The overall goal of this proposal is the characterization of cellular mechanisms which may play a role in the etiology such common colonic disorders as the irritable bowel syndrome and chronic constipation. Even though sustained contractile activity of myocytes from both longitudinal (L) and circular (C) muscle layer is largely dependent on Ca influx via voltage-sensitive Ca channels (VSCC), marked qualitative differences in underlying membrane potential changes, mechanisms of contraction coupling and contractile activity exist between the two muscle layers. In order to develop therapies targeted to specific muscle dysfunctions, an understanding of these differences in contraction coupling is essential. The proposed studies aim to test the hypothesis that differences in the interaction between Ca influx and intracellular Ca release result in distinct receptor-mediated changes in the subplasmalemmal space Ca concentration ([Ca]sub). Due to the close apposition of [Ca]sub and Ca- sensitive ion channels in the plasma membrane, differences in [Ca]sub regulation could explain the differences in patterns of membrane potential changes observed in L and C muscle. The central features of the proposed model are (a) differences between C and L muscle in the intracellular distribution of 1,4,5-trisphosphate (IP3) -sensitive and IP3-insensitive Ca release and reuptake mechanisms, and in the coupling of these mechanisms to plasma membrane receptors; (b) differences in the regulation of Ca-sensitive membrane conductances by regional [Ca]i gradients; (c) differences in intercellular propagation of [Ca]i gradients. According to the model, the differences between L and C muscle may result in muscle layer-specific and agonist-specific [Ca]i changes, such as [Ca]i oscillations and [Ca]i waves. The majority of the proposed experiments will be performed on freshly dispersed myoyctes from the L and C muscle layers of the rabbit colon. Using video imaging techniques with the fluorescent dyes fura-2 and fluo-3, we will first determine the role of ion channels mediating Ca influx in receptor-mediated regional increases in [Ca]i and contraction in C and L myocytes. Using [Ca]i imaging techniques in permeabilized myoyctes, we will characterize the role of the IP3-sensitive and the IP3-insensitive Ca release channel in the generation of distinct intra- and possibly intercellular [Ca]i patterns. By employing a pipette perfusion technique and using the Ca-activated K channel as a bioassay for [Ca]sub, and monitoring subcellular [Ca]i changes simultaneously with fluorescence imaging, we will characterize the kinetics of Ca release from and reuptake into subplasmalemmal stores. The questions addressed in this proposal focuses on how receptor-mediated Ca regulation within subcellular compartments ultimately results in receptor- and muscle-layer specific effector changes despite the ubiquitous involvement of extracellular Ca in contraction coupling. The elucidation of these mechanisms is a central problem in cell physiology. It involves several fundamental membrane processes such as ion channel regulation by Ca and IP3 in the plasma membrane and in the sarcoplasma membrane. The elucidation of these mechanisms may form the basis for future identifications of specific abnormalities associated with neuromuscular disorders of colonic function.
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