Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation.

A recent report from this laboratory (Chitaley K, Wingard C, Webb R, Branam H, Stopper V, Lewis R, and Mills T. Nature Medicine 7: 119-122, 2001) showed that inhibition of Rho-kinase increased the erectile response (intracavernosal pressure and mean arterial pressure) by a process that does not require nitric oxide or cGMP. The present study investigated whether vasoconstrictor agents, which are active in the penis, act via the Rho-kinase pathway. Western analysis revealed RhoA and Rho-kinase protein in the penis. Treatment with the selective Rho-kinase inhibitor Y-27632 significantly increased the magnitude of the erectile response. Intracavernous administration of endothelin-1 (ET-1; 50 pmol) or methoxamine (10 microg/kg) reduced the erectile response to autonomic stimulation. If Y-27632 was given before ET-1 or methoxamine, the vasoconstrictor effect was reduced, and intracavernosal pressure and mean arterial pressure remained elevated. However, when given after methoxamine, Y-27632 had a reduced vasodilatory effect, and Y-27632 had no vasodilatory effect when given after ET-1. These findings suggest that ET-1 and methoxamine increase Rho-kinase activity in the cavernous circulation and support the hypothesis that the vasoconstriction that maintains the penis in the nonerect state is mediated, in part, by the Rho-kinase pathway.     

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.     

RhoA-Rho kinase mediates synergistic ET-1 and phenylephrine contraction of rat corpus cavernosum

Maintenance of the detumescent state of the penis is believed to involve the actions of several vasoconstrictors. However, our mechanistic understanding of any synergistic vasoconstrictor influences is extremely limited. We tested the hypothesis that a vasoconstrictor combination of endothelin (ET-1) and phenylephrine (PE) augments the constrictor responses in rat corporal cavernosal tissues by a mechanism involving the RhoA-Rho kinase pathway. Independently, ET-1 (1 nM-30 microM) and PE (100 nM-100 microM) both caused dose-dependent contractions of isolated rat cavernosal tissues. In combination, ET-1 (30 nM) augmented the contractile effect of PE and shifted the calculated EC50 for PE (90 +/- 12 to 45 +/- 5 microM). The active stress generated by cavernosal strips during the ET-1 + PE combined stimulation (4.9 +/- 0.2 mN/mm2) was greater than the combined stress generated with ET-1 (0.4 +/- 0.1 mN/mm2) or PE (3.3 +/- 0.2 mN/mm2) stimulations alone. Blockade of ETA receptors (30 nM; A-127722) reversed the augmented stress generation and the Rho-kinase inhibitor Y-27632 differentially and dose-dependently relaxed the tissue. The combined constrictor effect was associated with a fourfold increase of RhoA in the membrane faction of the tissue homogenates. We conclude that the ET-1 + PE combination potentiate vasoconstriction through mutual activation of the RhoA-Rho kinase pathway. The interactions of these agonists likely play important roles in the maintenance of the flaccid state and contribute to some forms of erectile dysfunction.     

RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability

Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Adeno-associated viral gene transfer of dominant negative RhoA enhances erectile function in rats

We previously reported the inhibition of Rho-kinase to result in increased intracavernosal pressure (ICP) in an in vivo rat model of erection. Expression of an upstream activator of Rho-kinase, RhoA, has been demonstrated in the penile vasculature; however, the functional role of RhoA in the regulation of erection remains unknown. We used adeno-associated viral gene transfer of a dominant negative RhoA mutant (T19NRhoA) into rat cavernosum to test the hypothesis that RhoA activation is physiologically important for maintenance of the non-erect state and inhibition of this pathway leads to erection. Anesthetized, male, Sprague-Dawley rats transfected with the T19NRhoA mutant exhibited an elevated baseline ICP/mean arterial pressure (MAP) and nerve stimulation-induced ICP/MAP as compared with beta-galactosidase-transfected controls. The novel findings of this study demonstrate a functional role of RhoA in maintaining the flaccid penis and provide support for the inhibition of RhoA as a potential therapy for the enhancement of erectile function.     

Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the alpha1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (< or =3 microM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in alpha-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.     

Rho-kinase inhibitor retards migration and in vivo dissemination of human prostate cancer cells

The Rho-kinase inhibitor, Y-27632, inhibited in vitro chemotactic migration to bone marrow fibroblast conditioned media and metastatic growth in immune-compromised mice of highly invasive human prostatic cancer (PC3) cells. Y-27632 also reduced myosin light chain phosphorylation and markedly altered the morphology of cells that developed numerous processes containing microtubules. A strikingly different, rounded phenotype was induced by an inhibitor of myosin light chain kinase, ML9. The M(110-130) subunit of the myosin phosphatase that is regulated by Rho-kinase was present in PC3 cells that contained significantly more RhoA than the less invasive, LNCaP cells. Y-27632 also inhibited angiogenesis as measured by endothelial cell tube formation on Matrigel. We conclude that invasiveness of human prostate cancer is facilitated by the Rho/Rho-kinase pathway, and exploration of selective Rho-kinase inhibitors for limiting invasive progress of prostate cancer is warranted.     

Androgen maintenance of erectile function in the rat penis.

Previous research has shown that the frequency and duration of penile erection is diminished after castration and that replacement with testosterone will restore the process. Using rats, the present study was designed to confirm that erection is androgen-dependent and to determine whether castration and androgen replacement affect the penile vascular smooth muscle responsiveness to vasoactive drugs. Blood pressure in the corpus cavernosum was measured directly during erections induced by electrical stimulation of the autonomic innervation of the penis. Maximal cavernosal pressure was markedly reduced after castration but was returned to normal levels if the castrated animals were treated with testosterone. Infusion of nitroglycerin (vasodilator) or phenylephrine (vasoconstrictor) resulted in a decline in cavernosal pressure in androgen-treated animals but not in castrated animals, even though the mean arterial blood pressure was strongly affected in all treatment groups by these drugs. When an inhibitor of nitric oxide synthesis was infused, cavernosal pressure was decreased in all groups, indicating that this substance is involved in penile erection. Taken together, these results show that androgens maintain the erectile process and may act specifically to support the responsiveness of the vascular smooth muscle to vasoactive drugs.     

Receptor-specific influence of endothelin-1 in the erectile response of the rat

Specific receptor antagonists were used to examine the role of endothelin-1 (ET-1) in the erectile response of the rat. In these studies, intact rats were cannulated to permit the continuous recording of mean arterial pressure (MAP) and intracavernosal pressure (CCP). Erection was induced by electrical stimulation of the autonomic ganglion, which regulates blood flow to the penis. The animals were subjected to intracavernosal injection with vehicle only (Cont) or with an antagonist to the endothelin-A receptor (ET(A)) or to the endothelin-B receptor (ET(B)). Blockade of the ET(A) or the ET(B) had no effect on the erectile response (CCP/MAP) during maximal ganglionic stimulation. When ET-1 was injected into Cont rats, there was a marked vasoconstriction with a sharp rise in MAP and a decline in CCP as the cavernosal arterioles constricted and limited inflow. The injection of the ET(A) antagonist prevented the vasoconstriction after ET-1 injection into Cont rats, whereas blockade of the ET(B) had no effect on the vasoconstrictive effect to ET-1. Similar results were obtained during submaximal ganglionic stimulation. With minimal levels of ganglionic stimulation, ET-1 injection led to a moderated degree of vasodilation in the presence of the ET(A) antagonist. The ET(B) antagonist failed to alter the CCP response during minimal stimulation, but it did have a marked effect on the MAP response to ET-1 injection. The results of these studies confirm that cavernosal tissue of the rat penis is highly responsive to ET-1. However, the failure of the ET-1 antagonists to affect penile erection in response to ganglionic stimulation reflects a minimal role of ET-1 in the erectile response in the rat.     

Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.     

Rho-kinase mediates TNF-α-induced MCP-1 expression via p38 MAPK signaling pathway in mesangial cells

Macrophage accumulation has been implicated in the pathogenesis of inflammatory glomerular disease. Monocyte chemoattractant protein-1 (MCP-1) plays a central role in recruiting monocytes to the glomeruli. Tumor necrosis factor-α (TNF-α) has been shown to induce MCP-1 expression in mesangial cells, although the precise mechanisms remain unclear. We previously demonstrated that RhoA and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are involved in the pathogenesis of diabetic nephropathy. However, its role in MCP-1 induction by TNF-α has not been elucidated. In the present study, we investigated whether the Rho/Rho-kinase signaling pathway regulates the TNF-α-mediated induction of MCP-1 in mesangial cells. Exposure of mouse mesangial cells (MES-13) to TNF-α resulted in an increase of MCP-1 expression (by RT-PCR) and secretion into the medium (by ELISA). Pull down and Western blot analysis revealed that TNF-α activated RhoA and Rho-kinase. Based on these observations, we speculated that the Rho/Rho-kinase signaling pathway may be involved in MCP-1 induction by TNF-α. In agreement with this concept, Y-27632, a specific Rho-kinase inhibitor, attenuated TNF-α-mediated induction of MCP-1. We demonstrated that Y-27632 inhibited TNF-α-mediated monocyte migration and attenuated TNF-α-mediated p38 MAPK activation. Based on these data we infer that Y-27632 inhibits TNF-α-induced MCP-1 expression, secretion and function through inhibition of Rho-kinase and p38 MAPK activity. Our study suggests that Rho/Rho-kinase is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease.     

RhoA-mediated Ca2+ sensitization in erectile function

A Rho-kinase inhibitor increases corpus cavernosum (CC) pressure in an in vivo rat model (Chitaley, K., Wingard, C. J., Webb, R. C., Branam, H., Stopper, V. S., Lewis, R. W., and Mills, T. M. (2001) Nat. Med. 7, 119-122) suggesting that Rho-mediated Ca(2+) sensitization of CC smooth muscle maintains the flaccid (contracted) state. We directly demonstrate Ca(2+) sensitization of permeabilized rabbit and human CC and identify a highly expressed molecular component of this pathway. Ca(2+) sensitization of force induced by endothelin or GTPgammaS was significantly greater in CC than in rabbit ileum smooth muscle and was accompanied by a 17-fold higher RhoA content. Pull-down assays with the RhoA binding domain of mDia showed the high RhoA content of CC to be available for activation by GTPgammaS. Ca(2+) sensitization induced by endothelin, phenylephrine, or GTPgammaS was completely relaxed by the Rho kinase inhibitor Y-27632. Human and rabbit CC both express the phosphatase inhibitor CPI-17, the myosin phosphatase regulatory (MYPT-1) and catalytic (PP1delta) subunits, and two isoforms of Rho kinase. We suggest that high expression of RhoA contributes, through RhoA-mediated Ca(2+) sensitization, to the flaccid state of CC that can be reversed by a water-soluble, orally active Rho kinase inhibitor suitable for therapy of erectile dysfunction.     

Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15-500 microg) and HA-1077 (500 microg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 +/- 0.18 to 0.16 +/- 0.02 mmHg x ml(-1) x min(-1) (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 +/- 0.03 to 0.19 +/- 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15-30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 +/- 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.     

A novel role of Rho-kinase in the regulation of ligand-induced phosphorylated EGFR endocytosis via the early/late endocytic pathway in human fibrosarcoma cells

The small GTPase RhoA and its downstream effectors, the Rho-associated kinase (Rho-kinase) family, are known to regulate cell morphology, motility, and tumor progression via the regulation of actin cytoskeleton rearrangement. In the present study, we evaluated the role of Rho-kinase in the intracellular endocytic trafficking of ligand-induced phosphorylated epidermal growth factor receptor (pEGFR). We investigated the time course of the internalization fate of EGF-induced pEGFR via the early/late endocytic pathway in human fibrosarcoma cell line HT1080 cells using Y-27632, a selective Rho-kinase inhibitor. We found, using confocal immunofluorescence microscopy and Western blot analysis, a large accumulation of pEGFR in the nuclei of HT1080 cells. In contrast, we observed decreased amounts of the pEGFR-positive staining in the nuclei along with an accumulation of cytosolic pEGFR staining when the cells were incubated for 15–30 min in the presence of Y-27632, implying that an aberrant endocytic trafficking mechanism of pEGFR occurs in HT1080 cells whereby pEGFR might be selectively translocated into the nucleus. Moreover, we demonstrated that after 15-min of stimulation with Texas Red-EGF, increasing numbers of pEGFR-positive staining that had colocalized with Texas Red-EGF-positive punctate staining were seen in the cytoplasm of HT1080 cells but after 30-min of stimulation, most of this staining had disappeared from the cytoplasm and a large accumulation of pEGFR-positive staining appeared in the nucleus. Thus, nuclear accumulation of pEGFR appears to occur in an EGF-dependent manner. In contrast, such nuclear pEGFR-positive staining was not seen in the Y-27632-treated cells. Furthermore, silencing of RhoA or Rho-kinases I/II by sequence specific siRNAs considerably inhibited the EGF-dependent nuclear accumulation of pEGFR. Collectively, these results provide the first evidence that Rho-kinase signaling pathway plays a suppressive role in the intracellular vesicle trafficking of pEGFR via the endocytic pathway and that an increased Rho-kinase activity leads to the attenuation of the normal endocytic vesicular traffic of pEGFR via the early/late endocytic pathway, instead causing pEGFR to be trafficked out of the endocytic vesicles into the nucleus.     

Vasoconstrictor effect of endothelin-1 on hypertensive pulmonary arterial smooth muscle involves Rho-kinase and protein kinase C

Although one of the common characteristics of pulmonary hypertension is abnormal sustained vasoconstriction, the signaling pathways that mediate this heightened pulmonary vascular response are still not well defined. Protein kinase C (PKC) and Rho-kinase are regulators of smooth muscle contraction induced by G protein-coupled receptor agonists including endothelin-1 (ET-1), which has been implicated as a signaling pathway in pulmonary hypertension. Toward this end, it was hypothesized that both Rho-kinase and PKC mediate the pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle, and therefore, the purpose of this study was to determine the role of PKC and Rho-kinase signaling in ET-1-induced vasoconstriction in both normotensive (Sprague-Dawley) and hypertensive (Fawn-Hooded) rat pulmonary arterial smooth muscle. Results indicate that ET-1 caused greater vasoconstriction in hypertensive pulmonary arteries compared with the normal vessels, and treatment with the PKC antagonists chelerythrine, rottlerin, and G? 6983 inhibited the vasoconstrictor response to ET-1 in the hypertensive vessels. In addition, the specific Rho-kinase inhibitor Y-27632 significantly attenuated the effect of ET-1 in both normotensive and hypertensive phenotypes, with greater inhibition occurring in the hypertensive arteries. Furthermore, Western blot analysis revealed that ET-1 increased RhoA expression in both normotensive and hypertensive pulmonary arteries, with expression being greater in the hypertensive state. These results suggest that both PKC and Rho/Rho-kinase mediate the heightened pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

RhoA/Rho kinase and nitric oxide modulate the agonist-induced pulmonary artery diameter response time

We studied the amplitude and response time (RT; time to 50% of maximal response) of pulmonary vasoreactivity and investigated whether the characteristics of pulmonary vasoreactivity could be modulated by endothelium removal, nitric oxide (NO) synthase inhibition [N(G)-nitro-L-arginine (L-NNA)], RhoA activation [lysophosphatidic acid (LPA)] and Rho kinase inhibition (Y-27632). Slow acetylcholine-induced pulmonary vasodilation (262 +/- 5 s) was not due to the RT of endothelial NO release (45-55 s) and was always longer than RT in renal arteries (15 +/- 4 s). The rate-determining step is located in the smooth muscle cells. This was confirmed by the existing differences between the RT of the NO solution and KCl-induced renal and pulmonary vasoreactivity in endothelium-denuded arteries. We found that the pulmonary contractile amplitude increases and the RT decreases by L-NNA or LPA. In contrast, Y-27632 reduced the contractile amplitude and increased the RT in pulmonary arteries. These phenomena were dependent on the contractile stimulus (phenylephrine or KCl). In conclusion, slow pulmonary vasoreactivity is a smooth muscle cell characteristic that can be enhanced by RhoA and NO or endothelium removal. These effects were counteracted by Rho kinase inhibition. We show a role for RhoA/Rho kinase and NO in the modulation of pulmonary vascular reactivity.     

Androgens augment renal vascular responses to ANG II in New Zealand genetically hypertensive rats

Males develop higher blood pressure than do females. This study tested the hypothesis that androgens enhance responsiveness to ANG II during the development of hypertension in New Zealand genetically hypertensive (NZGH) rats. Male NZGH rats were obtained at 5 wk of age and subjected to sham operation (Sham) or castration (Cas) then studied at three age groups: 6-7, 11-12, and 16-17 wk. Mean arterial blood pressure (MAP), heart rate (HR), and renal blood flow (RBF) measurements were recorded under Inactin anesthesia. These variables were measured after enalapril (1 mg/kg) treatment and during intravenous ANG II infusion (20, 40, and 80 ng/kg/min). Plasma testosterone was measured by ELISA. Angiotensin type 1 (AT1) receptor expression was assessed by Western blot analysis and RT-PCR. ANG II-induced MAP responses were significantly attenuated in Cas NZGH rats. At the highest ANG II dose, MAP increased by 40+/-4% in Sham vs. 22+/-1% in Cas NZGH rats of 16-17 wk of age. Similarly, renal vascular resistance (RVR) responses to ANG II were reduced by castration (209+/-20% in Sham vs. 168+/-10% in Cas NZGH rats at 16-17 wk of age). Castration also reduced MAP recorded in conscious NZGH rats of this age group. Testosterone replacement restored baseline MAP and the pressor and RVR responses to ANG II. Castration reduced testosterone concentrations markedly. Testosterone treatment restored these concentrations. Neither castration nor castration+testosterone treatment affected AT1 receptor mRNA or protein expression. Collectively, these data suggest that androgens modulate renal and systemic vascular responsiveness to ANG II, which may contribute to androgen-induced facilitation of NZGH rat hypertension.