Role of Protein Kinase G in Regulating Vascular Tone in Hypoxic Ovine Cerebral Arteries
Nitric oxide (NO) and its precursors have been used clinically to induce vasorelaxation long before the specific mechanism of how this operates was understood. With the discovery in 1977 of the influence of NO on guanylate cyclase, and the subsequent elucidation of the NO pathway, the role of cyclic guanidine monophosphate (cGMP)-dependent protein kinase (PKG) has been shown to be the main mediator of NO-induced vasorelaxation. Less well understood is the behavior of this pathway in conditions of long term hypoxia (LTH). It was later shown that LTH attenuates the ability of PKG to mediate vasorelaxation. How this operates is the focus of this investigation. Here we tested the hypothesis that LTH attenuates NO-induced vasorelaxation by reducing the ability of PKG to phosphorylate its target proteins that mediate vascular relaxation and contraction. A prominent target of PKG involved with vasorelaxation is the BK channel alpha protein. To examine the influence of LTH on PKG-induced vasorelaxation, and to demonstrate the relative influence of the BK channel in these conditions, we used carotid and middle cerebral arteries from normoxic and chronically hypoxic fetal and adult sheep. These arteries were denuded of endothelium and used in concentration-response relations using 5-HT as a contractant and measured contractile force. To measure the relative influence of the BK channel in these arteries, we used the selective BK channel blocker iberiotoxin. From this we demonstrated that while hypoxia affects both BK and non-BK channel mechanisms, the influence of LTH on the BK channel is dominant in the loss of PKG-mediated vasorelaxation. We also examined whether hypoxia attenuates PKG expression and specific activity, as well as the BK channel alpha and beta proteins. Using Western blotting and enzyme kinase activity assays, we learned that LTH has a modest influence on BK channel proteins expression, and no influence on PKG expression or activity. We next examined whether LTH influences protein organization, which could potentially control PKG-mediated vasorelaxation in normoxic and hypoxic conditions. Using confocal microscopy, we learned that LTH strongly alters protein organization, and specifically the proximal association of three proteins associated with vasorelaxation, namely PKG, BK-alpha and BK-beta.
Medical Biochemistry, Medical Sciences, Medicine and Health Sciences, Protein Kinases; Hypoxia; Nitric Oxide -- Pharmacology; Cerebrovascular Circulation, Vasorelaxation; PKG-mediated vasorelaxation; BK channel blockers;
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