Role Of Rho-kinase Research Articles

ROK controls urethral tone, but by what mechanism?

the role of rho-kinase (ROK) in regulation of smooth muscle contraction has been an exciting area of investigation for reasons spanning interests in defining cellular signaling systems to insights into potential (and actual) therapies designed to treat smooth muscle contractile disorders. Smooth

Attenuation of pulmonary hypertension secondary to left ventricular dysfunction in the rat by Rho‐kinase inhibitor fasudil

Pulmonary hypertension (PH) in left ventricular dysfunction is attributable not only to backward failure of the left ventricle, but also to increased pulmonary vascular resistance (PVR) in some patients. Recently, Rho-kinase has been known as a potent growth stimulator and mediator of vasoconstriction, and Rho-kinase inhibitors could ameliorate PVR, little is known about the role of Rho-kinase in left ventricular dysfunction-induced PH. We utilized the ascending aortic-banded rat and assessed the effect of Rho-kinase inhibitor fasudil on the development of PH secondary to left ventricular dysfunction. Subsequently, in rats subjected to aortic banding for 6 weeks, there were increases in mean pulmonary arterial pressure, pulmonary arteriolar medial thickness, active RhoA, Rho-kinase II, Rho-kinase activity, endothelial nitric oxide synthase (eNOS) and endothelin-1(ET-1) concomitant with decreased levels in NO and cGMP in the lung. Treatment with fasudil at a dose of 30 mg/kg/day from days 1 to 28 or from days 29 to 42 decreased the mean pulmonary arterial pressure by 57% and 56%, right ventricular hypertrophy by 31% and 30%, pulmonary arteriolar medial thickness by 50% and 50%, and pulmonary expression of Rho-kinase II by 41% and 28%, respectively, as well as augmented pulmonary expression of eNOS by 16% and 31% and NO by 50% and 76%, respectively, when compared with the vehicle controls. In conclusion, these results suggest that inhibition of Rho-kinase may provide therapeutic potential for preventing and attenuating the development of PH in left ventricular dysfunction. Further translational study in human is needed to substantiate the findings.

Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation

The role of sphingosine 1-phosphate (S1P)-induced Rho kinase (ROCK) activation in the angiogenic responses of pulmonary artery-derived endothelial cells (PAEC) and smooth muscle cells (PASMC) was examined. S1P, a biologically active phospholipid that regulates angiogenesis, promoted PAEC chemotaxis and capillary morphogenesis; furthermore, this activity was unaltered by pretreatment with the pharmacological inhibitor of ROCK, H1152. In contrast, S1P (500 nM) significantly inhibited spontaneous PASMC chemotaxis and differentiation; however, this inhibition was eradicated upon H1152 pretreatment. Similarly, PASMCs transfected with ROCK II siRNA diminished S1P-induced inhibition of the development of multi-cellular structures. Analysis by RT-PCR identified the presence of S1P(1) and S1P(3) receptors on both PAECs and PASMCs, while S1P(2) receptor expression was confined to only PASMCs. Consistent with this observation, the S1P(1) and S1P(3) receptor antagonist, VPC23019, virtually abolished the S1P-initiated PAEC differentiation but did not impede the S1P-induced inhibition of PASMC differentiation. However, the S1P(2) receptor antagonist, JTE013, had no effect on S1P-mediated differentiation of PAECs but abolished the S1P-induced inhibition of PASMC function. Co-cultured endothelial and smooth muscle cells differentiated into "neovascular-like" networks, which were significantly inhibited by S1P. The inhibition of co-culture differentiation in both PAECs and PASMCs was negated by H1152 pretreatment. However, when smooth muscle cells were added to S1P-initiated endothelial cell networks, additional S1P treatment did not inhibit the cellular networks generated by these cells. In conclusion, S1P-induced PAEC angiogenic responses are regulated by S1P(1) and/or S1P(3) receptors independent of Rho kinase activation, whereas S1P(2) receptor-mediated curtailment of PASMC function by S1P.

Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression

Rho-kinase (ROK), downstream of the mevalonate pathway, is detrimental to vessels, and suppressing its activity is a target for the treatment of human disease such as coronary artery disease and pulmonary hypertension. Recent studies have shown that ROK has a crucial role in bone metabolism. However, the role of ROK in stromal cells is still unclear. The present study was undertaken to investigate the effect of a ROK inhibitor, fasudil hydrochloride, on stromal cell lines, C3H10T1/2 and ST2. In both cells, Fasudil significantly stimulated alkaline phosphatase activity and enhanced cell mineralization. Moreover, fasudil significantly increased the mRNA expression of collagen-I, osteocalcin, and bone morphogenetic protein-2 (BMP-2). Supplementation of noggin, a BMP-2 antagonist, significantly reversed the fasudil-induced collagen-I and osteocalcin mRNA expression in both cells. These findings suggest that fasudil induces the osteoblastic differentiation of stromal cells via enhancing BMP-2 expression, and that this drug might be beneficial for not only atherosclerosis but also osteoporosis by promoting bone formation.

Molecular Mechanism of Insulin Resistance in Obesity and Type 2 Diabetes

Insulin resistance is a major risk factor for developing type 2 diabetes caused by the inability of insulin-target tissues to respond properly to insulin, and contributes to the morbidity of obesity. Insulin action involves a series of signaling cascades initiated by insulin binding to its receptor, eliciting receptor autophosphorylation and activation of the receptor tyrosine kinase, resulting in tyrosine phosphorylation of insulin receptor substrates (IRSs). Phosphorylation of IRSs leads to activation of phosphatidylinositol 3-kinase (PI3K) and, subsequently, to activation of Akt and its downstream mediator AS160, all of which are important steps for stimulating glucose transport induced by insulin. Although the mechanisms underlying insulin resistance are not completely understood in skeletal muscle, it is thought to result, at least in part, from impaired insulin-dependent PI3K activation and downstream signaling. This review focuses on the molecular basis of skeletal muscle insulin resistance in obesity and type 2 diabetes. In addition, the effects of insulin-sensitizing agent treatment and lifestyle intervention of human insulin-resistant subjects on insulin signaling cascade are discussed. Furthermore, the role of Rho-kinase, a newly identified regulator of insulin action in insulin control of metabolism, is addressed.

Role of Rho kinase and oxidative stress in cardiac fibrosis induced by aldosterone and salt in angiotensin type 1a receptor knockout mice

Large clinical trials have shown that mineralocorticoid receptor (MR) antagonists improve cardiovascular or total mortality in patients with heart failure or myocardial infarction even though the patients were taking angiotensin-converting enzyme inhibitors or angiotensin II receptor (AT1R) antagonists. We previously reported that cardiac fibrosis induced by aldosterone and salt (Ald–NaCl) was exaggerated in AT1aR knockout mice (AT1aR-KOs). As the association of Rho kinase and oxidative stress was reported in Ald–NaCl-induced hypertension of rats, we investigated the effects of an MR antagonist (eplerenone) and a Rho kinase inhibitor (fasudil) on Ald–NaCl-induced cardiac fibrosis in AT1aR-KOs. AT1aR-KOs were administered aldosterone (0.15 μg/h) subcutaneously using an osmotic minipump and were provided with 1% NaCl drinking water for 4 weeks. AT1aR-KOs receiving Ald–NaCl were treated with a low (30 mg/kg/day) or high (100 mg/kg/day) dose of eplerenone or a fasudil (100 mg/kg/day). Systolic blood pressure (SBP), left ventricular weight/body weight (LVW/BW), histological examination and cardiac gene expression were evaluated on day 28. Ald–NaCl treatment caused increases in SBP and LVW/BW in AT1aR-KOs, and eplerenone dose-dependently decreased SBP, LVW/BW and cardiac fibrosis. Fasudil decreased LVW/BW and cardiac fibrosis without affecting SBP. The expressions of connecting tissue growth factor (CTGF) and nicotinamide adenine dinucleotide phosphate (NADPH) components (p22phox, p47phox and p67phox) were increased in Ald–NaCl-treated AT1aR-KOs, and eplerenone or fasudil decreased the expression of CTGF and NADPH components. Phosphorylated ERM (a marker of the phosphorylation of Rho kinase) was increased in Ald–NaCl-treated AT1aR-KOs and was decreased by eplerenone. Nitrotyrosine and 4-hydroxy-2-nonenal, which indicate tissue damage via oxidative stress, were increased in AT1aR-KO and were apparently attenuated by eplerenone or fasudil. These results suggested that the Rho kinase pathway was activated to induce cardiac fibrosis by Ald–NaCl via MR in AT1aR-KOs. A Rho kinase inhibitor as well as eplerenone might be useful for cardiac damage by Ald–NaCl.

Role of Rho-Kinase in the Pathogenesis of Coronary Hyperconstricting Responses Induced by Drug-Eluting Stents in Pigs In Vivo

This study examined whether the Rho-kinase pathway is involved in the pathogenesis of coronary hyperconstricting responses induced by drug-eluting stents (DES) in pigs in vivo. Recent studies showed that coronary vasoconstricting responses are enhanced at the edge of coronary segments implanted with DES compared with bare-metal stents (BMS) in humans. We have previously shown that the activated Rho-kinase pathway plays a central role in the molecular mechanism of coronary vasospasm in animals and humans. Human coronary artery smooth muscle cells (hCASMCs) were coincubated with various concentrations of paclitaxel (10(-9) to 10(-6) mol/l, corresponding levels reported in DES-implanted arterial tissue) for 24 h. A paclitaxel-eluting stent (PES), sirolimus-eluting stent (SES), and BMS were randomly implanted in the left coronary arteries in pigs for 4 weeks. In hCASMCs, paclitaxel significantly enhanced Rho-kinase expression and activity. In a porcine model, coronary vasoconstricting responses to serotonin (10 and 100 microg/kg intracoronary administration) were significantly enhanced at the PES site compared with the BMS site (45+/-4% vs. 30+/-3%; p<0.01; n=12 each), and were abolished by hydroxyfasudil (90 and 300 microg/kg intracoronary administration), a selective Rho-kinase inhibitor. The PES enhanced inflammatory responses and microthrombus formation at the stent edge, where immunoreactivities for Rho-kinase expression and activity were increased. In organ chamber experiments, serotonin-induced contractions were significantly enhanced in rings from the PES edge site compared with the BMS edge site. The SES also caused similar coronary hyperconstricting responses to serotonin in vivo. These results suggest that the Rho-kinase pathway plays an important role in the pathogenesis of DES-induced coronary hyperconstricting responses.

Role of rho-kinase in the regulation of vascular tone in hypertensive renal transplant recipients

Activation of rho-kinase (ROK) is involved in the development of hypertension as it is a potent regulator of vascular smooth muscle cell (VSMC) contractility. Here we evaluated whether activation of ROK is present in hypertensive kidney transplant recipients (NTX). We tested the effect of the ROK-inhibitor fasudil on the regulation of forearm blood flow (FBF) in NTX and in healthy control subjects (CTL). In addition potential modulating effects of ROK-inhibition on local vascular nitric oxide (NO) release were studied. The effect of intra-arterial infusion of fasudil on FBF was studied by venous-occlusion plethysmography in NTX and CTL. To unmask the role of NO fasudil was infused with/without clamping of vascular NO in NTX and CTL. To unravel the basal NO-mediated tone the NO-synthase inhibitor l-NMMA was infused. Fasudil markedly but comparably increased FBF in NTX and CTL. The vascular response to fasudil was blunted during NO-clamp in CTL (104 ± 18% vs. 244 ± 48% for NO-clamp + fasudil vs. fasudil alone; baseline = 0%, P < 0.05) but not in NTX. The l-NMMA-induced vasoconstriction was impaired in NTX compared to CTL. In NTX and CTL basal vascular tone equally depends on ROK. Fasudil-induced vasodilatation is partly mediated by vascular NO in CTL but not in NTX. The greater NO-insensitive fasudil-induced increase in FBF in NTX suggests an increased ROK-mediated VSMC constrictor tone in these patients. Basal NO-mediated tone is attenuated in hypertensive NTX.

Extraction of membrane cholesterol disrupts caveolae and impairs serotonergic (5-HT2A) and histaminergic (H1) responses in bovine airway smooth muscle: role of Rho-kinase

Some receptors and signaling molecules, such as Rho-kinase (ROCK), localize in caveolae. We asked whether the function of histamine receptors (H(1)) and 5-hydroxytryptamine (serotonin) receptors (5-HT(2A)) in bovine tracheal smooth muscle are modified after caveolae disruption and if so, whether the altered ROCK activity plays a role in this modification. Methyl-beta-cyclodextrin (MbetaCD), used to deplete membrane cholesterol, was shown to disrupt caveolae and diminish sustained contractions to histamine (approximately 80%), 5-HT (100%), alpha-methyl-5-HT (100%), and KCl (approximately 30%). Cholesterol-loaded MbetaCD (CL-MbetaCD) restored the responses to KCl and partially restored the responses to agonists. ROCK inhibition by Y-27632 diminished contractions to histamine (approximately 85%) and 5-HT (approximately 59%). 5-HT or histamine stimulation augmented ROCK activity. These increases were reduced by MbetaCD and partially reestablished by CL-MbetaCD. The increase in intracellular Ca(2+) that was induced by both agonists was reduced by MbetaCD. The presence of caveolin-1 (Cav-1), H1, 5-HT(2A), and ROCK1 was corroborated by immunoblotting of membrane fractions from sucrose gradients and by confocal microscopy. H(1) receptors coimmunoprecipitated with Cav-1 in caveolar and noncaveolar membrane fractions, whereas 5-HT(2A) receptors appeared to be restricted to noncaveolar membrane fractions. We conclude that caveolar and cholesterol integrity are indispensable for the proper functionality of the H(1) and 5-HT(2A) receptors through their Rho/ROCK signaling.

Role of RHO Kinase in Gastrointestinal Motility

Role of RHO Kinase in Gastrointestinal Motility

Rho-kinase inhibition reduces pressure-mediated autoregulatory adjustments in afferent arteriolar diameter.

Preglomerular resistance is regulated by calcium influx- and mobilization-dependent mechanisms; however, the role of Rho-kinase in calcium sensitization in the intact kidney has not been carefully examined. Experiments were performed to test the hypothesis that Rho-kinase inhibition blunts pressure-mediated afferent arteriolar autoregulatory behavior and vasoconstrictor responses evoked by angiotensin II and P2X1 receptor activation. Rat kidneys were studied in vitro using the blood-perfused juxtamedullary nephron technique. Autoregulatory behavior was assessed before and during Rho-kinase inhibition with Y-27632 (1.0 microM; n = 5). Control diameter averaged 14.3 +/- 0.8 microm and increased to 18.1 +/- 0.9 microm (P < 0.05) during Y-27632 treatment. In the continued presence of Y-27632, reducing perfusion pressure to 65 mmHg slightly increased diameter to 18.7 +/- 1.0 microm. Subsequent pressure increases to 130 and 160 mmHg yielded afferent arteriolar diameters of 17.5 +/- 0.8 and 16.6 +/- 0.6 microm (P < 0.05). This 11% decline in diameter is significantly smaller than the 40% decrease obtained in untreated kidneys. The inhibitory effects of Y-27632 on autoregulatory behavior were concentration dependent. Angiotensin II responses were blunted by Y-27632. Angiotensin II (1.0 nM) reduced afferent diameter by 17 +/- 1% in untreated arterioles and by 6 +/- 2% during exposure to Y-27632. The P2X1 receptor agonist, alpha, beta-methylene ATP, reduced afferent arteriolar diameter by 8 +/- 1% but this response was eliminated during exposure to Y-27632. Western blot analysis confirms expression of the Rho-kinase signaling pathway. Thus, Rho-kinase may be important in pressure-mediated autoregulatory adjustments in preglomerular resistance and responsiveness to angiotensin II and autoregulatory P2X1 receptor agonists.

Rho-Kinase, but not Protein Kinase C, is Involved in Generation of the Spontaneous Tone in the Resting Phase of the Isolated Pig Iris Sphincter Muscle

Purpose: The purpose of the present study was to clarify the role of Rho-kinase and/or protein kinase C in the resting tension of the isolated pig iris sphincter muscle. Materials and Methods: The motor activity of the isolated pig iris sphincter muscle was measured isometrically. Results: EGTA, a chelator of extracellular Ca2+, significantly reduced the resting tension. Y27632, a Rho-kinase inhibitor, significantly reduced the resting tension in a concentration-dependent manner. The resting tension diminished by Y27632 was significantly recovered by the addition of calyculin A, a myosin light chain phosphatase (MLCP) inhibitor, in a concentration-dependent manner. GF109203X, a protein kinase C inhibitor, had no effect on the resting tension. Conclusion: These results suggest that, in the isolated pig iris sphincter muscle, Rho-kinase plays an important role in the generation of spontaneous tone in the resting phase via the inhibition of MLCP activity.

Lysophosphatidylcholine induces glial cell activation: Role of rho kinase

Lysophosphatidylcholine (LPC), a major phospholipid component of atherogenic oxidized LDL, is implicated in atherosclerosis and, recently, in neurodegenerative diseases. We investigated the immunomodulatory functions of LPC in the central nervous system (CNS) using both an in vivo rat model, and in vitro culture systems of human primary astrocytes and a microglia cell line, HMO6. Compared with PBS injection, 20 nmol LPC-injection into the rat striatum increased astrocyte and microglial accumulation and elevated iNOS expression; concomitantly a time-dependent decrease in number of neurons was exhibited. In vitro studies on astrocytes and HMO6 cells showed that LPC increased the gene expression of proinflammatory factors IL-1beta, COX-2, and GM-CSF. LPC also induced chemotactic responses in HMO6 cells. Inhibition of rho kinase by fasudil, Y27632, or expressing a dominant negative form of rho kinase inhibited the LPC-induced IL-1beta mRNA expression in both astrocytes and HMO6. Moreover, intraperitoneal fasudil injection inhibited the LPC-induced microglial accumulation and iNOS expression and also was effective in protecting against neuronal loss. Silencing G2A, a specific receptor for LPC, inhibited proinflammatory gene expression and HMO6 migration. Overall, our results indicate that LPC induced considerable neuroinflammatory reactivity in glia mediated by rho kinase-dependent pathways with inhibition of these pathways conferring significant extents of neuroprotection.

Abstract 2474: Rho-Kinase Inhibition Improves Endothelium-Dependent Vasodilation during Hyperinsulinemia in Patients with Metabolic Syndrome

Impaired insulin-mediated vasodilation in the skeletal muscle may be involved in the development of hypertension in patients with metabolic syndrome (MetS) and contribute to insulin resistance by diminishing the glucose uptake. Rho-kinase, an effector of the small G protein Rho A, plays an important role in hypertension and is reported to interfere with insulin signaling through serine phosphorylation of insulin receptor substrate-1 in blood vessels. We therefore examined the role of Rho-kinase in the pathophysiology of impaired vascular reactivity in patients with MetS by evaluating the effect of Rho-kinase inhibition on NO-dependent vasodilation during hyperinsulinemia. Forearm blood flow (FBF) responses to acetylcholine (ACh), a stimulus for endothelial release of NO, and sodium nitroprusside (SNP), an exogenous NO donor, were assessed during insulin administration (0.1 mU/Kg/min) using the forearm perfusion technique in patients with MetS (n=10) and matched controls (n=10). Patients with MetS were then randomized to intra-arterial infusion of either fasudil (inhibitor of Rho-kinase, 200 μg/min) or placebo and reactivity to ACh and SNP was reassessed. During hyperinsulinemia, vasodilator responses to both ACh and SNP were blunted in patients with MetS (both P&gt;0.001 vs. controls). In patients who received fasudil, its administration did not change unstimulated FBF (P=0.75 vs. insulin alone); the vasodilator response to ACh, however, was significantly enhanced by fasudil (P=0.009 vs. insulin alone), while the response to SNP was not significantly changed (P=0.56). In patients with MetS who received placebo, vascular reactivity to both ACh and SNP was not different than before (both P&gt;0.05). In conclusion, Rho-kinase inhibition during hyperinsulinemia improves endothelium-dependent vasodilator responsiveness in patients with MetS. This suggests that, under those conditions, intravascular activation of Rho-kinase is involved in the pathophysiology of endothelial dysfunction and may constitute a critical mediator linking metabolic and hemodynamic abnormalities in insulin resistance. As a consequence, targeting Rho-kinase might beneficially impact both vascular function and insulin sensitivity in patients with MetS.

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.

Rho-kinase signalling mediates endotoxin hypersensitivity after partial hepatectomy

Excessive loss of functional liver mass results in hepatic dysfunction and is associated with an increased sensitivity to infection. This experimental study investigated the role of Rho-kinase in hepatectomy-induced sensitization to endotoxin. Male C57BL/6J mice underwent 68 per cent hepatectomy and were injected 24 h later with 100 microg Escherichia coli lipopolysaccharide (LPS). Simultaneously, animals received either fasudil or Y-27632 for Rho-kinase inhibition, or phosphate-buffered saline. Untreated hepatectomized animals served as positive controls and sham-operated animals as negative controls. Liver injury and inflammatory parameters were assessed 6 h after LPS challenge by serum alanine aminotransferase (ALT) levels, histomorphology and enzyme-linked immunosorbent assay. Hepatectomy resulted in a significant susceptibility to LPS, as indicated by inflammatory leucocyte recruitment (mean(s.e.m.) 10(1) leucocytes per high-power field), hepatocellular disintegration (ALT 22.4(3.1) microkat/l) and apoptotic cell death (3.8(0.2) per cent). Rho-kinase inhibition reduced leucocytic infiltration by more than 33 per cent, abolished hepatocellular apoptosis entirely, and reduced tumour necrosis factor alpha expression by more than 48 per cent and CXC chemokine expression by more than 36 per cent. Hepatectomy increased susceptibility to LPS by Rho-kinase-dependent mechanisms. Blocking Rho-kinase signalling decreased LPS-induced liver injury in hepatectomized mice.

Effect of Age on the Role of Rho-Kinase in Short-term Partial Bladder Outlet Obstruction

We examined the expression of Rho-kinase (ROK) isoforms in young and old rabbits' detrusor smooth muscles (SM) during the progression of short-term partial bladder outlet obstruction and correlated them with the time course of obstruction. We obtained detrusor samples from bladders after 1, 3, 7, and 14 days of obstruction and also sham-operated control rabbits. We used reverse transcriptase-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting to determine the relative levels of ROK isoform expression at the mRNA and protein levels. Bladder weight for young rabbits increased between 1 and 7 days' obstruction and came back toward control levels at 14 days' obstruction. In old rabbits, bladder weight increased after obstruction, reaching a maximum at 3 days and remaining at this level throughout the 14 days. In young rabbits, the expression of ROKalpha increased in 1- to 7-day obstructed groups and decreased in the 14-day group, whereas it increased progressively in the old rabbits at both the mRNA and protein levels. There was a significant decrease in the expression of ROKbeta in young obstructed rabbits, which gradually decreased during the course of 1- to 7-day obstruction period and increased after 14 days of obstruction. In old groups, there was a decrease in expression after 1 day of obstruction and values remained at a decreased level throughout the course of the study. Young rabbit bladders are better able to adapt to bladder outlet obstruction and ROK isoforms respond in a similar way.

Rho kinase and Ca2+ entry mediate increased pulmonary and systemic vascular resistance in l-NAME-treated rats

The small GTP-binding protein and its downstream effector Rho kinase play an important role in the regulation of vasoconstrictor tone. Rho kinase activation maintains increased pulmonary vascular tone and mediates the vasoconstrictor response to nitric oxide (NO) synthesis inhibition in chronically hypoxic rats and in the ovine fetal lung. However, the role of Rho kinase in mediating pulmonary vasoconstriction after NO synthesis inhibition has not been examined in the intact rat. To address this question, cardiovascular responses to the Rho kinase inhibitor fasudil were studied at baseline and after administration of an NO synthesis inhibitor. In the intact rat, intravenous injections of fasudil cause dose-dependent decreases in systemic arterial pressure, small decreases in pulmonary arterial pressure, and increases in cardiac output. L-NAME caused a significant increase in pulmonary and systemic arterial pressures and a decrease in cardiac output. The intravenous injections of fasudil after L-NAME caused dose-dependent decreases in pulmonary and systemic arterial pressure and increases in cardiac output, and the percent decreases in pulmonary arterial pressure in response to the lower doses of fasudil were greater than decreases in systemic arterial pressure. The Ca(++) entry blocker isradipine also decreased pulmonary and systemic arterial pressure in L-NAME-treated rats. Infusion of sodium nitroprusside restored pulmonary arterial pressure to baseline values after administration of L-NAME. These data provide evidence in support of the hypothesis that increases in pulmonary and systemic vascular resistance following L-NAME treatment are mediated by Rho kinase and Ca(++) entry through L-type channels, and that responses to L-NAME can be reversed by an NO donor.

Endogenous RhoA Inhibitor Protects Endothelial Barrier

See related article, pages 50–58 Vascular leakage is a hallmark of many, often life-threatening, inflammatory diseases, and contributes to disease severity in disorders such as sepsis, cancer, diabetes, and atherosclerosis.1 Despite the tremendous medical importance of vascular leakage, few specific therapies are available today to counteract it, and current therapies often fail to do so.2 This is in part because the in vivo molecular targets are incompletely identified, although a wealth of data obtained from in vitro studies is available on signal transduction pathways that regulate vascular permeability.3,4 Interestingly, among various new agents that potentially reduce endothelial hyperpermeability, the cholesterol-lowering statin drugs have been proposed to reduce vascular leakage by means of their inhibitory effects on RhoA proteins.5,6 In the current issue of Circulation Research Gorovoy et al provide an elegant proof-of-principle that increased RhoA activity results in vascular hyperpermeability in vivo.7 They show that elevation of RhoA activity by deletion of one of its inhibitory proteins, RhoGDI, causes a loss of endothelial junctional integrity and decrease in vascular barrier function. Rho GTPases, particularly RhoA, Rac-1 and cdc42 have received much attention as key regulators of cell shape, movement and proliferation. In vitro studies have shown that the balance of activities of these small G proteins determines the status of endothelial barrier8: Cdc42 enhancing recovery of a disturbed barrier,9 Rac-1 being required for establishing a tight barrier,8 and RhoA being involved in inducing endothelial hyperpermeability by various stimuli, such as thrombin, VEGF, angiopoietin-2 and LPA (see Figure).10–12 Similarly, these Rho GTPases are acting …

983: Long Term Partial Bladder Outlet Obstrcuction Induced Contractile Dysfunction in Male Rabbits: A Role for Rho-Kinase

983: Long Term Partial Bladder Outlet Obstrcuction Induced Contractile Dysfunction in Male Rabbits: A Role for Rho-Kinase