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
C. Malcolm et al., 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, Journal of neuroscience research, 45(2), 1996, pp. 129-142
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.