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.

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).

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.

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).

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.

The influence of the extracellular matrix on the morphology and intracellular pH of cultured astrocytes exposed to media lacking bicarbonate.

In previous work we showed that the polygonal shape of hippocampal astrocytes cultured on poly-L-lysine changes to a stellate morphology with loss of actinomyosin stress fibers on exchanging the culture medium for saline buffered with HEPES [Brain Res 946 (2002)12]. By contrast, in bicarbonate-buffered saline containing Ca(2+) astrocytes remained polygonal and continued to express stress fibers. Evidence suggests that stellation induced by saline buffered with HEPES is related to intracellular acidification due to the absence of bicarbonate.

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.

Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures.

Organotypic hippocampal cultures have been recently used to study in vitro ischaemic neuronal death. Sub-lethal periods of ischaemia in vivo confer resistance to lethal insults and many studies have demonstrated the involvement of heat shock proteins in this phenomenon. We used organotypic hippocampal cultures to investigate the involvement of heat shock protein (HSP) 27 in preconditioning to oxygen and glucose deprivation.

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.

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.

Effects of global cerebral ischemia and preconditioning on heat shock protein 27 immunocontent and phosphorylation in rat hippocampus.

Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus.

An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation.

Brain ischemia results in cellular degeneration and loss of function. Here we investigated the neuroprotective effect of lithium in an in vitro model of ischemia. Organotypic hippocampal slice cultures were exposed to oxygen and glucose deprivation.

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures.

ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes.

Protein kinase C-mediated in vitro invasion of human glioma cells through extracellular-signal-regulated kinase and ornithine decarboxylase.

We investigated the involvement of protein kinase C (PKC) in the in vitro invasiveness of the A-172, U-87 and U-373 human glioma cell lines, as well as the role of ornithine decarboxylase (ODC) and/or extracellular-signal-regulated kinase (ERK) in the actions of PKC. Thus, cells were treated under serum-free conditions with the PKC activator phorbol 12-myristate 13-acetate (PMA), or with the PKC inhibitors bisindolylmaleimide I (GF 109203X) or calphostin C in the absence or presence of the ODC inhibitor D,L-alpha-difluoromethylornithine (DFMO), and/or the mitogen-activated protein kinase/extracellular-signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD 098059). Subsequently, cells were assessed for membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA contents, 72-kD latent, and 59/62-kD activated matrix metalloproteinase 2 (MMP-2) in conditioned media, as well as invasiveness.

Identification of brain ecto-apyrase as a phosphoprotein.

Ecto-apyrase is a transmembrane glycoprotein that hydrolyzes extracellular nucleoside tri- or diphosphates. Apyrase activity is affected by several physiological and pathological conditions indicating the existence of regulatory mechanisms. Considering that apyrase presents consensus phosphorylation sites, we studied the phosphorylation of this enzyme.

Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology.

Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures.

Time-dependent enhancement of inhibitory avoidance retention and MAPK activation by post-training infusion of nerve growth factor into CA1 region of hippocampus of adult rats.

Several studies have demonstrated that chronic intracerebroventricular nerve growth factor (NGF) infusion has a beneficial effect on cognitive performance of lesioned as well as old and developing animals. Here we investigate: (i) the effect of post-training infusion of NGF into the CA1 region of hippocampus on inhibitory avoidance (IA) retention in rats; (ii) the extension of the effect, in time and space, of NGF infusion into CA1 on the activity of mitogen-activated protein kinase (MAPK, syn: ERK1/2, p42/p44 MAPK). NGF was bilaterally injected into the CA1 regions of the dorsal hippocampus (0.

Extracellular S100B protein modulates ERK in astrocyte cultures.

S100B is a calcium binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates the proliferation of astroglial cells and the survival of neurons. Extracellular signal regulated kinases (ERK) are involved in the transduction of proliferating signals in astrocytes.

P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes.

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek).

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions.

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca(2+)-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems.

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.

Effects of transient cerebral ischemia on glial fibrillary acidic protein phosphorylation and immunocontent in rat hippocampus.

Transient global cerebral ischemia induced in rats by four-vessel occlusion for 20 min produced an increase in the immunocontent of glial fibrillary acidic protein and a protein phosphorylation response that was different in the CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (one, four, seven, 14 and 30 days) and observed that the immunocontent and in vitro rate of phosphorylation of glial fibrillary acidic protein in the CA1 region was significantly increased at all intervals after the ischemic insult, indicating that the astrocytic response was maintained for at least 30 days. After reperfusion for 14 days a significant increase in the ratio "in vitro phosphorylation rate/immunocontent" in the CA1 region was observed when compared to control values, to other intervals and to the dentate gyrus, suggesting a hyperphosphorylation of this intermediate filament protein at this interval.

Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27).

The effect of external Ca2+ ([Ca2+]e) on the incorporation of [32P] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+]e increased total 32P-incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total 32P-incorporation between zero [Ca2+]e and 1 mM [Ca2+]e was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1-10 nM; higher concentrations of okadaic acid (50-100 nM) further increased total 32P-incorporation.

Lithium treatment causes gliosis and modifies the morphology of hippocampal astrocytes in rats.

The biological basis of the clinical efficacy of lithium in the treatment of mental illness has been extensively studied in neurones, but little is known about the effects of the drug on glia. Recently we showed that treatment of rats with clinically relevant doses of lithium chloride results in a 35% increase in the immunocontent of the astrocyte marker GFAP in the hippocampus. Here we studied the cytology of this phenomenon.

The S100B protein inhibits phosphorylation of GFAP and vimentin in a cytoskeletal fraction from immature rat hippocampus.

The S100B protein belongs to a family of small Ca2+-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S 100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 microM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca2+ alone, Ca2+ plus calmodulin or Ca2+ plus cAMP.