Dinitrophenol pretreatment of rat ventricular myocytes protects against damage by metabolic inhibition and reperfusion.
We have investigated the protective effects of pretreatment with the mitochondrial uncoupler 2,4-dinitrophenol on the cellular damage induced by metabolic inhibition (with cyanide and iodoacetic acid) and reperfusion in freshly isolated adult rat ventricular myocytes. Damage was assessed from changes in cell length and morphology measured using video microscopy. Intracellular Ca(2+), mitochondrial membrane potential, and NADH were measured using fura-2, tetramethylrhodamine ethyl ester and autofluorescence, respectively. During metabolic inhibition myocytes developed rigor, and on reperfusion 73.6+/-8.1% hypercontracted and 10.8+/-6.7% recovered contractile function in response to electrical stimulation. Intracellular Ca(2+) increased substantially, indicated by a rise in the fura-2 ratio (340/380 nm) on reperfusion from 0.86+/-0.04 to 1.93+/-0.18. Myocytes pretreated with substrate-free Tyrode containing 50 microm dinitrophenol showed reduced reperfusion injury: 29.0+/-7.4% of cells hypercontracted and 65.3+/-7.3% recovered contractile function (P<0.001 vs control). The fura-2 ratio on reperfusion was also lower at 1.01+/-0.08. Fluorescence measurements showed that dinitrophenol caused mitochondrial depolarisation, and decreased NADH. The presence of the substrates glucose and pyruvate reduced these effects, and abolished the protection against damage by metabolic inhibition and reperfusion. However protection was unaffected by block of ATP-sensitive potassium channels. Thus the protective effects of pretreatment with dinitrophenol may result from a reduction in NADH in response to mitochondrial depolarisation.
Rodrigo
G C
GC
Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester, LE1 9HN, UK. nbs@le.ac.uk
Lawrence
C L
CL
Standen
N B
NB
D000818:Animals; D003486:Cyanides / Q000633:toxicity; D004140:Dinitrophenols / Q000494:pharmacology*; D019807:Iodoacetic Acid / Q000633:toxicity; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000473:pathology; D009200:Myocardial Contraction / Q000187:drug effects; D015428:Myocardial Reperfusion Injury / Q000139:chemically induced / Q000517:prevention & control*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009243:NAD / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014475:Uncoupling Agents / Q000494:pharmacology*
D003486:Cyanides; D004140:Dinitrophenols; D014475:Uncoupling Agents; D009243:NAD; D019807:Iodoacetic Acid