SARCOPLASMIC-RETICULUM CA2-PUMP DYSFUNCTION IN RAT CARDIOMYOCYTES BRIEFLY EXPOSED TO HYDROXYL RADICALS()

The effects of hydroxyl radical exposure of intact cardiomyocytes on s arcoplasmic reticulum (SR) function were investigated. For this purpos e, isolated rat heart myocytes were exposed briefly (1 min) to the hyd roxyl radical generating system (H2O2/FeCl2 or FeSO4) or 5-5'-dithiobi s-nitrobenzoic acid (DTNB), a sulfhydryl oxidizing reagent, and follow ing this a SR-enriched fraction was isolated. Marked decreases in the SR calcium uptake activities were seen in the myocytes exposed to eith er the hydroxyl radical-generating system or DTNB. The exposure of myo cytes to the hydroxyl radical, but not DTNB, markedly increased the am ount of malonyldialdehyde (MDA) in the subsequently isolated SR. Total sulfhydryl group content in SR was decreased by exposure of myocytes to DTNB. Further, there was a significant decrease in [H-3]-NEM bindin g to SR isolated from the hydoxyl radical-treated myocytes indicating that sulfhydryl groups are affected (oxidized). Both mannitol and cata lase were found to offer complete protection against the inhibitory ef fect of peroxide +/- iron on calcium uptake. Also the above-mentioned alterations in both MDA and sulfhydryl group content were prevented by mannitol and catalase. Exogenously added cyclic AMP-dependent protein kinase (A-PK) or calmodulin (CAM) increased SR calcium uptake activit y. In the SR isolated from the treated myocytes, the stimulatory effec ts of A-PK and CAM were also seen, although under all assay conditions calcium uptakes were of lower magnitude. The findings are consistent with the view that the damaging effect of the hydroxyl radical and DTN B on the functioning of SR occurs rapidly in the intact cardiomyocytes . The hydroxyl radical-provoked damage involves both protein sulfhydry l and lipid oxidation. Copyright (C) 1996 Elsevier Science Inc.