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Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

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Brain astrocytes in primary culture from the rat or the mouse have been shown to possess ionotropic and metabotropic glutamatergic receptors. The activation of both types of receptors is responsible for a rise in the cytosolic concentration of calcium, while the stimulation of metabotropic receptors induces the accumulation of inositol phosphates. In the present study, it is demonstrated that in striatal astrocytes from mouse embryos, glutamate evokes a release of arachidonic acid. The nonionotropic receptors involved in this effect appeared to be pharmacologically distinct from those coupled to phospholipase C: (1) glutamate displayed different dose-response curves for the production of inositol phosphates (biphasic: EC50 = 25 and 300 microM) and the release of arachidonic acid (monophasic: EC50 = 200 microM); (2) L(+)-2-amino-4-phosphonobutyric acid (AP4) only antagonized the glutamate-evoked release of arachidonic acid without altering the production of inositol phosphates; (3) when used at a concentration of 0.1 mM, quisqualate induced a higher formation of inositol phosphates than glutamate (2 mM) while, in contrast to glutamate, it only weakly stimulated arachidonic acid release when used either at 0.1 mM or 1 mM. L(+)-2-amino-3-phosphonopropionic acid (AP3) suppressed both responses. The glutamate-evoked release of arachidonic acid seems to be oppositely regulated by protein kinases A and C. Indeed, the stimulation of adenylate cyclase by the beta-adrenergic agonist isoproterenol, vasoactive intestinal peptide, or pretreatment of striatal astrocytes with cholera toxin decreased the glutamate-evoked release of arachidonic acid. In contrast, ATP, which markedly stimulated inositol phosphate production, strongly potentiated the glutamate-evoked release of arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

During neuropathological states associated with inflammation, the levels of cytokines such as interleukin-1␤ (IL-1␤) are increased. Several studies have suggested that the neuronal damage observed in pathogenesis implicating IL-1␤ are caused by an alteration in the neurochemical interactions between neurons and astrocytes. We report here that treating striatal astrocytes in primary culture with IL-1␤ for 22-24 hr enhances the ATP-evoked release of arachidonic acid (AA) with no effect on the ATP-induced accumulation of inositol phosphates. The molecular mechanism responsible for this effect involves the expression of P 2Y2 receptors (a subtype of purinoceptor activated by ATP) and cytosolic phospholipase A2 (cPLA 2 , an enzyme that mediates AA release). Indeed, P 2Y2 antisense oligonucleotides reduce the ATP-evoked release of AA only from IL-1␤-treated astrocytes. Further, both the amount of cPLA2 (as assessed by Western blotting) and the release of AA resulting from direct activation of cPLA 2 increased fourfold in cells treated with IL-1␤. We also report evidence indicating that the coupling of newly expressed P 2Y2 receptors to cPLA 2 is dependent on PKC activity. These results suggest that during inflammatory conditions, IL-1␤ reveals a functional P 2Y2 signaling pathway in astrocytes that results in a dramatic increase in the levels of free AA. This pathway may thus contribute to the neuronal loss associated with cerebral ischemia or traumatic brain injury.Key words: purinoceptor; phospholipase; cytokine; inflammation; glutamate; neurotoxicity ATP acts both as an intracellular source of energy and an intercellular signaling molecule. Several studies carried out in smooth muscle nerve endings, peripheral ganglia, and brain have shown that ATP is (1) stored in neuronal vesicles; (2) released in a Ca 2ϩ -dependent manner; (3) able to activate specific receptors; and (4) hydrolyzed by ecto-ATPases (for review, see Zimmermann, 1994). By way of illustration, it is present in synaptic vesicles of cholinergic interneurones of the striatum where it is co-localized and co-released with acetylcholine (Richardson and Brown, 1987).This purine binds to and activates a family of purinoceptors (P 2 receptors) namely P 2X , P 2Y , P 2U , P 2Z , and P 2T , which have been classified based on the potencies of structural ATP analogs (Fredholm et al., 1994). For most of them, cDNAs have been cloned and characterized (Lustig et al

Sphingosine-1-phosphate (S1P) is a potent and pleiotropic bioactive lysophospholipid mostly released by activated platelets that acts on its target cells through its own G protein-coupled receptors. We have previously reported that mouse striatal astrocytes expressed mRNAs for S1P1 and S1P3 receptors and proliferate in response to S1P. Here, we investigated the effect of S1P on gap junctions. We show that a short-term exposure of astrocytes to S1P causes a robust inhibition of gap junctional communication, as demonstrated by dye coupling experiments and double voltage-clamp recordings of junctional currents. The inhibitory effect of S1P on dye coupling involves the activation of both Gi and Rho GTPases. Rho-associated kinase (ROCK) also plays a critical role. The capacity of S1P to activate a Rho/ROCK axis in astrocytes is demonstrated by the typical remodeling of actin cytoskeleton. Connexin43, the protein forming gap junction channels, is a target of the Gi- and Rho/ROCK-mediated signaling cascades. Indeed, as shown by Western blots and confocal immunofluorescence, its nonphosphorylated form increases following S1P treatment and this change does not occur when both cascades are disrupted. This novel effect of S1P may have an important physiopathological significance when considering the proposed roles for astrocyte gap junctions on neuronal survival.

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...

Evidence is presented for the simultaneous release of platelet-activating factor (PAF-acether) and of its deacetylated derivative (lyso-PAF-acether) from hog leukocytes.