REGULATION OF VASCULAR SMOOTH MUSCLE Ca2+ SENSITIVITY

血管平滑肌（VSM）收缩在亚细胞水平上受两种机制调节：[Ca 2 +]升高和收缩的Ca 2+敏感性增加。Ca ~（2+）敏感性可通过RhoA激酶（ROK）诱导的肌球蛋白轻链（MLC）磷酸酶活性的抑制而增加，并通过激活MLC磷酸酶活性的细胞信号而降低。钙敏感性调节的重要性，强调了最近的发现，过度活动的钙敏感性参与高血压的病理生理。本实验室的长期目标是研究亚细胞机制调节VSM对收缩刺激的反应性，重点是调节Ca 2+敏感性。我认为调节Ca 2+敏感性的一种机制涉及收缩受体刺激的最近历史。简而言之，先前强的VSM受体刺激诱导适应性反应，称为记忆，暂时降低后续刺激的能力，以提高Ca 2+的敏感性。拟议的研究项目将使用离体兔动脉来1）确定记忆是否调节小动脉中的肌源性张力，2）阐明记忆如何在亚细胞水平上运作。Ca 2+敏感性的增加在肌源性张力的调节中起着重要作用，目的1将检验记忆通过降低Ca 2+敏感性来降低肌源性张力的假设。在完整的生物体中，预期这种调节在强烈的VSM收缩受体刺激发作后的一段时间内提供血流的总体增加，所述强烈的VSM收缩受体刺激可能发生在交感神经活动升高期间，或在血管损伤期间当血管活性刺激被释放时。这些研究将使用插管加压小动脉进行。目的2将测试的假设，记忆减少刺激诱导的Ca 2+敏化减少ROK激活。另一种假设，记忆涉及激活的钙脱敏机制，涉及增加MLC磷酸酶活性，也将进行测试。将采用标准生物力学和生物化学技术，包括使用前表面荧光测定法和Ca 2+指示剂fura-2同时测量组织等长力和[Ca 2+]1。总的来说，这些研究将提供新的见解调节Ca 2+的敏感性和VSM收缩刺激的反应性的细胞机制。

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.