NMDA RECEPTOR-MEDIATED CGMP SYNTHESIS IN PRIMARY CULTURES OF MOUSE CEREBELLAR GRANULE CELLS APPEARS TO INVOLVE NEURON-ASTROCYTE COMMUNICATION WITH NO OPERATING AS THE INTERCELLULAR MESSENGER

The possibility that neuron-astrocyte communication may be responsible for glutamate (Glu)-stimulated cGMP formation even in relatively homo geneous primary cultures of mouse cerebellar granule cells (7 days in vitro) was investigated. Pharmacological analysis using selective exci tatory amino acid (EAA) receptor antagonists showed that cGMP producti on, stimulated in these cultures by Glu and a variety of endogenous EA As structurally-related to Glu (namely, L-aspartate, L-cysteine sulphi nate, L-homocysteate, S-sulpho-L-cysteine), was mediated wholly by N-m ethyl-D-aspartate (NMDA) receptor activation. Moreover, EAA-induced re sponses were dependent on the presence of extracellular calcium but un affected by addition of the L-type voltage-sensitive calcium channel b lockers nifedipine (10 mu M) or verapamil (5 mu M). The mode of calciu m entry was also shown to be important since the calcium ionophore, A2 3187 (10 mu M), was unable to stimulate cGMP levels above basal. cGMP formation was blocked by the competitive nitric oxide synthase inhibit or, L-N-G-nitroarginine (100 mu M), consistent with a role of nitric o xide (NO) in this signalling pathway. In the presence of added haemogl obin (1 mu M), acting as a membrane-impermeable NO scavenger, Glu-stim ulated cGMP formation was abolished implying that NO must act as an in tercellular messenger. When the neuronal population was destroyed foll owing a 24 hr exposure to the excitotoxin, S-sulpho-L-cysteine (200 mu M), Glu-stimulated cGMP formation was abolished; whereas responses to the NO donor, sodium nitroprusside (SNP), although markedly reduced w ere still double that stimulated by Glu in the absence of the excitoto xin, suggesting the presence of non-neuronal cells that can generate c GMP if supplied directly with NO. Consistent with this suggestion, low levels of the glial specific enzyme, glutamine synthetase, were detec ted in granule cell cultures. Furthermore, omission or delayed additio n of the antimitotic agent, cytosine arabinoside (20 mu M), to the gro wth medium caused a significant increase in the level of Glu-stimulate d cGMP formation. (C) 1996 Wiley-Liss, Inc.