REGULATION OF VASCULAR SMOOTH MUSCLE CA++ SENSITIVITY

人们普遍认为[Ca~(2+)]i的增加引发了 激活钙调蛋白依赖的肌球蛋白光引起的血管收缩 链(MLC)激酶和随后的20 kDa MLC的磷酸化。 然而，最近我们已经证明了GTP依赖的激动剂 刺激可以增加肌丝对钙离子的敏感性，而不是 伴随而来的是稳态MLC磷酸化或 跨桥循环表明MLC磷酸化并不是唯一的 交叉桥行为的决定因素。我们也证明了 钙离子和去甲肾上腺素对通透性组织的激活作用 推测的细丝调节蛋白的磷酸化， 钙粘蛋白。这不是单用钙离子就能观察到的。从长远来看 这项提案的目标是确定 血管平滑肌激动剂激活对肌丝钙离子的调节 敏感度。此应用程序基于这样的假设： 血管平滑肌收缩的钙敏感性增加是由于 主要是蛋白激酶C(PKC)的激活和由此产生的 细丝调节蛋白活性的改变， 钙粘蛋白。我们建议研究阿尔法毒素通透性动脉 与以前使用的蒙皮模型相比，具有明显的优势 血管平滑肌中受体的激活维持均匀 尽管细胞膜对小离子具有渗透性， 分子。这一模型将被用来确定 力、[Ca2+]i、蛋白质磷酸化(包括 磷酸盐)，以及激动剂过程中的跨桥循环速率和附着 诱发宫缩。具体目标要通过以上几点来解决 实验内容如下：1.确定PKC是否是激动剂的介体 测定肌丝对钙离子敏感性的变化 二酰甘油水平与PKC从胞浆到膜的转位 在钙敏感性增加之前和期间，并确定是否 抑制PKC活性可消除钙敏感性的增加； PKC直接或间接向收缩细丝的移位 调解其行动也将受到审查；2.确定是否 钙蛋白的磷酸化介导了钙敏感性的改变。 这将通过将钙蛋白磷酸化与蛋白激酶C相关联来实现。 激活并随钙离子敏感性增强的幅度增加； 确定cAMP和cGMP降低肌丝钙离子的机制 敏感度。环核苷酸诱导的恒定[Ca~(2+)]i的松弛 将与PKC和磷脂酶C的激活程度相关， 和收缩蛋白的磷酸化；4.测定 肌丝对钙离子敏感性的调节作用程度 血管运动。这将通过同时测量以下各项来实现 在完整的血管平滑肌上的力量和[Ca~(2+)]i。总而言之， 拟议的研究将确定导致 钙离子敏感性的调节及其潜在的生理效应 角色。

It is widely accepted that an increase in [Ca2+]i initiates vasoconstriction by activation of the calmodulin dependent myosin light chain (MLC) kinase and subsequent phosphorylation of the 20 kDa MLC. Recently however, we have demonstrated that GTP dependent agonist stimulation can increase myofilament Ca2+ sensitivity without a concomitant maintained increase in steady state MLC phosphorylation or crossbridge cycling suggesting that MLC phosphorylation is not the sole determinant of crossbridge behavior. We have also demonstrated that activation of permeabilized tissues by Ca2+ and NE induces phosphorylation of the putative thin filament regulatory protein, calponin. This is not observed with Ca2+ alone. The long term objectives of this proposal are to determine the mechanisms by which agonist activation of vascular smooth muscle modulate myofilament Ca2+ sensitivity. This application is based on the hypothesis that the increase in Ca2+ sensitivity of vascular smooth muscle contraction is due primarily to protein kinase C (PKC) activation and the resultant alteration in the activity of the thin filament regulatory protein, calponin. We propose to study the alpha toxin permeabilized artery which has the distinct advantage over previously used models of skinned vascular smooth muscle in that receptor activation is maintained even though the cell membranes have been made permeable to small ions and molecules. This model will be used to determine the relationships among force, [Ca2+]i, protein phosphorylation (including specific site of phosphate), and crossbridge cycling rate and attachment during agonist induced contractions. The specific aims to be addressed with the above experiments are: 1. To determine if PKC is the mediator of agonist induced changes in myofilament Ca2+ sensitivity by measuring diacylglycerol levels and translocation of PKC from cytosol to membrane prior to and during the increase in Ca2+ sensitivity and determining if inhibition of PKC activity abolishes the increase in Ca2+ sensitivity; translocation of PKC directly or indirectly to the contractile filaments to mediate its actions will also be examined; 2. To determine if phosphorylation of calponin mediates the change in Ca2+ sensitivity. This will be achieved by correlating calponin phosphorylation with PKC activation and with the magnitude of the enhanced Ca2+ sensitivity; 3. To determine the mechanism by which cAMP and cGMP decrease myofilament Ca2+ sensitivity. Cyclic nucleotide induced relaxation at constant [Ca2+]i will be correlated with the degree of PKC and phospholipase C activation, and with contractile protein phosphorylation; and 4. To determine the degree to which modulation of myofilament Ca2+ sensitivity contributes to vasomotion. This will be accomplished by simultaneous measurement of force and [Ca2+]i in intact vascular smooth muscle. In conclusion, the proposed research will determine the mechanisms responsible for modulation of Ca2+ sensitivity as well as its potential physiological role.