The permeability of gap junctions in cultured striatal astrocytes was investigated by the scrape-loading/dyetransfer technique. Prolonged application of norepinephrine (NE) (10 j)M) reduced by half the extent of dye (Lucifer yellow) spread. This effect was linked to the activation of a, -adrenergic receptors since it was mimicked by methoxamine and antagonized by prazosin. The adenosine agonist 2-chloroadenosine (10 ,uM), which potentiates the NE-evoked activation of phospholipase C (PLC) in striatal astrocytes, also potentiated the NE-evoked closure of gap junctions, the effect being as important as that observed with the uncoupling agent octanol. Measurements of inositol phospholipid turnover performed in identical experimental conditions revealed a close relationship between the extent of PLC activation and the magnitude of the uncoupling process. The effect of NE was mimicked by both phorbol ester and arachidonic acid, suggesting that biochemical events linked to PLC stimulation such as protein kinase C activation and/or eicosanoid production are likely involved in the NE-induced uncoupling. In addition, in the presence of a cAMP phosphodiesterase inhibitor, the stimulation of fl-adrenergic receptors by isoproterenol (10 jM) led to a large increase in cAMP accumulation correlated with an extension of dye diffusion. This observation suggests that junctional permeability could also be controlled by a cAMP-dependent mechanism. Altogether these results indicate that intercellular communication between cultured astrocytes can be regulated by different second messenger pathways as a result of the action of neurotransmitters on their receptors.

As previously shown with adenosine, somatostatin, which is ineffective alone, enhanced the a1-adrenergicagonist-stimulated production of inositol phosphates in cultured striatal astrocytes. This effect was suppressed in cells pretreated with pertussis toxin. It required external calcium and was selectively antagonized by both mepacrine, an inhibitor of phospholipase A2, and 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of arachidonic acid. In addition, a long-lasting elevation of cytosolic calcium and a release of arachidonic acid were observed only under the combined stimulation of somatostatin and a1-adrenergic receptors. Arachidonic acid could in turn inhibit glutamate uptake into astrocytes, and the resulting external accumulation of glutamate could account for the somatostatin-evoked amplifiation of the a1-adrenergic-agonist-stimulated hydrolysis of inositolphospholipids. The effect of somatostatin was indeed reproduced by glutamate or glutamate uptake inhibitors and uppressed by enzymatic removal of external glutamate. Thus, astrocytes may contribute to long-term plasticity events in glutamatergic synapses through regulation of external glutamate levels.Somatostatin (SRIF, somatotropin release-inhibiting factor, tetradecapeptide form) inhibits spontaneous neuronal firing and various secretory responses in different cell types (1-4). This could be related to its hyperpolarizing effect resulting from an increase in potassium conductances (5-10) and/or an inhibition of voltage-dependent calcium current (11)(12)(13) (19). In the present study, attempts were made to determine whether somatostatin, like the nucleoside, could enhance the a1-adrenergic-agonist-induced formation of IPs in striatal astrocytes from the mouse, since receptors for adenosine and somatostatin have generally been associated with similar transduction systems-i.e., guanine nucleotide-binding regulatory proteins (G proteins) sensitive to pertussis toxin (PTX) (20)(21)(22). As will be shown, the peptide increased the formation of IPs in the presence of methoxamine, a specific agonist of a1-adrenergic receptors.In hippocampal CAl pyramidal neurons, the hyperpolarizing effect of somatostatin seems to be mediated by a release of arachidonic acid (AA) (23). Particular attention was thus made to evaluate a possible role for AA in the potentiating effect of somatostatin seen in striatal astrocytes. In addition, as recently demonstrated in glial (Muller) cells, AA inhibits glutamate uptake (24). Furthermore, it has been shown that glutamate stimulates PLC activity in astrocytes (25). Therefore, the possible involvement of glutamate in the enhancing effect of somatostatin on the a1-adrenergic-agonist-evoked response was also investigated. (Dakopatts, Glostrup, Denmark). The remaining 5% of cells could be immature glioblasts, which are unlabeled by GFAP (26). The cultures were free of microglial cells, since no staining was observed when monoclonal antibody to mouse macrophages (anti-MAC 1; Serotec) was used. MATERIALS AND METHO...

In cultured striatal astrocytes, 2-chloroadenosine, an adenosine analog resistant to adenosine deaminase, although inactive alone, markedly potentiated the activation of phospholipase C induced by methoxamine, an alpha 1-adrenergic agonist. This effect was suppressed by antagonists of either A1 adenosine or alpha 1-adrenergic receptors. An influx of calcium and two distinct G-proteins are involved in this phenomenon since the potentiating effect of 2-chloradenosine was suppressed in the absence of external calcium or when cells were pretreated with pertussis toxin. In addition, arachidonic acid is likely involved in this potentiating effect. This was shown first by examining the effects of inhibitors of phospholipase A2 or arachidonic metabolism, then by examining the action of arachidonic acid on the production of inositol phosphates in either the presence or absence of methoxamine, and finally by measuring the release of arachidonic acid. The sequential activation of phospholipase C and of protein kinase C is required for the 2-chloroadenosine-induced activation of phospholipase A2 since 2-chloroadenosine markedly stimulated phospholipase C activity in the absence of methoxamine when protein kinase C was activated by a diacylglycerol analog. Finally, the enhancing effect of 2- chloroadenosine on the methoxamine-evoked response seems to result from an inhibition of glutamate reuptake into astrocytes by arachidonic acid. Indeed, the potentiating effect of 2-chloroadenosine was suppressed when external glutamate was removed enzymatically and mimicked by either selective inhibitors of the glutamate reuptake process or direct application of glutamate.