Studies will be performed to determine how Ca2+ is regulated in smooth muscle and how in turn Ca2+ regulates contraction of these cells. To this end studies will be performed to characterize the mechanisms responsible for the storage and release of Ca2+ from internal storage compartments. The spatial organization of these compartments and their functional and structural interconnection will be determined. Sites of Ca2+ entry into cells, as well as mechanisms linking transmembrane Ca2+ entry with release of Ca2+ from internal stores will be determined. Studies into the mechanisms responsible for removing Ca2+ from the cell, and for modulating the activity of these processes will be carried out. The role of protein kinase C as a feedback regulator of Ca2+ signalling mechanisms will also be determined. These studies will be performed on single isolated smooth muscle cells, thereby avoiding the complexities of intact multicellular preparations and affording a direct view into these cellular/subcellular issues. Single smooth muscle cells will be isolated by enzymatic disaggregation of the stomach muscularis of the toad, Bufo Marinus. This system is a well=characterized model system for studying smooth muscle physiology at the cellular level. The properties of Ca2+ transport and release by internal compartments will be investigated using a saponin skinned preparation measuring ion fluxes by techniques involving isotopes or fluorescent indicators. The 3D molecular cytoarchitecture of these compartments, will be determined by fluorescent antibodies in conjunction with the digital imaging microscope. Sudden changes in the level of key molecules suspected to be involved in Ca2+ regulation in smooth muscle will be accomplished by their photolytic generation from caged precursors. The role of protein kinases will be tested by injection of peptide modulators of specific kinases. The Ca2+ sensitivity of activation of smooth muscle contraction will be assessed by simultaneous measurements of Ca2+ and force in a single smooth muscle cell. The proposed studies employing tools of biophysics, molecular biology, cell biology and computer science are part of a long-term effort to determine the mechanism underlying the generation and regulation of force in smooth muscle. These basic studies of normal function of smooth muscle should provide much needed insight into derangements of smooth muscle function in diseases such as hypertension, asthma, and spastic disorders of the G.I. System.
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