Richard Rodnight

P2X7 receptors stimulate AKT phosphorylation in astrocytes

Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3‐kinase (PI3K)/Akt pathway in astrocytes. 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 P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 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. 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. In conclusion, our data indicate that stimulation of astrocytic P2X7 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.

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. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process‐bearing phenotype of astrocytes.

P2Y 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 P2Y receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P2Y receptors expressed in astrocytes have not been defined and it is not known whether all P2Y receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P2Y receptor agonists ATP, ADP, UTP and 2‐methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf‐1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf‐1 was not stimulated by ATP. Furthermore, ATP did not activate B‐Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase‐polymerase chain reaction (RT–PCR) studies using primer pairs for cloned rat P2Y receptors revealed that rat cortical astrocytes express P2Y1, a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P2Y2 and P2Y4, subtypes in rats for which ATP and UTP are equipotent. Transcripts for P2Y6, a pyrimidine‐preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P2Y11, an ATP‐preferringz receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT–PCR experiments reveal differences in the activation of cRaf‐1 by P2Y receptor agonists that are inconsistent with properties of the P2Y1, P2Y2 and P2Y4 receptors shown to be expressed in astrocytes, i.e. ATP≠UTP; ATP≠2MeSATP, ADP. This suggests that the properties of the native P2Y receptors coupled to the Erk cascade differ from the recombinant P2Y receptors or that astrocytes express novel purine‐preferring and pyrimidine‐preferring receptors coupled to the ERK cascade.

Protein phosphorylation in nervous tissue: possible involvement in nervous tissue function and relationship to cyclic nucleotide metabolism

lntroductlon I, Protein phosphorylation and presynaptic events I .l. Phosphorylation and calcium 1.2. Protein phosphorylation in relation to the brain actomyosin system and microtubular function 1.3. Involvement of cyclic AMP in presynaptic events 2. Protein phosphorylation and postsynaptic events 2.1. Interactions of putative transmitters and other agents with cyclic AMP in nervous tissue preparations 2.2. In vitro studies of membrane-bound protein phosphorylation 22.1. Cyclic AMP-stimulated protein kinases and membrane phosphorylation 2.2.2. Cyclic GMP-stimulated protein kinases and membrane phosphorylation 2.2.3. Other factors regulating protein kinase activity 2.3. Electrophysiological correlates of cyclic nucleotlde action in nervous tissue and possible relation to protein phosphorylation 2.3.1. The cerebellar Purkinje cell system 2.3.2. The superior cervical ganglion system 2.3.3. Other electrophysiological observations 2.4. Transmitter-related increases in nervous tissue protein phosphorylation 2.4.1. Protein phosphorylation in brain slices 7.4.2. Protein phosphorylation and cyclic nucleotides in retina 2.4.3. Protein phosphorylation in the pineal gland 2.4.4. Protein phosphorylation and cyclic nucleotides in invertebrate nervous tissue 2.5. Protein phosphorylation and altered enzyme activity in nervous tissue 2.5.1. The glycogen phosphorylase system 2.5.2. Cyclic nucleotide phosphodiesterase 2.5.3. Tyrosine mono-oxygenase (tyrosine hydroxylase) 2.5.4. Enzymes of carbohydrate metabolism 2.6. Phosphorylation of structural proteins in nervous tissue 2.6.1. Phosphorylation of microtubular protein 2.6.2. Phosphorylation of ribosomal protein 2.6.3. Phosphorylation of myelin protein 3. Protem phosphorylation, cyclic AMP and behavioral correlates Conclusions Acknowledgements References

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. Neuronal damage was assessed using propidium iodide fluorescence; HSP27 phosphorylation and immunocontent were obtained using (32)Pi labelling followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. We observed that immunocontent of HSP27 was increased after lethal or sub-lethal treatment, indicating it is a response to metabolic stress. Treatments with 5 or 10 min of oxygen and glucose deprivation (OGD) or 1- microM N-methyl-D-aspartate (NMDA) induced tolerance to 40 min of OGD associated with an increase in HSP27 immunocontent and phosphorylation. These data suggest that, in vitro, phosphorylated HSP27 might be involved in preconditioning, probably acting as a modulator of actin filaments or by the blockage of neurodegenerative processes.

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 48h 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 ≅ guanosine ≅ inosine ≅ adenosine > UTP > ADP while ATPγS 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.

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. Cellular death was quantified by measuring uptake of propidium iodide (PI). Lithium chloride (0.2-1.2 mM) was added to the medium before, during and after lesion induction. A decrease in incorporation of PI was observed, indicating a neuroprotective effect in all doses tested. We also studied the effect of lithium on the phosphorylation of HSP27, a heat shock protein involved in cellular protection in its dephosphorylated state. In the lesioned hippocampus, 0.4 mM lithium chloride decreased the proportion of phosphorylated HSP27 to total HSP27. These results suggest that lithium may be useful in the treatment of brain ischemia.

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. Here we report that S100B significantly increases the activity of ERK in primary cultures of astrocytes, a result which may be related to previous observations of the effect of this protein on glial proliferation. We further confirm that conversion of S100B to its covalent dimer by oxidation of cysteine residues increases its extracellular activity. Although we cannot exclude S100B involvement in other mechanisms of signal transduction, these results suggest that ERK activity in astrocytes is modulated by extracellular S100B.

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 S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 μM 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. Analysis by two dimensional electrophoresis revealed a Ca2+/calmodulin-dependent and a Ca2+/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.

Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate.

Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by guanine nucleotides. Recent work has shown that guanine nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that guanine nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.