PKC Signaling in cAMP-Induced Pulmonary Vasodilation

描述(申请人提供)：特发性肺动脉高压(IPAH)是一种原因不明的疾病，导致肺动脉狭窄，引起肺动脉高压，通常导致心力衰竭。目前，对IPAH的细胞和分子基础知之甚少。正常情况下，升高肺血管内cAMP和cGMP的信号机制维持低压力、高灌流环境。大电导、钙和电压激活钾(BKCa)通道的激活在调节肺动脉压中起重要作用，而BKCa通道的抑制与肺动脉高压有关。本实验室已发表的关于肺动脉高压动物模型--FHR的研究表明，cAMP是cAMP依赖的蛋白激酶(PKA)的激活剂，通过交叉激活cGMP依赖的蛋白激酶(PKG)打开BKCa通道，而PKG被蛋白激酶C(PKC)激活剂抑制。相反，在正常血压对照的SD大鼠(SDR)的PASMC中，PKC激活开放了BKCa通道。新的初步数据表明，特定的PKC同工酶在正常血压和高血压PASMC中有不同的表达，而PKC和PKG对BKCa通道活性的调节需要BKCa通道亚基上特定位点的表达和磷酸化。因此，本研究提出的假设是，特定的PKC同工酶通过靶向BKCa通道亚基上的PKC和PKG磷酸化位点来不同地调节正常血压和高血压的肺动脉平滑肌的BKCa通道活性。这一假说将利用现有的电生理学、血管收缩和生物化学/分子生物学技术来验证，以确定在正常血压和高血压PASMC中：1)BKCa通道的PKC调节机制，2)BKCa通道亚单位表达与蛋白激酶(PKA、PKC、PKG)介导的功能的关系，以及3)特定亚基位点的磷酸化机制，从而引起PKA、PKC和PKG对BKCa通道活性的调节作用。长期目标是确定PKC如何调节肺动脉平滑肌中cAMP提高剂的作用。这些研究的进展将为开发新的治疗药物提供重要的知识，有助于降低与肺动脉高压相关的发病率和死亡率。

DESCRIPTION (provided by applicant): Idiopathic Pulmonary Arterial Hypertension (IPAH) is a disease of unknown origin that results in narrowing of the pulmonary arteries causing high pulmonary blood pressure often leading to heart failure. Currently, there is little knowledge on the cellular and molecular foundation of IPAH. Normally, signaling mechanisms which elevate cAMP and cGMP in the pulmonary vasculature maintain a low pressure, high perfusion environment. Activation of large-conductance, calcium- and voltage-activated potassium (BKCa) channels is important in the regulation of pulmonary arterial pressure and inhibition of BKCa channels has been implicated in pulmonary hypertension. Published studies from our laboratory in pulmonary arterial smooth muscle cells (PASMC) of the Fawn-Hooded rat (FHR), an animal model of pulmonary hypertension, show that cAMP, an activator of cAMP-dependent protein kinase (PKA), opens BKCa channels through "cross-activation" of cGMP-dependent protein kinase (PKG), which is inhibited by protein kinase C (PKC) activators. In contrast, PKC activation opens BKCa channels in (normotensive control) PASMC of Sprague-Dawley rats (SDR). New preliminary data indicate that specific PKC isozymes are differentially expressed in normotensive and hypertensive PASMC, and that PKC and PKG regulation of BKCa channel activity requires the expression and phosphorylation of specific sites on BKCa channel subunits. Therefore, the hypothesis of the proposed studies is that specific PKC isozymes differentially regulate BKCa channel activity in normotensive and hypertensive pulmonary arterial smooth muscle via targeted PKC and PKG phosphorylation sites on BKCa channel subunits. This hypothesis will be tested using current techniques of electrophysiology, vascular contraction, and biochemistry/molecular biology to determine in normotensive and hypertensive PASMC: 1) mechanisms of PKC regulation of BKCa channels, 2) the relationship of BKCa channel subunit expression to protein kinase (PKA, PKC, PKG)-mediated function, and 3) mechanisms of phosphorylating specific subunit sites that elicit regulatory effects of PKA, PKC and PKG on BKCa channel activity. The long-term goal is to determine how PKC regulates the effect of cAMP-elevating agents in pulmonary arterial smooth muscle. Progress of these studies will provide important knowledge towards the development of novel therapeutic agents that will help reduce the morbidity and mortality associated with pulmonary hypertension.