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 μM ONOO−). This inhibition is irreversible and mostly due to a decreased Vmax, and to the 2-fold increase of the K0.5 for Ca2+ stimulation and about 5-fold increase of the KM 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.