Trophic functions of nucleotides in the central nervous system.

In addition to short-term effects, one of the fundamental roles of extracellular nucleotides in the central nervous system involves long-term trophic effects. Physiological outcomes include neurogenesis, neuronal differentiation, glial proliferation, migration, growth arrest and apoptosis. Nucleotides exert these functions via P2-receptor-mediated mechanisms that can also interact with polypeptide-growth-factor-mediated or integrin-mediated signaling pathways. In addition, pathogenic roles for extracellular nucleotides in response to central nervous system injury including trauma and ischemia have been observed after the release of nucleotides by damaged and dying cells and in the development of neuropathic and inflammatory pain. Here, we illuminate the contribution of extracellular nucleotides to the development, growth, cellular plasticity and death of neural cells and the mechanisms regulating these trophic effects.

Opposing effects of P2X(7) and P2Y purine/pyrimidine-preferring receptors on proliferation of astrocytes induced by fibroblast growth factor-2: implications for CNS development, injury, and repair.

Extracellular nucleotides play important trophic roles in development and central nervous system (CNS) injury, but the functions of distinct purinergic receptors and related signaling pathways have not been fully elucidated. In the present study we identified opposing effects of P2X and P2Y receptors on the ability of FGF2 to induce proliferation in primary cultures of rat cortical astrocytes. Low concentrations of ATP enhanced DNA synthesis induced by FGF2, whereas high concentrations inhibited FGF2-induced proliferation. Comparison of concentration-response experiments with ATP and 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) indicated that the inhibitory effect was mediated by P2X(7) receptors. Interestingly, activation of P2X(7) receptors led to a state of reversible growth arrest rather than cell death. Selectivity studies showed that proliferation evoked by epidermal growth factor and platelet-derived growth factor was also inhibited by P2X(7) receptors, but P2X(1) or P2X(3) receptors did not inhibit proliferation induced by FGF2. A marker of mitosis, phosphohistone-3, was reduced by BzATP and increased by UTP, suggesting that the enhancing effect of ATP on FGF2-induced proliferation was mediated by P2 purine/pyrimidine receptors. Phosphorylation of the growth arrest-related protein kinases p38/MAPK and SAPK/JNK was strongly increased by BzATP but only weakly affected by UTP. We conclude that P2Y purine/pyrimidine receptors enhance proliferation induced by FGF2 in astrocytes, whereas stimulation of P2X(7) receptors inhibits proliferation by shifting cells to a state of reversible growth arrest that may be mediated by protein kinase signaling. These trophic actions of P2X(7) and P2Y purine/pyrimidine receptors may contribute to the regulation of CNS development, adult neurogenesis, and the response of astrocytes to injury.

A new in vitro model of the glial scar inhibits axon growth.

Astrocytes respond to central nervous system (CNS) injury with reactive astrogliosis and participate in the formation of the glial scar, an inhibitory barrier for axonal regeneration. Little is known about the injury-induced mechanisms underlying astrocyte reactivity and subsequent development of an axon-inhibitory scar. We combined two key aspects of CNS injury, mechanical trauma and co-culture with meningeal cells, to produce an in vitro model of the scar from cultures of highly differentiated astrocytes. Our model displayed widespread morphological signs of astrocyte reactivity, increases in expression of glial fibrillary acidic protein (GFAP), and accumulation of GFAP in astrocytic processes. Expression levels of scar-associated markers, phosphacan, neurocan, and tenascins, were also increased. Importantly, neurite growth from various CNS neuronal populations was significantly reduced when neurons were seeded on the scar-like cultures, compared with growth on cultures of mature astrocytes. Quantification of neurite growth parameters on the scar model demonstrated significant reductions in neuronal adhesion and neurite lengths. Interestingly, neurite outgrowth of postnatal neurons was reduced to a greater extent than that of embryonic neurons, and outgrowth inhibition varied among neuronal populations. Scar-like reactive sites and neurite-inhibitory patches were found throughout these cultures, creating a patchwork of growth-inhibitory areas mimicking a CNS injury site. Thus, our model showed relevant aspects of scar formation and produced widespread inhibition of axonal regeneration; it should be useful both for examining mechanisms underlying scar formation and to assess various treatments for their potential to improve regeneration after CNS injury. (c) 2008 Wiley-Liss, Inc.

Purinergic receptor signaling regulates N-cadherin expression in primary astrocyte cultures.

Extracellular ATP exerts both short-term and long-term effects in the CNS by stimulating cell-surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N-cadherin expression, a calcium-dependent cell adhesion molecule involved in many processes, including glia-glia and axon-glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N-cadherin protein expression increased in a time- and concentration-dependent manner. In addition, ATP treatment caused an increase in N-cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N-cadherin to the cell surface membrane were independent of protein synthesis. The ATP-induced increase in N-cadherin protein expression was blocked by reactive blue 2 and 8-(p-sulfophenyl)-theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N-cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N-cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N-cadherin expression and suggest a role for this mechanism in cell-cell interactions.

P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells.

Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.

P2X(7) receptors: properties and relevance to CNS function.

Among seven members of P2X ionotropic receptors activated by extracellular ATP, the P2X(7) subtype is unique in that it can function as a cation channel, a nonselective pore, or even a signaling complex coupled with multiple downstream components. Several roles of P2X(7) receptors have been described in CNS cells in the past decade, including release of cytokines and transmitters, modulation of presynaptic transmitter release, and activation of multiple signaling pathways. The finding that P2X(7) pores may directly mediate efflux of cytosolic glutamate, GABA, and ATP in glial cells is particularly interesting, as it provides a novel mechanism of glial transmitter release that may play important roles not only in physiological intercellular communication but also in pathological neural injury.

P2 purinergic receptors signal to glycogen synthase kinase-3beta in astrocytes.

Glycogen synthase kinase (GSK)-3 was identified initially as an enzyme that regulates glycogen synthesis in response to insulin, but more recent studies indicate that it is also involved in numerous cellular processes, including cell survival, cell cycle regulation, proliferation, and differentiation. Because extracellular ATP exerts trophic actions on astrocytes, we investigated a possible signaling linkage from P2 purinergic receptors to GSK3beta. Addition of ATP to primary cultures of rat cortical astrocytes resulted in phosphorylation of Ser9 on GSK3beta and a concomitant decrease in GSK3 activity. UTP and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) increased phosphorylation of Ser9 on GSK3beta indicating that metabotropic P2Y and ionotropic P2X receptors are coupled to GSK3beta. Signaling studies showed that phosphorylation of Ser9-GSK3beta in response to ATP was inhibited by downregulation of protein kinase C (PKC) but not by blockade of Akt or p70 S6 kinase pathways. PKC also links P2 receptors to ERK in astrocytes, but inhibition of ERK signaling did not block phosphorylation of Ser9-GSK3beta stimulated by P2 receptors. Mechanical strain, which releases ATP, also stimulated Ser9 phosphorylation and this was attenuated by hydrolysis of extracellular ATP with apyrase or by blockade of P2 receptors. We conclude that P2 receptors are coupled to GSK3beta by a PKC-dependent pathway that is independent of Akt, p70 S6 kinase, and ERK pathways. These findings suggest that purinergic signaling contributes to the regulation of GSK3beta functions, one of which may be the response of astrocytes to CNS injury on release of ATP.

Purinergic signaling induces thrombospondin-1 expression in astrocytes.

Thrombospondin (TSP)-1, a multidomain glycoprotein, is secreted from astrocytes and promotes synaptogenesis. However, little is known about the mechanisms regulating its expression and release. In this article, we report that purinergic signaling participates in the production and secretion of TSP-1. Treatment of primary cultures of rat cortical astrocytes with extracellular ATP caused an increase in TSP-1 expression in a time- and concentration-dependent manner and was inhibited by antagonists of P2 and P1 purinergic receptors. Agonist studies revealed that UTP, but not 2',3'-O-(4-benzoyl)benzoyl-ATP, 2-methylthio-ADP, adenosine, or 5'-N-ethyl-carboxamidoadenosine, caused a significant increase in TSP-1 expression. In addition, release of TSP-1 was stimulated by ATP and UTP but not by 2-methylthio-ADP or adenosine. Additional studies indicated that P2Y(4) receptors stimulate both TSP-1 expression and release. P2Y receptors are coupled to protein kinase cascades, and signaling studies demonstrated that blockade of mitogen-activated protein kinases or Akt inhibited ATP- and UTP-induced TSP-1 expression. Using an in vitro model of CNS trauma that stimulates release of ATP, we found that TSP-1 expression increased after mechanical strain and was completely blocked by a P2 receptor antagonist and by inhibition of p38/mitogen-activated protein kinase and Akt, thereby indicating a major role for P2 receptor/protein kinase signaling in TSP-1 expression induced by trauma. We conclude that TSP-1 expression can be regulated by activation of P2Y receptors, particularly P2Y(4), coupled to protein kinase signaling pathways and suggest that purinergic signaling may be an important factor in TSP-1-mediated cell-matrix and cell-cell interactions such as those occurring during development and repair.

P2 receptor signalling, proliferation of astrocytes, and expression of molecules involved in cell-cell interactions.

Growing evidence indicates that trophic actions of extracellular nucleotides are involved in CNS development, injury and repair. For example, upon CNS injury, ATP is released and contributes to the formation of reactive astrocytes, cells that produce molecules that can impede or promote axonal regeneration. Proliferation is one of the features of reactive astrogliosis, particularly in traumatic injury. Fibroblast growth factor (FGF)2 is also increased after injury and can stimulate astrocyte proliferation. Extracellular ATP enhances FGF2-induced proliferation in a process mediated by P2Y receptors and increased cyclin expression. However, when P2X receptors are activated, FGF2-induced proliferation is inhibited. P2 receptors are coupled to extracellular signal regulated protein kinase (ERK), and differences in the extent and duration of ERK activation by P2Y and P2X receptors may mediate the opposing effects of these receptors on FGF2-induced mitogenesis. Trauma also activates P2 receptor/ERK signalling, and stimulation of this and other protein kinase pathways by extracellular ATP increases expression of cell adhesion and extracellular matrix molecules involved in migration, glial contact formation, neuronal guidance and synapse formation. These findings support the hypothesis that purinergic signalling via protein kinase cascades plays a key role in astrocyte proliferation, glia-glia connections, and neuron-glia interactions in both normal and pathological conditions.

Signaling from P2 nucleotide receptors to protein kinase cascades induced by CNS injury: implications for reactive gliosis and neurodegeneration.

Gliosis is a hypertrophic and hyperplastic response to many types of central nervous system injury, including trauma, stroke, seizure, as well as neurodegenerative and demyelinating disorders. Reactive astrocytes, a major component of the glial scar, express molecules that can both inhibit and promote axonal regeneration. ATP, which is released upon traumatic injury, hypoxia, and cell death, contributes to the gliotic response by binding to specific cell surface astrocytic P2 nucleotide receptors and evoking characteristic features of gliosis such as increased expression of glial fibrillary acidic protein (GFAP), generation and elongation of astrocytic processes, and cellular proliferation. Here, we review recent studies that demonstrate that (1) metabotropic, P2Y, and ionotropic, P2X, receptors expressed in astrocytes are coupled to protein kinase signaling pathways that regulate cellular proliferation, differentiation, and survival such as ERK and protein kinase B/Akt and (2) these P2 receptor/protein kinase cascades are activated after trauma induced by mechanical strain. We suggest that P2 receptor/protein kinase signaling pathways might provide novel therapeutic targets to regulate the formation of reactive astrocytes and the production of molecules that affect axonal regeneration and neurodegeneration.

Traumatic injury activates protein kinase B/Akt in cultured astrocytes: role of extracellular ATP and P2 purinergic receptors.

Protein kinase B/Akt is a key signaling molecule that regulates cell survival, growth, and metabolism, and inhibits apoptosis. Traumatic brain injury (TBI) activates Akt, and Akt has been implicated in neuronal survival after TBI, but little is known about injury-induced Akt activation in astrocytes, cells that exhibit hypertrophic and hyperplastic responses to CNS injury. Here we have investigated the effect of mechanical strain on Akt activation in primary cultures of rat cortical astrocytes growing on deformable Silastic membranes. When astrocytes were subjected to mechanical strain (50 msec; 5-7.5 mm displacement), we observed an increase in phosphorylation of serine 473, a key indicator of Akt activation. Akt phosphorylation was increased at 3 min postinjury, was maximal from 5 to 10 min, and declined gradually thereafter. Akt activation was also dependent on the severity of the injury. Stretch-induced Akt phosphorylation was attenuated by blocking calcium influx and phosphoinositide 3-kinase (PI3K), an upstream activator of Akt. In addition, we found that ATP is rapidly released after mechanical strain and that the P2 purinergic receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',5'disulfonate (PPADS) attenuated trauma-induced Akt activation. We conclude that mechanical strain causes activation of Akt in astrocytes via stimulation of P2 receptors. This suggests that P2 receptor/Akt signaling promotes astrocyte survival and growth, and this process may play a role in the generation of reactive gliosis after TBI.

Bi-functional effects of ATP/P2 receptor activation on tumor necrosis factor-alpha release in lipopolysaccharide-stimulated astrocytes.

Neuroinflammation is associated with a variety of CNS pathologies. Levels of tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory cytokine, as well as extracellular ATP, are increased following various CNS insults. Here we report on the relationship between ATP/P2 purinergic receptor activation and lipopolysaccharide (LPS)-induced TNF-alpha release from primary cultures of rat cortical astrocytes. Using ELISA, we confirmed that treatment with LPS stimulated the release of TNF-alpha in a concentration and time dependent manner. ATP treatment alone had no effect on TNF-alpha release. LPS-induced TNF-alpha release was attenuated by 1 mm ATP, a concentration known to activate P2X7 receptors. Consistent with this, 3'-O-(4-Benzoyl)benzoyl-ATP (BzATP), a P2X7 receptor agonist, also attenuated LPS-induced TNF-alpha release. This reduction in TNF-alpha release was not due to loss of cell viability. Adenosine and 2-chloroadenosine were ineffective, suggesting that attenuation of LPS-induced TNF-alpha release by ATP was not due to ATP breakdown and subsequent activation of adenosine/P1 receptors. Interestingly, treatment of astrocyte cultures with 10 microm or 100 microm ATP potentiated TNF-alpha release induced by a submaximal concentration of LPS. UTP and 2methylthioADP (2-MeSADP), P2Y receptor agonists, also enhanced this LPS-induced TNF-alpha release. Our observations demonstrate opposing effects of ATP/P2 receptor activation on TNF-alpha release, i.e. P2X receptor activation attenuates, whereas P2Y receptor activation potentiates TNF-alpha release in LPS-stimulated astrocytes. These observations suggest a mechanism whereby astrocytes can sense the severity of damage in the CNS via ATP release from damaged cells and can modulate the TNF-alpha mediated inflammatory response depending on the extracellular ATP concentration and corresponding type of astrocyte ATP/P2 receptor activated.

Cell cycle regulation of astrocytes by extracellular nucleotides and fibroblast growth factor-2.

Extracellular ATP enhances the mitogenic activity of fibroblast growth factor-2 (FGF2) in astrocytes, but the molecular mechanism underlying this synergistic interaction is not known. To determine whether the potentiating effect of extracellular ATP involves cell cycle control mechanisms, we have measured the expression of cyclins that are induced in different phases of the cell cycle in primary cultures of rat cortical astrocytes. We found that ATP potentiated the ability of FGF2 to stimulate expression of cyclin D1, a regulator of cell cycle entry, as well as cyclin A, a regulator of DNA replication. Because FGF2 and P2 purinergic receptors are coupled to extracellular signal regulated protein kinase (ERK), a key member of a signaling cascade that regulates proliferation, we also investigated the role of ERK in regulating cyclin expression induced by FGF2 and ATP. We found that the potentiating effect of ATP on cyclin expression was significantly reduced by U0126, an inhibitor of MEK, the upstream activator of ERK. P2 receptor agonist studies revealed that UTP enhanced FGF2-induced cyclin expression and mitogenesis whereas 2-methylthioADP was ineffective. By contrast, 2',3'-O-(4-benzoyl)-benzoyl-ATP markedly inhibited FGF2-induced mitogenesis. Consistent with opposing effects of P2Y and P2X receptors on mitogenesis, UTP stimulated a transient activation of ERK whereas BzATP stimulated a more sustained ERK signal. These findings suggest that signaling by P2Y receptors, most likely of the purine/pyrimidine subtype, enhance the ability of FGF2 to stimulate entry into a new cell cycle, as well as DNA replication, by an ERK-dependent mechanism, whereas signaling by P2X receptors, possibly the P2X7 subtype, inhibits FGF2-induced mitogenesis in astrocytes. Interactions between P2Y, P2X and polypeptide growth factor signaling pathways may have important implications for CNS development as well as injury and repair.

Protein kinase signaling cascades in CNS trauma.

Advances in our understanding of the signaling pathways and cellular functions regulated by protein kinase cascades have paved the way to study their role in the response of brain and spinal cord to traumatic injury. Mechanical forces imparted by trauma stimulate mitogen-activated protein kinases and protein kinase B/Akt as well as cause changes in the state of phosphorylation of glycogen synthase kinase-3beta. Extracellular ATP released by mechanical strain stimulates P2 purinergic receptors that are coupled to these protein kinase signaling pathways. These kinases regulate gene expression, cell survival, proliferation, differentiation, growth arrest, and apoptosis, thereby affecting cell fate, repair and plasticity after trauma. Elucidation of the molecular responses of protein kinase cascades to mechanical strain and the genes regulated by these signaling pathways may lead to therapeutic opportunities to minimize losses in motor skills and cognitive function caused by trauma to the central nervous system.

P2Y receptors activate neuroprotective mechanisms in astrocytic cells.

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.

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.

Signaling from nucleotide receptors to protein kinase cascades in astrocytes.

In the CNS, extracellular ATP can function as an excitatory neurotransmitter as well as a trophic factor. These short-term and long-term actions are mediated by nucleotide receptors. Extracellular ATP can also act as a co-mitogen in conjunction with polypeptide growth factors such as basic fibroblast growth factor (FGF2). Cellular proliferation, differentiation and survival are regulated by signaling cascades composed of protein kinases, including extracellular signal regulated protein kinase (ERK) and protein kinase B (also called Akt). Here we summarize recent studies on nucleotide receptor signaling to ERK and Akt in astrocytes and the role of protein kinase cascades in mediating the trophic actions of extracellular ATP, alone or together with FGF2. Because extracellular ATP and FGF2 contribute to the hyperplastic and hypertrophic response of astrocytes to CNS injuries, an understanding of their protein kinase signaling mechanisms may lead to novel therapeutic approaches for neurological conditions that involve gliosis and the generation of reactive astrocytes, such as trauma, stroke, seizure and neurodegenerative and demyelinating disorders.

Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors.

Gliosis is characterized by hypertrophic and hyperplastic responses of astrocytes to brain injury. To determine whether injury of astrocytes produced by an in vitro model of brain trauma activates extracellular signal-regulated protein kinase (ERK), a key regulator of cellular proliferation and differentiation, astrocytes cultured on deformable SILASTIC membranes were subjected to rapid, reversible strain (stretch)-induced injury. Activation of ERK was observed 1 min after injury, was maximal from 10 to 30 min, and remained elevated for 3 hr. Activation of ERK was dependent on the rate and magnitude of injury; maximum ERK activation was observed after a 20-60 msec, 7.5 mm membrane displacement. ERK activation was blocked by inhibiting MEK, the upstream activator of ERK. Activation of ERK was reduced when calcium influx was diminished. When extracellular ATP was hydrolyzed by apyrase or ATP/P2 receptors were blocked, injury-induced ERK activation was significantly reduced. P2 receptor antagonist studies indicated a role for P2X2 and P2Y1, but not P2X1, P2X3, or P2X7, receptors in injury-induced ERK activation. These findings demonstrate for the first time that ATP released by mechanical injury is one of the signals that triggers ERK activation and suggest a role for extracellular ATP, P2 purinergic receptors, and calcium-dependent ERK signaling in the astrocytic response to brain trauma.

Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells.

Astrocytes are involved in normal and pathological brain functions, where they become activated and undergo reactive gliosis. Astrocytes have been shown to respond to extracellular nucleotides via the activation of P2 receptors, either G protein-coupled P2Y receptors or P2X receptors that are ligand-gated ion channels. In this study, we have examined the manner in which activation of the P2X(7) nucleotide receptor, an extracellular ATP-gated ion channel expressed in astrocytes, can lead to the phosphorylation of ERK1/2. Results showed that the P2X(7) receptor agonist 2',3'-O-(4-benzoyl)benzoyl-ATP induced ERK1/2 phosphorylation in human astrocytoma cells overexpressing the recombinant rat P2X(7) receptor (rP2X(7)-R), a response that was inhibited by the P2X(7) receptor antagonist, oxidized ATP. Other results suggest that rP2X(7)-R-mediated ERK1/2 phosphorylation was linked to the phosphorylation of the proline-rich/Ca(2+)-activated tyrosine kinase Pyk2, c-Src, phosphatidylinositol 3'-kinase, and protein kinase Cdelta activities and was dependent on the presence of extracellular Ca(2+). These results support the hypothesis that the P2X(7) receptor and its signaling pathways play a role in astrocyte-mediated inflammation and neurodegenerative disease.