No abstract

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.

Plasma membrane Na؉ /Ca 2؉ -exchangers play a predominant role in Ca 2؉ extrusion in brain. Neurons express several different Na ؉ /Ca 2؉ -exchangers belonging to both the K ؉ -independent NCX family and the K ؉ -dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca 2؉ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K ؉ -dependent Na ؉ /Ca 2؉ -exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca 2؉ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca 2؉ ] that is essential for the development of synaptic plasticity associated with learning and memory.

A high cytoplasmic Na(+) concentration may contribute to N-methyl-D-aspartate (NMDA)-induced excitotoxicity by promoting Ca(2+) influx via reverse operation of the Na(+)/Ca(2+) exchanger (NaCaX), but may simultaneously decrease the electrochemical Ca(2+) driving force by depolarizing the plasma membrane (PM). Digital fluorescence microscopy was used to compare the effects of Na(+) versus ions that do not support the NaCaX operation, i.e., N-methyl-D-glucamine(+) or Li(+), on: PM potential; cytoplasmic concentrations of Ca(2+), H(+), and K(+); mitochondrial Ca(2+) storage; and viability of primary cultures of cerebellar granule cells exposed to NMDA receptor agonists. In the presence of Na(+) or Li(+), NMDA depolarized the PM and decreased cytoplasmic pH (pH(C)); in the presence of Li(+), Ca(2+) influx was reduced, mitochondrial Ca(2+) overload did not occur, and the cytoplasm became more acidified than in the presence of Na(+). In the presence of N-methyl-D-glucamine(+), NMDA instantly hyperpolarized the PM, but further changes in PM potential and pH(C) were Ca-dependent. In the absence of Ca(2+), hyperpolarization persisted, pH(C) was decreasing very slowly, K(+) was retained in the cytoplasm, and cerebellar granule cells survived the challenge; in the presence of Ca(2+), pH(C) dropped rapidly, the K(+) concentration gradient across the PM began to collapse as the PM began to depolarize, and Ca(2+) influx and excitotoxicity greatly increased. These results indicate that the dominant, very likely excitotoxic, component of NMDA-induced Ca(2+) influx is mediated by reverse NaCaX and that direct Ca(2+) influx via NMDA channels is curtailed by Na-dependent PM depolarization.

] i ) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn 2+ ] i and intracellular Ca 2+ concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH i ) was studied in parallel experiments using 2¢,7¢-bis-(2-carboxyethyl)-5 (6)