Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB337

Poster Communications

Novel insights into the role of oxidant-activation of Protein Kinase G within the vascular endothelium

S. Syed A Kadir1,2, M. Nelson3,1, A. S. Greenstein1

1. Division of Cardiovascular Sciences, University of Manchester, Greater Manchester, United Kingdom. 2. Department of Pharmacology, University of Malaya, Kuala Lumpur, Malaysia. 3. Dept. of Pharmacology, University of Vermont, Burlington, Vermont, United States.

A healthy endothelium is vital for cardiovascular health. Within the endothelium of small resistance arteries, vasodilatory pathways are predominantly dependent on localised calcium (Ca2+) signalling. These endothelial cells (ECs) Ca2+ signals control potassium (K+) channel activity, and this regulates arterial membrane potential. The principal of endothelial vasodilatory Ca2+ mechanisms is controlled by inositol trisphosphate-induced Ca2+release from the endoplasmic reticulum1 and extracellular Ca2+ entry through TRPV4 channels2. In the vascular smooth muscle cells, Protein Kinase G (PKG) plays an important role in K+ dilation mechanism. Our previous study has shown that oxidants generated in response to the intraluminal pressure are able to activate PKG3. However, the crosstalk between PKG and Ca2+ pathways in the endothelium reminds unclear. 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 (PKG[C42S]KI). EC Ca2+ signals were visualised using high-speed (50Hz) spinning disc confocal microscopy of third order mesenteric arteries which were slit open and imaged in an ‘en-face' configuration. Results were correlated with diameter changes via the same Ca2+ induced pathways using pressure myography. 10µM Acetylcholine (ACh) increased IP3 (Pulsar frequency WT=6; 15.37±3.64 vs KI=n;7.99±1.55) and TRPV4 (Sparklets activity 34.92±7.35 vs 22.43±2.37) Ca2+ signalling pathways with intact vascular endothelium compared to PKG[C42S]KI arteries. Similarly, ACh dilated WT arteries but not in the oxidant resistant model (0.75±0.15% vs 0.16±0.08%). Vasodilation to TRPV4 agonists (10nM GSK1016790A) was also impaired in the PKG[C42S]KI arteries. In contrast, direct K+ channel vasodilation using 0.3µM NS309 was preserved (0.89±0.18% vs 0.91±0.27%). The principal vasodilatory pathways of the resistance artery endothelium are critically dependent on oxidant generation which activate PKG. The oxidant-activation of PKG controls Ca2+ signal generation within the EC rather than the vasodilatory K channel function.

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