Loss of nitroglycerin-induced blood pressure lowering in redox-dead Cys42Ser PKG1α knock-in mouse

Abstract

Nitroglycerin has remained in clinical use since 1879 however the mechanism by which it relaxes blood vessels to lower blood pressure remains incompletely understood. Metabolism of nitroglycerin generates several reaction products, including oxidants, and this ‘bioactivation’ process is essential for vasodilation. Protein kinase G (PKG) mediates classical nitric oxide-dependent vasorelaxation, but the 1α isoform is also independently activated by oxidation involving interprotein disulfide formation within this homodimeric protein complex. We hypothesised that nitroglycerin-induced vasodilation is mediated by disulfide activation of PKG1α. Treating smooth muscle cells or isolated blood vessels with nitroglycerin caused PKG1α disulfide dimerization. PKG1α disulfide formation was increased in wild-type mouse aortae by in vivo nitroglycerin treatment, but this oxidation was lost as tolerance developed. To establish whether kinase oxidation underlies nitroglycerin-induced vasodilation in vivo we employed a Cys42Ser PKG1α knock-in mouse that cannot transduce oxidant signals as it does not contain the vital redox-sensing thiol. This ‘redox-dead’ knock-in mouse was substantively deficient in hypotensive response to nitroglycerin compared to wild-type littermates as measured in vivo using radiotelemetry. Resistance blood vessels from knock-ins were markedly less sensitive to nitroglycerin-induced vasodilation (EC50 = 39.2 ± 10.7 μM) than wild-types (EC50 = 12.1 ± 2.9 μM). Furthermore, after 24 h of treatment wild-type controls stopped vasodilating to nitroglycerin and the vascular sensitivity to nitroglycerin was decreased, whereas this ‘tolerance’ phenomenon that routinely hampers the management of hypertensive patients was absent in knock-ins. We conclude that PKG1α disulfide formation is a significant mediator of nitroglycerin-induced vasodilation and tolerance to nitroglycerin is associated with loss of kinase oxidation.