivity have been suggested; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of specific protein kinase C (PKC) isoforms in vascular smooth muscle. Aortic strips were isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Isometric contraction was measured in endothelium-denuded aortic strips. PKC activity was measured in the cytosolic and particulate fractions, and the amount of PKC was measured using Western blots and isoform-specific anti-PKC antibodies. In intact male WKY rats, phenylephrine (Phe, 10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated contraction to 0.37 +/- 0.02 and 0.42 +/- 0.02 g/mg tissue wt, respectively. The basal particulate/cytosolic PKC activity ratio was 0.86 +/- 0.06, and Western blots revealed alpha-, delta-, and zeta-PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of alpha- and delta-PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of alpha- and delta-PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe and PDBu contraction, and PKC activity and alpha- and delta-PKC translocation were greater in SHR than WKY rats. The reduction in Phe and PDBu contraction and PKC activity in intact females compared with intact males was greater in SHR ( approximately 30%) than WKY rats ( approximately 20%). Phe and PDBu contraction and PKC activity were not significantly different between castrated males and intact males but were greater in ovariectomized (OVX) females than intact females. Treatment of OVX females or castrated males with 17 beta-estradiol, but not 17 alpha-estradiol, subcutaneous implants caused significant reduction in Phe and PDBu contraction and PKC activity that was greater in SHR than WKY rats. Phe and PDBu contraction and PKC activity in OVX females or castrated males treated with 17 beta-estradiol plus the estrogen receptor antagonist ICI-182,780 were not significantly different from untreated OVX females or castrated males. Thus a gender-related reduction in vascular smooth muscle contraction in female WKY rats with intact gonads compared with males is associated with reduction in the expression and activity of vascular alpha-, delta-, and zeta-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are augmented in the SHR and are possibly mediated by estrogen.     

Protein kinase C regulates vascular myogenic tone through activation of TRPM4

Myogenic vasoconstriction results from pressure-induced vascular smooth muscle cell depolarization and Ca(2+) influx via voltage-dependent Ca(2+) channels, a process that is significantly attenuated by inhibition of protein kinase C (PKC). It was recently reported that the melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced smooth muscle depolarization and constriction in cerebral arteries. Interestingly, PKC activity enhances the activation of cloned TRPM4 channels expressed in cultured cells by increasing sensitivity of the channel to intracellular Ca(2+). Thus we postulated that PKC-dependent activation of TRPM4 might be a critical mediator of vascular myogenic tone. We report here that PKC inhibition attenuated pressure-induced constriction of cerebral vessels and that stimulation of PKC activity with phorbol 12-myristate 13-acetate (PMA) enhanced the development of myogenic tone. In freshly isolated cerebral artery myocytes, we identified a Ca(2+)-dependent, rapidly inactivating, outwardly rectifying, iberiotoxin-insensitive cation current with properties similar to those of expressed TRPM4 channels. Stimulation of PKC activity with PMA increased the intracellular Ca(2+) sensitivity of this current in vascular smooth muscle cells. To validate TRPM4 as a target of PKC regulation, antisense technology was used to suppress TRPM4 expression in isolated cerebral arteries. Under these conditions, the magnitude of TRPM4-like currents was diminished in cells from arteries treated with antisense oligonucleotides compared with controls, identifying TRPM4 as the molecular entity responsible for the PKC-activated current. Furthermore, the extent of PKC-induced smooth muscle cell depolarization and vasoconstriction was significantly decreased in arteries treated with TRPM4 antisense oligonucleotides compared with controls. We conclude that PKC-dependent regulation of TRPM4 activity contributes to the control of cerebral artery myogenic tone.     

Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by MCh in tracheal smooth muscle

We examined the effects of Rho kinase on contraction and intracellular Ca2+ concentration ([Ca2+](i)) in guinea pig trachealis by measuring isometric force and the fura 2 signal [340- to 380-nm fluorescence ratio (F340/F380)]. A Rho kinase inhibitor, Y-27632 (1-1,000 microM), inhibited methacholine (MCh)-induced contraction, with a reduction in F340/F380 in a concentration-dependent manner. The values of EC(50) for contraction and F340/F380 induced by 1 microM MCh with Y-27632 were 27.3 +/- 5.1 and 524.1 +/- 31.0 microM, respectively. With 0.1 microM MCh, the values for these parameters were decreased to 1.0 +/- 0.1 and 98.2 +/- 6.2 microM, respectively. Tension-F340/F380 curves for MCh indicated that Y-27632 caused an ~50% inhibition of MCh-induced contraction, without a reduction in F340/F380. These effects of Y-27632 were not inhibited by a protein kinase C inhibitor, GF-109203X. Our results indicate that inhibition of Rho kinase attenuates both Ca2+ sensitization and [Ca2+](i).     

Sex-specific differences in cerebral arterial myogenic tone in hypertensive and normotensive rats

Cerebral blood flow (CBF) is maintained constant despite changes in systemic blood pressure (BP) through multiple mechanisms of autoregulation such as vascular myogenic reactivity. Our aim was to determine myogenic characteristics of cannulated middle cerebral arteries (MCA) in male and female stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) at 12 wk of age under pressurised no-flow conditions. MCA pressure-diameter relationships (20-200 mmHg) were constructed in active (with calcium) and passive (without calcium) conditions, and myogenic and mechanical properties were determined. Myogenic reactivity in WKY (P < 0.05) and SHRSP (P < 0.05) males was impaired compared with their female counterparts. Comparison of SHRSP with WKY in males revealed similar myogenic reactivity, but in females SHRSP exhibited augmented myogenic reactivity (P < 0.05). In both sexes, myogenic tone yielded at lower pressure in SHRSP compared with WKY vessels (120-140 vs. 140-180 mmHg). Stress-strain relationships and elastic moduli in WKY rats showed that vessels were stiffer in females than in males. Conversely, in SHRSP, male vessels were stiffer than female vessels. Comparison of strains in males indicated that stiffness was increased in SHRSP compared with WKY vessels, whereas the converse was observed in females. These findings demonstrate that MCA myogenic and distensibility characteristics exhibit significant sex- and strain-dependent differences. Inappropriate myogenic adaptation and augmented vascular stiffness, particularly in male SHRSP, are potential limiting factors in blood flow autoregulation and may increase the predisposition for stroke-related cerebrovascular events.     

Endothelin-1 modulates hemoglobin-mediated signaling in cerebrovascular smooth muscle via RhoA/Rho kinase and protein kinase C

Endothelin-1 (ET-1) and oxyhemoglobin (OxyHb) have been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. However, the contribution of ET-1 to this condition has not been definitely established. In this study, we investigated whether threshold concentration of ET-1 enhances cerebrovascular smooth muscle (CVSM) contraction to OxyHb by activating the RhoA/Rho kinase and protein kinase C (PKC) pathways. CVSM contraction was measured in endothelium-denuded rabbit basilar arteries. Cytosolic and particulate fractions of CVSM cells were examined for RhoA and PKC reactivity with specific antibodies using immunoblotting procedures. ET-1 (0.1 nM) alone did not produce any significant contraction, but it markedly potentiated the magnitude (223% of control) and rate (149% of control) of contraction in response to OxyHb, which was attenuated by the inhibitors of Rho kinase Y-27632 and HA-1077. ET-1-mediated potentiation of the contraction was also inhibited by inhibitors of PKC, Ro-32-0432, and GF-109203X. BQ-123 prevented potentiation of vasoconstriction mediated by ET-1, indicating that the action of ET-1 was mediated by the endothelin type A receptor. Pretreatment with ET-1 significantly enhanced OxyHb-mediated RhoA translocation in CVSM cells and intact basilar arteries. ET-1 also caused potentiation of PKC-epsilon expression in membranes of CVSM cells exposed to OxyHb for 10 and 60 min but did not markedly change the distribution of PKC-alpha. Thus, in CVSM, threshold concentration of ET-1 potentiates contraction induced by OxyHb via RhoA/Rho kinase- and PKC-epsilon-dependent mechanisms. This process may contribute to the pathological contraction of cerebral arteries observed after subarachnoid hemorrhage.