Morphological plasticity of rodent astroglia.

In the past two decades, there has been an explosion of research on the role of neuroglial interactions in the control of brain homeostasis in both physiological and pathological conditions. Astrocytes, a subtype of glia in the central nervous system, are dynamic signaling elements that regulate neurogenesis and development of brain circuits, displaying intimate dynamic relationships with neurons, especially at synaptic sites where they functionally integrate the tripartite synapse. When astrocytes are isolated from the brain and maintained in culture, they exhibit a polygonal shape unlike their precursors in vivo. However, cultured astrocytes can be induced to undergo morphological plasticity leading to process formation, either by interaction with neurons or by the influence of pharmacological agents. This review highlights studies on the molecular mechanisms underlying morphological plasticity in astrocyte cultures and intact brain tissue, both in situ and in vivo.

P2X7 receptors stimulate AKT phosphorylation in astrocytes.

1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

Astrocyte stellation in saline media lacking bicarbonate: possible relation to intracellular pH and tyrosine phosphorylation.

Primary cultures of astrocytes exhibit a polygonal morphology, but on treatment with agents that increase cAMP they change to stellate cells. We found that astrocyte stellation also occurred on replacing the culture medium with saline buffered with HEPES. However, stellation did not occur when the medium was replaced with saline buffered with bicarbonate/CO(2) provided Ca(2+) was present. Since exposure of astrocytes to media lacking bicarbonate results in a decrease in intracellular pH (pH(i)) we sought evidence for an association between pH(i) and morphology. Astrocytic pH(i) was monitored for 60 min after transferring the cells to HEPES or bicarbonate-buffered saline. HEPES-induced stellation was associated with transient acidification which coincided with the morphological changes. Acidification was not observed in cells transferred to bicarbonate-saline. However when cytoplasmic acidification of cells in bicarbonate-saline was induced pharmacologically, rapid stellation occurred. Stellation induced by cAMP is reversed by activation of the RhoA pathway with lysophosphatidic acid (LPA). Here we found that LPA inhibited HEPES-induced stellation, but only with Ca(2+) present. Inhibition of stellation by LPA+Ca(2+) was associated with transient acidification followed by modest alkanization. A close association of tyrosine phosphorylation with stellation and pH(i) was observed. Thus incubation of astrocytes in HEPES-saline with orthovanadate to inhibit dephosphorylation abolished stellation and acidification; conversely incubation of cells in bicarbonate-saline with genistein to inhibit tyrosine kinases caused stellation and major acidification. Acidification may be one of several factors resulting in stellation, but it is not a necessary factor since stellation without acidification was observed in bicarbonate-saline lacking Ca(2+).

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.

Signal transduction mechanisms involved in the proliferation of C6 glioma cells induced by lysophosphatidic acid.

We studied pathways involved in the proliferation of rat C6 glioma cells induced by lysophosphatidic acid (LPA), a phospholipid with diverse biological functions. LPA induced a dose-responsive proliferation of C6 cells after 48 h. Proliferation was blocked by inhibitors of the sodium/proton exchanger type 1 (NHE1), Rho-associated kinase, the phosphatidylinositol 3-kinase/Akt pathway (PI3K/Akt), protein kinase C (PKC) and extracellular signal regulated kinase kinase (MEK). Phospho-specific antibodies were used to investigate the pathways involved. LPA induced transient (10 min) phosphorylations of ERK 1/2, Akt and the transcription factor CREB. The LPA-induced phosphorylation of ERK 1/2 and CREB was blocked by inhibition of PI3K, PKC and MEK, but that of Akt was only inhibited by wortmannin, the PI3K inhibitor. Inhibition of Rho kinase or NHE1 did not reduce the LPA-induced phosphorylation of ERK, Akt or CREB. The results were compared with the effects of LPA on transduction pathways in other cell types.

ERK, PKC and PI3K/Akt pathways mediate extracellular ATP and adenosine-induced proliferation of U138-MG human glioma cell line.

OBJECTIVE: Extracellular nucleotides and nucleosides induce proliferation in a set of human glioma cell lines. In this study we investigate the signal transduction pathways involved in ATP and adenosine-mediated proliferation in U138-MG human glioma cells. METHODS: Cell proliferation was accessed through [(3)H]thymidine incorporation, cell counting and flow cytometry. Protein phosphorylation was detected through Western blotting. RESULTS: ATP or adenosine (100 microM) induced extracellular signal-regulated protein kinase (ERK), Akt and GSK3beta phosphorylation. The increase in [(3)H]thymidine incorporation induced by ATP or adenosine was decreased when cells were incubated with LY 294002 (by +/-90%), GF 109203X (by +/-76%) or PD 098059 (by +/-63%). The increase in cell numbers with ATP or adenosine was less after a 48-hour treatment of cells with ATP or adenosine plus GF 109203X (by +/-66%) or LY 294002 (by +/-83%). Percentage of cells in S phase was decreased in cells treated with LY 294002 plus ATP when compared to ATP- treated cells. CONCLUSION: Stimulation of purinergic receptors in U138-MG cells leads to cell proliferation mediated by PI3K/Akt, ERK and PKC signaling. It may be clinically important for pharmacological intervention in gliomas to associate purinergic receptor antagonists and signal transduction pathways blockers.

Extracellular nucleotides and nucleosides induce proliferation and increase nucleoside transport in human glioma cell lines.

Extracellular purines (adenosine triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine) and pyrimidines (uridine 5'-triphosphate (UTP) and UDP) are important signaling molecules that mediate diverse biological effects via P1 and P2 purinergic receptors. The human glioma cell lines U87 MG, U251 MG and U138 MG were treated with purines and pyrimidines for 24 or 48 h and proliferation was measured by [3H]-thymidine incorporation, flow cytometry and cell counting. The studies showed that extracellular nucleotides and nucleosides induce proliferation of the studied glioma cells. Incorporation of [3H]-thymidine followed the order of ATP approximately equal to guanosine approximately equal to inosine approximately equal to adenosine > UTP > ADP while ATPgammaS and 2MeSATP had no effect. The effect of ATP was partially inhibited by suramin and by reactive blue 2 (RB2). Co-treatment with the following antagonists of P1 purinoreceptors DPCPX, CPT or 8PT did not block the effect of adenosine while a specific antagonist of the A3 receptor, MRS1220, totally blocked the effect of adenosine. ATP and adenosine also increased the overall uptake of [3H]-thymidine into the cell, producing a positive effect on the [3H]-thymidine incorporation measurements. These data indicate that the uptake of thymidine and proliferation of gliomas can be induced by purines and pyrimidines via both P1 and P2 purinoceptors.