Heterogeneity of hypoxic Ca2+ release in pulmonary and *

描述(由申请人提供)： 低氧可引起肺动脉平滑肌细胞(PASMCs)[Ca~(2+)]i升高及相应的血管收缩反应，但对体动脉肌细胞无明显影响。此外，低氧钙离子和收缩反应的阻力比导管PASMCs大得多。然而，人们对这些功能差异背后的细胞和分子机制知之甚少。我们和其他研究人员一直证明，Ryanodine受体(RyR)钙释放在PASMCs缺氧性[Ca2+]i增加和相关的血管收缩中起关键作用。我们的初步研究结合以前的发现表明，特定的RyR亚型(RyR1、RyR2和RyR3)在血管系统中有不同的表达。这种表达的异质性可能有助于观察到不同血管中兴奋-收缩偶联以及低氧钙和收缩反应的差异。此外，内源性调节分子FKBP12.6(RyR抑制物)和cADPR(RyR激活剂)与不同RyR亚型的特异性相互作用也可能在生理性和缺氧性钙释放和血管收缩的异质性中发挥重要作用。为了解决这些假说，本研究试图检验以下三个问题(具体目的)：RyR亚型在阻力和导管肺和肠系膜动脉SMC中是否异质性表达？2)RyR钙释放在阻力和管道性肺和肠系膜动脉SMC中是否异质性？3)RyR亚型表达和RyR偶联FKBP12.6和cADPR的异质性是否解释了阻力和管道性肺和肠系膜动脉SMC之间低氧钙释放的差异？这些目标将通过使用最先进的生物物理学(共聚焦显微镜、膜片钳等)、分子和遗传方法(基因敲除和过度表达)来实现。因此，这一研究结果将扩大我们对生理和低氧钙及收缩反应差异和异质性的细胞和分子机制的理解，并可能确定新的治疗肺动脉高压的靶点。这些研究对于调节其他类型细胞的钙释放也具有重要的生理学意义。

DESCRIPTION (provided by applicant): Hypoxia induces [Ca2+]I increase and associated vasoconstriction in pulmonary artery smooth muscle cells (PASMCs), but not in systemic artery myocytes. Futhermore, hypoxic Ca2+ and contractile responses are much greater in resistance than conduit PASMCs. However, very little is known about the cellular and molecular mechanisms underlying these functional differences. We and other investigators have consistently demonstrated that ryanodine receptor (RyR) Ca2+ release is critical for hypoxic [Ca2+]I increase and associated vasoconstriction in PASMCs. Our preliminary studies, together with previous findings, suggest that specific RyR subtypes (RyR1, RyR2 and RyR3) are differentially expressed in the vasculature. This heterogeneous nature of expression may contribute to observed differences in excitation-contraction coupling and hypoxic Ca2+ and contractile responses in various blood vessels. In addition, specific interactions of endogenous regulatory molecules FKBP12.6 (RyR inhibitor) and cADPR (RyR activator with distinct RyR subtypes may also play an important role in the heterogeneity of physiological and hypoxic Ca2+ release and vasoconstriction. To address these hypotheses, this proposal seeks to examine the following three questions (Specific Aims): Are RyR subtypes heterogeneously expressed in resistance and conduit pulmonary and mesenteric artery SMCs? 2) Is RyR Ca2+ release heterogeneous in resistance and conduit pulmonary and mesenteric artery SMCs? 3) Do the heterogeneities of RyR subtypes expression and RyR coupling to FKBP12.6 and cADPR explain differences in hypoxic Ca2+ release between resistance and conduit pulmonary and mesenteric artery SMCs? These aims will be pursued by using the state-of-the-art biophysical (confocal microscopy, patch clamp, etc), molecular and genetic approaches (gene knockout and over-expression). Thus, the findings from this proposal will extend our understanding of the cellular and molecular mechanisms that contribute to the differences and heterogeneity of physiological and hypoxic Ca2+ and contractile responses, and may identify novel therapeutic targets for pulmonary hypertension. The proposed studies are also of fundamental physiological significance with respect to the regulation of Ca2+ release in other cell types.