Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells.

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Abstract
Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to 1/3 their normal length, provided Ca2+ and ATP are present. Ca2+-independent contraction was obtained by preincubation in Ca2+ and ATP.gamma.S, or by addition of trypsin-treated myosin L chain kinase (MLCK) that no longer requires Ca2+ for activation. In the absence of Ca2+, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions simliar to those previously reported for the in vitro diassembly of [chicken] gizzard myosin filaments. Ca2+-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP) followed by addition of MLCK, calmodulIn, Ca2+ and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA [ethyleneglycolbis-(.beta.-amino-ethyl-ether)-N,N,N'',N''-tetraacetic acid] and ATP) and then initiating contraction by Ca2+ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca2K+-dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in thin filament-intermediate filament organization.

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