We plan to evaluate some of the regulatory mechanisms governing contraction in vascular smooth muscle. Phosphorylation of the 20,000 dalton light chain of myosin permits rapid crossbridge cycling (shortening velocity) and thus rapid stress development. Another form of crossbridge interaction, termed """"""""latch,"""""""" allows attached crossbridges to maintain stress with greatly reduced crossbridge cycling and ATP consumption. Both forms of crossbridge interactions are dependent on (Ca2+)i. We have techniques to measure myosin phosphorylation in intact tissues and to biophysically quantitate crossbridge interactions. Correlation of these measurements with estimates of intracellular (Ca2+) in smooth muscle is the goal of this project. Estimation of intracellular free Ca2+ involves loading the photoprotein aequorin intracellularly and measuring the photon emission as aequorin binds Ca2+. We already have reported a correlation of light estimated (Ca2+)i with myosin phosphorylation in K+ depolarized swine carotid media. This correlation supports the fourth criterion of Krebs and Beavo for acceptance of Ca2+- stimulated myosin phosphorylation as physiologically significant. The (Ca2+) sensitivity for stress maintenance was higher than for myosin phosphorylation or shortening velocity. We plan to (1) further test the validity of the aequorin method by simultaneously measuring (Ca2+)i in single cells using aequorin and the fluorescent dyes. (2) Test the universality of the (Ca2+)- myosin phosphorylation and (Ca2+)-stress relationships. This involves measuring (Ca2+), myosin phosphorylation, shortening velocity, and stress during contraction with several agonists and during cyclic nucleotide-dependent vasodilation. (3) Test the effect of mechanical perturbations, which could mediate myogenic responses, on (Ca2+)i and the (Ca2+) sensitivity of the contractile apparatus.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038918-02
Application #
3355382
Study Section
Physiology Study Section (PHY)
Project Start
1987-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
van Riper, D A; McDaniel, N L; Rembold, C M (1997) Myosin light chain kinase phosphorylation in nitrovasodilator induced swine carotid artery relaxation. Biochim Biophys Acta 1355:323-30
Chen, X L; Panek, K; Rembold, C M (1997) Metformin relaxes rat tail artery by repolarization and resultant decreases in Ca2+ influx and intracellular [Ca2+]. J Hypertens 15:269-74
Rembold, C M; Weaver, B A (1997) Tyrosine phosphorylation and regulation of swine carotid artery contraction. J Vasc Res 34:1-10
Chen, X L; Rembold, C M (1996) Nitroglycerin relaxes rat tail artery primarily by lowering Ca2+ sensitivity and partially by repolarization. Am J Physiol 271:H962-8
Van Riper, D A; Chen, X L; Gould, E M et al. (1996) Focal increases in [Ca2+]i may account for apparent low Ca2+ sensitivity in swine carotid artery. Cell Calcium 19:501-8
Gould, E M; Rembold, C M; Murphy, R A (1995) Genistein, a tyrosine kinase inhibitor, reduces Ca2+ mobilization in swine carotid media. Am J Physiol 268:C1425-9
Van Riper, D A; Weaver, B A; Stull, J T et al. (1995) Myosin light chain kinase phosphorylation in swine carotid artery contraction and relaxation. Am J Physiol 268:H2466-75
Rembold, C M; Van Riper, D A; Chen, X L (1995) Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery. J Physiol 488 ( Pt 3):549-64
Chen, X L; Rembold, C M (1995) pHi, [Ca2+]i, and myosin phosphorylation in histamine- and NH4(+)-induced swine carotid artery contraction. Hypertension 25:482-9
Chen, X L; Rembold, C M (1995) Phenylephrine contracts rat tail artery by one electromechanical and three pharmacomechanical mechanisms. Am J Physiol 268:H74-81

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