Synaptosomal plasma membrane Ca2+ pump activity inhibition by repetitive micromolar ONOO- pulses

A sustained increase of intracellular free [Ca2+] ([Ca2+](i)) has been shown to be an early event of neuronal cell death induced by peroxynitrite (ONOO-). In this paper, chronic exposure to ONOO- has been simulated by treatment of rat brain synaptosomes or plasma membrane vesicles with repetitive pulses of ONOO- during at most 50 min, which efficiently produced nitrotyrosine formation in several membrane proteins (including the Ca2+-ATPase). The plasma membrane Ca2+-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca2+, and millimolar Mg2+-ATP concentrations), which plays a major role in the control of synaptic [Ca2+](i), can be more than 75% inhibited by a sustained exposure to micromolar ONOO- (e.g., to 100 pulses of 10 muM ONOO-). This inhibition is irreversible and mostly due to a decreased V-max, and to the 2-fold increase of the K-0.5 for Ca2+ stimulation and about 5-fold increase of the K-M for Mg2+-ATP. [Ca2+](i) increases to > 400 nM when synaptosomes are subjected to this treatment. Reduced glutathione can afford only partial protection against the inhibition produced by micromolar ONOO- pulses. Therefore, inhibition of the plasma membrane Ca2+-pump activity during chronic exposure to ONOO- may account by itself for a large and sustained increase of intracellular [Ca2+](i) in synaptic nerve terminals. (C) 2001 Elsevier Science Inc.