Attenuation of contractility in rat epididymal vas deferens by Rho kinase inhibitors

Summary 1 The involvement of Ca2+ 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 Ca2+‐free/ethyleneglycol‐bis‐(β‐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 (IC50, 1.08 and 1.75 μm respectively). The Ca2+‐free contraction was reduced by wortmannin (10 μm) or ML‐9 (10 μm) 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 μm) or 1,6‐bis(cyclohexyloximinocarbonylamino)hexane (RHC‐80267) (10 μm) or by the phospholipase A2 (PLA2) inhibitor, quinacrine (up to 100 μm) or tyrosine kinase inhibitor, genistein (30 μm). 3 In the presence of Ca2+ (2.5 mm), noradrenaline (100 μm) evoked rhythmic activity and biphasic tonic contractions. Y‐27632 (1–10 μm) or HA 1077 (1–10 μm) reduced the amplitude of rhythmic activity and tonic contractions. ML‐9 (10 μm) attenuated the occurrence of rhythmic activity and modestly reduced the tonic contractions. ML‐9 (10 μm) combined with Y‐27632 (10 μm) significantly reduced the tonic contractions. ML‐9 (30 μm) alone (or combined with Y‐27632 10 μm) suppressed the rhythmic activity and substantially reduced (or abolished) the tonic contractions. 4 Contractions evoked by high [K+]o (120 mm) or α,β‐methylene ATP (10 μm) were reduced significantly by Y‐27632 (1–3 μm) indicating that the Rho kinase signalling pathway is activated by direct tissue depolarization or by stimulation of ligand‐gated P2X purinoceptors. 5 Collectively, these results indicate that Ca2+‐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 Ca2+‐free responses. It contributes to Ca2+‐dependent rhythmic contractility and optimizes the development of full contractile tension triggered through c