Rho kinase expression and its central role in ovine gallbladder contractions elicited by a variety of excitatory stimuli.
Rho kinase has contractile activity, which induces Ca2+ sensitization in various cells. Several receptors are linked to the Rho/Rho-kinase pathway. Therefore, in this study we aimed to demonstrate the central importance of this novel pathway for diverse excitatory stimuli in the smooth muscle of the sheep gallbladder. Accordingly, the effects of a Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-8)-3 x 10(-5) M), were investigated on cholecystokinin-8 (CCK-8, 10(-8) M), endothelin-1 (10(-8) M), carbachol (10(-6)-10(-5) M), 5-hydroxytryptamine (5-HT, 10(-6)-10(-5) M), histamine (10(-6)-10(-5) M), phenylephrine (10(-5)-10(-4) M), neurokinin A (10(-7)-10(-6) M), electrical field stimulation (40 V, 0.5 ms, 2, 4, 8, 16, 32 Hz, 15 s, 3 min intervals) and potassium chloride (KCl, 25-50 mM)-induced contractions as well as spontaneous contractile activity. Electrical field stimulation evoked tetrodotoxin (3 x 10(-6) M)-sensitive reproducible contractions, which were inhibited by atropine (2 x 10(-6) M) and potentiated by eserine (5 x 10(-7) M). EFS-induced contraction was significantly inhibited by Y-27632 (10(-5) M). In addition, spontaneous contractile activity was suppressed in the presence of the compound (10(-6)-10(-5) M). This Rho kinase inhibitor also dramatically decreased the contractions elicited by 5-HT, neurokinin A and carbachol. KCl-induced contraction, which was not atropine-sensitive, was also conspicuously attenuated by Y-27632. Moreover, Y-27632 (10(-8)-3 x 10(-5) M) relaxed gallbladder strips that were contracted by histamine, endothelin-1, CCK-8 and phenylephrine in a concentration-dependent manner. pEC50 values for Y-27632 were 6.25+/-0.10, 5.79+/-0.12, 5.83+/-0.09 and 5.70+/-0.13 for the contraction elicited by histamine, CCK-8, endothelin-1 and phenylephrine, respectively. Furthermore, we also demonstrated Rho kinase protein expression (ROCK-1 and ROCK-2) by Western blot analysis. In conclusion, ROCK is expressed in the smooth muscle of the ovine gallbladder, and it has a central role in the contractile activity induced by diverse excitatory stimuli.
Sahan-Firat
Seyhan
S
Department of Pharmacology Medical Faculty Mersin University Campus Yenişehir 33169 Mersin, Turkey.
Tiftik
Rukiye Nalan
RN
Nacak
Muradiye
M
Büyükafşar
Kansu
K
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D005704:Gallbladder / Q000187:drug effects / Q000201:enzymology*; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D015288:Neurokinin A / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012701:Serotonin / Q000494:pharmacology; D012756:Sheep; D015398:Signal Transduction; D054460:rho-Associated Kinases
D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D011725:Pyridines; C108830:Y 27632; D012701:Serotonin; D015288:Neurokinin A; D002217:Carbachol; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases