Selective Rho Kinase Inhibitor Y-27632 Research Articles

Anesthetic Sevoflurane Causes Rho-Dependent Filopodial Shortening in Mouse Neurons.

Early postnatal anesthesia causes long-lasting learning and memory impairment in rodents, however, evidence for a specific neurotoxic effect on early synaptogenesis has not been demonstrated. Drebrin A is an actin binding protein whose localization in dendritic protrusions serves an important role in dendritic spine morphogenesis, and is a marker for early synaptogenesis. We therefore set out to investigate whether clinically-relevant concentrations of anesthetic sevoflurane, widely- used in infants and children, alters dendritic morphology in cultured fetal day 16 mouse hippocampal neurons. After 7 days in vitro, mouse hippocampal neurons were exposed to four hours of 3% sevoflurane in 95% air/5% CO2 or control condition (95% air/5% CO2). Neurons were fixed in 4% paraformaldehyde and stained with Alexa Fluor555-Phalloidin, and/or rabbit anti-mouse drebrin A/E antibodies which permitted subcellular localization of filamentous (F)-actin and/or drebrin immunoreactivity, respectively. Sevoflurane caused acute significant length-shortening in filopodia and thin dendritic spines in days-in-vitro 7 neurons, an effect which was completely rescued by co-incubating neurons with ten micromolar concentrations of the selective Rho kinase inhibitor Y27632. Filopodia and thin spine recovered in length two days after sevoflurane exposure. Yet cluster-type filopodia (a precursor to synaptic filopodia) were persistently significantly decreased in number on day-in-vitro 9, in part owing to preferential localization of drebrin immunoreactivity to dendritic shafts versus filopodial stalks. These data suggest that sevoflurane induces F-actin depolymerization leading to acute, reversible length-shortening in dendritic protrusions through a mechanism involving (in part) activation of RhoA/Rho kinase signaling and impairs localization of drebrin A to filopodia required for early excitatory synapse formation.

Expression and function of c-Rel in choriocarcinoma cells

Both villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown.IL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used.We found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR1, induced IL-8 gene expression but not protein secretion. The specific S1PR2 inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR1/S1PR3 antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells.In this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR1 and its secretion is regulated through S1PR2 receptor subtype. Rho GTPases signaling is essential for S1P-induced IL-8 secretion.

Sphingosine 1-phosphate regulates IL-8 expression and secretion via S1PR1 and S1PR2 receptors-mediated signaling in extravillous trophoblast derived HTR-8/SVneo cells

IntroductionBoth villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown. MethodsIL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used. ResultsWe found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR1, induced IL-8 gene expression but not protein secretion. The specific S1PR2 inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR1/S1PR3 antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells. DiscussionIn this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR1 and its secretion is regulated through S1PR2 receptor subtype. Rho GTPases signaling is essential for S1P-induced IL-8 secretion.

Antitumoral effect of a selective Rho-kinase inhibitor Y-27632 against Ehrlich ascites carcinoma in mice

BackgroundThe Rho proteins and Rho-kinase (ROCK) enzymes are responsible for signal transduction, and cause cell permeability, contractility, differentiation, migration, proliferation or apoptosis depending on cell types. All of these functions are vital for cancer initiation and progression. In this study, the preventive and protective effects of a selective ROCK inhibitor Y-27632 against Ehrlich ascites carcinoma in Swiss albino mice were investigated. MethodsAdult male albino mice were divided into five equal groups, and Y-27632 (0.1, 1, and 10mg/kg) was given to groups as two steps; before (pre-carcinoma) and after inoculation of carcinoma cell suspensions (post-carcinoma). At the end of the experiments (at day 15), cardiac blood samples, the ascitic fluid, and intestinal specimens were collected for histopathology and biochemical investigation. ResultsSignificant decreases in the body weight and immunostaining scores in small and large intestine for ROCK2, preservation of serum glutathione (GSH) levels, and an increase in tumor level of nitric oxide were recorded in groups pretreated with Y-27632. However, treatment with Y-27632 after tumor inoculation did not affect body weight and ROCK2 immunostaining scores, increased serum MDA levels, and decreased GSH levels. ConclusionsThis is the first study on the effectiveness of Y-27632 in this experimental tumor model. Our findings provided direct evidence for ROCK involvement in tumor development. These data suggest that pretreatment with Y-27632 has a protective effect against tumor formation.

Anti-Neurotrophic Effects from Autoantibodies in Adult Diabetes Having Primary Open Angle Glaucoma or Dementia

Aim: To test for anti-endothelial and anti-neurotrophic effects from autoantibodies in subsets of diabetes having open-angle glaucoma, dementia, or control subjects.Methods: Protein-A eluates from plasma of 20 diabetic subjects having glaucoma or suspects and 34 age-matched controls were tested for effects on neurite outgrowth in rat pheochromocytoma PC12 cells or endothelial cell survival. The mechanism of the diabetic glaucoma autoantibodies’ neurite-inhibitory effect was investigated in co-incubations with the selective Rho kinase inhibitor Y27632 or the sulfated proteoglycan synthesis inhibitor sodium chlorate. Stored protein-A eluates from certain diabetic glaucoma or dementia subjects which contained long-lasting, highly stable cell inhibitory substances were characterized using mass spectrometry and amino acid sequencing.Results: Diabetic primary open angle glaucoma (POAG) or suspects (n = 20) or diabetic dementia (n = 3) autoantibodies caused significantly greater mean inhibition of neurite outgrowth in PC12 cells (p < 0.0001) compared to autoantibodies in control diabetic (n = 24) or non-diabetic (n = 10) subjects without glaucoma (p < 0.01). Neurite inhibition by the diabetic glaucoma autoantibodies was completely abolished by 10 μM concentrations of Y27632 (n = 4). It was substantially reduced by 30 mM concentrations of sodium chlorate (n = 4). Peak, long-lasting activity survived storage ×5 years at 0–4°C and was associated with a restricted subtype of Ig kappa light chain. Diabetic glaucoma or dementia autoantibodies (n = 5) caused contraction and process retraction in quiescent cerebral cortical astrocytes effects which were blocked by 5 μM concentrations of Y27632.Conclusion: These data suggest that autoantibodies in subsets of adult diabetes having POAG (glaucoma suspects) and/or dementia inhibit neurite outgrowth and promote a reactive astrocyte morphology by a mechanism which may involve activation of the RhoA/p160 ROCK signaling pathway.

The Uremic Retention Solute p-Cresyl Sulfate and Markers of Endothelial Damage

Cardiovascular disease is highly prevalent in patients with chronic kidney disease. In hemodialysis patients, the protein-bound uremic retention solute p-cresol is independently associated with cardiovascular disease. The underlying mechanisms have not been elucidated. (1) Prospective observational study of humans and (2) in vitro study in human umbilical vein endothelial cells. Hemodialysis patients. p-Cresol and its main derivative p-cresyl sulfate. Endothelial dysfunction. We studied: (1) the relation between p-cresol and blood markers of endothelial dysfunction, including soluble P-selectin and endothelial microparticles; and (2) direct effects of p-cresol and p-cresyl sulfate on endothelial cell cultures. (1) In a cohort of 100 maintenance hemodialysis patients, free serum p-cresol concentrations (median, 11.7 micromol/L; interquartile range, 15.2) were directly associated with circulating endothelial microparticles (P = 0.007), but not with soluble P-selectin (mean, 37.7 +/- 14.4 [SD] pg/mL). Other independent determinants of the degree of circulating microparticles were greater serum phosphorus (mean, 4.8 +/- 1.5 mg/dL; P = 0.008) and serum calcium concentrations (mean, 9.3 +/- 0.8 mg/dL; P = 0.03), whereas treatment with active vitamin D (P = 0.008) and vintage (median, 25 months; P = 0.04) were inversely associated. (2) In vitro, p-cresyl sulfate induced a dose-dependent increase in the shedding of endothelial microparticles (P < 0.001) by human umbilical vein endothelial cells. Shedding was reduced, but not completely aborted, in the presence of albumin, whereas the selective Rho kinase inhibitor Y-27632 abrogated the p-cresyl sulfate-induced generation of endothelial microparticles. The relationship between p-cresyl sulfate and shedding of endothelial microparticles in vivo was not mechanistically explored. p-Cresyl sulfate induces shedding of endothelial microparticles in the absence of overt endothelial damage in vitro and is independently associated with the number of endothelial microparticles in hemodialysis patients. These findings suggest that p-cresyl sulfate alters endothelial function in hemodialysis patients.

Sevoflurane, but Not Propofol, Prevents Rho Kinase-Dependent Contraction Induced by Sphingosylphosphorylcholine in the Porcine Coronary Artery

Sphingosylphosphorylcholine may induce coronary vasospasm by the activation of Rho kinase. We designed the current study to examine the differential effects of anesthetics on Rho kinase activation induced by sphingosylphosphorylcholine in porcine coronary arteries. Rings of porcine coronary artery without endothelium were prepared in tissue bath containing modified Krebs-Ringer bicarbonate solution. Using isometric force recording, concentration-response curves in response to sphingosylphosphorylcholine were obtained in the absence or in the presence of sevoflurane, propofol, or a selective Rho kinase inhibitor Y27632, which was added 15 min before the application of sphingosylphosphorylcholine. Intracellular translocation of Rho A toward the plasma membrane and phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase were also evaluated by Western blotting. Sphingosylphosphorylcholine (10(-7) to 10(-5) M) produced contraction of the porcine coronary artery, which was abolished by a selective Rho kinase inhibitor Y27632 (2 x 10(-6) M). Sevoflurane (1.7%) reduced sphingosylphosphorylcholine-induced coronary artery constriction, and the higher concentration (3.4%) abolished it (P < 0.05). In contrast, propofol (3 x 10(-6) M and 10(-5) M) had no effect on coronary artery constriction due to sphingosylphosphorylcholine. Sevoflurane, but not propofol, reduced intracellular translocation of Rho A toward the plasma membrane. Sevoflurane and Y27632, but not propofol, similarly decreased (64.4% or 70.8% reduction, respectively, P < 0.05) phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase. Sphingosylphosphorylcholine induces coronary vasocontriction via activation of Rho kinase. Sevoflurane, but not propofol, inhibits this pathway, resulting in prevention of vasoconstriction.

A Rho-kinase inhibitor, Y-27632, reduces cholinergic contraction but not neurotransmitter release

This study examined the effects of the selective Rho-kinase inhibitor Y-27632 [(+)-( R)- trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) on cholinergic nerve-mediated contraction and neurotransmitter release in murine and guinea-pig isolated tracheal preparations. In tracheal preparations obtained from both species, Y-27632 shifted carbachol concentration–effect curves to the right and reduced the maximal contractile response. Repeated electrical field stimulation (EFS) evoked transient, consistent and reproducible contractions in murine and guinea-pig tracheal preparations. Y-27632 inhibited these cholinergic nerve-mediated contractions in a concentration-dependent manner. EFS (0.1–30 Hz) elicited frequency-dependent cholinergic nerve-mediated contractile responses. In murine tracheal preparations, Y-27632 (3 μM and 10 μM) shifted frequency–response curves to EFS to the right by 5.5 and 13.0 fold respectively and markedly reduced the maximal contractile response. In murine and guinea-pig tracheal preparations loaded with [ 3H]-choline, Y-27632 (10 μM) significantly increased the EFS-induced outflow of radioactivity from airway cholinergic nerves by 27% and 54% respectively. Thus, Y-27632 inhibited both carbachol-induced and cholinergic nerve-mediated contractile responses. Conversely, Y-27632 increased neurotransmitter release from airway cholinergic nerves. However, since antagonism of acetylcholine-induced contraction by Y-27632 overwhelmed the increased neurotransmitter release, the overall effect of this Rho-kinase inhibitor was to inhibit cholinergic nerve-mediated contraction.

Differential Rho‐kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.

Tubuloglomerular feedback-dependent modulation of renal myogenic autoregulation by nitric oxide

Nonselective inhibition of nitric oxide (NO) synthase (NOS) augments myogenic autoregulation, an action that implies enhancement of pressure-induced constriction and dilatation. This pattern is not explained solely by interaction with a vasoconstrictor pathway. To test involvement of the Rho-Rho kinase pathway in modulation of autoregulation by NO, the selective Rho kinase inhibitor Y-27632 and/or the NOS inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) were infused into the left renal artery of anesthetized rats. Y-27632 and l-NAME were also infused into isolated, perfused hydronephrotic kidneys to assess myogenic autoregulation over a wide range of perfusion pressure. In vivo, l-NAME reduced renal vascular conductance and augmented myogenic autoregulation, as shown by increased slope of gain reduction and associated phase peak in the pressure-flow transfer function. Y-27632 (10 mumol/l) strongly dilated the renal vasculature and profoundly inhibited autoregulation in the absence or presence of l-NAME in vivo and in vitro. Afferent arteriolar constriction induced by 30 mmol/l KCl was reversed (-92 +/- 3%) by Y-27632. Phenylephrine caused strong renal vasoconstriction but did not affect autoregulation. Inhibition of neuronal NOS by N(5)-(1-imino-3-butenyl)-l-ornithine (l-VNIO) did not cause significant vasoconstriction but did augment myogenic autoregulation. Thus vasoconstriction is neither necessary (l-VNIO) nor sufficient (phenylephrine) to explain the augmented myogenic autoregulation induced by l-NAME. The effect of l-VNIO implicates tubuloglomerular feedback (TGF) and neuronal NOS at the macula densa in regulation of the myogenic mechanism. This conclusion was confirmed by the demonstration that systemic furosemide removed the TGF signature from the pressure-flow transfer function and significantly inhibited myogenic autoregulation. In the presence of furosemide, augmentation of myogenic autoregulation by l-NAME was significantly reduced. These results provide a potential mechanism to explain interaction between myogenic and TGF-mediated autoregulation.

Short Synthesis of the Selective Rho-Kinase Inhibitor Y-27632 and of Its Enantiomer

Y-27632 and its enantiomer were synthesized in 8 steps from trans-1,4-cyclohexanedicarboxylic acid by using a diastereoselective reductive amination of a ketone. Keywords: kinase inhibitor, rho-associated kinase, reductive amination

Abstracts to be presented at the 12th Congress of the Japan Placenta Association 18-19 November 2004, Beppu City, Japan

Both villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown.IL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used.We found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR1, induced IL-8 gene expression but not protein secretion. The specific S1PR2 inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR1/S1PR3 antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells.In this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR1 and its secretion is regulated through S1PR2 receptor subtype. Rho GTPases signaling is essential for S1P-induced IL-8 secretion.

Astrocyte stellation induced by Rho kinase inhibitors in culture

To understand the role of Rho kinases in regulation of astrocyte morphology, we investigated the effects of Rho kinase inhibitors on the morphology of cultured rat cortical astrocytes. Cultured astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but changed into process-bearing stellate cells following treatment with the selective Rho kinase inhibitor Y-27632 (1–10 μM). The Y-27632-induced astrocyte stellation was abolished by treatment with colchicine, indicating that the response requires reorganization of cytoskeletal elements. The effect of Y-27632 was mimicked by another Rho kinase inhibitor HA1077, but not by the protein kinase C inhibitor GF-109203X or the protein kinase A inhibitor KT5720. These results suggest that Rho kinases are in an activated state in the absence of stimuli and contribute to the maintenance of polygonal morphology of cultured astrocytes.

Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway

Urokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.

Circumferential stretching of saphenous vein smooth muscle enhances vasoconstrictor responses by Rho kinase-dependent pathways.

Surgical preparation and/or pulsatile arterial perfusion of saphenous vein increases the sensitivity of vein rings to calcium mobilising agonists such as phenylephrine. We have investigated the mechanism(s) underlying this effect. We have used an ex vivo flow circuit, with simulated arterial or venous flows (mean pressure 100 and 20 mmHg, respectively), to investigate the sensitivity of human saphenous vein to phenylephrine, 5-hydroxytryptamine (5-HT) and KCl, using organ chamber pharmacology. After 90 min of pulsatile arterial perfusion the mean maximum tension induced by KCl had increased from 4.7 to 11.1 g (n=5), by phenylephrine had increased from 4.4 to 10.2 g (n=8) and by 5-HT had increased from 4.4 to 6.7 g (n=10), all P<0.01. Phenylephrine did not augment the tension in vein rings maximally precontracted with KCl (n=4). The EC(50) for KCl was unchanged after pulsatile arterial perfusion (n=5), but for phenylephrine and 5-HT there were significant reductions from 14+/-5 to 2+/-1 microM (n=8) and from 1.0+/-0.4 to 0.20+/-0.06 microM (n=10), respectively. The rate of contraction (in response to 3 microM phenylephrine) increased from 0.11 g/min to 0.37 g/min, P<0.02, after arterial perfusion (n=4). These changes in contractile properties (to phenylephrine) were endothelium-independent, evident within 5 min of simulated arterial perfusion. The changes in contractile properties could be abrogated by external stenting of the vein (to attenuate circumferential deformation) or inclusion in the perfusate of a vasodilator, e.g., cromakalim (5 microM) or the selective Rho kinase inhibitor Y-27632 (20 microM). The heightened sensitivity and contractility to phenylephrine was maintained after inclusion of adenosine (100 microM), gadolinium (10 microM) or cycloheximide (10 microM) in the vein perfusate. The circumferential deformations imposed by simulated arterial perfusion alter the vasomotor responses of saphenous vein smooth muscle. These effects are independent of new protein synthesis or the activation of stretch activated cation channels. The Rho kinase pathway appears to mediate the signalling mechanisms leading to increased agonist-induced tension and the increased sensitivity to vasoconstrictors.

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.

Kinases, myosin phosphatase and Rho proteins: curiouser and curiouser.

Kinases, myosin phosphatase and Rho proteins: curiouser and curiouser.