Smooth muscle cells in the colon perform the mechanical work required for colonic motility. At the molecular level phosphorylation of the 20,000 dalton myosin light chain in some smooth muscles appears to be both necessary and sufficient for contraction. However, work in our laboratory and in other laboratories suggests there may be several calcium-dependent systems regulating the contractile element.
We aim to test the hypothesis that contraction of intact strips of canine colonic smooth muscle is regulated by three distinct molecular mechanisms; 1) A thick filament mechanism involving myosin phosphorylation, 2) A thin filament mechanism involving calcium/calmodulin dependent phosphorylation of caldesmon, and 3) Maintenance of tonic contraction by actin crosslinking regulated by phosphorylation of one or more cytoskeletal proteins. Thick filament regulation will be investigated by comparing the dependence of electrical activity, contraction, myosin phosphorylation and shortening velocity on time, stimulant concentration and external Ca2+ concentration. Phasic contractions induced by several agonists and tonic contractions induced by potassium depolarization in the absence or presence of other agonists will be studied The hypotheses to be tested are: 1) Dephosphorylated, noncycling crossbridges (""""""""latchbridges"""""""") contribute to maintenance of tonic contraction of the colon and 2) Myosin phosphorylation controls the rate of crossbridge cycling in colon smooth muscle. Thin filament and cytoskeletal regulatory mechanisms will be investigated by measuring phosphorylation of caldesmon, filamin and desmin in isolated colonic smooth muscle. Activation by receptor-mediated agonists will be compared to potassium depolarization,. The rats, stoichiometry and sites of phosphorylation will be determined for each putative regulatory protein by peptide mapping and protein sequencing. Selective protein kinase inhibitors will be introduced into reversibly permeabilized smooth muscle cells to test for a role for C-kinase in controlling each of the three myofilament systems. Changes in myoplasmic calcium activity will be monitored by measuring indo-1 or fluo-3 fluorescence to correlate with mechanical, electrical and biochemical events during contraction and relaxation. The results will contribute to our understanding of the cellular and molecular mechanisms of smooth muscle contraction and gastrointestinal motility.
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