Characterization of ionotropic glutamate receptor-mediated nitric oxide production in vivo in rats

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 [¹⁴C]L-arginine. Recovery of [¹⁴C]L-citrulline in the effluent was used as a marker of NO production. In 13 groups of rats, increases in [¹⁴C]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-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 [¹⁴C]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 [¹⁴C]L-citrulline recovery. Perfusion with 1 mmol/L MK-801 or I mmol/L CNQX reduced [¹⁴C]L-citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [¹⁴C]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 [¹⁴C]L-citrulline recovery, and this enhancement was also attenuated by L-NAME, MK801, 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.