The origin of calcium overload in rat cardiac myocytes following metabolic inhibition with 2,4-dinitrophenol.
We have investigated the characteristics of the rise in cytoplasmic calcium that occurs when rat isolated cardiac ventricular myocytes are exposed to 2,4-dinitrophenol using conventional and confocal fluorescence microscopy and patch clamp. 2,4-dinitrophenol (200 microM) caused cytoplasmic calcium to increase in two phases: (1) an initial rise in fluo-3 fluorescence of 36+/-2% that was maintained until rigor contraction; (2) a further progressive rise so that fluo-3 fluorescence had increased by 177+/-12% 535 s after 2,4-dinitrophenol addition. Both phases were unaffected by removal of external Ca(2+). 2,4-dinitrophenol caused mitochondrial depolarization, measured using tetramethyl rhodamine ethyl ester fluorescence. Mitochondrial depolarization was associated with a decrease in intra-mitochondrial calcium measured with rhod-2, and experiments on myocytes loaded with both fluo-3 and rhod-2 showed that fluo-3 fluorescence increased as rhod-2 fluorescence fell. The correlation of the onset of the second phase of the increase in cytoplasmic calcium with rigor suggested that this phase was consequent on ATP depletion. DNP also caused activation of an ATP-sensitive potassium current. Depletion of sarcoplasmic reticulum calcium stores by pretreatment with ryanodine, thapsigargin and caffeine prior to the addition of 2,4-dinitrophenol did not affect the initial increase in cytoplasmic calcium, but abolished phase 2. Our results suggest that the initial rise in cytoplasmic calcium seen on application of 2,4-dinitrophenol results from release of mitochondrial calcium because of mitochondrial depolarization, while the second phase is caused by progressive release of calcium from the sarcoplasmic reticulum following depletion of intracellular ATP.
Hudman
Diane
D
Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN, UK.
Rainbow
Richard D
RD
Lawrence
Chris L
CL
Standen
Nicholas B
NB
D019297:2,4-Dinitrophenol / Q000494:pharmacology*; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000502:physiology; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000502:physiology; D014475:Uncoupling Agents / Q000494:pharmacology*; D014966:Xanthenes / Q000494:pharmacology
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014475:Uncoupling Agents; D014966:Xanthenes; C059715:Fluo-3; D019297:2,4-Dinitrophenol; D002118:Calcium