REGULATION OF VASCULAR SMOOTH MUSCLE Ca2+ SENSITIVITY

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Vascular smooth muscle (VSM) contractions are regulated at the subcellular level by two mechanisms, an elevation in [Ca2+], and an increase in the Ca2+ sensitivity of contractions. Ca2+ sensitivity may be increased by RhoA kinase (ROK)-induced inhibition of myosin light chain (MLC) phosphatase activity, and decreased by cell signals that activate MLC phosphatase activity. The importance of regulation of Ca2+ sensitivity is underscored by the recent finding that overactivity of Ca2+ sensitization is involved in the pathophysiology of hypertension. The long-term goal of my laboratory is to investigate subcellular mechanisms regulating VSM reactivity to contractile stimuli, focusing on regulation of Ca2+ sensitivity. I propose that one mechanism regulating Ca2+ sensitivity involves the recent history of contractile receptor stimulation. In short, prior strong receptor stimulation of VSM induces an adaptive response, termed memory, that temporarily reduces the ability of subsequent stimuli to elevate Ca2+ sensitivity. The proposed research project will use isolated rabbit arteries to 1) determine whether memory regulates myogenic tone in small arteries, and 2) elucidate how memory operates at the subcellular level. An increase in Ca2+ sensitivity plays a prominent role in regulation of myogenic tone, and aim 1 will test the hypothesis that memory reduces myogenic tone by reducing Ca2+ sensitivity. In the intact organism, such regulation would be expected to provide an overall increase in blood flow for some time after an episode of strong VSM contractile receptor stimulation that may occur during elevated sympathetic activity, or during blood vessel injury when vasoactive stimuli are released. These studies will be conducted using cannulated, pressurized small arteries. Aim 2 will test the hypothesis that memory reduces stimulus-induced Ca2+ sensitization by reducing ROK activation. An alternate hypothesis, that memory involves activation of a Ca2+ desensitization mechanism involving increased MLC phosphatase activity, will also be tested. Standard biomechanical and biochemical techniques will be employed, including simultaneous measurement of tissue isometric force and [Ca 2+]1 using front-surface fluorimetry and the Ca2+ indicator, fura-2. Collectively, these studies will provide new insights into the cellular mechanisms regulating Ca2+ sensitivity and VSM reactivity to contractile stimuli.