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的调节，一种是[Ca~(2+)]升高，另一种是对Ca~(2+)的敏感性增强。RhoA激酶(RhoA Kinase，ROK)对肌球蛋白轻链(MLC)磷酸酶活性的抑制可增强细胞对钙离子的敏感性，而激活MLC磷酸酶活性的细胞信号则可降低细胞对钙离子的敏感性。最近的研究发现，钙离子敏感性的过度活跃参与了高血压的病理生理过程，从而强调了调节钙离子敏感性的重要性。我的实验室的长期目标是研究调节VSM对收缩刺激的反应性的亚细胞机制，重点是调节钙敏感性。我认为，调节钙敏感性的一种机制与收缩受体刺激的近期历史有关。简而言之，先前对VSM的强烈受体刺激诱导了一种被称为记忆的适应性反应，这种反应暂时降低了随后刺激提高钙敏感性的能力。这项拟议的研究项目将使用分离的兔动脉来1)确定记忆是否调节小动脉的肌源性张力，以及2)阐明记忆如何在亚细胞水平上运作。钙敏感性的增加在肌源性张力的调节中起着显著作用，目标1将检验记忆通过降低钙敏感性而降低肌源性张力的假说。在完整的生物体中，这种调节有望在VSM收缩受体强烈刺激后的一段时间内提供整体血流量的增加，这种刺激可能发生在交感神经活动升高的过程中，或者发生在血管活动刺激释放时的血管损伤期间。这些研究将使用插管、加压的小动脉进行。目的2将验证记忆通过减少韩国的激活来减少刺激诱导的钙敏化的假设。另一种假设，即记忆涉及激活钙离子脱敏机制，包括增加的MLC磷酸酶活性，也将得到测试。将使用标准的生物力学和生化技术，包括使用前表面荧光法和钙指示剂Fura-2同时测量组织等长力和[Ca2+]1。总之，这些研究将为调节钙敏感性和VSM对收缩刺激的反应性的细胞机制提供新的见解。

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.