Effects of reactive oxygen species on brain synaptic plasma membrane Ca2+-ATPase

The regulation of free intracellular calcium [Ca2+](i) is altered in neuron s from the aged brain, possibly due to reductions in the activity of Ca2+ t ransporters. The plasma membrane Ca2+-ATPase (PMCA) plays a critical role i n Ca2+ homeostasis, and its kinetic properties change in aged rat brain. Th ese changes could be due to oxidative modification of PMCA as a result of a ge-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of s ynaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examin ed the effects of three oxidants including peroxyl radicals generated by az o-initiators, 2,2 '-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4 '-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxyn itrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant: loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in V-max without significant changes in K-act. Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducin g conditions and completely reversed with addition of urea. The PMCA appear s to be very sensitive to inhibition by ROS and hence may be a target of ox idative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+](i) regulation. (C) 1999 Else vier Science Inc.