We propose to better understand the mechanism of excitation- contraction coupling during neurohormonal stimulation of smooth muscle from different segments of the colon. The studies will be performed on tissue from the rabbit which has both a taeniated proximal colon and a non-taeniated distal colon and from humans who have diseases associated with normal or abnormal colonic motility. Electrophysiologic studies using the partition- stimulation chamber will be used to determine the contribution of the transmembrane flux of Ca2+ to neurohumoral stimulation of the colonic muscle. The contribution of Na+, K+ or Cl- fluxes in mediating the neurohormonal excitation of the muscle membrane also will be measured. The quantity of Ca2+ stored in the sarcoplasmic reticulum (SR) in different colonic muscles will be estimated by determining the effect of inositol trisphosphate (InsP) or caffeine, on isometric contraction. The quantity of Ca2+3 released from the SR will be measured with a Ca2+ electrode and 43Ca efflux after stimulation by caffeine, InsP3 or the neuropeptides. The role in InsP3 in neurohumoral stimulation of colonic muscle will be determined by measuring an increase in InsP3 from phosphotidylinositol bisphosphate. The goal of these studies is to determine the mechanism of regulation of (Ca2+)i; which initiates muscle contraction after neuropeptide administration. The techniques to be used include 1) measurement of membrane potential using intracellular microelectrodes, 2) measurement of changes of membrane resistance using the double sucrose gap and partition stimulation chamber, 3) measurement of Ca2+ dependence of contraction using saponin-permeabilized strips of colonic muscle, 4) measurement of Ca2+ efflux into extracellular space by a Ca2+ electrode and 45Ca efflux. 5) measurement of InsP3 by labeling phosphotidylinositol with (H) myo-inositol and measuring the generation of inositol metabolities using HPLC. The information obtained from this proposal should enhance our understanding of the smooth muscle abnormalities in patients with disorders of colonic motility.
