REGULATION OF VASCULAR SMOOTH MUSCLE CA++ SENSITIVITY

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It is widely accepted that an increase in [Ca2+]i initiates vasoconstriction by activation of the calmodulin dependent myosin light chain (MLC) kinase and subsequent phosphorylation of the 20 kDa MLC. Recently however, we have demonstrated that GTP dependent agonist stimulation can increase myofilament Ca2+ sensitivity without a concomitant maintained increase in steady state MLC phosphorylation or crossbridge cycling suggesting that MLC phosphorylation is not the sole determinant of crossbridge behavior. We have also demonstrated that activation of permeabilized tissues by Ca2+ and NE induces phosphorylation of the putative thin filament regulatory protein, calponin. This is not observed with Ca2+ alone. The long term objectives of this proposal are to determine the mechanisms by which agonist activation of vascular smooth muscle modulate myofilament Ca2+ sensitivity. This application is based on the hypothesis that the increase in Ca2+ sensitivity of vascular smooth muscle contraction is due primarily to protein kinase C (PKC) activation and the resultant alteration in the activity of the thin filament regulatory protein, calponin. We propose to study the alpha toxin permeabilized artery which has the distinct advantage over previously used models of skinned vascular smooth muscle in that receptor activation is maintained even though the cell membranes have been made permeable to small ions and molecules. This model will be used to determine the relationships among force, [Ca2+]i, protein phosphorylation (including specific site of phosphate), and crossbridge cycling rate and attachment during agonist induced contractions. The specific aims to be addressed with the above experiments are: 1. To determine if PKC is the mediator of agonist induced changes in myofilament Ca2+ sensitivity by measuring diacylglycerol levels and translocation of PKC from cytosol to membrane prior to and during the increase in Ca2+ sensitivity and determining if inhibition of PKC activity abolishes the increase in Ca2+ sensitivity; translocation of PKC directly or indirectly to the contractile filaments to mediate its actions will also be examined; 2. To determine if phosphorylation of calponin mediates the change in Ca2+ sensitivity. This will be achieved by correlating calponin phosphorylation with PKC activation and with the magnitude of the enhanced Ca2+ sensitivity; 3. To determine the mechanism by which cAMP and cGMP decrease myofilament Ca2+ sensitivity. Cyclic nucleotide induced relaxation at constant [Ca2+]i will be correlated with the degree of PKC and phospholipase C activation, and with contractile protein phosphorylation; and 4. To determine the degree to which modulation of myofilament Ca2+ sensitivity contributes to vasomotion. This will be accomplished by simultaneous measurement of force and [Ca2+]i in intact vascular smooth muscle. In conclusion, the proposed research will determine the mechanisms responsible for modulation of Ca2+ sensitivity as well as its potential physiological role.