A delicate balance must be maintained between colonic contractions that are too forceful (causing obstructive disorders of the colon) and those that are ineffective in promoting water absorption and movement of fecal material. As in other tissues calcium is believed to play a central role in the process of excitation-contraction coupling to regulate the force and timing of contractions. The focus of this project is to determine the sources of calcium and the basic mechanisms which regulate intracellular calcium in colonic smooth muscle. Recently developed calcium sensitive, fluorescent dyes will be used to optically measure cytoplasmic concentrations of calcium in tissue segments and isolated smooth muscle fibers. Calcium measurements will be correlated with contractile forces, intracellular potentials, pharmacological agents that affect calcium transport or release from intracellular stores, the presence of transmitters, and whole cell calcium currents measured using the patch clamp technique. In addition, calcium-dependent mechanisms at each phase during the slow wave cycle will be actively probed using chelators that release or sequester intracellular calcium upon photo-activation. The colon provides a unique opportunity to study excitation-contraction coupling because of the diversity of electrical activities. There are 2 distinct rhythmic patterns of excitation which originate in discrete layers of the colon with different spontaneous frequencies; as well as gradients in resting membrane potential, slow wave amplitude and degree of innervation. Rhythmic patterns will be studied in isolation and collectively, so that an integrative approach can be taken to describe overall colonic electromechanical activity. This proposal is central to the overall theme of this program project. It relates to each of the other proposals. Since calcium entry appears to be: via ionic channels modulated by neural inputs, varied as a function of thickness through the colon, a major contributor to the waveform of rhythmic electrical events, and a signal for activation of intracellular processes including contraction. These experiments will further our understanding of the basic mechanisms that control rhythmicity and the patterns of contractions responsible for colonic motility.
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