Oxidant activation of endothelial PKG regulates TRPV4-mediated vasodilation in mesenteric and pulmonary resistance arteries

Abstract

Smooth muscle protein kinase G (PKG) mediates vasodilation whether activated by the NO/cGMP signalling pathway or by oxidation of cysteine residues in the PKGIα subunit1. PKG is also present in endothelial cells, where it is proposed to inhibit Ca2+ influx through heteromeric TRPV4-TRPC channels2. We tested the hypothesis that oxidant activation of PKG regulates vasodilation by modulating endothelial TRPV4 channel activity. Endothelium-dependent dilation was evoked in pulmonary arteries (PA), which employ NO as the mediator, and mesenteric arteries (MA), where Ca2+ entry via TRP4 channels leads to endothelial and smooth muscle hyperpolarisation. Arteries from PKG[C42S]KI mice, which lack the cysteine based PKG oxidant sensor1, were compared with wild type (WT) mice using pressure (MA) and wire (PA) myography and en face imaging of fluo-4 loaded endothelium with spinning disc confocal microscopy. Ca2+ signals were measured in the absence (pulsars) and presence (sparklets) of 1 μM cyclopiazonic acid to deplete Ca2+ stores2. TRPV4 currents were recorded from mesenteric endothelial cells using whole-cell patch-clamp. Muscarinic agonists caused concentration-dependent dilation of both arteries from WT mice, as well as PA, but not MA, from PKG[C42S]KI mice. They also stimulated the activity of Ca2+ pulsars in the endothelium of both WT arteries and in PA, but not MA, from PKG[C42S]KI mice. The response in WT MA was suppressed by reducing the availability of endogenous H2O2 with PEG-catalase. The TRPV4 agonist, GSK1016709A (1-100 nM), evoked endothelium and concentration-dependent relaxation of PA and MA from WT mice but was nearly 10-fold less potent in both vessels from PKG[C42S]KI mice. At 10 nM, GSK1016709A activated endothelial TRPV4 sparklets in PA and MA, but was less effective in vessels from PKG[C42S]KI mice. GSK1016709A was also 10-fold less potent at activating TRPV-4 current in mesenteric endothelial cells from PKG[C42S]KI mice, compared with WT mice.The loss of endothelium-dependent relaxation to GSK1016790A in PKG[C42S]KI arteries, along with the reduced ability of GSK1016790A to generate endothelial currents and Ca2+ sparklets, indicates that vasodilation mediated by endothelial TRPV4 channels requires PKG activation by endogenous oxidants. The differential ability of muscarinic agonists to dilate MA and PA from PKG[C42S]KI mice is consistent with the central role of TRP4 channels in endothelium-dependent dilation of MA, compared with a lack of TRPV4 contribution in PA.1. Burgoyne JR, Madhani M, Cuello F, Charles RL, Brennan JP, Schroder E, Browning, DD, Eaton P (2007). Cysteine redox sensor in PKGIα enables oxidant-induced activation. Science 317: 1393-13972. Zhang, P., Mao, Aq., Sun, Cy. et al. Translocation of PKG1α acts on TRPV4-C1 heteromeric channels to inhibit endothelial Ca2+ entry. Acta Pharmacol Sin 37, 1199–1207 (2016).3. Sonkusare SK, Bonev AD, Ledoux J, Liedtke W, Kotlikoff MI, Heppner TJ, Hill-Eubanks DC, Nel British Heart Foundation; Amman Arab University, University of Malaya, Ministry of Higher Education Malaysia This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.