Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild-type but not connexin40 null mice in oxidant- and PKA-dependent manner.
Although electrical coupling along the arteriolar endothelium is central in arteriolar conducted response and in control of vascular resistance, little is known about the pathophysiological effect of hypoxia and reoxygenation (H/R) on this coupling. We examined this effect in a monolayer of cultured microvascular endothelial cells (ECs) derived from wild-type (WT) or connexin (Cx)40-/- mice (Cx40 is a key gap junction protein in ECs). To assess electrical coupling, we used a current injection technique and Bessel function model to compute the monolayer intercellular resistance. Hypoxia (0.1% O2, 1 h) followed by abrupt reoxygenation (5-90 min) reduced coupling (i.e., increased resistance) in WT but not in Cx40-/- monolayer. H/R increased superoxide production and reduced protein kinase A (PKA) activity in both monolayers. Activation of PKA by 8-bromo-cAMP prevented the reduction in coupling. Preloading of the WT monolayer with the antioxidant ascorbate prevented reductions in both PKA activity and cell coupling. Inhibition of PKA with 6-22 amide during normoxia mimicked the reduction in coupling. Finally, hypoxia followed by slow reoxygenation caused no change in superoxide level, PKA activity, or coupling. Using intravital microscopy, we assessed the physiological relevance of these findings in terms of KCl-induced conducted vasoconstriction in arterioles of WT mouse cremaster muscle in vivo. Ischemia (1 h) followed by abrupt reperfusion (15-30 min) reduced conduction. 8-bromo-cAMP prevented this reduction, while 6-22 amide mimicked this reduction in control nonischemic arterioles. We propose that abrupt reoxygenation reduces interendothelial electrical coupling via oxidant- and PKA-dependent signaling that targets Cx40. We suggest that this mechanism contributes to compromised arteriolar function after H/R.
Bolon
Michael L
ML
Lawson Health Research Institute, London, Ontario, Canada.
Ouellette
Yves
Y
Li
Fuyan
F
Tyml
Karel
K
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D017630:Connexins / Q000235:genetics* / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000473:pathology; D000860:Hypoxia; D007511:Ischemia / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D017382:Reactive Oxygen Species; D013997:Time Factors; D000097003:Gap Junction alpha-5 Protein
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000975:Antioxidants; D017630:Connexins; D017868:Cyclic AMP-Dependent Protein Kinases; D016877:Oxidants; D010100:Oxygen; D011189:Potassium Chloride; D017382:Reactive Oxygen Species; D000097003:Gap Junction alpha-5 Protein