Expression of COX-2 by normal and reactive astrocytes in the adult rat central nervous system.

We have used a previously characterized antiserum against cycloxygenase-2 (COX-2) together with cold methanol fixation to immunohistochemically locate the protein in astrocytes in rat brain. Although in cerebral cortex most enzyme was located in neuronal perikarya as previously described, a number of glial fibrillary acidic protein (GFAP)-positive astrocytes were also labeled. No COX-2-positive neurons were seen in the cerebellum, but here also a subset of GFAP+ astrocytes was present which contained the enzyme. The number of COX-2-positive astrocytes increased considerably after injection of the neurotoxin kainate into the cerebellum. These immunohistochemical data were supported by semiquantitative RT-PCR results, which were used to assess the levels of COX-2 mRNA relative to the housekeeping gene hypoxanthine phosphoribosyl transferase. PGE2 levels were measured in contralateral and lesioned cerebellum to correlate changes in COX-2 immunoreactivity and mRNA with physiological events. PGE2 levels increased by 230% in the lesioned cerebellar hemispheres in comparison to the contralateral ones. We discuss the possibility that the targets for astrocytic prostaglandins might include both autocrine effects and paracrine responses of neurons, lymphocytes and capillary endothelial cells.

Preprotachykinin-A and substance P receptor (NK1) gene expression in rat astrocytes in vitro.

The presence of mRNA transcripts coding for preprotachykinin-A and the substance P receptor in cultured astrocytes is demonstrated by a combination of reverse transcription/polymerase chain reaction (PCR) and Southern blotting. These findings showed that astrocytes in culture are capable of synthesing both the precursor of substance P (preprotachykinin-A) and the cognate receptor, substance P receptor (NK1). The simultaneous presence of both the ligand (substance P) and the receptor (NK1) may indicate an autocrine nature of astrocyte communication.

Up-regulation of lipocortin-1 and its mRNA in reactive astrocytes in kainate-lesioned rat cerebellum.

We have used a combined molecular and immunocytochemical approach to examine the expression of lipocortin-1 (LC-1) in kainate-lesioned rat cerebellum. Using immunocytochemistry, Western and Northern blotting, we have shown upregulation of LC-1 mRNA and expression of LC-1 localised specifically to reactive astrocytes. These studies suggest that reactive astrocytes are a major synthetic compartment for the expression of LC-1. The well-reported immuno-suppressive effects of lipocortin(s), suggests that reactive astrocytes could serve to negatively modulate inflammatory reactions in the central nervous system.

Substance P receptors on O-2A progenitor cells and type-2 astrocytes in vitro.

Bradykinin- and substance P (SP)-stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin-stimulated synthesis of phosphoinositides (PI) in type-1 astrocytes and oligodendrocytes. SP-stimulated PI accumulation was restricted to oligodendrocyte/type-2 astrocyte progenitor cells and type-2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP-stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type-2 astrocyte type-2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.

Electrophysiological and biochemical evidence for bradykinin receptors on cultured rat cortical oligodendrocytes.

The effects of the neuropeptide bradykinin (Bk) were examined on antigenically-identified rat cortical oligodendrocytes. Bk significantly increased the incorporation of [3H]myo-inositol into phospholipids, indicating the turnover of phosphatidyl inositol (PI). Ca2+ flux analysis experiments confirmed that this effect was accompanied by an increase in intracellular Ca2+. Using the whole-cell patch clamp technique, Bk was shown to induce an inward current associated with a decrease in membrane conductance, indicating a closure of ion channels. The reversal potential of the current was close to the potassium equilibrium potential, consistent with an effect on a K+ conductance in these cells. These results show that oligodendrocytes possess Bk receptors that may be of functional relevance.

Preincubation with substance P induces substance P-stimulated phosphatidylinositol turnover in cultured cerebellar astrocytes.

In this study we have investigated the effects of preincubation of cultured astrocytes with substance P (SP) on subsequent 125I-Bolton-Hunter conjugated SP (125I-BHSP) receptor binding, and SP-stimulated phosphatidyl-inositol (PI) accumulation. Spinal cord astrocytes preincubated for up to 96 h with SP (0.001-1,000 nM) suffered a dose-dependent decrease in both subsequent 125I-BHSP and SP-stimulated PI turnover. In contrast, preincubation of cerebellar astrocytes with SP resulted in an increase in SP-stimulated PI turnover, with no change in 125I-BHSP receptor binding. SP-induced PI turnover in cerebellar astrocytes was maximal after 72 h of preincubation with 0.1 nM SP. These data suggest that increased coupling between receptor and second messenger occurs in response to chronic exposure to SP.

Lack of effect of focally administered prostaglandins on electrically kindled seizure activity.

Several previous studies have demonstrated increased synthesis of cerebral prostaglandins (PGs) following convulsive activity. In addition, it has been proposed that endogenous prostanoids have anticonvulsive properties and may act to attenuate or limit seizure activity in vivo. In this study we have used focal injections of prostaglandins (PGs) to examine their potential modulatory effects on electrically kindled seizure activity. We report that the intra-amygdaloid administration of PGD2, PGE2 or PGF2a, (1-10 micrograms) showed no significant effects on any of the kindled seizure parameters studied. The highest dose of PGF2a was ineffective at all pretreatment times between 2-30 mins. Our data is inconsistent with the view that PGs exert protective effects against seizure activity, at least within the amygdala against electrically kindled seizures.

Substance P-induced release of prostaglandins from astrocytes: regional specialisation and correlation with phosphoinositol metabolism.

Addition of substance P (SP) to astrocytes cultured from rat neonatal spinal cord evoked a time- and concentration-dependent increase in the accumulation of phosphoinositol and the release of prostaglandin (PG) D2 and PGE2. Both basal and stimulated releases were reduced to similar levels by indomethacin. In contrast, astrocytes cultured from cerebral cortex and cerebellum showed no SP-stimulated increase in phosphoinositol accumulation or release of PGs. Release of PGD2 and PGE2 was, however, stimulated by the calcium ionophore A23187, and both phosphoinositol accumulation and PG release were stimulated from cortical astrocytes incubated in the presence of serum. The results from this study suggest that SP-stimulated phosphoinositol accumulation and release of PGs from cultured rat neonatal astrocytes are regionally specialised in favour of cells derived from spinal cord.