REGULATION OF ENERGY TRANSDUCTION IN SMOOTH MUSCLE

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The overall goal of this proposal is to elucidate the mechanism(s) responsible for regulation of contractile protein interaction and the resulting mechanical output in normally functioning smooth muscle. The mechanism of regulation of smooth muscle is not fully understood, nor is it known how this process is modified in pathological conditions such as hypertension and asthma. Such information on normal function will provide the framework for future studies on the etiology of altered smooth muscle function as well as therapeutic strategies. The specific questions deal with how phosphorylation of the myosin light chain regulates the number of myosin molecules which are activated as well as the cycling rate and force output from an activated myosin crossbridge. The first specific aim is to determine whether there are different pools of activated crossbridges with different cycling rates, and, if so, how myosin light chain phosphorylation affects the distribution and the cycling rate of each pool. How myosin light chain phosphorylation mediates an increase in the cycling rate of unphosphorylated myosin will be determined. Isometric conditions and mechanical perturbations such as shortening and stretching will be studied. This will give insight into how the kinetics of crossbridge cycling are controlled by the mechanical state of the muscle, as well as the effects of smooth muscle's unique regulatory system on this process. Other experiments will specifically test whether the degree of Cooperative activation of unphosphorylated myosin can be modulated by receptor activation, and whether the known activation mechanisms can account for the regulation of number of crossbridges cycling and their cycling rate during calcium-mediated contractions. The main experimental approach 'will be a single turnover protocol to determine the rate at which ADP bound to myosin is replaced with 3H-ADP from splitting of 3H-ATP formed by photolysis of "caged" 3H-ATP. The second aim is to quantitate the interaction of myosin light chain kinase and myosin light chain phosphatase with the crossbridge cycle in smooth muscle. The action of these enzymes on intermediates in the crossbridge cycle may alter the kinetics of the completion of the cycle. A method will be developed to permit measurement of the rate of phosphate turnover in the myosin light chain. This will involve measurement of the rate at which 33P-phosphate from gamma33P-ATP (derived from photolysis of caged gamma33P-ATP) appears in the light chain. The experiments will give a quantitative assessment of the rates of kinase and phosphatase activities compared to the kinetics of the crossbridge cycle. The final specific aim will determine how the ATPase of smooth muscle is controlled by mechanical conditions. Specifically, ATPase measurements will be made during brief shortenings and stretches and will be compared to isometric conditions. The experiments will allow determination of the relationship between ATP utilization by myosin and work output from the muscle, and will provide evidence as to whether the variation in velocity of shortening under different conditions results from an internal loading of fast cycling crossbridges by those with slower cycling rates.