Acquired resistance of a mammalian cell line to hypoxia-reoxygenation through cotransfection of Kir6.2 and SUR1 clones.
Reoxygenation after transient hypoxia is a common clinical condition that often causes greater tissue damage than persistent hypoxia itself. This warrants the development of a means to protect cells against hypoxia-reoxygenation injury. Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels have been proposed to play an essential role in the mechanisms of endogenous cellular protection. Thus far, however, KATP channel proteins have not been exploited to generate an injury-resistant cellular phenotype by delivering KATP channel genes into injury-prone cells. A first step in this direction is the evaluation of the outcome of transferring genes encoding KATP channels into a KATP channel-deficient cell type exposed to metabolic stress. Untransfected COS-7 monkey kidney cells, which natively lack KATP channels, were found to be vulnerable to hypoxia-reoxygenation injury, which induced cytosolic Ca2+ loading, as measured by digital epifluorescent imaging. COS-7 cells cotransfected with KATP channel genes, Kir6.2 and SUR1, gained resistance to hypoxia-reoxygenation. This acquired resistance was abolished by glyburide, the KATP channel antagonist. We have previously shown that Kir6.2 and SUR1 physically associate to form a functional KATP channel, not reconstituted by either of the subunits alone. Transfection with individual channel subunits, Kir6.2 or SUR1, failed to produce resistance to hypoxia-reoxygenation induced Ca2+ loading. This is a first demonstration that transfer of KATP channel subunits can generate an injury-resistant cellular phenotype. The findings from this study may, thus, provide a framework for future therapeutic strategies based on gene delivery of KATP channel subunits in cells and tissues vulnerable to hypoxia-reoxygenation insults.
Department of Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
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Jovanovic
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Jovanovic
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Carrasco
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Terzic
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D005905:Glyburide / Q000494:pharmacology; D016695:Glycosyltransferases; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology*; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D008565:Membrane Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D016695:Glycosyltransferases; C092031:SUR1 protein, S cerevisiae; D005905:Glyburide; D002118:Calcium