Daniela Mendes Oppelt

Phosphorylation of glial fibrillary acidic protein is stimulated by glutamate via NMDA receptors in cortical microslices and in mixed neuronal/glial cell cultures prepared from the cerebellum

In previous work we showed that phosphorylation of glial fibrillary acidic protein (GFAP), an astrocyte marker, is increased by glutamate in hippocampal slices from immature rats via a type II metabotropic receptor. In the present work we show that glutamate also stimulates GFAP phosphorylation in microslices prepared from immature cerebellar cortex, but by a different receptor mechanism from that observed in the hippocampus. Thus, in cerebellar microslices, NMDA consistently stimulated GFAP phosphorylation, whereas no effect of metabotropic or non-NMDA ionotropic agonists was observed. Glutamate and NMDA also stimulated GFAP phosphorylation in mixed neuronal/glial cell cultures from the cerebellum, although no effect of these agonists was observed in primary cultures of cerebellar astrocytes. In both models, the effects of glutamate and NMDA were dependent on external Ca(2+), were reversed by the NMDA receptor antagonist AP5 and were not blocked by tetrodotoxin. In the slice study the effect of NMDA was confined to a period starting with the first detectable expression of GFAP at 10 days and finishing at 16 days postnatal, as previously observed with metabotropic agonists in hippocampal slices. This period in the rat corresponds to the start of synaptogenesis when astrocyte hypertrophy is occurring. The results are discussed in the light of information in the literature on the occurrence of functional NMDA receptor subunits in glia.

The mGluR stimulating GFAP phosphorylation in immature hippocampal slices has some properties of a group II receptor.

In a previous study we showed that phosphorylation of the astrocytic marker glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats (P12-P16) is regulated by a metabotropic glutamate receptor (mGluR). The subtypes of these receptors are divided into three groups and exhibit two distinct transduction signals: activation of phospholipase C and liberation of internal calcium (group I) or modulation of cAMP synthesis (groups II and III). Here we investigated the subtype of mGluR involved. Phosphorylation was strongly stimulated by the selective group II agonists DCG IV, L-CCG-I and 1S,3S-ACPD, whereas the group I agonist 3,5-DHPG and the group III agonist L-AP4 had no effect. These results show that the receptor regulating GFAP phosphorylation in the immature hippocampus has some of the properties of a group II mGluR.

Role of intracellular calcium stores on the effect of metabotropic glutamate receptors on phosphorylation of glial fibrillary acidic protein in hippocampal slices from immature rats.

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517–523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca2+-entry through L-type Ca2+ channels and down-regulates a Ca2+-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca2+-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor–agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.