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Ca2+ Sensitization (ca2+ + sensitization)
Selected AbstractsSphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004L. Formigli We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+ -independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca2+ -independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC, and PKC,, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+ -independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction. J. Cell. Physiol. 198: 1,11, 2004© 2003 Wiley-Liss, Inc. [source] Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin IITHE JOURNAL OF PHYSIOLOGY, Issue 2 2000Andrew P. Somlyo We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non-muscle cells, by modulating the Ca2+ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca2+ sensitization of smooth muscle contraction and non-muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non-muscle. The active, GTP-bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho-kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G-protein-coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho-kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI-17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho-kinase pathway and plays only a minor, transient role in the G-protein-coupled mechanism of Ca2+ sensitization. Ca2+ sensitization by the Rho/Rho-kinase pathway contributes to the tonic phase of agonist-induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis. [source] Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestationBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2003Hiroshi Ozaki Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 ,M) induced sustained contractions with no increase in [Ca2+]i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser19. The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 ,M). LY333531 (1 ,M), a specific inhibitor of PKC,, also inhibited the PDBu-induced contraction in the pregnant myometrium. In the pregnant myometrium permeabilized with , -toxin, PDBu increased the contractions induced at fixed Ca2+ concentration (0.3 ,M) both in nonpregnant and pregnant myometria, indicating Ca2+ sensitization of contractile elements. Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (,, ,, ,), novel PKC (,, ,, ,), and atypical PKC (, but not , and ,). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of , isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the , PKC isoform and CPI-17. British Journal of Pharmacology (2003) 140, 1303,1312. doi:10.1038/sj.bjp.0705552 [source] Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscleBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2003T Unno In guinea-pig ileal longitudinal muscle, muscarinic partial agonists, 4-(N -[3-chlorophenyl]-carbomoyloxy)-2-butynyl-trimethylammonium (McN-A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca2+ concentration ([Ca2+]c) with a higher EC50 than that of the full agonist carbachol. The maximum response of [Ca2+]c or tension was not much different among the three agonists. The Ca2+ channel blocker nicardipine markedly inhibited the effects of all three agonists The contractile response to any agonist was antagonized in a competitive manner by M2 receptor selective antagonists (N,N,- bis[6-[[(2-methoyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride and 11-[[2-[(diethlamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one), and the apparent order of M2 antagonist sensitivity was McN-A343>pilocarpine>carbachol. M3 receptor selective antagonists, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN-A343, while darifenacin had a similar action in the case of pilocarpine. Both M3 antagonists behaved in a competitive manner in the case of the carbachol response. McN-A343 failed to release Ca2+ from the intracellular stores, and the Ca2+ -releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca2+ sensitivity of the contractile proteins. McN-A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. The results suggest that muscarinic agonist-evoked contractions result primarily from the integration of Ca2+ entry associated with the increased spike discharge and myofilaments Ca2+ sensitization, and that Ca2+ store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M2 and M3 receptors plays a crucial role in mediating the contraction response. British Journal of Pharmacology (2003) 139, 337,350. doi:10.1038/sj.bjp.0705267 [source] Role of Ca2+ mobilization and Ca2+ sensitization in 8-iso-PGF2, -induced contraction in airway smooth muscleCLINICAL & EXPERIMENTAL ALLERGY, Issue 2 2009A. Shiraki Summary Background Isoprostanes are prostaglandin (PG)-like compounds synthesized by oxidative stress, not by cyclooxygenase, and increase in bronchoalveolar lavage fluid of patients with asthma. The airway inflammation implicated in this disease may be amplified by oxidants. Although isoprostanes are useful biomarkers for oxidative stress, the action of these agents on airways has not been fully elucidated. Objective This study was designed to determine the intracellular mechanisms underlying the effects of oxidative stress on airway smooth muscle, focused on Ca2+ signalling pathways involved in the effect of 8-iso-PGF2,. Methods Using simultaneous recording of isometric tension and F340/F380 (an indicator of intracellular concentrations of Ca2+, [Ca2+]i), we examined the correlation between tension and [Ca2+]i in response to 8-iso-PGF2, in the fura-2 loaded tracheal smooth muscle. Results Augmented tension and F340/F380 by 8-iso-PGF2, were attenuated by ICI-192605, an antagonist of thromboxane A2 receptors (TP receptors). Moreover, D609, an antagonist of phosphatidylcholine-specific phospholipase C, markedly reduced both the tension and F340/F380 induced by 8-iso-PGF2,, whereas U73122, an antagonist of phosphatidylinositol-specific phospholipase C, modestly inhibited them by 8-iso-PGF2,. SKF96365, a non-selective antagonist of Ca2+ channels, markedly reduced both tension and F340/F380 by 8-iso-PGF2,. However, diltiazem and verapamil, voltage-dependent Ca2+ channel inhibitors, modestly attenuated tension although their reduction of F340/F380 was not different from that by SKF96365. Y-27632, an inhibitor of Rho-kinase, significantly attenuated contraction induced by 8-iso-PGF2, without reducing F340/F380, whereas GF109203X and Go6983, protein kinase C inhibitors, did not markedly antagonize them although reducing F340/F380 with a potency similar to Y-27632. Conclusion 8-iso-PGF2, causes airway smooth muscle contraction via activation of TP receptors. Ca2+ mobilization by SKF96365- and D609-sensitive Ca2+ influx and Ca2+ sensitization by Rho-kinase contribute to the intracellular mechanisms underlying the action of 8-iso-PGF2,. Rho-kinase may be a therapeutic target for the physiologic abnormalities induced by oxidative stress in airways. [source] Roles of P2X receptors and Ca2+ sensitization in extracellular adenosine triphosphate-induced hyperresponsiveness in airway smooth muscleCLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2007T. Oguma Summary Background The release of adenosine triphosphate (ATP) from the airway epithelial cells during the inflammatory process is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. Objective This study was designed to determine whether extracellular ATP is involved in the bronchial hyperresponsiveness as an interaction between epithelium and smooth muscle in the airways. Methods We examined the contractile response to methacholine (MCh) before and after exposure to low concentrations (10 ,m) of ATP in isolated, epithelium-denuded guinea-pig tracheal smooth muscle by measuring isometric tension. Intracellular Ca2+ concentrations ([Ca2+]i) were assessed by fluorescent intensities of fura-2. Results MCh-induced contractile force was increased with no change in [Ca2+]i after exposure to 10 ,m ATP for 15 min. The ability of ATP to enhance the MCh-induced contraction was markedly attenuated by suramin, a non-selective P2 receptor inhibitor. Pre-incubation with ATP,S, a non-hydrolysable analogue of ATP and ,,,-meATP, a P2X agonist, also enhanced the MCh-induced contraction. In contrast, uracil triphosphate, a P2Y agonist, did not affect the MCh-induced contraction. Y-27632, a Rho-kinase inhibitor, suppressed the ability of ATP to enhance the MCh-induced contraction. Moreover, PP1 and PP2, Src tyrosin kinase inhibitors, suppressed the enhancement of MCh-induced contraction by ATP. Conclusion Pre-treatment with ATP induces hyperresponsiveness to MCh mediated by Ca2+ sensitization via the P2X receptor in airway smooth muscle. The present findings suggest the possible involvement of both the Rho-kinase and Src pathways in the intracellular mechanism of this phenomenon. [source] Rho kinase inhibitors reduce neurally evoked contraction of the rat tail artery in vitroBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Melanie Yeoh The effects of Rho kinase inhibitors (Y27632, HA-1077) on contractions to electrical stimulation and to application of phenylephrine, clonidine or ,,, -methylene adenosine 5,-triphosphate (,,, -mATP) were investigated in rat tail artery in vitro. In addition, continuous amperometry and intracellular recording were used to monitor the effects of Y27632 on noradrenaline (NA) release and postjunctional electrical activity, respectively. Y27632 (0.5 and 1 ,M) and HA-1077 (5 ,M) reduced neurally evoked contractions. In contrast, the protein kinase C inhibitor, Ro31-8220 (1 ,M), had little effect on neurally evoked contraction. In the absence and the presence of Y27632 (0.5 ,M), the reduction of neurally evoked contraction produced by the , -adrenoceptor antagonists prazosin (10 nM) and idazoxan (0.1 ,M) was similar. The P2-purinoceptor antagonist, suramin (0.1 mM), had no inhibitory effect on neurally evoked contraction in the absence or the presence of Y27632 (1 ,M). In the presence of Y27632, desensitization of P2X-purinoceptors with ,,, -mATP (10 ,M) increased neurally evoked contractions. Y27632 (1 ,M) and H-1077 (5 ,M) reduced sensitivity to phenylephrine and clonidine. In addition, Y27632 reduced contractions to ,,, -mATP (10 ,M). Y27632 (1 ,M) had no effect on the NA-induced oxidation currents or the purinergic excitatory junction potentials and NA-induced slow depolarizations evoked by electrical stimulation. Rho kinase inhibitors reduce sympathetic nerve-mediated contractions of the tail artery. This effect is mediated at a postjunctional site, most likely by inhibition of Rho kinase-mediated ,Ca2+ sensitization' of the contractile apparatus. British Journal of Pharmacology (2005) 146, 854,861. doi:10.1038/sj.bjp.0706377 [source] | |||||||||||||