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 P2Y2 and P2Y4, but not P2Y6, receptors as well as a partial role for P2Y1 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.

Molecular determinants of P2Y2 nucleotide receptor function: implications for proliferative and inflammatory pathways in astrocytes.

In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.

Effects of marine sponge extracts on mitogen-activated protein kinase (MAPK/ERK(1,2)) activity in SW-13 human adrenal carcinoma cells.

Some species of marine sponge have been shown to produce metabolites with endocrine-altering and cell growth regulatory properties. Since cell division and differentiation are controlled, in part, by the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK/ERK) cascade, we tested extracts (1.0mg/ml) from six shallow water marine species obtained in the Florida Keys for effects on MAPK/ERK(l,2) (sub-variant of EC 2.7.1.37) activity in incubations with SW-13 human adrenal carcinoma cells in culture. In these short-term incubations, extracts from two species, the purple bleeding sponge (Iotrochota birotulata) and the West Indian bath sponge (Spongia barbara), significantly inhibited MAPK/ERK(1,2) activity (to 51 and 44% of control levels, respectively) without altering cell survival. Western blots for phosphorylated and total ERK showed that ERK(2) predominated over ERK(1) by a factor of about 4:1 and that the phosphorylated forms of these isozymes were strongly suppressed by active extracts from both sponges. Another species, the green sponge (Haliclona veridis), whose extract has been shown previously to activate guanylate cyclase and to inhibit adenylate cyclase in a variety of mammalian tissues, was found not to affect MAPK/ERK(1,2) in human adrenal carcinoma cultures but did lyse and kill most of these cultured cells. Extracts from the sheepswool sponge (Hippospongia lachne) and the bleeding sponge (Oligoceras hemorrhages) did not significantly affect either MAPK/ERK(1,2) activity or the survival of attached cells. An extract from the fire sponge (Tedania ignis) did not alter MAPK/ERK(1,2) activity but did modestly decrease cell viability. These studies document for the first time species-specifc effects of marine sponge extracts on the MAPK/ERK(1,2) cascade and on the growth and survival of human adrenal carcinoma cells in culture.

Biochemical mechanisms of action of Hypericum LI 160 in glial and neuronal cells: inhibition of neurotransmitter uptake and stimulation of extracellular signal regulated protein kinase.

We have investigated biochemical mechanisms that may underlie the antidepressant effects of Hypericum LI 160. We found that LI 160 inhibits uptake of serotonin and norepinephrine in cultures of rat cortical astrocytes. Observed differences in the kinetic parameters Km and Vmax as well as in the recovery of uptake after removal of Hypericum indicates that LI 160 does not affect serotonin and norepinephrine transport in the same manner. This suggests that multiple components of the extract can mediate inhibition of these neurotransmitter transporters. Hypericum LI 160 also inhibits serotonin uptake in neuronal cultures from serotonergic-rich raphe nuclei; concentration-response studies indicate LI 160 is 25 times more potent in terms of inhibition of serotonin uptake in neurons than in astrocytes. In addition, Hypericum LI 160 inhibits norepinephrine uptake in SK-N-SH cells, a human neuroblastoma cell line enriched in norepinephrine transporters. A chemical constituent of LI 160, hyperforin, is about 10 to 20 times more potent than LI 160 in inhibiting neurotransmitter uptake in astrocytes and neuronal cells; this finding is consistent with the observation that hyperforin comprises 5% of LI 160 extracts. As several weeks are needed to achieve a clinical response with antidepressants, we have also investigated whether Hypericum LI 160 affects biochemical mechanisms that underlie long-term changes such as gene expression. We found that LI 160 stimulates a sustained activation of extracellular signal regulated protein kinase (ERK), a key component of a signal transduction pathway involved in gene expression. Taken together, our findings suggest that Hypericum LI 160 can affect rapidly-acting as well as slower-developing, long-term biochemical mechanisms related to depressive disorders.

Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C.

The mitogen-activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one-trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5-hydroxytryptamine (5-HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by 'upstream' signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5-HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA-dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5-HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA-AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca(2+)-dependent PKC activation contributes to ERK phosphorylation, although a PKC-independent pathway is also involved in 5-HT-dependent ERK phosphorylation and activation.

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. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS:

Trophic actions of extracellular ATP: gene expression profiling by DNA array analysis.

In addition to Professor Burnstock's work on the short-term signaling actions of extracellular nucleotides and nucleosides, Geoff has had a long-standing interest in trophic actions of purines in development and in pathophysiological conditions which has been instrumental in encouraging my work in this area. The trophic actions of extracellular ATP, alone or in combination with polypeptide growth factors, may play an important role in brain development and may contribute to the reactive gliosis that accompanies brain injury and neurodegeneration. P2Y receptors in astrocytes are coupled to the ERK/MAPK cascade, a signal transduction mechanism crucial for cellular proliferation and differentiation. The mitogenic signaling pathway from P2Y receptors to ERK involves phospholipase D and a calcium-independent PKC isoform, PKCdelta. DNA array analysis reveals a number of changes in gene expression after P2Y receptor occupancy, indicating that this methodology will be a powerful tool in understanding the mechanisms underlying the trophic actions of extracellular nucleotides and nucleosides.

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). 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 P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring 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 P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.

Mitogenic signaling by ATP/P2Y purinergic receptors in astrocytes: involvement of a calcium-independent protein kinase C, extracellular signal-regulated protein kinase pathway distinct from the phosphatidylinositol-specific phospholipase C/calcium pathway.

Activation of ATP/P2Y purinergic receptors stimulates proliferation of astrocytes, but the mitogenic signaling pathway linked to these G-protein-coupled receptors is unknown. We have investigated the role of extracellular signal-regulated protein kinase (ERK) in P2Y receptor-stimulated mitogenic signaling as well as the pathway that couples P2Y receptors to ERK. Downregulation of protein kinase C (PKC) in primary cultures of rat cerebral cortical astrocytes greatly reduced the ability of extracellular ATP to stimulate ERK. Because occupancy of P2Y receptors also leads to inositol phosphate formation, calcium mobilization, and PKC activation, we explored the possibility that signaling from P2Y receptors to ERK is mediated by a phosphatidylinositol-specific phospholipase C (PI-PLC)/calcium pathway. However, neither inhibition of PI-PLC nor chelation of calcium significantly reduced ATP-stimulated ERK activity. Moreover, a preferential inhibitor of calcium-dependent PKC isoforms, Gö 6976, was significantly less effective in blocking ATP-stimulated ERK activity than GF102903X, an inhibitor of both calcium-dependent and -independent PKC isoforms. Furthermore, ATP stimulated a rapid translocation of PKCdelta, a calcium-independent PKC isoform, but not PKCgamma, a calcium-dependent PKC isoform. ATP also stimulated a rapid increase in choline, and inhibition of phosphatidylcholine hydrolysis blocked ATP-evoked ERK activation. These results indicate that P2Y receptors in astrocytes are coupled independently to PI-PLC/calcium and ERK pathways and suggest that signaling from P2Y receptors to ERK involves a calcium-independent PKC isoform and hydrolysis of phosphatidylcholine by phospholipase D. In addition, we found that inhibition of ERK activation blocked extracellular ATP-stimulated DNA synthesis, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y receptors.

Hypericum LI 160 inhibits uptake of serotonin and norepinephrine in astrocytes.

Extracts of Hypericum perforatum, commonly known as St. John's wort, are frequently used in Germany and other European countries to treat mild to moderately severe depression, but the mechanism of antidepressant activity of Hypericum is not understood. Because known mechanisms of antidepressant activity include inhibition of serotonin and/or norepinephrine uptake, we investigated the effects of standardized extracts of Hypericum LI 160 on the transport of these monoamine neurotransmitters into astrocytes, cells which surround synaptic terminals and regulate neurotransmission by means of their uptake systems. We found that LI 160 inhibited both serotonin and norepinephrine uptake in a dose-dependent manner. The two monoamine transport systems were affected differently by LI 160: for serotonin, the main effect was a 50% decrease in the rate of maximal transport, whereas for norepinephrine, the main effect was a 4.5 fold reduction in the apparent affinity of norepinephrine for its uptake sites. Upon removal of LI 160, uptake was restored, thereby indicating that the inhibition was not due to a toxic effect of Hypericum on the cells. These findings suggest that the ability of LI 160 to inhibit serotonin and norepinephrine uptake may underlie the antidepressant activity of this Hypericum extract.

Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

Mitogenic signaling from P1 and P2 purinergic receptors to mitogen-activated protein kinase in human fetal astrocyte cultures.

To investigate potential trophic actions of extracellular ATP in human astrocytes, we have examined mitogenic signaling by purinergic receptors in cultures prepared from first trimester rostral central nervous system tissue. We found that ATP and ATPgammaS, a hydrolysis-resistant analog, stimulated DNA synthesis, thereby indicating that P2 purinergic receptors can stimulate mitogenic signaling in these cells. In addition, ATP activated a mitogen-activated protein kinase (MAPK) termed ERK (extracellular signal-regulated protein kinase), a key component of signal transduction pathways involved in cellular proliferation and differentiation. The activation of MAPK was mediated at least in part by P2 purinergic receptors, because a P2 purinoceptor antagonist, suramin, inhibited the ATP-evoked stimulation by 50%, whereas a P1 purinergic-receptor antagonist, 8-(para-sulfonphenyl)-theophylline, was without effect. In contrast to rat astrocytes, adenosine/P1 purinergic-receptor agonists, 2-chloroadenosine and 5'-N-ethylcarboxyamidoadenosine, stimulated MAPK activity and DNA synthesis in human astrocytes. A selective inhibitor of protein kinase C, Ro 31-8220, blocked the ability of ATP and adenosine analogs to stimulate MAPK, thereby indicating that protein kinase C is upstream of MAPK in both P2- and P1-receptor signaling pathways. An inhibitor of the MAPK activator MEK, PD 098059, effectively blocked ATP- and 2-chloroadenosine-induced DNA synthesis, thereby indicating that the ERK/MAPK cascade mediates mitogenic signaling by P2 and P1 purinergic receptors in human fetal astrocytes. These findings suggest a role for P1 and P2 purinergic receptors in the proliferation of human fetal astrocytes.

The glial glutamate transporter in hyperammonemia and hepatic encephalopathy: relation to energy metabolism and glutamatergic neurotransmission.

Abnormalities in glutamate metabolism and glutamatergic neurotransmission appear to play a major role in the pathogenesis of hyperammonemia and hepatic encephalopathy. Astrocytes may be involved in these derangements as ammonia has been shown to impair the ability of these cells to take up glutamate. This study presents a northern blot analysis of the GLT-1 glutamate transporter in hyperammonemic rats, and in rats with thioacetamide-induced acute liver failure. Our findings demonstrate a downregulation of GLT-1 mRNA in both conditions. This article examines the potential impact of deficits in glutamate uptake on energy metabolism and glutamatergic neurotransmission in the context of abnormalities in glial-neuronal interactions. We propose that an ammonia-induced abnormality in astroglial glutamate uptake constitutes a critical aspect in the pathogenesis of hepatic encephalopathy and other hyperammonemic conditions.

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

Extracellular ATP induces formation of AP-1 complexes in astrocytes via P2 purinoceptors.

The transcription activator protein-1 (AP-1) complex is a heterodimer consisting of Fos and Jun family members. We found that extracellular ATP stimulated AP-1 DNA binding activity in cerebral cortical astrocyte cultures. This activity was maximal at 1 h and persisted for at least 3 h post-treatment. Shift-Western blotting indicated the presence of c-Fos in the AP-1 complexes. Stimulation of AP-1 binding by ATP was due to activation of P2 rather than P1 purinoceptors. The protein kinase C (PKC) inhibitor Ro 31-8220 markedly reduced P2 purinoceptor-mediated AP-1 induction. The induction of AP-1 complexes by ATP may contribute to changes in gene expression which underlie the trophic effects of extracellular ATP on astrocytes.

P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies.

Extracellular ATP is known to activate intracellular enzymes in astrocytes via P2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (ICl,Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P2 purinoceptors. These results indicated that the two P2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P2 purinoceptor antagonist (suramin) but not by the P1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase.

Time-dependent increase in protein phosphorylation following one-trial enhancement in Hermissenda.

One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of 32 PO4 into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins, detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time-dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one-trial conditioning.

Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells.

In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.