Ischemic preconditioning: a potential role for protein S-thiolation?
Oxidant stress plays a crucial role in the triggering of cardioprotection involving ischemic preconditioning (IPC). We have used biotin-tagged cysteine to probe for redox-modified proteins in IPC protocols. Cysteine was biotinylated and introduced into isolated rat hearts. S-Thiolated proteins were detected and quantified using nonreducing western blots probed with streptavidin-horseradish peroxidase. Controls (15 min of aerobic perfusion plus 5 min of 0.5 mM biotin-cysteine plus 5 min of aerobic perfusion) showed low-level protein S-thiolation. Hearts preconditioned with 5 min of ischemia and reperfused for 5 min with biotin-cysteine plus 5 min of aerobic perfusion showed increased thiolation (160%) that was fully blocked by the antioxidant mercaptopropionylglycine, which is also known to block IPC. "Preconditioning" agonists (phorbol 12-myristate 13-acetate or phenylephrine) or oxidants (hydrogen peroxide or diamide) administered during aerobic preparations to biotin-cysteine-loaded hearts induced efficient protein S-thiolation. Preconditioning agonist-induced S-thiolation was significantly attenuated by diphenyleneiodonium (a flavoprotein inhibitor) or by the protein kinase C inhibitor bisindolylmaleimide I. Additional studies testing the role of a Nox2-containing NAD(P)H oxidase as the source of the oxidant stress essential to the triggering IPC showed that protein S-thiolation was the same in wild-type and Nox2 knockout mice.
Eaton
Philip
P
Department of Cardiology, King's College London, London, UK. philip.eaton@kcl.ac.uk
Bell
Robert M
RM
Cave
Alison C
AC
Shattock
Michael J
MJ
D000818:Animals; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D010656:Phenylephrine / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D017293:Protein S / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D008562:Membrane Glycoproteins; D016877:Oxidants; D017293:Protein S; D013438:Sulfhydryl Compounds; D010656:Phenylephrine; D015240:Phorbol 12,13-Dibutyrate; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D011493:Protein Kinase C