Synaptosomal plasma membrane Ca 2+ pump activity inhibition by repetitive micromolar ONOO − pulses

Abstract

A sustained increase of intracellular free [Ca 2+] ([Ca 2+] 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 Ca 2+-ATPase). The plasma membrane Ca 2+-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca 2+, and millimolar Mg 2+-ATP concentrations), which plays a major role in the control of synaptic [Ca 2+] i, can be more than 75% inhibited by a sustained exposure to micromolar ONOO − (e.g., to 100 pulses of 10 μM 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 Ca 2+ stimulation and about 5-fold increase of the K M for Mg 2+-ATP. [Ca 2+] 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 Ca 2+-pump activity during chronic exposure to ONOO − may account by itself for a large and sustained increase of intracellular [Ca 2+] i in synaptic nerve terminals.