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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL046704-01A2
Application #
3365825
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
1993-05-01
Project End
1997-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Graduate Hospital (Philadelphia)
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19146
Su, X; Pott, J W; Moreland, R S (1999) Effect of Mg2+ on stress, myosin phosphorylation, and ATPase activity in detergent-skinned swine carotid media. Am J Physiol 276:H1416-24
Katoch, S S; Su, X; Moreland, R S (1999) Ca(2+)- and protein kinase C-dependent stimulation of mitogen-activated protein kinase in detergent-skinned vascular smooth muscle. J Cell Physiol 179:208-17
Rokolya, A; Walsh, M P; Singer, H A et al. (1998) Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate. J Cell Physiol 176:545-52
Ahtaridis, S A; Katoch, S S; Moreland, R S (1998) Mechanism of galanin-induced contraction of longitudinal smooth muscle of the rat jejunum. Am J Physiol 274:G306-13
Earley, J J; Su, X; Moreland, R S (1998) Caldesmon inhibits active crossbridges in unstimulated vascular smooth muscle: an antisense oligodeoxynucleotide approach. Circ Res 83:661-7
Gorenne, I; Su, X; Moreland, R S (1998) Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery. Am J Physiol 275:H131-8
Ahn, H Y; Chang, K C; Chung, M H et al. (1997) Cyclic AMP and cyclic GMP relax phorbol ester-induced contractions of rat aorta by different mechanisms. Life Sci 60:2333-40
Moreland, R S; Lichtenstein, A H; Chobanian, A V (1996) Effects of hypertension on hypercholesterolemia-induced changes in contraction of rabbit aorta and carotid artery. Eur J Pharmacol 307:55-64
Rokolya, A; Walsh, M P; Moreland, R S (1996) Calcium-and phorbol ester-dependent calponin phosphorylation in homogenates of swine carotid artery. Am J Physiol 271:H776-83
Katoch, S S; Moreland, R S (1995) Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. Am J Physiol 269:H222-9

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