Redox signaling is recently emerging as a fundamental mechanism regulating vascular function, particularly mediating the vasodilator action of nitric oxide (NO). There is also accumulating evidence indicating that NAD(P)H oxidase-derived superoxide (O2"""""""") is importantly involved in various vasomotor responses. However, it remains unknown how O2"""""""" is produced in response to different physiological stimuli and how the NO-O2"""""""" interaction works to control vascular tone under physiological conditions. This grant proposal will test the hypothesis that an NAD(P)H oxidase-mediated redox amplifying system in vascular smooth muscle cells importantly contributes to the control of vascular tone and vasomotor responses through cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling and serves as a critical target for the action of NO in coronary arteries.
In Aim 1, we will first characterize a membrane-associated NAD(P)H oxidase (mNOX) activity in coronary arterial myocytes (CAMs) by measuring its transmembrane electron currents and coupled proton channels and then determine its physiological response to vasoconstrictor agonists using patch clamp technique.
In Aim 2, we will determine whether this mNOX-derived O2~ enhances cADPR production and stimulates Ca2+ release via ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) by biochemical analyses and fluorescent microscopy of intracellular Ca2+ in coronary arterial smooth muscle. In these experiments, we will address whether cADPR production and Ca2+ release in CAMs occur due to accumulation of NAD+ and internalization-dimerization of CD38 (ADP-ribosylcyclase) associated with mNOX activation.
Aim 3 will explore the mechanisms increasing intracellular O2' levels in CAMs by confocal microscopic colocalization of O2~ and SR and by HPLC and ESR analysis of srNOX activity. We will further determine srNOX-mediated regulation of RyR function by lipid bilayer channel reconstitution. A Ca2+-sensitive srNOX will be demonstrated as a critical redox regulator on the SR in CAMs. Finally, in Aim 4 we will determine whether NO-induced vasodilation is associated with targeting this redox amplifying mechanism using video microscopy of isolated small coronary arteries. This proposal will clarify how oxygen free radicals and calcium signaling receptors interact in coronary arterial muscle cells. Understanding of this interaction will help develop new therapy for heart diseases since blockade of this interaction may open coronary arteries and increase blood flow to the heart. ? ? ?
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