Abstract 19562: PKG-I[[Unable to Display Character: ɑ]] Oxidative Activation Regulates Small Artery Myogenic Tone Through Calcium Sparks and the BK Channel

Abstract

Small resistance arteries function in a state of partial constriction generated by vascular smooth muscle cells (VSMC) in response to intraluminal pressure (‘myogenic constriction’). Collectively, the degree of myogenic constriction determines peripheral resistance and so influences systemic blood pressure. cGMP-dependent protein kinase (PKG) is widely implicated in vascular physiology, but until now has been assumed to play no role in modulation of this important vascular function on the basis that inhibitors of cGMP have no influence on pressure-induced tone. Recent identification of a novel redox mechanism by which PKG can be activated independently of the ‘classical’ NO-sGC-cGMP pathway made it important to revisit PKG’s role in these processes. To examine the relative contribution of these pathways, a transgenic mouse model was utilized, in which a single point mutation precludes activation via oxidative dimerization of PKG, whist preserving classical activation by cGMP. Diameter studies of small arteries from this ‘knock-in’ model were investigated in a pressure-myograph system, and demonstrated reduced constitutively functioning BKCa channel activity in these animals, and in consequence markedly enhanced myogenic constriction. Through imaging of calcium sparks with a spinning disc confocal microscope, the loss of BKCa channel function was shown to be due to diminished VSMC calcium sparks. Patch clamp electrophysiological studies showed that the amplitude and frequency of spontaneous transient outward currents were also reduced, consistent with these findings. Pharmacological studies using both pressure and wire myography with the application of the oxidant hydrogen peroxide provided a signal that myogenic tone initiates oxidant-induced activation of PKG. Western blot analysis of vessels fixed during relevant functional responses confirmed that generation of pressure-induced tone triggers oxidant activation of PKG. Finally, optical monitoring confirmed elevated ROS production with the induction of tone. Our results provide a conceptual framework by which vascular tone is modulated through release of ROS and oxidant-activation of PKG through calcium signalling. This may have important implications for blood pressure.