PKC dependent pathways appear to be of particular importance in the tonic contraction of smooth muscle. The calmodulin dependent pathway has been well characterized in gastrointestinal (GI) muscle while the PKC-dependent pathway has not. We propose to study the intracellular PKC-dependent, calmodulin independent mechanisms responsible for regulation of circular smooth muscle contraction in the rectosigmoid colon. Preliminary data indicate that in response to contractile agonists that seem to activate the PKC-dependent pathway, like carbachol, endothelin and bombesin, different molecular species of diacylglycerol (DAG) are produced and may derive from phosphatidylcholine, phosphatidylinositol and phosphatidylserine. In addition, these same agonists cause a dose- dependent increase in production of ceramide which results from hydrolysis of the membrane sphingolipid sphingomyelin. The involvement of the sphingomyelin pathway in GI smooth muscle contraction has not been examined previously. In addition, the PKC-mediated contraction induced by the same agonists ((carbachol, endothelin and bombesin), by ceramide or through direct activation of PKC by TPA causes a dose-dependent increase in mitogen activated protein (MAP) kinase activity. MAP kinase has been suggested as a mediator of PKC induced smooth muscle contraction. The observed increase in MAP kinase activity may be derived from DAG induced activation of PKC, from ceramide induced activation of PKC, or from their interactions. Furthermore, PKC-mediated contraction results in phosphorylation of proteins which are different from those phosphorylated during calmodulin dependent contraction. One of these proteins is a heat shock-like 27kd protein (HSP 27). Preliminary studies suggest that bombesin and ceramide induce phosphorylation of HSP27, cellular translocation and redistribution of MAP kinase associated with HSP27. The relaxant neuropeptide VIP causes a reduction in bombesin induced contraction, a reduction in MAP kinase activation and a reduction in HSP phosphorylation, suggesting that phosphorylation of HSP and PKC mediated contraction may be related. In addition there is a strong association and colocalization between HSP 27 and actin. We therefore propose to examine DAG and ceramide, kinases, and phosphorylated substrates which are activated in response to peptide induced, PKC-mediated contraction. These pathways which are responsible for a calmodulin independent contraction have not been clearly defined in gastrointestinal smooth muscle.
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