Vasoregulation by IP3 receptor coupling to TRPC channels

动脉直径是全身血压和器官血流的主要调节器，由 动脉肌细胞收缩性能的变化。动脉肌细胞的收缩状态由以下因素决定 几个局部和全局的细胞内钙([Ca~(2+)]i)信号。Ca~(2+)信号与肌醇的直径调节 1，4，5-三磷酸(IP3)是一种由磷脂酶C产生的第二信使，人们对此知之甚少。这个 传统观点认为，IP3通过刺激肌浆网(SR)钙离子释放而收缩动脉 在肌细胞中。我们最近发表的数据表明，IP3诱导的血管收缩有一种新的机制 与肌浆网钙离子释放无关，并通过IP3受体(IP3R)和典型的瞬时受体发生 电位(TRPC)3依赖的阳离子电流(ICAT)激活。然而，IP3R激活的机制 刺激动脉肌细胞中的TRPC通道以调节动脉直径的机制尚不清楚。初步数据 提示心肌细胞IP3Rs和TRPC3通道之间的物理偶联调节IP3诱导 血管收缩。数据还表明，动脉肌细胞小窝促进了这一IP3R-TRPC通道 血管调节机制。这一建议的中心假设是，在大脑动脉肌细胞中，小凹 促进TRPC3通道与IP3Rs偶联介导血管收缩和IP3诱导的膜 去极化、电压依赖性钙通道激活、[钙]i升高和收缩。这项建议 将研究三个特定目标：目标1将检验假设，在大脑动脉肌细胞中，IP3R到TRPC3 IP3诱导的ICAT激活需要通道物理耦合。目标2将检验IP3R到 TRPC3偶联介导IP3诱导的脑膜去极化、[Ca~(2+)]i升高和收缩 动脉。目标3将检验动脉肌细胞小窝调节生理和功能的假设。 将TRPC3通道耦合到IP3R。研究这些目标的实验将结合在 分子、细胞和完整组织水平，包括钙离子成像、FRET、电生理学、加压动脉 肌电扫描和基因抑制。

Arterial diameter, a principal modulator of systemic blood pressure and organ blood flow is regulated by changes in the contractility of arterial myocytes. The contractile status of arterial myocytes is determined by several local and global intracellular calcium ([Ca2+]i) signals. Ca2+ signal and diameter regulation by inositol 1,4,5-trisphophate (IP3), a phospholipase C-generated second messenger is poorly understood. The conventional view has been that IP3 constricts arteries by stimulating sarcoplasmic reticulum (SR) Ca2+ release in myocytes. Our recently published data indicated a novel mechanism of IP3-induced vasoconstriction that occurred independently of SR Ca2+ release, and via IP3 receptor (IP3R)- and canonical transient receptor potential (TRPC) 3-dependent cation current (ICat) activation. However, mechanisms by which IP3R activation stimulates TRPC channels in arterial myocytes to regulate arterial diameter are unclear. Preliminary data suggest that physical coupling between myocyte IP3Rs and TRPC3 channels regulates IP3-induced vasoconstriction. Data also indicate that arterial myocyte caveolae facilitate this IP3R-TRPC channel vasoregulatory mechanism. The central hypothesis of this proposal is that in cerebral artery myocytes, caveolae facilitate TRPC3 channel coupling with IP3Rs to mediate vasoconstrictor and IP3-induced membrane depolarization, voltage-dependent Ca2+ channel activation, [Ca2+]i elevation, and contraction. This proposal will investigate 3 specific aims: Aim 1 will test the hypothesis that in cerebral artery myocytes, IP3R to TRPC3 channel physical coupling is required for IP3-induced ICat activation. Aim 2 will test the hypothesis that IP3R to TRPC3 coupling mediates IP3-induced membrane depolarization, [Ca2+]i elevation, and constriction in cerebral arteries. Aim 3 will examine the hypothesis that arterial myocyte caveolae mediate physical and functional coupling of TRPC3 channels to IP3Rs. Experiments to study these aims will integrate techniques performed at molecular, cellular, and intact tissue levels, including Ca2+ imaging, FRET, electrophysiology, pressurized artery myography, and gene suppression.