Rho Kinase, Ca2+-Sensitization Mediated Arterial Smooth Muscle Contractionin Hypertension.

It is recognized recently that excitatory agonist can induced significant vascular smooth muscle contraction under constant free Ca 2+ through a mechanism called Ca 2+ -sensitization. Rho kinase (ROK), a serine and threonine kinase, plays an important role mediating agonist-induced Ca 2+ -sensitization of contraction. Increase of ROK and increase of Ca 2+ -sensitization of contraction have been implicated in the pathophysiology of hypertension. In preliminary studies, we obseNed an increased agonist and GTP-induced Ca 2+ -sensitization of contraction and increased ROK protein in Spontaneouly Hypertensive Rat (SHR). However, it is not known whether the increased Ca 2+ -sensitization of contraction in SHR contributes to the cause and/or maintenance of hypertension and whether the increased ROK is responsible for the increase of Ca 2+ -sensitiztion of contraction. To address these two questions, we propose to test the hypotheisis that increased ROK enhances the Ca2+-sensitization mechanism mediated arterial smooth muscle contraction and promotes hypertension. Specific aims are: Aim 1. To determine whether the increase of ROK protein level, the Ca 2+ -sensitizationmediated contraction and MLC20 phosphorylation increase precedes the rise of blood pressure in spontaneously hypertensive rat. We will determine, in pre-hypertensive and hypertensive SHR, the blood pressure, the amount of ROK protein, the amplitude of GTP and agonist-induced Ca2+-sensitization of contraction, and the level of 20 kDa myosin light chain (MLC20) phosphorylation. Aim 2. To test the hypothesis that increased ROK protein expression is responsible for the increased Ca 2+ -sensitization of contraction. We will use adenoviral mediated gene transfer to determine that if ROK expression causes increased Ca2+-sensitization mediated contractions in isolated smooth muscle strips. Isolated intact (non-permeabilized) and a-toxin permeabilized small mesenteric arteries will be used. Our standard alpha-toxin permeabilized preparation is appropriate for the proposed study as it retains the receptor, G-protein coupled signal transduction mechanism, and it allows for selective detection of the Ca2+-sensitizing effect over the Ca2+-mobilizing effect of agonists