PHOSPHORYLATION CALCIUM AND SMOOTH MUSCLE CONTRACTION

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Phosphorylation of the 20000 dalton myosin light chain (MLC) by a Ca-calmodulin-MLC kinase complex is the most widely accepted mechanism hypothesized to initiate contraction in smooth muscle. However it has been shown that stress can be maintained without high levels of MLC phosphorylation. This state of stress maintenace has been termed "latch" and is characterized by low levels of MLC phosphorylation and low shortening velocities. This suggests stress is maintained by dephosphorylated, slowly or non-cycling cross bridges. Although the concept of latch is currently gaining acceptance, several major questions remain unanswered about the mechanism of formation of this state. This application will address these questions. Specifically: 1) is MLC phosphorylation a prerequisite for latch state formation as suggested by Chatterjee and Murphy (Science 211:464,1983) or can latch be formed by a direct mechanism?; and 2) if the MLC phosphorylated, is dephosphorylation necessary for the formation of latch? The hyperpermeable, detergent skinned fiber of the swine carotid artery will be used to answer these questions. Skinned fibers will be exposed to Mg2+, suggested to be a MLC phosphorylation independent contraction, or to Ca2+ in the presence of calmodulin inhibiters. Measurement of MLC phosphorylation, isotonic shortening velocity (indicative of average cross bridge cycling rate), and stiffness and peak resistance to stretch (indicative of average number of attached cross bridges) during these conditions will determine if slowly cycling or non-cycling attached latch bridges can be formed without MLC phosphorylation. The MLC will also be irreversibly thiophosphorylated (MLC phosphatase resistant) and the [Ca2+] varied. The parameters of phosphorylation, velocity, and stiffness will be measured to determine if latch bridges can be formed during constant level of MLC phosphorylation. These studies will add significant information about the Ca2+ regulation of vascular smooth muscle. This initial proposal will begin a long term program to relate the basic physiology of the contractile machinery in vascular smooth muscle to alterations that occur in this tissue in hypertension. If a latch state can be formed without prior MLC phosphorylation resulting in an increase in stress and/or stiffness, then the increase Ca2+ "leak" into the muscle cell demonstrated during hypertension may activate this state. This would result in an increase in vascular resistance and an increase in arterial pressure. This liason will aid in the understanding of the changes that occur in hypertension.