The involvement of Rho-associated kinase in rabbit urethral smooth muscle contraction

The mechanism of activation of urethral smooth muscle contraction was investigated using freshly-isolated rabbit urethral strips mounted on a force transducer and perfused with Krebs solution at 37 oC. The involvement of Rho-associated kinase (ROK) in urethral smooth muscle contraction was investigated by examining the effects of pre-incubation with two structurally distinct inhibitors of ROK, Y27632 (10 M) and H1152 (1 M), on the contractile response to electric field stimulation (EFS; 4 Hz), membrane depolarization with KCl (80 mM) and 1-adrenoceptor stimulation with phenylephrine (10 M). Both compounds markedly inhibited contractions elicited by all three stimuli, implicating ROK in urethral smooth muscle contractile responses. Addition of H-1152 at the plateau of a phenylephrine-induced contraction evoked relaxation. The protein kinase C inhibitor, GF109203X (0.2 M), on the other hand, had no effect on the contractile response to EFS, KCl or phenylephrine. For biochemical analyses, urethral muscle strips connected to a force transducer were incubated in Krebs solution in a 1-ml cuvette at 21 oC. Tissue strips were quick frozen at selected times for analysis of phosphorylation of three potential direct or indirect substrates of ROK: (i) myosin regulatory light chain at S19 and T18; (ii) the myosin targeting subunit of myosin light chain phosphatase, MYPT1, at T697 and T855; and (iii) cofilin at S3. The following results were obtained: (i) under resting tension (0.5 g), LC20 was phosphorylated to ~0.5 mol Pi / mol LC20 at S19; (ii) LC20 phosphorylation did not change in response to KCl or phenylephrine; (iii) ROK inhibition had no effect on LC20 phosphorylation in the absence or presence of contractile stimuli; (iv) under resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin at S3; (v) phosphorylation of MYPT1 and cofilin was unaffected by KCl or phenylephrine; and (vi) KCl- and phenylephrine-induced contraction-relaxation cycles did not corrrelate 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. The high level of LC20 phosphorylation at resting tension suggests that activation of ROK leads to alleviation of inhibition of cross-bridge cycling