Francesco Caciagli

Trophic effects of purines in neurons and glial cells.

In addition to their well known roles within cells, purine nucleotides such as adenosine 5 triphosphate (ATP) and guanosine 5 triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimers disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.

Involvement of astrocytes in purine-mediated reparative processes in the brain

Astrocytes are involved in multiple brain functions in physiological conditions, participating in neuronal development, synaptic activity and homeostatic control of the extracellular environment. They also actively participate in the processes triggered by brain injuries, aimed at limiting and repairing brain damages. Purines may play a significant role in the pathophysiology of numerous acute and chronic disorders of the central nervous system (CNS). Astrocytes are the main source of cerebral purines. They release either adenine‐based purines, e.g. adenosine and adenosine triphosphate, or guanine‐based purines, e.g. guanosine and guanosine triphosphate, in physiological conditions and release even more of these purines in pathological conditions. Astrocytes express several receptor subtypes of P1 and P2 types for adenine‐based purines. Receptors for guanine‐based purines are being characterised. Specific ecto‐enzymes such as nucleotidases, adenosine deaminase and, likely, purine nucleoside phosphorylase, metabolise both adenine‐ and guanine‐based purines after release from astrocytes. This regulates the effects of nucleotides and nucleosides by reducing their interaction with specific membrane binding sites. Adenine‐based nucleotides stimulate astrocyte proliferation by a P2‐mediated increase in intracellular [Ca2+] and isoprenylated proteins. Adenosine also, via A2 receptors, may stimulate astrocyte proliferation, but mostly, via A1 and/or A3 receptors, inhibits astrocyte proliferation, thus controlling the excessive reactive astrogliosis triggered by P2 receptors. The activation of A1 receptors also stimulates astrocytes to produce trophic factors, such as nerve growth factor, S100β protein and transforming growth factor β, which contribute to protect neurons against injuries. Guanosine stimulates the output of adenine‐based purines from astrocytes and in addition it directly triggers these cells to proliferate and to produce large amount of neuroprotective factors. These data indicate that adenine‐ and guanine‐based purines released in large amounts from injured or dying cells of CNS may act as signals to initiate brain repair mechanisms widely involving astrocytes.

Activation of A1 adenosine or mGlu3 metabotropic glutamate receptors enhances the release of nerve growth factor and S-100β protein from cultured astrocytes

Pharmacological activation of A1 adenosine receptor with 2‐chloro‐N6‐cyclopentyladenosine (CCPA) or mGlu3 metabotropic glutamate receptors with (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV) or aminopyrrolidine‐2R,4R‐dicarboxylate (2R,4R‐APDC) enhanced the release of nerve growth factor (NGF) or S‐100β protein from rat cultured astrocytes. Stimulation of release by CCPA and DCG‐IV or 2R,4R‐APDC was inhibited by the A1 adenosine receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine and by the mGlu2/3 receptor antagonist (2S,1′S,2′S,3′R)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine (PCCG‐4), respectively. Time‐course studies revealed a profound difference between the release of S‐100β protein and the release of NGF in response to extracellular signals. Stimulation of S‐100β protein exhibited rapid kinetics, peaking after 1 h of drug treatment, whereas the enhancement of NGF release was much slower, requiring at least 6 h of A1 adenosine or mGlu3 receptor activation. In addition, stimulation of NGF but not S‐100β release was substantially reduced in cultures treated with the protein synthesis inhibitor cycloheximide. In addition, a 6–8 h treatment of cultured astrocytes with A1 or mGlu3 receptor agonists increased the levels of both NGF mRNA and NGF‐like immunoreactive proteins, including NGF prohormone. We conclude that activation of A1 adenosine or mGlu3 receptors produces pleiotropic effects in astrocytes, stimulating the synthesis and/or the release of protein factors. Astrocytes may therefore become targets for drugs that stimulate the local production of neurotrophic factors in the CNS, and this may provide the basis for a novel therapeutic strategy in chronic neurodegenerative disorders. GLIA 27:275–281, 1999.

Rat astroglial P2Z (P2X7) receptors regulate intracellular calcium and purine release.

Treatment of rat astrocyte cultures with 2- and 3-O-(4-benzoylbenzoyl)-adenosine 5-triphosphate (BzATP), a P2X7 agonist, but not with adenosine 5-[alpha, beta methylene] triphosphate (alpha, beta meATP), a P2X agonist, increased influx of extracellular Ca2+ and [Ca2+]i. Lucifer yellow, a small molecule which permeates P2X7 receptor-induced pores, entered BzATP-treated but not control astrocytes. BzATP also stimulated efflux of [3H]purine from cultured astrocytes. The P2X7 receptor antagonist oxidized ATP abolished the effects of BzATP on [Ca2+]i, lucifer yellow permeation and [3H]purine release, indicating that these effects were due to P2X7 receptor activation. In neurological diseases or injuries extracellular ATP may activate P2X7 receptors further enhancing [3H]purine release, with important pathophysiological consequences.

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.

Glial cells express multiple ATP binding cassette proteins which are involved in ATP release

Rat brain astrocyte and microglia cultures express different members of ATP-binding-cassette (ABC) proteins. RT-PCR analysis showed that astrocytes are equipped with P-glycoprotein (mdr1a, mdr1b), multidrug resistance-associated-protein (mrp1, mrp4, mrp5) and cystic fibrosis transmembrane conductance regulator (CFTR). No transcripts for mrp5 and CFTR were detected in microglia. The ABC protein functional activities are shown by the following results: (i) cyclosporin A (50u2009μM), verapamil (50u2009μM), probenecid (1u2009mM) or sulfinpyrazone (2u2009mM) enhanced [3H]vincristine accumulation; (ii) cyclosporin A or verapamil but not probenecid or sulfinpyrazone enhanced [3H]digoxin accumulation; (iii) glibenclamide (100u2009μM) inhibited 36Clefflux from astrocytes. ATP release from glial cells was inhibited by the pretreatment with ABC protein inhibitors indicating that ABC proteins are involved in nucleotide efflux from glial cells which represent the main source of cerebral extracellular purines.

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).

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.