Role of Oxidant-activation of Protein Kinase G on Small Resistance Artery Vasodilation

Abstract

Protein Kinase G (PKG) is an essential and most abundant kinase in the vasculature system. PKG is known to be activated by cyclic guanosine monophosphate (cGMP) until the recent discovery of a novel mode of activation by oxidant. The general objective of this thesis is to understand the role of oxidant-activation of PKG on Calcium (Ca2+) signalling within endothelial and vascular smooth muscle cells of small resistance arteries. Effects of oxidant-activation of PKG on Ca2+ signalling pathways were assessed via the use of a genetically modified mouse model whereby an amino acid switch (Cysteine to Serine) renders PKG insensitive to oxidant activation referred as PKG[C42S]KI. The primary aim of this project is to scrutinise the involvement of PKG in the vasodilatory endothelial mechanism involving Inositol trisphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry through TRPV4 channels. Endothelial Ca2+ signals were imagined using high-speed (50Hz) spinning disc confocal microscopy of third-order mesenteric arteries which were slit open and imaged in an ‘en-face’ configuration. M3 muscarinic receptor activation by Acetylcholine (ACh) on the endothelial cells revealed an increase in Ca2+ kinetics in both IP3 and TRPV4 Ca2+ events in the healthy wildtype mice compared to significantly reduced Ca2+ events in the PKG[C42S]KI mice. The reduction in Ca2+ kinetics from the PKG[C42S]KI further supports the observation of attenuated vasodilatory capacity of the oxidant-sensitive mice, PKG[C42S]KI in the pressure myography experiments. Vasodilation to TRPV4 agonists was also impaired in the PKG[C42S]KI arteries. Hence, the principal vasodilatory pathways of small artery endothelium are critically dependent on oxidant generation, which activate PKG. The secondary aim of this project is to delineate the vasodilatory mechanism of Ca2+ sparks within VSMCs of small arteries, which are vital for microcirculatory homeostasis. Ca2+ sparks are generated by intraluminal pressure, a process ascribed to depolarisation of the VSMC membrane. However, we recently showed that intraluminal pressure within small arteries regulates Ca2+ spark signalling by pressure-dependent oxidative activation of PKG. Using high speed intracellular Ca2+ imaging, pressure and wire myography and molecular approaches, we examined the effects of varying intraluminal pressure and pharmacological stimulation on the regulation of small artery tone by oxidant-activation of PKG. Intraluminal pressure, specifically, rather than a generic stretch of the artery, was necessary to activate the oxidative pathway. Results demonstrated a steep activation profile for the activation of Ca2+ sparks and also a functional ‘ceiling’ for this pressure-sensitive oxidative pathway. These findings contextualise the role of pressure-induced oxidants in the regulation of small artery pressure induced-constriction and further highlight the importance of oxidant-activation of PKG in maintaining small artery function.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Eisner (Supervisor) & Adam Greenstein (Supervisor)