PKC Signaling in cAMP-Induced Pulmonary Vasodilation

描述（由申请人提供）：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，引起肺动脉血压升高，通常导致心力衰竭。目前，对IPAH的细胞和分子基础知之甚少。通常，在肺血管系统中升高cAMP和cGMP的信号传导机制维持低压、高灌注环境。大电导、钙和电压激活的钾（BKCa）通道的激活在肺动脉压的调节中是重要的，并且BKCa通道的抑制与肺动脉高压有关。我们实验室在肺动脉高压动物模型Fawn-Hooded大鼠（FHR）的肺动脉平滑肌细胞（PASMC）中发表的研究表明，cAMP（cAMP依赖性蛋白激酶（PKA）的激活剂）通过cGMP依赖性蛋白激酶（PKG）的“交叉激活”打开BKCa通道，而PKG被蛋白激酶C（PKC）激活剂抑制。相比之下，PKC激活打开了Sprague-Dawley大鼠（SDR）PASMC（血压正常对照）中的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中差异表达，PKC和PKG调节BKCa通道活性需要BKCa通道亚基上特定位点的表达和磷酸化。因此，提出的研究的假设是，特定的PKC同工酶差异调节BKCa通道活性在正常血压和高血压肺动脉平滑肌通过靶向的PKC和PKG磷酸化位点的BKCa通道亚基。将使用电生理学、血管收缩和生物化学/分子生物学的当前技术来检验这一假设，以确定血压正常和高血压PASMC：1）PKC对BKCa通道的调控机制; 2）BKCa通道亚单位表达与蛋白激酶的关系（PKA，PKC，PKG）介导的功能，和3）磷酸化特定亚基位点的机制，引发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cAMP升高剂在肺动脉平滑肌中的作用。这些研究的进展将为开发新的治疗药物提供重要的知识，这将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.