Amobi N I B, Chung I-P, Smith I C H
King's College London, Division of Biomedical Science Research, Guys Campus, London SE1 1UL, UK.
Auton Autacoid Pharmacol. 2006 Apr;26(2):169-81. doi: 10.1111/j.1474-8673.2006.00367.x.
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(+) (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+).
使用Rho激酶抑制剂反式-4-[(1R)-1-氨基乙基]-N-4-吡啶基环己烷甲酰胺二盐酸盐（Y-27632）和1-(5-异喹啉磺酰基)高哌嗪（HA 1077），与肌球蛋白轻链激酶（MLCK）抑制剂渥曼青霉素和1-(5-氯萘磺酰基)高哌嗪（ML-9）以及影响蛋白激酶C（PKC）和非受体酪氨酸激酶细胞内信号传导的药物相比，研究了通过Rho激酶介导的Ca(2+)致敏在大鼠附睾输精管收缩性中的作用。2在无Ca(2+)/乙二醇双(β-氨基乙醚)N,N,N',N'-四乙酸（EGTA）（1 mM）培养基中，去甲肾上腺素引起持续收缩。Y-27632和HA 1077引起浓度依赖性抑制并完全松弛（IC(50)分别为1.08和1.75 microM）。渥曼青霉素（10 microM）或ML-9（10 microM）可降低无Ca(2+)收缩，但二酰基甘油代谢抑制剂3-[2-[4[双(4-氟苯基)亚甲基]-1-哌啶基]-2,3-二氢-2-硫代-4(H)-喹唑啉酮（R59949）（10 microM）或1,6-双(环己基氧代羰基氨基)己烷（RHC-80267）（10 microM）或磷脂酶A(2)（PLA(2)）抑制剂奎纳克林（高达100 microM）或酪氨酸激酶抑制剂染料木黄酮（30 microM）则不能。3在存在Ca(2+)（2.5 mM）的情况下，去甲肾上腺素（100 microM）引起节律性活动和双相强直性收缩。Y-27632（1 - 10 microM）或HA 1077（1 - 10 microM）降低了节律性活动和强直性收缩的幅度。ML-9（10 microM）减弱了节律性活动的发生并适度降低了强直性收缩。ML-9（10 microM）与Y-27632（10 microM）联合使用显著降低了强直性收缩。单独使用ML-9（30 microM）（或与Y-27632 10 microM联合使用）抑制了节律性活动并大幅降低（或消除）了强直性收缩。4高K(+)（120 mM）或α,β-亚甲基ATP（10 microM）引起的收缩被Y-27632（1 - 3 microM）显著降低，表明Rho激酶信号通路通过直接组织去极化或通过刺激配体门控P(2X)嘌呤受体而被激活。5总体而言，这些结果表明，由Rho激酶介导的Ca(2+)致敏参与了大鼠附睾输精管中激动剂或去极化诱导的收缩。它是去甲肾上腺素诱导的无Ca(2+)反应的主要收缩机制。它有助于Ca(2+)依赖性节律性收缩，并优化通过Ca(2+)刺激内流激活钙调蛋白/MLCK触发的完全收缩张力的发展。
