The objective of this proposal is to study the intracellular control mechanisms responsible for regulation of smooth muscle in the internal anal sphincter (IAS) and colon. We will test the hypothesis that different agonists may preferentially activate different intracellular pathways through mechanisms initiated at the membrane level and phosphorylate different sets of contraction-related proteins. In particular bombesin and substance P will be used as tools to examine two such pathways. Pilot studies indicate that substance P-induced contraction of the IAS and colon is a transient contraction that is mediated by intracellular calcium release and a calmodulin dependent pathway, whereas bombesin induces a sustained contraction that is initiated by extracellular calcium influx and is mediated by PKC through a calmodulin independent pathway. Pilot studies also indicate that the IP3/calmoodulin dependent contraction induced by substance P results in phosphorylation of the 20,000 Dalton myosin light chain, as has been previously demonstrated by numerous studies. The bombesin /PKC induced contraction, however, results in a pattern of protein phosphorylation which includes the 20,000 Dalton myosin light chain, but differs from the substance P- induced IP3/calmodulin dependent contraction as it phosphorylates several different proteins. One of the proteins phosphorylated by the PKC-dependent pathway and not by the IP3dependent pathway is a heat shock-like 27kd protein (HSP27) identified by a specific monoclonal antibody. Preliminary data suggest that HSP27 plays a role in contraction mediated by the PKC dependent pathway, as the monoclonal antibody to HSP27 blocks contraction induced by bombesin and by PKC, but has no effect on contraction induced by substance P or by IP3. We now propose to confirm this initial observation, and to focus on HSP27 as a possible mediator of contraction in response to PKC activation. In summary we will focus on the hypothesis that different agonists may preferentially activate different intracellular pathways. We can follow the chain of events leading to contraction from binding of the agonists to specific receptor sites, to phosphorylation of specific contraction-related proteins. We believe that this type of expertise is unique in the field of gastrointestinal motility, and we hope the studies outlined may shed some new light on the regulatory mechanisms responsible for contraction and maintenance of tone in the anorectal region.
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