Patrizia Di Iorio

Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia

Brain ischemia stimulates release from astrocytes of adenine‐based purines, particularly adenosine, which is neuroprotective. Guanosine, which has trophic properties that may aid recovery following neurological damage, is present in high local concentrations for several days after focal cerebral ischemia. We investigated whether guanine‐based purines, like their adenine‐based counterparts, were released from astrocytes and whether their release increased following hypoxia/hypoglycemia. HPLC analysis of culture medium of rat astrocytes showed spontaneous release of endogenous guanine‐based purines at a higher rate than their adenine‐based counterparts. The concentration of guanosine (≈120 nM) and adenosine (≈43 nM) in the culture medium remained constant, whereas concentrations of adenine and guanine nucleotides, particularly GMP, and their metabolites increased with time. Exposure of the cultures to hypoxia/hypoglycemia for 30 min increased the extracellular concentration of adenine‐based purines by 2.5‐fold and of guanine‐based purines by 3.5‐fold. Following hypoxia/hypoglycemia extracellular adenine nucleotide levels increased further. Adenosine concentration increased, but not proportionally to nucleotide levels. Accumulation of adenosine metabolites indicated it was rapidly metabolized. Conversely, the concentrations of extracellular guanine‐based nucleotides remained elevated and the concentration of guanosine continued to increase. These data indicate that astrocytes are a major source of guanine‐based purines, the release of which is markedly increased following hypoxia/hypoglycemia, permitting them to exert neurotrophic effects. GLIA 25:93–98, 1999.

Opposite influence of the metabotropic glutamate receptor subtypes mGlu3 and -5 on astrocyte proliferation in culture

In non‐synchronized, subconfluent secondary cultures of rat cortical astrocytes, the selective group‐I metabotropic glutamate (mGlu) receptor agonist 3,5‐dihydroxyphenylglycine (DHPG) increased [methyl‐3H]‐thymidine incorporation. This effect was mediated by the activation of the mGlu5 receptor, which was shown to be present by either RT‐PCR or Western blot analysis. The mixed mGlu receptor antagonist (+)‐α‐methyl‐4‐carboxyphenylglycine reduced the increase in both intracellular Ca2+ and [methyl‐3H]‐thymidine incorporation produced by DHPG. In contrast, (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycylopropyl)glycine (DCG‐IV), a potent and selective agonist of group‐II mGlu receptors, reduced [methyl‐3H]‐thymidine incorporation in non‐synchronized astrocyte cultures. The antiproliferative effect of DCG‐IV was prevented by the selective group‐II mGlu receptor antagonist (2S,1′S,2′S,3′R)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine (PCCG‐IV). The opposite effect of DHPG and DCG‐IV on astrocyte proliferation was confirmed in cultures deprived of serum for 48 hours and then stimulated to proliferate with either epidermal growth factor (EGF) or the metabolically stable ATP analogue adenosine 5′‐(β,γ‐imido)‐triphosphate (AMP‐PNP).

Mechanisms of apoptosis induced by purine nucleosides in astrocytes

Astrocytes release adenine‐based and guanine‐based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration‐dependent manner up to 100 μM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 μM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2–3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine‐based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 μM) or propentophylline (100 μM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A1 or A2 adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A3 adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S‐adenosyl‐L‐homocysteine (SAH) and greatly reduced the ratio of S‐adenosyl‐L‐methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A3 adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A3 adenosine receptors. GLIA 38:179–190, 2002.

Specific [3H]‐guanosine binding sites in rat brain membranes

Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine‐based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G‐protein linked cell membrane receptors. Specific binding sites for [3H]‐guanosine were detected on membrane preparations from rat brain. The kinetics of [3H]‐guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122×107 M−1 min−1 and 1.69 min−1, respectively. A single high affinity binding site for [3H]‐guanosine with a KD of 95.4±11.9 nM and Bmax of 0.57±0.03 pmol mg−1 protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [3H]‐guanosine was: guanosine=6‐thio‐guanosine>inosine>6‐thio‐guanine>guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well‐characterized receptors for adenosine and purines. The addition of GTP produced a small concentration‐dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G‐protein. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.

Cultured astrocyte proliferation induced by extracellular guanosine involves endogenous adenosine and is raised by the co‐presence of microglia

Extracellular adenosine (Ado) and ATP stimulate astrocyte proliferation through activation of P1 and P2 purinoceptors. Extracellular GTP and guanosine (Guo), however, that do not bind strongly to these receptors, are more effective mitogens than ATP and Ado. Exogenous Guo, like GTP and 5′‐guanosine‐βγ‐imidotriphosphate (GMP‐PNP), dose‐dependently stimulated proliferation of rat cultured astrocytes; potency order GMP‐PNP > GTP ≥ Guo. The mitogenic effect of Guo was independent of the extracellular breakdown of GTP to Guo, because GMP‐PNP, a GTP analogue resistant to hydrolysis, was the most mitogenic. In addition to a direct effect on astrocytes, Guo exerts its proliferative activity involving Ado. Exogenous Guo, indeed, enhanced the extracellular levels of endogenous Ado assayed by HPLC in the medium of cultured astrocytes. Culture pretreatment with Ado deaminase (ADA), that converts Ado into inosine, reduced but did not abolish Guo‐induced astrocyte proliferation whereas erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA), that inhibits ADA activity, amplified Guo effect. Moreover, the mitogenic activity of Guo was partly inhibited by 8‐cyclopentyl‐1,3‐dipropylxanthine and alloxazine, antagonists of Ado A1 and A2B receptors, respectively. Also microglia seem to be a target for the action of Guo. Indeed, the mitogenic effect of Guo on astrocytes was: i) increased proportionally to the number of microglial cells present in the astrocyte cultures; ii) amplified when purified cultures of astrocytes were supplemented with conditioned medium deriving from Guo‐pretreated microglial cultures. These data indicate that the mitogenic effects exerted by exogenous Guo on rat astrocytes are mediated via complex mechanisms involving extracellular Ado and microglia‐derived soluble factors. GLIA 29:202–211, 2000.

The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase‐3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (∼ 10‐fold over basal apoptotic values) in 18–24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration‐dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 × 10−5 M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi‐proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3‐kinase (PI3K; LY294002, 30 μM) or the MAPK pathway (PD98059, 10 μM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase‐3β (GSK‐3β) and induced an upregulation of Bcl‐2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine‐induced apoptosis by activating multiple pathways, and these are mediated by a Gi‐protein‐coupled putative guanosine receptor.

Molecular signalling mediating the protective effect of A1 adenosine and mGlu3 metabotropic glutamate receptor activation against apoptosis by oxygen/glucose deprivation in cultured astrocytes

Astrocyte death may occur in neurodegenerative disorders and complicates the outcome of brain ischemia, a condition associated with high extracellular levels of adenosine and glutamate. We show that pharmacological activation of A1 adenosine and mGlu3 metabotropic glutamate receptors with N6-chlorocyclopentyladenosine (CCPA) and (–)2-oxa-4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), respectively, protects cultured astrocytes against apoptosis induced by a 3-h exposure to oxygen/glucose deprivation (OGD). Protection by CCPA and LY379268 was less than additive and was abrogated by receptor blockade with selective competitive antagonists or pertussis toxin. Both in control astrocytes and in astrocytes exposed to OGD, CCPA and LY379268 induced a rapid activation of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) pathways, which are known to support cell survival. In cultures exposed to OGD, CCPA and LY379268 reduced the activation of c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the proapoptotic protein Bad, increased the levels of the antiapoptotic protein Bcl-XL, and were highly protective against apoptotic death, as shown by nuclear 4′-6-diamidino-2-phenylindole staining and measurements of caspase-3 activity. All of these effects were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit the MAPK and the PI3K pathways, respectively. These data suggest that pharmacological activation of A1 and mGlu3 receptors protects astrocytes against hypoxic/ischemic damage by stimulating the PI3K and ERK1/2 MAPK pathways.

Cysteinyl-leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via cys-LT1 receptors and mitogen-activated protein kinase pathway

Cysteinyl‐leukotrienes (cys‐LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5′‐lipoxygenase and LTC4 synthase) required for cys‐LT production, and release cys‐LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen–glucose deprivation or 2‐methyl‐thioATP, a selective P2Y1/ATP receptor agonist. MK‐886, a LT synthesis inhibitor, prevented basal and evoked cys‐LT release. In addition, 2‐methyl‐thioATP‐induced cys‐LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys‐LT release. We also showed that astrocytes express cys‐LT1 and not cys‐LT2 receptors. The stimulation of these receptors by LTD4 activated the mitogen‐activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor‐coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK‐571, a cys‐LT1 selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin‐dependent kinase II (KN‐93), Ca2+‐dependent and ‐independent (GF102903X) or Ca2+‐dependent (Gö6976) protein kinase C isoforms. LTD4 also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK‐571 or PD98059. Thus, cys‐LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar.

Staurosporine-induced apoptosis in astrocytes is prevented by A1 adenosine receptor activation

Astrocyte apoptosis occurs in acute and chronic pathological processes at the central nervous system and the prevention of astrocyte death may represent an efficacious intervention in protecting neurons against degeneration. Our research shows that rat astrocyte exposure to 100 nM staurosporine for 3h caused apoptotic death accompanied by caspase-3, p38 mitogen-ed protein kinase (MAPK) and glycogen synthase kinase-3beta (GSK3beta) activation. N(6)-chlorocyclopentyladenosine (CCPA, 2.5-75 nM), a selective agonist of A(1) adenosine receptors, added to the cultures 1h prior to staurosporine, induced a dose-dependent anti-apoptotic effect, which was inhibited by the A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. CCPA also caused a dose- and time-dependent phosphorylation/activation of Akt, a downstream effector of cell survival promoting phosphatidylinositol 3-kinase (PI3K) pathway, which in turn led to inhibition of staurosporine-induced GSK3beta and p38 MAPK activity. Accordingly, the anti-apoptotic effect of CCPA was abolished by culture pre-treatment with LY294002, a selective PI3K inhibitor, pointing out the prevailing role played by PI3K pathway in the protective effect exerted by A(1) receptor activation. Since an abnormal p38 and GSK3beta activity is implicated in acute (stroke) and chronic (Alzheimers disease) neurodegenerative diseases, the results of the present study provide a hint to better understand adenosine relevance in these disorders.

Altered distribution and function of A2A adenosine receptors in the brain of WAG/Rij rats with genetic absence epilepsy, before and after appearance of the disease

The involvement of excitatory adenosine A2A receptors (A2ARs), which probably contribute to the pathophysiology of convulsive seizures, has never been investigated in absence epilepsy. Here, we examined the distribution and function of A2ARs in the brain of Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, a model of human absence epilepsy in which disease onset occurs 2–3 months after birth. In the cerebral areas that are mostly involved in the generation of absence seizures (somatosensory cortex, reticular and ventrobasal thalamic nuclei), A2AR density was lower in presymptomatic WAG/Rij rats than in control rats, as evaluated by immunohistochemistry and western blotting. Accordingly, in cortical/thalamic slices prepared from the brain of these rats, A2AR stimulation with the agonist 2‐[4‐(‐2‐carboxyethyl)‐phenylamino]‐5′‐N‐ethylcarboxamido‐adenosine failed to modulate either cAMP formation, mitogen‐activated protein kinase system, or K+‐evoked glutamate release. In contrast, A2AR expression, signalling and function were significantly enhanced in brain slices from epileptic WAG/Rij rats as compared with matched control animals. Additionally, the in vivo injection of the A2AR agonist CGS21680, or the antagonist 5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐fuyl)‐pyrazolo‐(4,3‐c)1,2,4‐triazolo(1,5‐c)‐pyrimidine, in the examined brain areas of epileptic rats, increased and decreased, respectively, the number/duration of recorded spontaneous spike–wave discharges in a dose‐dependent manner during a 1–5 h post‐treatment period. Our results support the hypothesis that alteration of excitatory A2AR is involved in the pathogenesis of absence seizures and might represent a new interesting target for the therapeutic management of this disease.