The overal hypothesis being tested in this PPG is that loss of the normal function of the small group of critical proteins that regulate VSMC relaxation can cause abnormalities of blood pressure regulation. Cyclic GMP-dependent protein kinase Ialpha (PKGIalpha)0 mediates vascular relaxation by endothelial-derived nitric oxide and related nitrovasodilators. Extensive preliminary data from our laboratory show that the N-terminal leucine zipper (LZ) domain of PKGIalpha mediates interaction of PKGIalpha with two key proteins that cause vascular relaxation. The specific objective of Project 2 of this PPG is to test the hypothesis that PKGIalpha, through its LZ domain, binds to and activates two enzymes, myosin phosphatase (PP1M) and the regulator of G-protein signaling 2 (RGS2), which directly regulate VSMC tone and systemic blood pressure in vivo. To test the hypothesis, the importance and relative contributions of RGS2 and PP1M to VSMC relaxation in intact cells and blood vessels will be explored using molecular, cellular and physiological approaches. A major focus of this project will be the study of LZ mutant 'knock-in' (LZM KI) mice we have recently created by gene targeting. LZM KI mice, which express solely mutant PKGIalpha protein that lacks a functional LZ domain, develop marked vascular dysfunction and hypertension. Based on extensive preliminary data and using a number of novel reagents developed in our laboratory, we propose to test the hypothesis in three inter-related specific aims: SA1: Explore the contribution of PKG-mediated PP1M activation to myosin light chain dephosphorylation and VSMC relaxation; SA2: Explore the contribution of PKG-mediated RGS2 activation to myosin light chain dephosphorylation and VSMC relaxation; and SA3: Study the effect of tissue (VSMC)-specific disruption of PKGIalpha on vascular function and blood pressure in genetically modified mice we will create. To explore the hypothesis, we propose a broad spectrum of state-of-the-art molecular signal transduction methods, as well as ex vivo vascular ring contraction and relaxation bioassays, and creation and study of genetically modified mice. The studies of Project 2 are highly integrated with the other projects of this PPG and are expected to advance our understanding of vascular regulation in health and disease. They also provide a strong test of the hypothesis that hypertension can be caused by primary disorders of vascular smooth muscle, which has important implications for the diagnosis and treatment of hypertension and ischemic cardiovascular diseases in general.
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