Studies will be performed to determine the manner in which contractile proteins are organized into a contractile apparatus in smooth muscle, and how changes in this order might account for active cell shortening as well as the length dependence of active force development. Studies will also be performed to characterize the mechanical events underlying force development, and to analyze the role played by Ca+2 in regulating these processes. These studies will all be performed on single isolated smooth muscle cells, thereby avoiding the complexities of intact multicellular preparations and affording a direct view of cellular properties. Single smooth muscle cells will be isolated by enzymatic disaggregation of the stomach of Bufor marinus. The distribution of contractile proteins will be analyzed using flourescently labelled protein specific probes in both fixed and living cells. Fluorescent tags will be attached to either antibodies (both mono- and polyclonal) or purified native contractile protein; the former will be used to localize contractile protein in fixed cells and either one of these may be used in living cells into which they will be introduced by microinjection. The distribution of fluorescence will be assessed utilizing a low light level TV whose output is subject to digital image processing to obtain a computerized reconstruction of the distribution of contractile protein within a single isolated smooth muscle cell. Contractile state of these cells will be monitored by isometric measurement of force by tying a single cell to a force transducer. Ca+2 will be measured using a new class of highly fluorescent Ca+2 indicators which are trapped inside the smooth muscle cell by the action of cellular esterases. The proposed studies employing tools of biophysics, biochemistry, cell biology and computer science are part of long term effort to determine the mechanisms 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 inside function in diseases such as hypertension, asthma, and spastic disorders of the G. I. system.
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