Attenuation of contractility in rat epididymal vas deferens by Rho kinase inhibitors.
The involvement of Ca(2+) sensitization mediated through Rho kinase in the contractility of rat epididymal vas deferens was investigated using Rho kinase inhibitors, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinilcyclohexanecarboxamide dihydrochloride (Y-27632) and 1-(5-isoquinolinesulphonyl)homopiperazine (HA 1077), in comparison with myosin light chain kinase (MLCK) inhibitors, wortmannin and 1-(5-chloronaphthalenesulphonyl)homopiperazine (ML-9) and agents that affect protein kinase C (PKC) and non-receptor tyrosine kinase intracellular signalling. 2 In Ca(2+)-free/ethyleneglycol-bis-(beta-aminoethylether)N,N,N('),N(')-tetraacetic acid (EGTA) (1 mM) medium, noradrenaline evoked sustained contractions. Y-27632 and HA 1077 caused a concentration-dependent inhibition and complete relaxation (IC(50), 1.08 and 1.75 microM respectively). The Ca(2+)-free contraction was reduced by wortmannin (10 microM) or ML-9 (10 microM) but not by inhibitors of diacylglycerol metabolism, 3-[2-[4[bis(4-Fluoropheny)methylene]-1-piperidinyl]-2,3-dihydro-2-thioxi-4(H)-quinazolinone (R59949) (10 microm) or 1,6-bis(cyclohexyloximinocarbonylamino)hexane (RHC-80267) (10 microM) or by the phospholipase A(2) (PLA(2)) inhibitor, quinacrine (up to 100 microM) or tyrosine kinase inhibitor, genistein (30 microM). 3 In the presence of Ca(2+) (2.5 mM), noradrenaline (100 microM) evoked rhythmic activity and biphasic tonic contractions. Y-27632 (1-10 microM) or HA 1077 (1-10 microM) reduced the amplitude of rhythmic activity and tonic contractions. ML-9 (10 microM) attenuated the occurrence of rhythmic activity and modestly reduced the tonic contractions. ML-9 (10 microM) combined with Y-27632 (10 microM) significantly reduced the tonic contractions. ML-9 (30 microM) alone (or combined with Y-27632 10 microM) suppressed the rhythmic activity and substantially reduced (or abolished) the tonic contractions. 4 Contractions evoked by high [K(+)](o) (120 mM) or alpha,beta-methylene ATP (10 microM) were reduced significantly by Y-27632 (1-3 microM) indicating that the Rho kinase signalling pathway is activated by direct tissue depolarization or by stimulation of ligand-gated P(2X) purinoceptors. 5 Collectively, these results indicate that Ca(2+)-sensitization mediated by Rho kinase is involved in agonist- or depolarization-induced contraction of rat epididymal vas deferens. It is the major contractile mechanism underlying noradrenaline-induced Ca(2+)-free responses. It contributes to Ca(2+)-dependent rhythmic contractility and optimizes the development of full contractile tension triggered through calmodulin/MLCK activation by stimulated influx of Ca(2+).
King's College London, Division of Biomedical Science Research, Guys Campus, London SE1 1UL, UK.
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D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058469:Receptors, Purinergic P2X; D014649:Vas Deferens / Q000187:drug effects* / Q000502:physiology; D054460:rho-Associated Kinases
D000577:Amides; D001381:Azepines; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D058910:Purinergic P2 Receptor Agonists; D011725:Pyridines; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; C056218:ML 9; C108830:Y 27632; D004533:Egtazic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; C049347:fasudil; D011188:Potassium; D002118:Calcium; D009638:Norepinephrine