The long-term objective of the proposed work is to describe events involved in the physiological regulation of the contraction (relaxation) cycle of the bovine tracheal smooth muscle cell and, eventually, alterations in these processes associated with disease. In order to avoid large diffusional delays in excitation seen with addition of agonist to the bath, cells of the trachealis muscle will be more synchronously activated with the use of selective electrical stimulation of nerves and muscle cells. Entire stimulus response relations will be described for eventual comparisons of matched contractions utilizing intracellular (neural excitation) and extracellular (cell depolarization) sources of calcium. Stimulus frequency will be modulated to simulate activation during physiological rates and types of nervous stimulation. Mechanical characterization (passive and active length-force relations, force-velocity relations and muscle stiffness) of the trachealis preparation will be done to define criteria for estimating degrees of activation of the muscle cells. Muscle strips will be quick-frozen under defined conditions of activation and analyzed using two-dimensional electrophoresis for phosphate content of the 20,000 dalton myosin light chain. Calcium-calmodulin initiation of myosin light chain kinase activity and resultant phosphorylation of myosin is the proposed biochemical mechanism of activation of smooth muscle contraction. Experiments are set forth to test the correlation between mechanical and biochemical activation of cells in neurally stimulated, directly depolarized and paced spontaneously active tissues. An electronic rapid freeze hammer will be used to freeze synchronously activated muscle strips at various times during the first second after stimulation in order to test the hypothesis that rapid muscle shortening (analogous with high actomyosin ATPase rates) will not occur before both 20,000 dalton light chains of myosin are phosphorylated.
