Regulation of nitric oxide synthase activity in cortical slices by excitatory amino acids and calcium

Nitric oxide synthase (NOS) activity was determined in adult rat frontal cortex and hippocampus by measuring the conversion of L-[³H]arginine to L- [³H]citrulline. N-methyl-D-aspartate (NMDA), but not kainate or α-amino-3- hydroxy-5-methylisoxazole-4-propionate (AMPA), stimulated NOS activity. This effect was concentration dependent (EC₅₀ ≃ 30 μM) and was inhibited by tetrodotoxin, EGTA, N(ω)-nitro-L-arginine (NOARG), Mg²⁺, phencyclidine, and (cis)-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). NOS activity was increased to an even greater extent by the calcium ionophores ionomycin and A23187 and by depolarization with 50 mM K⁺. Interestingly, neither caffeine nor 1- aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), drugs that would be expected to increase intracellular Ca²⁺ concentration by release of Ca²⁺ from intracellular ryanodine- and inositol-1,4,5-trisphosphate-sensitive stores, respectively, had any significant effect on NOS activity. It is concluded that NOS can be activated by NMDA binding to a classic NMDA glutamate receptor subtype as well as by depolarization or other agents that increase the influx of extracellular Ca²⁺. The paradoxical lack of effect of caffeine, as well as the inhibitory effect of tetrodotoxin, are discussed.