Characterization of Ionotropic Glutamate Receptor–Mediated Nitric Oxide Production In Vivo in Rats

Abstract

Background and Purpose Glutamate receptor activation can stimulate nitric oxide (NO) production and possibly play a role in long-term potentiation and excitotoxic-mediated injury. We studied the differential effect of agonist-induced activation of ion channel–linked N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtypes on NO production in vivo in rat hippocampus. We also studied whether dantrolene, a ryanodine calcium channel inhibitor previously shown to attenuate metabotropic glutamate receptor stimulation of NO production, also attenuated ionotropic glutamate receptor–mediated stimulation of NO production. Methods Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of pentobarbital-anesthetized adult Sprague-Dawley rats and were perfused for 5 hours with artificial cerebrospinal fluid (CSF) containing 3 μmol/L [ 14 C] l -arginine. Recovery of [ 14 C] l -citrulline in the effluent was used as a marker of NO production. In 13 groups of rats, increases in [ 14 C] l -citrulline recovery were compared between right- and left-sided probes perfused with no additional drugs versus combinations of NMDA, AMPA, the NO synthase inhibitor N ω -nitro- l -arginine methyl ester (L-NAME), the noncompetitive glutamate receptor blocker MK-801, the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and dantrolene. Results Recovery of [ 14 C] l -citrulline during perfusion with artificial CSF progressively increased to 272±73 fmol/min (±SEM) over 5 hours. Contralateral perfusion with 1 mmol/L L-NAME inhibited [ 14 C] l -citrulline recovery. Perfusion with 1 mmol/L MK-801 or 1 mmol/L CNQX reduced [ 14 C] l -citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [ 14 C] l -citrulline recovery, and this enhancement was attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Perfusion with 1 mmol/L AMPA enhanced [ 14 C] l -citrulline recovery, and this enhancement was also attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Conclusions Through an indirect method of assessing NO production in vivo, results with MK-801 and CNQX indicate that NMDA and AMPA receptor activation contribute to basal NO production in the rat hippocampus. Enhanced NO production with NMDA and AMPA agonists appears to involve a complex neuronal interaction because the effect of NMDA was attenuated by both MK-801 and CNQX and because the effect of AMPA was attenuated by both CNQX and MK-801. In contrast to metabotropic glutamate receptor activation, release of calcium from intracellular ryanodine calcium channels does not appear to be a prominent mediator of ionotropic glutamate receptor stimulation of NO production.