A Role for Oxidant-activated Protein Kinase G in Pulmonary Artery Dilation

Abstract

In the pulmonary vasculature, the endothelial and smooth muscle cells are two key cell types that regulate vascular tone. Protein Kinase G (PKG) is the end effector kinase in blood vessel dilation and the most abundant kinase in the vascular system. PKGI is activated by the classical pathway involving nitric oxide (NO) and cyclic guanosine monophosphate (cGMP). An alternative pathway of PKGI activation, independent of cGMP, involves the oxidation of cysteine 42 residues on adjacent chains in the PKGIα homodimer complex was discovered. Oxidants cause disulfide bond formation, which also activates the kinase to phosphorylate target proteins. This study aims to elucidate the role of PKGIα oxidant activation in mediating pulmonary artery dilation. Functional studies on isolated arteries employed wire myography and Ca2+ imaging of endothelial cells in intact arteries to investigate the effects of oxidant-activated PKGIα. Inositol trisphosphate-induced Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry through TRPV4 channels were monitored as Ca2+ pulsars and sparklets, respectively. Comparisons were made between wild-type (WT) mice and the transgenic Cys42Ser knock-in (KI) mouse model, in which PKGIα lacks the cysteine-based oxidant sensor, rendering it insensitive to activation by oxidation: it can only be activated through the NO/cGMP pathway. No difference was found between WT and Cys42Ser KI mice in the relaxation of pulmonary arteries to sodium nitroprusside, glyceryltrinitrate or sildenafil, which all stimulate cGMP signalling. Endothelium-dependent relaxation to carbachol was also similar in WT and KI mice. The TRPV4 agonist, GSK1016790A, evoked endothelium-dependent relaxation at nanomolar concentrations. Cys42Ser KI arteries demonstrated reduced relaxation and endothelial Ca2+ sparklet activity in response to GSK1016790A, compared with WT arteries. From these results, it is proposed that oxidation of PKGIα is specific to the endothelium in the pulmonary artery and is essential for the activation of TRPV4 channels, endothelial Ca2+ influx and the resulting smooth muscle relaxation. It is proposed that the oxidation of PKGIα prevents cGMP from activating the kinase, thereby relieving its inhibition of TRPV4 activity.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alison Gurney (Supervisor) & Adam Greenstein (Supervisor)