Contribution of plasma membrane and endoplasmic reticulum Ca 2+-ATPases to the synaptosomal [Ca 2+] i increase during oxidative stress

Abstract

In the present study we analyzed the effect of ascorbate (0.8 mM)/Fe 2+ (2.5 μM)-induced membrane lipid peroxidation on the levels of intracellular free calcium, [Ca 2+] i, and on the possible mechanisms involved in the perturbation of intracellular calcium homeostasis during oxidative stress. For this purpose, the influence of the ascorbate/iron oxidant system on the plasma membrane and endoplasmic reticulum Ca 2+-dependent ATPases of brain cortical synaptosomes was studied. In addition, the influence of the peroxidative process on the uptake of calcium ( 45Ca 2+) and on the Na +/Ca 2+ exchange activity at the plasma membrane was evaluated. After ascorbate/Fe 2+-induced membrane lipid peroxidation of the order of 18.05 ± 4.20 nmol TBARS/mg protein, an increase in [Ca 2+] i occured, under basal or depolarizing conditions (30 mM KCI), which was dependent on the extracellular calcium concentration. Thus, for 1 and 3 mM extracellular calcium concentration, an increase of the resting [Ca 2+] i values of 19.8% and 33.7% was observed, while after the K +-depolarization the enhancement of the [Ca 2+] i was 18.4% and 29.5%, respectively. The Na +/Ca 2+ exchange activity and the time-dependent influx of 45 Ca 2+, observed in basal conditions and after the 30 mM K +-depolarization, were not affected under the peroxidative conditions. The Ca 2+-dependent ATPase activity of the synaptosomal plasma membrane was significantly depressed following peroxidation of membrane lipids, decreasing the V max by 48.1%, without significant changes in the affinity of the enzyme for calcium ( K m for Ca 2+ was 0.54 ± 0.04 μM in control conditions and 0.56 ± 0.034 μM in peroxidized conditions). The Ca 2+-ATPase activity of the endoplasmic reticulum was also affected during ascorbate/iron-induced oxidative stress; thus, an inhibition of 45.2% was observed 5 min after adding ATP. These data suggest that the increase in synaptosomal [Ca 2+] i due to oxidative stress may result from the inhibition of the plasma membrane and the endoplasmic reticulum membrane Ca 2+-ATPase activities, probably as a result of the alteration of the lipid environment required for the maximal activity of these membrane enzymes. The consequent increase in [Ca 2+] i may be responsible for the injury of the nervous tissue observed during several pathological conditions in which free radical generation seems to be involved.