Cultured Rat Cortical Astrocytes Research Articles

Characterization of guanine and guanosine transport in primary cultured rat cortical astrocytes and neurons

In this study, we examined the transport mechanisms for guanine and guanosine in rat neurons and astrocytes, and compared their characteristics. In the both types of cell, the uptake of [(3)H]guanine and [(3)H]guanosine was time-, temperature-, and concentration-dependent, and Na(+)-independent. Their uptake decreased on the addition of purine and pyrimidine nucleobases or nucleosides, and the inhibitory effect of the purine analogues was greater than that of the pyrimidine ones. In both cell types, equilibrative nucleoside transporter (ENT) 1 and ENT2 expression was confirmed at the mRNA level, and nitrobenzylmercaptopurine riboside, a representative inhibitor for ENT, decreased their uptake at concentrations of over 10 microM. Comparing uptake characteristics between the substrates, [(3)H]guanine uptake exhibited higher affinity and clearance than [(3)H]guanosine uptake in each type of cell. Although between neurons and astrocytes, there was no difference in the apparent uptake clearance for [(3)H]guanine and [(3)H]guanosine, which was calculated based upon the cellular protein content, the cellular uptake clearance was significantly greater in astrocytes than in neurons. These findings indicate that guanine and guanosine, of which the former is a preferable substrate, are taken up into both neurons and astrocytes via ENT2, and that the extracellular concentrations of guanine and guanosine are mainly regulated by astrocytes to maintain brain physiology.

A pharmacological activator of AMP-activated protein kinase (AMPK) induces astrocyte stellation

AMP-activated protein kinase (AMPK) represents a key energy-sensing molecule in many cell types. Because astrocytes are key mediators of metabolic signaling in the brain, we have initiated studies on the expression and activation of AMPK in these cells. Treatment of cultured rat cortical astrocytes with a pharmacological AMPK activator, AICA-riboside (AICAR) resulted in a time- and concentration-dependent increase in phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a direct substrate. AICAR treatment also induced a transition from epithelioid to stellate morphology in a time- and concentration-dependent manner. As stellation is indicative of actin cytoskeletal reorganization, the formation of stress fibers and focal adhesions in response to AICAR was assessed. AICAR-induced stellation correlated with F-actin disassembly and focal adhesion dispersal. Furthermore, transient transfection of an activated RhoA construct prevented AICAR-induced stellation, indicating a mechanism upstream of RhoA. Use of pharmacological inhibitor compound C prevented AICAR-induced stellation demonstrating necessity of AMPK activity for the response. Our findings suggest that AMPK mediates morphological alterations of astrocytes in response to energy depletion.

Cultured rat cortical astrocytes synthesize melatonin: absence of a diurnal rhythm

Melatonin not only plays a major role in the regulation of circadian rhythms, but is also involved in antioxidative defense and immunomodulation. Circulating melatonin levels are derived primarily from the pineal gland while other sources of melatonin have also been reported. Here, we show for the first time that astrocytes from the rat cortex and glioma C6 cell line synthesize melatonin in vitro. In addition, we show the presence of serotonin, the precursor of melatonin and the two key enzymes in the pathway of melatonin synthesis, i.e. N-acetyltransferase and hydroxyndole-O-methyltransferase in the cultured rat cortical astrocytes. Release of melatonin into the culture medium showed no diurnal changes. These point to astrocytes as a local source of melatonin in the rat brain. Its exact physiological function remains a topic for future studies.

Activator protein-1 responsive to the group II metabotropic glutamate receptor subtype in association with intracellular calcium in cultured rat cortical neurons

Activation of ionotropic glutamate (Glu) receptors, such as N-methyl- d-aspartate receptors, is shown to modulate the gene transcription mediated by the transcription factor activator protein-1 (AP1) composed of Fos and Jun family proteins in the brain, while little attention has been paid to the modulation of AP1 expression by metabotropic Glu receptors (mGluRs). In cultured rat cortical neurons, where constitutive expression was seen with all groups I, II and III mGluR subtypes, a significant and selective increase was seen in the DNA binding activity of AP1 120 min after the brief exposure to the group II mGluR agonist (2 S,2′ R,3′ R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) for 5 min. In cultured rat cortical astrocytes, by contrast, a significant increase was induced by a group I mGluR agonist, but not by either a group II or III mGluR agonist. The increase by DCG-IV was significantly prevented by a group II mGluR antagonist as well as by either an intracellular Ca 2+ chelator or a voltage-sensitive Ca 2+ channel blocker, but not by an intracellular Ca 2+ store inhibitor. Moreover, DCG-IV significantly prevented the increase of cAMP formation by forskolin in cultured neurons. Western blot analysis revealed differential expression profiles of Fos family members in neurons briefly exposed to DCG-IV and NMDA. Prior or simultaneous exposure to DCG-IV led to significant protection against neuronal cell death by NMDA. These results suggest that activation of the group II mGluR subtype would modulate the gene expression mediated by AP1 through increased intracellular Ca 2+ levels in cultured rat cortical neurons.

Cytoprotective properties of phenolic antidiarrheic ingredients in cultured astrocytes and neurons of rat brains

We have previously shown that particular phenolic antidiarrheic ingredients, including 2-methoxy-4-methylphenol (2M4MP) and 2-methoxy-4-ethyphenol (2M4EP), but not 2-methoxyphenol (2MP), significantly inhibit cellular maturation and differentiation of the bone-resorbing osteoclasts with concomitant protection of the bone-forming osteoblasts against oxidative stress by hydrogen peroxide (H 2O 2). In the present study, we evaluated the pharmacological actions of these three major phenolic antidiarrheic ingredients on the cellular viability in cultured astrocytes and neurons of the rat brain in vitro. Both 2M4MP and 2M4EP induced more efficient prevention of cell death induced by the brief exposure to 0.1 mM H 2O 2 for 2 h than 2MP upon simultaneous exposure in cultured rat cortical astrocytes. Similarly, both 2M4MP and 2M4EP were more effective than 2MP in significantly protecting the cytotoxicity by brief exposure to 0.1 mM H 2O 2 for 6 h in cultured rat hippocampal neurons, with concomitant suppression of the generation of intracellular reactive oxygen species in neurons exposed to H 2O 2. Moreover, the three ingredients not only significantly prevented cell death in hippocampal neurons exposed to 0.1 mM glutamate for 1 h when determined 48 h after the brief exposure, but also inhibited the generation of intracellular reactive oxygen species and the elevation of intracellular Ca 2+ ions in neurons exposed to glutamate. These results suggest that particular phenolic antidiarrheic ingredients may prevent cell death through a mechanism related to diminution of the neurotoxicity of glutamate in neurons, in addition to eliciting cytoprotection against oxidative stress in astrocytes and neurons, in the rat brain.

Cannabinoids inhibit sodium-dependent, high-affinity excitatory amino acid transport in cultured rat cortical astrocytes

Cannabinoids have been shown to increase the extracellular levels of glutamate in vivo and in vitro, but no studies have evaluated the possible involvement of glial glutamate reuptake system. The present study investigates whether cannabinoids and endocannabinoid, anandamide have an effect on astroglial excitatory amino acid (EAA) transport. The kinetics of glutamate transport was studied in rat cortical astrocytes, using the radiolabeled, non-metabolized amino acid, d-[ 3H] aspartate in the absence or presence of cannabinoid receptor agonists. The results show that in vehicle controls the uptake of d-aspartate was rapid, sodium-dependent and saturated within the first 5 min, resulting in a K m 7.365 ± 1.16 μmol/L ( n = 5) and the maximum velocity ( V max) 1207 ± 51 nmol/mg protein/min. Addition of the synthetic cannabinoid analog R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2; 3 μmol/L) increased the K m (26.25 ± 4.84 μmol/L) without affecting the V max (1122 ± 77 nmol/mg protein/min), suggesting the inhibition was competitive and reversible. Various other cannabinoid agonists also inhibited d-aspartate uptake in a dose-dependent and stereospecific manner. The cannabinoid inhibition of EAA transport was partially blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 100 nmol/L). The inhibitory effects of WIN 55,212-2, or its endogenous counterpart anandamide were reversed by 98,059, an inhibitor of mitogen-activated kinase (MAPK) kinase (MEK). These results suggest that cannabinoids and endocannabinoids may constitute a novel class of inhibitors of astroglial glutamate transport system.

MAPKs are key players in mediating cytokine release and cell death induced by unconjugated bilirubin in cultured rat cortical astrocytes

When activated by unconjugated bilirubin (UCB), astrocytes are important sources of inflammatory mediators such as TNF-alpha, IL-1beta and IL-6, which may contribute for the neurotoxicity observed during severe neonatal hyperbilirubinemia. In the present study, we have addressed the role of the mitogen-activated protein kinases (MAPKs) p38, Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)1/2 pathways and their relation with the nuclear factor kappaB (NF-kappaB) cascade in the signalling events involved in cytokine release and cell death caused by UCB in primary cultures of rat astrocytes. Stimulation of astrocytes with UCB in the presence of all the MAPK inhibitors prevented UCB-induced release of TNF-alpha and IL-6, while IL-1beta secretion was only reduced by JNK1/2 and ERK1/2 inhibitors. In addition, activation of the NF-kappaB transcription factor, needed for cytokine release by UCB-stimulated astrocytes, was shown to be dependent on JNK1/2 and ERK1/2 phosphorylation. Moreover, all MAPK inhibitors prevented astroglial apoptosis triggered by UCB. Interestingly, UCB-induced lactate dehydrogenase release was prevented by blockade of JNK1/2, ERK1/2 and NF-kappaB cascades but enhanced by p38 inhibition. Taken together, our data demonstrate for the first time that MAPK transduction pathways are key players in the UCB-induced inflammatory response and cell death in astrocytes, probably also involving NF-kappaB modulation. These findings contribute to unraveling the complex mechanisms of astrocyte reactivity to UCB and may ultimately prove useful in the development of new therapeutic strategies to prevent nerve cell damage during acute bilirubin encephalopathy.

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Functional down‐regulation of volume‐regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl(-) current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl(-) current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.

P2 purinergic receptors signal to STAT3 in astrocytes: Difference in STAT3 responses to P2Y and P2X receptor activation

Extracellular ATP, released upon tissue damage to the CNS, can evoke reactive astrogliosis. The released ATP activates P2 purinergic receptors associated with the proliferation of normally quiescent astrocytes, although the underlying mechanisms remain to be fully elucidated. Signal transducer and activator of transcription 3 (STAT3) has been implicated in reactive astrogliosis and plays an important role in cell cycle regulation. Therefore, we investigated whether extracellular ATP and purinergic receptors regulate STAT3 signaling. Using immunoblot analysis, we found that addition of ATP to primary cultures of rat cortical astrocytes increased Ser-727 phosphorylation of STAT3 in a time-sensitive and concentration-dependent manner. ATP-stimulated Ser-727 STAT3 phosphorylation was mediated through P2 receptor activation since suramin, an antagonist of P2 receptors, diminished this response, whereas 8-(para-sulfo-phenyl)-theophylline (8PSTP), an antagonist of P1 receptors, did not. We found that UTP, an agonist of P2Y 2/4/6 receptors, stimulated rapid and robust phosphorylation of Ser727-STAT3, whereas BzATP, an agonist for P2X receptors, exhibited a delayed and weaker response. In contrast, both P2Y and P2X agonists stimulated phosphorylation of Tyr705-STAT3 to a similar extent. P2 receptors can couple to extracellular signal-regulated protein kinases (ERK) and we found that inhibition of ERK signaling blocked phosphorylation of Ser727-STAT3. Further characterization of the Ser727-STAT3 phosphorylation response to P2Y receptor activation supported a role for P2Y 2 and P2Y 4, but not P2Y 6, receptors as well as a partial role for P2Y 1 receptors. Because phosphorylation of Ser727-STAT3 can promote DNA transcriptional activity of cell cycle regulatory genes, the differences in phosphorylation of Ser727-STAT3 may contribute to the mechanism by which P2Y receptors promote, whereas P2X receptors inhibit, astrocyte proliferation. In support of this hypothesis, inhibition of STAT3 activation by cucurbitacin I prevented ATP-stimulated mitogenesis. We conclude that P2 receptors stimulate STAT3 activation and suggest that P2 receptor/STAT3 signaling may play an important role in astrocyte proliferation and reactive astrogliosis.

P2 purinergic receptors signal to glycogen synthase kinase‐3β 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.

Identity of SMCT1 (SLC5A8) as a neuron‐specific Na+‐coupled transporter for active uptake ofl‐lactate and ketone bodies in the brain

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.

Guanosine promotes the up‐regulation of inward rectifier potassium current mediated by Kir4.1 in cultured rat cortical astrocytes

Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long-term effects on both neurones and astroglial cells. Whether Guo also modulates the activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 microm) promotes the functional expression of an inward rectifier K+ (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up-regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo-induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo-induced up-regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that down-regulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K+ homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K+-buffering impairment of the astroglial syncytium that occurs in pathological conditions.

Critical Role of Phospholipase Cγ1 in the Generation of H2O2-evoked [Ca2+]i Oscillations in Cultured Rat Cortical Astrocytes

Reactive oxygen species, such as the superoxide anion, H2O2, and the hydroxyl radical, have been considered as cytotoxic by-products of cellular metabolism. However, recent studies have provided evidence that H2O2 serves as a signaling molecule modulating various physiological functions. Here we investigated the effect of H2O2 on the regulation of intracellular Ca2+ signaling in rat cortical astrocytes. H2O2 triggered the generation of oscillations of intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner over the range 10-100 microM. The H2O2-induced [Ca2+]i oscillations persisted in the absence of extracellular Ca2+ and were prevented by depletion of intracellular Ca2+ stores with thapsigargin. The H2O2-induced [Ca2+]i oscillations were not inhibited by pretreatment with ryanodine but were prevented by 2-aminoethoxydiphenyl borate and caffeine, known antagonists of inositol 1,4,5-trisphosphate receptors. H2O2 activated phospholipase C (PLC) gamma1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H2O2-induced [Ca2+]i oscillations. In addition, RNA interference against PLCgamma1 and the expression of the inositol 1,4,5-trisphosphate-sequestering "sponge" prevented the generation of [Ca2+]i oscillations. H2O2-induced [Ca2+]i oscillations and PLC1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H2O2 production, PLCgamma1 activation, and [Ca2+]i increases, which were attenuated by N-acetylcysteine and diphenyleneiodonium and by the overexpression of peroxiredoxin type II. Therefore, we conclude that low concentrations of exogenously applied H2O2 generate [Ca2+]i oscillations by activating PLCgamma1 through sulfhydryl oxidation-dependent mechanisms. Furthermore, we show that this mechanism underlies the modulatory effect of endogenously produced H2O2 on epidermal growth factor-induced Ca2+ signaling in rat cortical astrocytes.

Functional expression of the organic cation/carnitine transporter 2 in rat astrocytes

In this study, we sought to identify the transporters that mediate the uptake of L-carnitine and acetyl-L-carnitine in cultured rat cortical astrocytes. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake were both saturable, and mediated by a single Na(+)-dependent transport system. Uptake of both was inhibited by L-carnitine, D-carnitine, acetyl-L-carnitine and various organic cations. Acylcarnitines (acetyl-, butyryl-, hexanoyl-, octanoyl- and palmitoyl-L-carnitine) also interacted with L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine transport. 2-Amino-2-norbornane carboxylic acid, a known inhibitor of amino acid transporter B(0,+) (ATB(0,+)), did not cause any significant inhibition. A highly significant correlation was found between the potencies of acylcarnitines in the inhibition of L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake and the acyl chain length of acylcarnitines. The expression of mRNA for organic cation/carnitine transporters (OCTNs), carnitine transporter 2 (CT2) and ATB(0,+) in astrocytes was investigated by reverse transcription (RT)-PCR. OCTN2 mRNA was expressed in astrocytes, whereas the expression of OCTN1, OCTN3 and CT2 mRNA could not be detected. ATB(0,+) mRNA was expressed at very low levels in astrocytes. Western blotting analysis indicated that anti-OCTN2 polyclonal antibody recognized a band of 70 kDa in both kidney and astrocyte preparations. OCTN2 immunoreactivity was detected in rat astrocytes by immunocytochemical staining. Inhibition of OCTN2 expression by RNA interference significantly inhibited L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake into astrocytes. These results suggest that OCTN2 is functionally expressed in rat astrocytes, and is responsible for L-carnitine and acetyl-L-carnitine uptake in these cells.

P2 purinergic receptors and mechanical strain signal to glycogen synthase kinase‐3beta independently of protein kinase B/Akt in astrocytes

Glycogen synthase kinase (GSK)-3 was initially identified as an enzyme that regulates glycogen synthesis in response to insulin, but more recent studies indicate that it is also involved in a number of 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 GSK3β. When ATP was added to primary cultures of rat cortical astrocytes, phosphorylation of Ser9 on GSK3β was increased and GSK3 activity was decreased. Agonist studies revealed that that both metabotropic P2Y and ionotropic P2X receptors signal to Ser9-GSK3β. Signaling studies revealed that P2 receptors are independently coupled to GSK3β, Akt and ERK, and that phosphorylation of Ser9-GSK3β is dependent on protein kinase C (PKC). Mechanical strain, which releases ATP, also stimulated Ser9 phosphorylation and this was attenuated by blockade of P2 receptors. These studies indicate that P2 receptors are coupled to GSK3β by a PKC-dependent pathway and suggest that purinergic signaling contributes to the regulation of GSK3β functions such as glycogen metabolism in astrocyte-neuron interactions as well as the response of astrocytes to CNS injury upon release of ATP. (Supported by Dept. Veteran Affairs & NIH.)

Impact of the gliotoxin l-serine- O-sulphate on cellular metabolism in cultured rat astrocytes

l-Serine- O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that l-serine- O-sulphate, at a sub-toxic concentration (400 μM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using 13C NMR spectroscopy a significant reduction in labelled end products from [1- 13C]glucose and [3- 13C]alanine was found in the presence of l-serine- O-sulphate. Additionally, using [2- 13C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with l-serine- O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, l-serine- O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.

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.