The long-range goals of the research described in this application are to define intracellular processes regulating contractility in smooth muscle with emphasis on the mechanistic role for myosin phosphorylation, and eventually to define alterations in these processes associated with disease. The experiments described are designed (1) to improve mechanical estimates of contractility in smooth muscle tissues by measurement of complex stiffness, (2) to measure mechanical properties of skinned tracheal muscle under conditions where the composition of the medium surrounding the contractile elements can be controlled and altered and (3) to use these findings to investigate physiological modes of regulation of contractility in intact muscle. Elastic and viscous stiffness will be measured in tonically contracted bovine tracheal smooth muscle by analysis of the force amplitude and phase angle between force and applied sinusoidal length perturbations at a number of frequencies. This method will be used to test the hypothesis that the muscle exhibits an altered frequency response function for states in which myosin cross bridges are phosphorylated and rapidly cycling or dephosphorylated and slowly cycling (latch). A skinned tracheal smooth muscle preparation will be characterized. By measuring the dependence of force on calcium and calmodulin concentrations following dephosphorylation of myosin by addition of phosphatase or specific inhibitors of myosin light chain kinase (synthetic peptides) it will be determined if a latch state can be obtained in skinned trachealis. The frequency response function of the skinned muscle in rigor and latch will be compared to that in the living muscle. The ionic environment of the skinned muscle in latch will be manipulated to reproduce mechanical behavior observed in the intact muscle. The force-velocity relation will be measured in skinned fibers following treatments with myosin light chain kinase and/or protein kinase C in order to test the hypothesis that phosphorylation of specific combinations of amino acids on light chain can modulate crossbridge cycling rates differentially. The rate of inactivation of myosin light chain kinase, rate and kinetic mechanism of myosin dephosphorylation by phosphatase and rate of decline in stiffness will be measured in intact trachealis muscle which is rapidly relaxing from neurally stimulated contraction.
