Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

Abstract

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca²⁺-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, N^G-monomethyl-L-arginine (MeArg) and N^G-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [³H]arginine uptake. Kainate (300 μM) stimulated ⁴⁵Ca²⁺ influx to the same extent as 100 μM 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.