title: Rho-associated kinase plays a role in rabbit urethral smooth muscle contraction, but not via enhanced myosin light chain phosphorylation.
abstract: The involvement of Rho-associated kinase (ROK) in activation of rabbit urethral smooth muscle contraction was investigated by examining the effects of two structurally distinct inhibitors of ROK, Y27632 and H1152, on the contractile response to electric field stimulation, membrane depolarization with KCl, and α1-adrenoceptor stimulation with phenylephrine. Both compounds inhibited contractions elicited by all three stimuli. The protein kinase C inhibitor GF109203X, on the other hand, had no effect. Urethral smooth muscle strips were analyzed for phosphorylation of three potential direct or indirect substrates of ROK: 1) myosin regulatory light chains (LC20) at S19, 2) the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) at T697 and T855, and 3) cofilin at S3. The following results were obtained: 1) under resting tension, LC20 was phosphorylated to 0.65±0.02 mol Pi/mol LC20 (n=21) at S19; 2) LC20 phosphorylation did not change in response to KCl or phenylephrine; 3) ROK inhibition had no effect on LC20 phosphorylation in the absence or presence of contractile stimuli; 4) under resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin at S3; 5) phosphorylation of MYPT1 and cofilin was unaffected by KCl or phenylephrine; and 6) KCl- and phenylephrine-induced contraction-relaxation cycles did not correlate with actin polymerization-depolymerization. We conclude that ROK plays an important role in urethral smooth muscle contraction, but not via inhibition of MLCP or polymerization of actin.
authors.affiliation: Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1. walsh@ucalgary.ca
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.forename: Keith
authors.initials: K
authors.lastname: Thornbury
authors.forename: William C
authors.initials: WC
authors.lastname: Cole
authors.forename: Gerard
authors.initials: G
authors.lastname: Sergeant
authors.forename: Mark
authors.initials: M
authors.lastname: Hollywood
authors.forename: Noel
authors.initials: N
authors.lastname: McHale
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D004558:Electric Stimulation; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects*; D018994:Myosin Light Chains / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D014521:Urethra / Q000502:physiology*; D054460:rho-Associated Kinases / Q000502:physiology*
chemical_list: C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000577:Amides; D018994:Myosin Light Chains; D011725:Pyridines; C108830:Y 27632; D010656:Phenylephrine; D011189:Potassium Chloride; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase