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Rho Kinase (rho + kinase)
Terms modified by Rho Kinase Selected AbstractsMechanistic studies of blood pressure in rats treated with a series of cholesteryl ester transfer protein inhibitors,DRUG DEVELOPMENT RESEARCH, Issue 1 2009Michael DePasquale Abstract ILLUMINATE, the Phase 3 clinical trial of morbidity and mortality (M&M) with the cholesteryl ester transfer protein inhibitor (CETPi), torcetrapib (CP-529,414), was terminated in December 2006 due to an imbalance in all cause mortality. The underlying cause of the M&M remains undetermined. While torcetrapib produced dose-related increases in blood pressure in clinical trials, the mechanism of the increase in blood pressure is also undetermined. The pressor effects of torcetrapib and structurally related compounds were studied in several pathways involved in blood pressure control. Studies were conducted in rats treated with a series of structurally related molecules (CP-529,414, CP-532,623, PF-868,348, CP-746,281, CP-792,485, PF-868,343, and CE-308,958). CP-529,414, CP-532,623, CP-868,343, and CP-792,485 are potent CETP inhibitors; PF-868,348 is weakly potent and CP-746,281 and CE-308,958 are CETP-inactive. Changes in blood pressure were determined in conscious animals in conjunction with pharmacologic blockade of numerous pressor agents/pathways. Torcetrapib and CP-532,623 increased blood pressure following both chronic PO and acute IV administration. The CETP-inactive enantiomer of CP-532,623, CP-746,281 failed to raise blood pressure. PF-868,348, a structural analogue with ,50-fold lower CETPi activity also displayed pressor activity. Blockade of adrenergic, cholinergic, angiotensin, endothelin, NOS, Rho kinase, and thromboxane pathways failed to attenuate the pressor response. These data demonstrate that the blood pressure activity seen with torcetrapib can be dissociated from CETP inhibitor pharmacology and numerous pharmacology pathways can be discounted in the attempt to understand the molecular basis of the pressor pharmacology. Drug Dev Res 70:2009 © 2009 Wiley-Liss, Inc. [source] Angiotensin-(1,7) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stimulating STAT3 and STAT5a/b phosphorylation and inhibiting angiotensin II-stimulated ERK1/2 and Rho kinase activityEXPERIMENTAL PHYSIOLOGY, Issue 5 2008Jorge F. Giani Angiotensin (ANG) II contributes to cardiac remodelling by inducing the activation of several signalling molecules, including ERK1/2, Rho kinase and members of the STAT family of proteins. Angiotensin-(1,7) is produced in the heart and inhibits the proliferative actions of ANG II, although the mechanisms of this inhibition are poorly understood. Accordingly, in the present study we examined whether ANG-(1,7) affects the ANG II-mediated activation of ERK1/2 and Rho kinase, STAT3 and STAT5a/b in rat heart in vivo. We hypothesized that ANG-(1,7) inhibits these growth-promoting pathways, counterbalancing the trophic action of ANG II. Solutions of normal saline (0.9% NaCl) containing ANG II (8 pmol kg,1) plus ANG-(1,7) in increasing doses (from 0.08 to 800 pmol kg,1) were administered via the inferior vena cava to anaesthetized male Sprague,Dawley rats. After 5 min, hearts were removed and ERK1/2, Rho kinase, STAT3 and STAT5a/b phosphorylation was determined by Western blotting using phosphospecific antibodies. Angiotensin II stimulated ERK1/2 and Rho kinase phosphorylation (2.3 ± 0.2- and 2.1 ± 0.2-fold increase over basal values, respectively), while ANG-(1,7) was without effect. The ANG II-mediated phosphorylation of ERK1/2 and Rho kinase was prevented in a dose-dependent manner by ANG-(1,7) and disappeared in the presence of the Mas receptor antagonist d -Ala7 -ANG-(1,7). Both ANG II and ANG-(1,7) increased STAT3 and STAT5a/b phosphorylation to a similar extent (130,140% increase). The ANG-(1,7)-stimulated STAT phosphorylation was blocked by the AT1 receptor antagonist losartan and not by d -Ala7 -ANG-(1,7). Our results show a dual action of ANG-(1,7), that is, a stimulatory effect on STAT3 and 5a/b phosphorylation through AT1 receptors and a blocking action on ANG II-stimulated ERK1/2 and Rho kinase phosphorylation through Mas receptor activation. The latter effect could be representative of a mechanism for a protective role of ANG-(1,7) in the heart by counteracting the effects of locally generated ANG II. [source] Simvastatin regulates oligodendroglial process dynamics and survivalGLIA, Issue 2 2007Veronique E. Miron Abstract Simvastatin, a lipophilic statin that crosses the blood-brain barrier, is being evaluated as a potential therapy for multiple sclerosis (MS) due to its anti-inflammatory properties. We assessed the effects of simvastatin on cultures of rat newborn and human fetal oligodendrocyte progenitor cells (OPCs) and human adult mature oligodendrocytes (OLGs) with respect to cellular events pertaining to myelin maintenance and repair. Short-term simvastatin treatment of OPCs (1 day) induced robust process extension, enhanced differentiation to a mature phenotype, and decreased spontaneous migration. These effects were reversed by isoprenoid products and mimicked with an inhibitor of Rho kinase (ROCK), the downstream effector of the isoprenylated protein RhoA GTPase. Prolonged treatment (2 days) caused process retraction that was rescued by cholesterol, and increased cell death (4 days) partially rescued by either cholesterol or isoprenoid co-treatment. In comparison, simvastatin treatment of human mature OLGs required a longer initial time course (2 days) to induce significant process outgrowth, mimicked by inhibiting ROCK. Prolonged treatment of mature OLGs was associated with process retraction (6 days) and increased cell death (8 days). Human-derived OPCs and mature OLGs demonstrated an increased sensitivity to simvastatin relative to the rodent cells, responding to nanomolar versus micromolar concentrations. Our findings indicate the importance of considering the short- and long-term effects of systemic immunomodulatory therapies on neural cells affected by the MS disease process. © 2006 Wiley-Liss, Inc. [source] RhoA/ROK Pathway Related to the Mechanism of Higher Susceptibility to Spasm in RA Than in IMAJOURNAL OF CARDIAC SURGERY, Issue 6 2009Xia Kun M.D. Methods: RA, IMA, and GSV that would otherwise have been discarded were collected from 25 patients who underwent coronary artery bypass grafting. Eleven matched rings of RA, IMA, and GSV were used to evaluate the vasodilatory properties of 10,7,10,4 mol/l of fasudil, a Rho-kinase inhibitor, by using in vitro organ chambers. Another 14 matched RA, IMA, and GSV were used to demonstrate the immunohistochemistry (IHC) of RhoA and mRNA of RhoA and Rho kinase. Results: The maximal vasodilation of RA to fasudil was significantly greater than IMA. RhoA protein IHC staining was different in IMA, RA, and GSV (RA > GSV >IMA). The expression of RhoA and Rho kinase mRNA in the RA was significantly greater than in the IMA. Conclusions: The expression of RhoA/Rho kinase mRNA and protein and function in the RA were significantly stronger than in the IMA, suggesting that RhoA/Rho kinase pathway may be one mechanism by which RA is more susceptible to spasm than IMA. Rho kinase inhibitors can be effective drug candidates to prevent and treat vasospasm. [source] Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdownJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Y. Baumer Barrier stabilizing effects of cAMP as well as of the small GTPase Rac 1 are well established. Moreover, it is generally believed that permeability-increasing mediators such as thrombin disrupt endothelial barrier functions primarily via activation of Rho A. In this study, we provide evidence that decrease of both cAMP levels and of Rac 1 activity contribute to thrombin-mediated barrier breakdown. Treatment of human dermal microvascular endothelial cells (HDMEC) with Rac 1-inhibitor NSC-23766 decreased transendothelial electrical resistance (TER) and caused intercellular gap formation. These effects were reversed by addition of forskolin/rolipram (F/R) to increase intracellular cAMP but not by the cAMP analogue 8-pCPT-2,-O-Methyl-cAMP (O-Me-cAMP) which primarily stimulates protein kinase A (PKA)-independent signaling via Epac/Rap 1. However, both F/R and O-Me-cAMP did not increase TER above control levels in the presence of NSC-23766 in contrast to experiments without Rac 1 inhibition. Because Rac 1 was required for maintenance of barrier functions as well as for cAMP-mediated barrier stabilization, we tested the role of Rac 1 and cAMP in thrombin-induced barrier breakdown. Thrombin-induced drop of TER and intercellular gap formation were paralleled by a rapid decrease of cAMP as revealed by fluorescence resonance energy transfer (FRET). The efficacy of F/R or O-Me-cAMP to block barrier-destabilizing effects of thrombin was comparable to Y27632-induced inhibition of Rho kinase but was blunted when Rac 1 was inactivated by NSC-23766. Taken together, these data indicate that decrease of cAMP and Rac 1 activity may be an important step in inflammatory barrier disruption. J. Cell. Physiol. 220: 716,726, 2009. © 2009 Wiley-Liss, Inc. [source] Rho kinase activates ezrin-radixin-moesin (ERM) proteins and mediates their function in cortical neuron growth, morphology and motility in vitroJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2007Matilda A. Haas Abstract The ezrin-radixin-moesin (ERM) family of proteins contribute to cytoskeletal processes underlying many vital cellular functions. Their previously elucidated roles in non-neuronal cells are an indication of their potential importance in CNS neurons. The specific mechanisms of their activation are unknown, but are likely to depend on factors such as the cell type and biological context. For ERM proteins to become active they must be phosphorylated at a specific C-terminal threonine residue. In non-neuronal cells, several kinases, including the Rho GTPase family member Rho kinase, have been identified as capable of phosphorylating the C-terminal threonine. In these experiments we have investigated specifically the potential role of Rho kinase mediated ERM activation in cortical neurons, utilizing a new pharmacologic inhibitor of Rho kinase and quantitative analysis of aspects of neuronal functions potentially mediated by ERM proteins. Rho kinase inhibition significantly suppressed aspects of neuronal development including neurite initiation and outgrowth, as well as growth cone morphology, with a concomitant loss of phosphorylated ERM immunolabeling in areas associated with neuronal growth. The ability of the Rho kinase inhibitor to decrease the amount of pERM protein was shown by immunoblotting. Post-injury responses were negatively affected by Rho kinase inhibition, namely by a significant decrease in the number of regenerative neurites. We investigated a novel role for ERM proteins in neuron migration using a post-injury motility assay, where Rho kinase inhibition resulted in significant and drastic reduction in neuron motility and phosphorylated ERM immunolabeling. Thus, Rho kinase is an important activator of ERMs in mediating specific neuronal functions. © 2006 Wiley-Liss, Inc. [source] The cytoplasmic tail of the ,3 integrin subunit promotes neurite outgrowth in PC12 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Nadja Mechai Abstract Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein,protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the ,3 integrin subunit of ,3,1 integrin is required for ,3,1 integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) , subunit and the cytoplasmic sequence of the ,3 integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic ,3 tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic ,3 sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-,3 can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase ,. © 2005 Wiley-Liss, Inc. [source] c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aortaTHE JOURNAL OF PHYSIOLOGY, Issue 16 2008Rong Lu Serotonin (5-hydroxytryptamine, 5-HT) receptors (5-HTRs) play critical roles in brain and cardiovascular functions. In the vasculature, 5-HT induces potent vasoconstrictions, which in aorta are mainly mediated by activation of the 5-HT2AR subtype. We previously proposed that one signalling mechanism of 5-HT-induced vasoconstriction could be c-Src, a member of the Src tyrosine kinase family. We now provide evidence for a central role of c-Src in 5-HT2AR-mediated contraction. Inhibition of Src kinase activity with 10 ,m 4-amino-5-(4-chlorophenyl)-7-(t -butyl)pyrazolo[3,4-d]pyrimidine (PP2) prior to contraction resulted in ,90,99% inhibition of contractions induced by 5-HT or by ,-methyl-5-HT (5-HT2R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A2 mimetic, U46619, and had no significant action on phenylephrine-induced contractions. 5-Hydroxytryptamine increased Src kinase activity and PP2-sensitive tyrosine-phosphorylated proteins. As expected for c-Src identity, PP2 pretreatment inhibited 5-HT-induced contraction with an IC50 of ,1 ,m. Ketanserin (10 nm), a 5-HT2A antagonist, but not antagonists of 5-HT2BR (100 nm SB204741) or 5-HT2CR (20 nm RS102221), prevented 5-HT-induced contractions, mimicking PP2 and implicating 5-HT2AR as the major receptor subtype coupled to c-Src. In HEK 293T cells, c-Src and 5-HT2AR were reciprocally co-immunoprecipitated and co-localized at the cell periphery. Finally, 5-HT-induced Src activity was unaffected by inhibition of Rho kinase, supporting a role of c-Src upstream of Rho kinase. Together, the results highlight c-Src activation as one of the early and pivotal mechanisms in 5-HT2AR contractile signalling in aorta. [source] cGMP-enhancing- and ,1A/,1D -adrenoceptor blockade-derived inhibition of Rho-kinase by KMUP-1 provides optimal prostate relaxation and epithelial cell anti-proliferation efficacyTHE PROSTATE, Issue 13 2007Chi-Ming Liu Abstract Background Soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) and Rho kinase (ROCK2) pathways are important in the regulation of prostate smooth muscle tone. This study is aimed to examine the relaxation activities of a sGC activator and PDE5A/ROCK2 inhibitor KMUP-1 in rat prostate and associated anti-proliferation activity in human prostatic epithelial cells. Methods The action characteristics of KMUP-1 were identified by isometric tension measurement, receptor binding assay, Western blotting and radioimmunoassay in rat prostate. Anti-proliferation activity of KMUP-1 in human prostatic epithelial PZ-HPV-7 cells was identified using flow cytometry and real time QRT-PCR. Results KMUP-1 inhibited phenylephrine-induced contractility in a concentration-dependent manner. KMUP-1 possessed potent ,1A/,1D -adrenoceptor binding inhibition activity, increased cAMP/cGMP levels and increased the expression of sGC, PKG, and PKA protein in rat prostate. Moreover, KMUP-1 inhibited phenylephrine-induced ROCK2 expression. KMUP-1 inhibited cell growth, arrested the cell cycle at G0/G1 phase and increased the expression of p21 in PZ-HPV-7 cells. Conclusions These results broaden our knowledge of sGC/cGMP/PKG and ROCK2 regulation on the relaxation and proliferation of prostate, which may help in the design of benign prostate hyperplasia (BPH) therapies that target these signaling pathways. KMUP-1 possesses the potential benefit in the treatment of BPH by its ,1A/,1D -adrenoceptor blockade, sGC activation, inhibition of PDE5A and ROCK2 and p21 protein enhancement, leading to attenuation of the smooth muscle tone and the proliferation of epithelial PZ-HPV-7 cells. The synergistic contribution of these pathways by KMUP-1 may benefit BPH patients with lower urinary tract symptoms. Prostate 67: 1397,1410, 2007. © 2007 Wiley-Liss, Inc. [source] Mast cell,derived tryptase inhibits apoptosis of human rheumatoid synovial fibroblasts via rho-mediated signalingARTHRITIS & RHEUMATISM, Issue 4 2010Norifumi Sawamukai Objective An abundance of mast cells are found in the synovium of patients with rheumatoid arthritis (RA). However, the role of mast cells in the pathogenesis of RA remains unclear. This study was undertaken to elucidate a role for mast cells in RA by investigating the antiapoptotic effects of tryptase, a major product of mast cells, on RA synovial fibroblasts (RASFs). Methods RA synovial tissue was obtained from RA patients during joint replacement surgery, and histologic changes in the tissue were examined. The expression of cell surface molecules and apoptotic markers on RASFs were detected by flow cytometry. Rho activation was determined using a pull-down assay. Results Mast cells, bearing both c-Kit and tryptase, accumulated in the sublining area of proliferating synovial tissue from RA patients. Protease-activated receptor 2 (PAR-2), a receptor for tryptase, was expressed on RASFs in the lining area, close to tryptase-positive mast cells in the RA synovium. Fas-mediated apoptosis of RASFs was significantly inhibited, in a dose-dependent manner, by the addition of tryptase, and this effect correlated with increased activation of Rho kinase. Furthermore, Y27632, a Rho kinase inhibitor, reduced the antiapoptotic effect of tryptase on RASFs, suggesting that Rho was responsible for the antiapoptotic effects of tryptase. Conclusion These results demonstrate that tryptase has a strong antiapoptotic effect on RASFs through the activation of Rho. Thus, we propose that the release of tryptase by mast cells leads to the binding of tryptase to PAR-2 on RASFs and inhibits the apoptosis of RASFs via the activation of Rho. Such mechanisms could play a pivotal role in the marked proliferation of RASFs and hyperplasia of synovial tissue seen in RA synovium. [source] Rho kinase,dependent activation of SOX9 in chondrocytesARTHRITIS & RHEUMATISM, Issue 1 2010Dominik R. Haudenschild Objective The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. Methods Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. Results SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser181, and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9Ser181 phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor , treatment and mechanical compression. Conclusion These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation. [source] Interleukin-1 receptor phosphorylation activates Rho kinase to disrupt human gastric tight junctional claudin-4 during Helicobacter pylori infectionCELLULAR MICROBIOLOGY, Issue 5 2010Tamia K. Lapointe Summary Helicobacter pylori infects more than half of the human population worldwide. In the absence of treatment, this persistent infection leads to asymptomatic gastritis, which in some cases can progress into gastric ulcers and adenocarcinomas. The host,microbial interactions that govern the clinical outcome of infection remain incompletely understood. H. pylori is known to disrupt gastric epithelial tight junctions, which may represent a significant component of disease pathogenesis. The present study demonstrates that H. pylori disrupt epithelial tight junctional claudin-4 in a Rho kinase (ROCK)-dependent manner in human gastric epithelial (HGE-20) cell monolayers, independently of the virulence factors CagA and VacA, and without altering claudin-4 transcription. In the same epithelial cell model, interleukin (IL)-1,, mediated a similar ROCK-dependent pattern of tight junction disruption. Further experiments revealed that H. pylori infection induced IL-1 receptor type I (IL-1RI) phosphorylation, independently of epithelial secretion of its endogenous ligands IL-1,, IL-1, or IL-18. Finally, inhibition of IL-1RI activation prevented H. pylori -induced ROCK activation and claudin-4 disruption. Taken together, these findings identify a novel pathophysiological mechanism by which H. pylori disrupts gastric epithelial barrier structure via IL-1RI-dependent activation of ROCK, which in turn mediates tight junctional claudin-4 disruption. [source] Protein phosphorylation and kinome profiling reveal altered regulation of multiple signaling pathways in B lymphocytes from patients with systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 8 2010Taher E. Taher Objective The cause of B lymphocyte hyperactivity and autoantibody production in systemic lupus erythematosus (SLE) remains unclear. Previously, we identified abnormalities in the level and translocation of signaling molecules in B cells in SLE patients. The present study was undertaken to examine the extent of signaling abnormalities that relate to altered B cell responses in SLE. Methods B lymphocytes from 88 SLE patients and 72 healthy controls were isolated from blood by negative selection. Protein tyrosine phosphorylation and cellular kinase levels were analyzed by Western blotting, flow cytometry, and a kinome array protocol. Changes in protein phosphorylation were determined in ex vivo B cells and following B cell receptor engagement. Results Differences in tyrosine phosphorylation in B cells from patients with SLE, compared with matched controls, were demonstrated. Further, the kinome array analysis identified changes in the activation of key kinases, i.e., the activity of phosphatidylinositol 3-kinase, which regulates survival and differentiation, was up-regulated and the activity of Rac and Rho kinases, which regulate the cytoskeleton and migration, was increased. In contrast, the activity of ATR, which regulates the cell cycle, was down-regulated in SLE patients compared with controls. Differences in signaling pathways were seen in all SLE B lymphocyte subsets that manifested phenotypic features of immature, mature, and memory cells. Conclusion This study revealed dysregulation in multiple signaling pathways that control key responses in B cells of SLE patients. Data generated in this study provide a molecular basis for further analysis of the altered B lymphocyte responses in SLE. [source] Implication of Rho-associated kinase in the elevation of extracellular dopamine levels and its related behaviors induced by methamphetamine in ratsJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Minoru Narita Abstract A growing body of evidence suggests that several protein kinases are involved in the expression of pharmacological actions induced by a psychostimulant methamphetamine. The present study was designed to investigate the role of the Rho/Rho-associated kinase (ROCK)-dependent pathway in the expression of the increase in extracellular levels of dopamine in the nucleus accumbens and its related behaviors induced by methamphetamine in rats. Methamphetamine (1 mg/kg, subcutaneously) produced a substantial increase in extracellular levels of dopamine in the nucleus accumbens, with a progressive augmentation of dopamine-related behaviors including rearing and sniffing. Methamphetamine also induced the decrease in levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Both the increase in extracellular levels of dopamine and the induction of dopamine-related behaviors by methamphetamine were significantly suppressed by pretreatment with an intranucleus accumbens injection of a selective ROCK inhibitor Y-27632. In contrast, Y-27632 had no effect on the decrease in levels of DOPAC and HVA induced by methamphetamine. Under these conditions, there were no changes in protein levels of membrane-bound RhoA in the nucleus accumbens following methamphetamine treatment. It is of interest to note that the microinjection of Y-27632 into the nucleus accumbens failed to suppress the increases in extracellular levels of dopamine, DOPAC, and HVA in the nucleus accumbens induced by subcutaneous injection of a prototype of µ-opioid receptor agonist morphine (10 mg/kg). Furthermore, perfusion of a selective blocker of voltage-dependent Na+ channels, tetrodotoxin (TTx) into the rat nucleus accumbens did not affect the increase in extracellular levels of dopamine in the rat nucleus accumbens by methamphetamine, whereas the morphine-induced dopamine elevation was eliminated by this application of TTx. The extracellular level of dopamine in the nucleus accumbens was also increased by perfusion of a selective dopamine re-uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]-4-(3-phenylpropyl)piperazine (GBR-12909) in the nucleus accumbens. This effect was not affected by pretreatment with intranucleus accumbens injection of Y-27632. These findings provide first evidence that Rho/ROCK pathway in the nucleus accumbens may contribute to the increase in extracellular levels of dopamine in the nucleus accumbens evoked by a single subcutaneous injection of methamphetamine. In contrast, this pathway is not essential for the increased level of dopamine in this region induced by morphine, providing further evidence for the different mechanisms of dopamine release by methamphetamine and morphine in rats. [source] BXL-628, a vitamin D receptor agonist effective in benign prostatic hyperplasia treatment, prevents RhoA activation and inhibits RhoA/Rho kinase signaling in rat and human bladderTHE PROSTATE, Issue 3 2007Annamaria Morelli Abstract BACKGROUND BXL-628 is a calcitriol analog shown to decrease prostate growth in preclinical and clinical studies. BPH symptoms are generated not only by prostate overgrowth but also by bladder overactivity, resulting from an increased RhoA/Rho-kinase signaling. Because bladder smooth muscle cells express VDR, we studied effects of BXL-628 on this pathway. METHODS RhoA and Rho-kinase gene expression and functional activity were studied in rat and human bladder smooth muscle by real-time RT-PCR, immuno-kinase assays, western blot analysis, confocal microscopy, in vitro contractility, and cell migration. RESULTS In bladder smooth muscle, carbachol responsiveness was delayed and Rho-kinase activity reduced by BXL-628 treatment because of impaired RhoA membrane translocation and activation. Accordingly, RhoA-mediated biological functions, such as cell migration and cytoskeleton remodeling were also inhibited by BXL-628. CONCLUSIONS BXL-628 inhibits RhoA/Rho-kinase signaling, a calcium sensitizing pathway, suggesting its possible clinical use in the treatment of altered bladder contractility often associated with BPH-induced lower urinary tract symptoms. Prostate 67:234,247, 2007. © 2006 Wiley-Liss, Inc. [source] Acute and Chronic Alcohol Exposure Impair the Phagocytosis of Apoptotic Cells and Enhance the Pulmonary Inflammatory ResponseALCOHOLISM, Issue 10 2010Darren M. Boé Background:, Alcohol abuse increases the risk for acute respiratory distress syndrome (ARDS). Efferocytosis, the clearance of apoptotic cells, is important in the resolution of inflammation and is regulated by RhoA and rho kinase (ROCK) activation. The effects of alcohol on pulmonary Rho pathway activation and efferocytosis have not been determined. We hypothesize that acute and chronic alcohol exposure impair pulmonary efferocytosis, leading to heightened inflammation during ARDS. Methods:, For in vivo experiments, C57BL/6 mice received either a single intraperitoneal injection of alcohol or chronic ethanol-in-water for 8 weeks prior to intratracheal instillation of apoptotic cells or lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed for cells counts, calculation of the phagocytic index (PI), and Rho activity measurements. For in vitro studies, primary alveolar macrophages were cultured in alcohol (25,100 mM) and then co-cultured with apoptotic cells. RhoA activity was determined following alcohol exposure, and the PI was determined before and after treatment with the ROCK inhibitor, Y27632. Results:, Acute alcohol exposure was associated with impaired efferocytosis. Following LPS exposure, acute alcohol exposure was also associated with increased BAL neutrophils. Chronic alcohol exposure alone did not alter efferocytosis. However, following exposure to LPS, chronic alcohol exposure was associated with both impaired efferocytosis and increased BAL neutrophils. In vitro alcohol exposure caused a dose-dependent decrease in efferocytosis. Despite the fact that RhoA activity was decreased by alcohol exposure and RhoA inhibition did not alter the effects of alcohol on efferocytosis, treatment with the Rho kinase inhibitor, Y27632, reversed the effects of alcohol on efferocytosis. Conclusions:, Acute alcohol exposure impairs pulmonary efferocytosis, whereas exposure to chronic alcohol is only associated with impaired efferocytosis following LPS-induced lung injury. Both forms of alcohol exposure are associated with increased alveolar neutrophil numbers in response to LPS. The acute effects of alcohol on efferocytosis appear to be mediated, at least in part, by RhoA-independent activation of ROCK. Further studies are needed to dissect the differences between the effects of acute and chronic alcohol exposure on efferocytosis and to determine the effects of alcohol on alternative activators of ROCK. [source] | |||||||||||||||||||||||||