Reduction of electrical coupling between microvascular endothelial cells by NO depends on connexin37.
We have previously shown that increased nitric oxide (NO) production in sepsis impairs arteriolar-conducted vasoconstriction cGMP independently and that the gap junction protein connexin (Cx) 37 is required for this conducted response. In the present study, we hypothesized that NO impairs interendothelial electrical coupling in sepsis by targeting Cx37. We examined the effect of exogenous NO on coupling in monolayers of cultured microvascular endothelial cells derived from the hindlimb skeletal muscle of wild-type (WT), Cx37 null, Cx40 null, and Cx43(G60S) (nonfunctional mutant) mice. To assess coupling, we measured the spread of electrical current injected in the monolayer and calculated the monolayer intercellular resistance (inverse measure of coupling). The NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA) rapidly and reversibly reduced coupling in cells from WT mice, cGMP independently. NO scavenger HbO(2) did not affect baseline coupling, but it eliminated DETA-induced reduction in coupling. Reduced coupling in response to DETA was also seen in cells from Cx40 null and Cx43(G60S) mice, but not in cells from Cx37 null mice. DETA did not alter the expression of Cx37, Cx40, and Cx43 in WT cells analyzed by immunoblotting and immunofluorescence. Furthermore, neither the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III), superoxide scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, nor preloading of WT cells with the antioxidant ascorbate affected this reduction. We conclude that NO-induced reduction of electrical coupling between microvascular endothelial cells depends on Cx37 and propose that NO in sepsis impairs arteriolar-conducted vasoconstriction by targeting Cx37 within the arteriolar wall.
Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada.
Rebecca L
RL
McKinnon
Michael L
ML
Bolon
Hong-Xing
HX
Wang
Scott
S
Swarbreck
Gerald M
GM
Kidder
Alexander M
AM
Simon
Karel
K
Tyml
D000818:Animals; D015153:Blotting, Western; D002196:Capillaries / Q000166:cytology / Q000502:physiology; D002469:Cell Separation; D018031:Connexin 43 / Q000235:genetics / Q000502:physiology; D017630:Connexins / Q000235:genetics / Q000502:physiology*; D004594:Electrophysiology; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D007202:Indicators and Reagents; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009573:Nitrites / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism; D000097003:Gap Junction alpha-5 Protein; D000097006:Gap Junction alpha-4 Protein
D018031:Connexin 43; D017630:Connexins; D007202:Indicators and Reagents; D009569:Nitric Oxide; D020030:Nitric Oxide Donors; D009573:Nitrites; D030421:Peroxynitrous Acid; D000097003:Gap Junction alpha-5 Protein; D000097006:Gap Junction alpha-4 Protein