Pressure-induced oxidative activation of Protein Kinase G enables Ca2+ spark/BK channel-mediated vasoregulation of myogenic tone in resistance arteries
Dr Majid Ahmed MBChB MRes (Distinction)
British Heart Foundation Clinical Research Fellow, Department of Cardiovascular Sciences, University of Manchester. UK.
Activation of Ca2+-sensitive, large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) by local, ryanodine receptor-mediated Ca2+ signals (Ca2+ sparks) acts as a brake on pressure induced (myogenic) vasoconstriction—a fundamental mechanism that regulates blood flow in resistance arteries. Here, we report a novel mechanism linking physiological intraluminal pressure within small arteries to Ca2+ spark/BK channel vasodilation: oxidative activation of VSMC cGMP-dependent protein kinase (PKG) through formation of an oxidant-induced disulphide bond between cysteine residues (Cys42).
Third-order mesenteric arteries were studied from transgenic knock‑in mice expressing a PKG variant in which Cys42 is replaced with serine; the resulting PKG[C42S]KI variant is resistant to oxidant-induced activation but can still be activated normally by cGMP. PKG[C42S]KI arteries displayed significantly enhanced intraluminal pressure-induced constriction compared with WT arteries and almost entirely absent functional BK vasodilation. Epifluorescent imaging of oxidant-sensitive CM-H2DCFDA loaded arteries demonstrated pressure-induced oxidant production and Western blot protocols demonstrated both oxidant- and pressure-induced PKG dimerization in mesenteric arteries. Perforated patch clamp studies of mesenteric VSMCs revealed absence of spontaneous transient outward currents from PKG[C42S]KI VSMCs at -40mV, but conversely, whole cell voltage step protocols indicated equivalent BK channel I/V characteristics. High speed confocal microscopy of pressurised arteries loaded with the Ca2+ indicator Fluo-4 revealed significant reduction in Ca2+ sparks in PKG[C42S]KI arteries compared with WT (see figure below). Importantly, exogenous H2O2 increased Ca2+ spark frequency in unpressurised WT arteries but not in PKG[C42S]KI arteries.
Our interpretation is that disablement of the oxidant activating mechanism in the PKG[C42S]KI mice reduces Ca2+ spark frequency, decreasing pressure-induced BK channel activation and thereby deactivating the BK channel-mediated negative feedback regulation of vasoconstriction leading to a higher degree of myogenic tone. Therefore, our results support the novel concept of a negative feedback control mechanism that regulates arterial diameter through pressure-induced mechanosensitive production of oxidants to activate PKG and enhance the Ca2+ spark-BK channel axis of vasodilation.