Experiments are proposed to study the basic electrophysiology and morphology of canine and human colonic smooth muscles. The electrical activity of the colon is complicated and has not been adequately described by extracellular recordings. We have developed cross-sectional preparations of the colonic muscularis which allow precise placement of intracellular microelectrodes to characterize the electrical activity at any point through the thickness of the muscle. Previous studies have revealed 2 pacemaker zones: one in the region of the myenteric plexus and another at the extreme submucosal surface of the circular layer. The origin and mechanisms of propagation of electrical events will be investigated further. We will also attempt to correlate electrical activity with morphology by investigating the structural features of pacemaker and non-pacemaker regions. We have also observed a large gradient in resting membrane potential across the circular layer which may influence electrical activity and excitation-contraction coupling. The basis for this potential gradient will be investigated. Electrical events appear to passively spread through the circular muscle from the pacemaker regions and summate, and it is probably this summation that results in excitation-contraction coupling in the proximal colon. We will investigate the regulation of this summation by physiological neurotransmitters. Finally, we have isolated a cell-type from pacemaker regions that may have structural similarities to interstitial cells of Cajal (ICC). These cells will be characterized morphologically and their ultrastructure will be compared to ICC found in situ. If the isolated cells are identified as ICC, then extensive electrical studies will be performed to determine whether these cells may generate pacemaker activity. Batch=P01DK41315,proj=0003 The ionic basic of electrical slow waves and pacemaker activity will be examined by voltage clamping enzymatically dispersed smooth muscle cells from dog colon. We plan to characterize the properties of whole-cell macroscopic Ca++ and K+ currents in these cells, examine the effects of autonomic transmitters on these currents and study possible second messenger systems involved in their regulation. In addition, we plan to test the hypothesis that a Na/Ca-exchange mechanism exists in these cells which is electrogenic and may be detected and characterized using a combination of electrophysiological and intracellular Ca++ measurement techniques. These experiments will contribute to a better understanding of ionic channels and carriers in colonic smooth and their role in excitation-contraction coupling.
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