Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries

Abstract

Long-term hypoxia (LTH) attenuates nitric oxide-induced vasorelaxation in ovine middle cerebral arteries. Because cGMP-dependent protein kinase (PKG) is an important mediator of NO signaling in vascular smooth muscle, we tested the hypothesis that LTH diminishes the ability of PKG to interact with target proteins and cause vasorelaxation. Prominent among proteins that regulate vascular tone is the large-conductance Ca-sensitive K (BK) channel, which is a substrate for PKG and is responsive to phosphorylation on multiple serine/threonine residues. Given the influence of these proteins, we also examined whether LTH attenuates PKG and BK channel protein abundances and PKG activity. Middle cerebral arteries were harvested from normoxic and hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep. These arteries were denuded and equilibrated with 95% O-5% CO in the presence of -nitro-l-arginine methyl ester (l-NAME) to inhibit potential confounding influences of events upstream from PKG. Expression and activity of PKG-I were not significantly affected by chronic hypoxia in either fetal or adult arteries. Pretreatment with the BK inhibitor iberiotoxin attenuated vasorelaxation induced by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate in normoxic but not LTH arteries. The spatial proximities of PKG with BK channel α- and β1-proteins were examined using confocal microscopy, which revealed a strong dissociation of PKG with these proteins after LTH. These results support our hypothesis that hypoxia reduces the ability of PKG to attenuate vasoconstriction in part through suppression of the ability of PKG to associate with and thereby activate BK channels in arterial smooth muscle. Using measurements of contractility, protein abundance, kinase activity, and confocal colocalization in fetal and adult ovine cerebral arteries, the present study demonstrates that long-term hypoxia diminishes the ability of cGMP-dependent protein kinase (PKG) to cause vasorelaxation through suppression of its colocalization and interaction with large-conductance Ca-sensitive K (BK) channel proteins in cerebrovascular smooth muscle. These experiments are among the first to demonstrate hypoxic changes in BK subunit abundances in fetal cerebral arteries and also introduce the use of advanced methods of confocal colocalization to study interaction between PKG and its targets.

Prenatal metyrapone treatment modulates neonatal cerebrovascular structure, function, and vulnerability to mild hypoxic-ischemic injury

“…The analysis involved calculation of the number of colocalized pixels in each category as a percentage of the total number of colocalized pixels for both markers across all 3 categories (defined as 100%). Previous publications include detailed descriptions of all of these methods (13,40) and validation by comparison with other established methods for quantitation of protein colocalization via confocal microscopy (63).…”

Calcium- and voltage-gated BK channels in vascular smooth muscle

“…Likewise, chronic intermittent hypoxia leads to increased vasodilation, which is mediated by SM BK channels [62]. In contrast, in adult middle cerebral arteries, chronic hypoxia reduces the ability of PKG to activate SM BK channels [177]. Obesity and diabetes .…”

Fetal Cerebrovascular Maturation: Effects of Hypoxia

“…The net result of these opposing effects is that myofilament calcium sensitivity is increased by chronic hypoxia in fetal cerebral arteries 34 . Chronic hypoxia also increases the agonist binding affinity of serotonergic receptors 41 and decreases the ability of calcium-sensitive and ATP-sensitive potassium channels to mediate vasorelaxation 42,43 . The integrated result of these effects is that reactivity to both contractile and relaxant stimuli is attenuated by chronic hypoxia in fetal cerebral arteries.…”

“…In fetal cerebral arteries, chronic hypoxia increases water content, total protein content, and wall thickness 30 . Accompanying these significant changes in structure and composition are depressed overall contractility and depressed endothelial vasodilator capacity 30,31 that is attributable both to a decreased capacity for endothelial NO synthesis, reduced soluble guanylate cyclase activity within cerebrovascular smooth muscle, and an attenuated ability of PKG to act on BK channels 43,182,183 . Chronic hypoxia also modulates the abundances, organization and function of contractile proteins, due partially to the effects of VEGF, resulting in a less efficient contractile apparatus 15,34,184 .…”