Abstract
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that contracts most smooth muscles. Although S1P has been shown to contract bladder smooth muscle, the mechanism(s) by which S1P initiates contraction has not been extensively investigated. The goal of this study was to determine if S1P-induced force generation and myosin light chain (MLC) phosphorylation are dependent on calcium sensitization pathways mediated by protein kinase C (PKC) and Rho kinase (ROCK) and which S1P receptor is important in this response. Bladder smooth muscle strips from rabbit and rat were mounted for isometric force recording and contracted in response to carbachol or S1P in the presence and absence of an inhibitor of PKC (3 µM Bisindolylmaleimide-1) or ROCK (1 µM H-1172). 10 µM S1P produced approximately 40% of the force generated in response to 110 mM KCl in rabbit bladder smooth muscle. S1P, up to 100 µM, did not produce a response in rat bladder smooth muscle, any response evoked was due to solvent (NaOH). S1P-dependent force development was associated with a concomitant increase in Ser(19), but not dual Thr(18)/Ser(19) MLC phosphorylation. Inhibition of PKC decreased force development, whereas inhibition of ROCK abolished S1P-induced force. An inhibitor of the S1P2 receptor, JTE-013, relaxed a S1P-induced contraction; whereas, an agonist with low affinity to the S1P2 receptor, dihydro-S1P, did not elicit a contraction. Our results suggest that S1P contracts rabbit, but not rat, bladder smooth muscle via the S1P2 receptor and is dependent on MLC phosphorylation and myofilament calcium sensitization primarily in response to ROCK activation.