REGULATION OF VASCULAR SMOOTH MUSCLE CA++ SENSITIVITY

血管平滑肌 CA 敏感性的调节

• 批准号: 2223148
• 负责人: Robert S Moreland
• 金额: $ 24.8万
• 依托单位: GRADUATE HOSPITAL (PHILADELPHIA)
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2223148
• 关键词: calcium flux; cyclic AMP; cyclic GMP; diacylglycerols; enzyme activity; laboratory rabbit; muscle contraction; phospholipase C; phosphorylation; protein kinase C; second messengers; vascular smooth muscle; vasomotion; western blottings

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.

人们普遍认为，[Ca 2 +]i的增加会引发 通过激活钙调蛋白依赖性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依赖性激动剂 刺激可以增加肌丝Ca 2+敏感性， 伴随的稳态MLC磷酸化持续增加，或 交叉桥循环表明MLC磷酸化不是唯一的 天桥行为的决定因素。 我们还证明， Ca ~（2+）和NE激活透化组织， 假定的细丝调节蛋白的磷酸化， 钙调蛋白。 这在单独的Ca 2+中没有观察到。 长期 该提案的目的是确定机制， 激动剂激活血管平滑肌调节肌丝Ca 2 + 灵敏度 本申请基于以下假设： 血管平滑肌收缩的Ca 2+敏感性增加是由于 主要是蛋白激酶C（PKC）激活， 细丝调节蛋白活性的改变， 钙调蛋白。 我们建议研究α毒素透化动脉， 与以前使用的皮肤模型相比， 血管平滑肌，因为即使受体激活也能维持， 尽管细胞膜已经被制成可渗透小离子， 分子。 该模型将用于确定 力，[Ca 2 +]i，蛋白磷酸化（包括特定位点的 磷酸盐），以及激动剂期间的交联循环速率和附着 诱发收缩。 上述措施所要达到的具体目标 实验是：1.确定PKC是否是激动剂的介导剂， 通过测量诱导的肌丝Ca 2+敏感性变化 甘油二酯水平和PKC从胞浆到膜的转运 在Ca 2+敏感性增加之前和期间， 抑制PKC活性可消除Ca ~（2+）敏感性的增加; PKC直接或间接转位到收缩丝 调解其行动也将被审查; 2.以确定是否 钙调蛋白的磷酸化介导Ca 2+敏感性的变化。 这将通过将钙调蛋白磷酸化与PKC 激活和增强的Ca 2+敏感性的大小; 3.到 确定cAMP和cGMP降低肌丝Ca 2+的机制 灵敏度 在恒定[Ca 2 +]i下环核苷酸诱导的弛豫 将与PKC和磷脂酶C激活的程度相关， 和收缩蛋白磷酸化;和4.确定 肌丝Ca 2+敏感性的调节有助于 血管舒缩 这将通过同时测量 力和[Ca 2 +]i。 最后 拟议的研究将确定负责的机制 调节Ca 2+敏感性以及其潜在的生理 作用