This multi-disciplinary program will elucidate the signal transduction pathways that regulate vascular smooth muscle development, hypertrophy, gene expression and contraction-processes that are subject to abnormalities contributing to high blood pressure, atherosclerosis, vasospasm, post-angioplasty restenosis and shock. The Projects are mutually interdependent, converge on investigation of a common theme of understanding smooth muscle cell contraction and growth, and require the complementary expertise available in molecular biology (Project 3), biochemistry (Project 2), physiology (Project 1) and biophysics and generation of novel probes (Core A). Newly developed tools and methods will be utilized for identifying and characterizing protein kinases and phosphatases, their upstream and downstream targets, and crossbridge- state kinetics. New reagents and methods include gamma-phosphate- activated ATP for affinity purification of protein kinases, mixed peptide sequencing of fmol level proteins, environmentally sensitive fluorescent nucleotide analogous, caged nucleotides, peptides and fatty acids, techniques for measuring contractility of embryonic smooth muscle, and the exchange of myosin light chains in situ. Project 1 and Core A will measure the kinetics of product release from myosin and test the hypothesis that a combination of variable expression of essential light chain and myosin and test the hypothesis that a combination of variable expression of essential light chain and myosin heavy chain isoforms determines the variable affinity of smooth muscles for MgADP and crossbridge kinetics. Project 2 and Core A will identify protein kinases and phosphatases that regulate smooth muscle myosin sequencing. In conjunction with the functional methods of Project 1, Project 3 will use these methods to identify kinases, phosphatases and their transcriptional targets that regulate angiotensin-induced smooth muscle hypertrophy, also testing the hypothesis that angiotensin-II plays an important role in control of smooth muscle cell differentiation and maturation during vascular development and mediates adaptive changes in contractile mass of SMC in adult animals. The effects of knockout of angiotensinogen, the angiotensin AT2 or AT1 receptors and MHox on SMC investment and/or growth differentiation/maturation during development will be determined in Project 2. The physiological role of telokin, a putative smooth muscle myosin phosphate activator, will be evaluated in transgenic and knockout animals (Projects 1, 2 and 3).
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