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Apoptosis is implicated in the pathophysiology of Alzheimer's disease. Extracellular guanosine inhibits staurosporine-induced apoptosis in astrocytes. We examined whether guanosine protects SH-SY5Y human neuroblastoma cells against beta-amyloid(betaA)-induced apoptosis. Addition of betaA (fragment 25-35, 5 microM for 24 h) to SH-SY5Y cells increased the number of apoptotic cells, as evaluated by oligonucleosome ELISA. Guanosine pre-treatment decreased betaA-induced apoptosis (maximal effect after 24 h, 300 microM, p<0.05). The anti-apoptotic effect of guanosine was reduced by LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) (p<0.05). Guanosine increased phosphorylation of Akt/PKB, and this was abolished by inhibiting PI3K or MEK, (p<0.001, 5 min). Thus, the protective effect of guanosine against betaA-induced apoptosis of SH-SY5Y cells is mediated via activation of the PI3K/Akt/PKB and MAPK pathways.

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Administration of guanosine (8 mg/kg/day, i.p.) for 7 consecutive days, starting 5 weeks after moderate SCI in rats, improved locomotor function and spinal cord remyelination. Myelinogenesis was associated with an increase in the number of mature oligodendrocytes detected in guanosine-treated spinal cord sections in comparison with controls. These data indicate that guanosine-induced remyelination resulted, at least in part, from activation of endogenous oligodendrocyte lineage cells. These findings may have significant implications for chronic demyelinating diseases.

Transected dorsal root axons of adult rats can be induced to regenerate through the normally non-permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals 1, 2 and 3 months, respectively, after injury. In contrast, the CTM reflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.

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 (50 microM), verapamil (50 microM), probenecid (1 mM) or sulfinpyrazone (2 mM) enhanced [3H]vincristine accumulation; (ii) cyclosporin A or verapamil but not probenecid or sulfinpyrazone enhanced [3H]digoxin accumulation; (iii) glibenclamide (100 microM) inhibited 36Cl efflux 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., (Copyright 2002 Lippincott Williams & Wilkins)

This article reviews the effects of extracellular purine bases, nucleosides, and nucleotides as intracellular signaling molecules with trophic effects on cells after insults to the brain and spinal cord. Astrocytes are the principal source of extracellular purines in brain after injury, ischemia, or trauma. In vitro and in vivo extracellular purines have both immediate and long-term trophic effects, including stimulation of astrocyte and neuronal differentiation, mitosis, morphogenesis, apoptosis, and stimulation of growth and trophic factor synthesis. The effects of the nucleoside adenosine and the nucleotide adenosine triphosphate (ATP) are mediated principally via specific receptors on the cell surface coupled to a series of signaling cascades. Unlike adenosine and ATP, guanosine and guanosine triphosphate (GTP) do not act at classical purine receptors. However, they exert similar effects on astrocytes, apparently by causing the astrocytes to release large amounts of adenosine and ATP over prolonged periods. The release of adenosine and ATP may be related to the effects of guanosine on the purine nucleoside transporters in the cell membrane, whereas the release of ATP may be due to the effects of GTP on the ATP-binding cassette (ABC) proteins. Physiologically, the effects of guanosine are important because this nucleoside, unlike adenosine, remains elevated for prolonged periods after brain injury.

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.

Guanosine at 30 and 300 microM elicited the de novo extension of neurites from PC12 cells. With saturating concentrations of NGF, guanosine acted in a synergistic manner to enhance neuritogenesis. Adenosine alone also stimulated neurite outgrowth, but did not enhance NGF-induced neuritogenesis. 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog and A1/A2 receptor agonist, also alone had neuritogenic effects. It enhanced NGF-induced neuritic outgrowth but not to the same extent as guanosine. However, when NECA was added together with guanosine in the presence of NGF, these compounds elicited a greatly enhanced neuritogenic response. This suggested that the mechanisms through which NECA modulates the neuritogenic effects may be different from those of guanosine and NGF.

Both extracellular guanosine and adenosine stimulated astrocyte proliferation in vitro and increased intracellular cAMP 6-fold within 2 min. The effects of both guanosine and adenosine on proliferation and cAMP levels were inhibited by antagonists of adenosine A2 receptors but augmented by A1 receptor antagonists. The correlation between cAMP accumulation and stimulation of cell proliferation by adenosine and guanosine indicates that increased intracellular cAMP may be one of the second messengers involved in these effects. Guanosine is not an adenosine A2 receptor agonist and does not activate adenylate cyclase. It may exert its effects indirectly by increasing the endogenous extracellular adenosine concentration.

The activities of the membrane-bound protein kinases of the human erythrocytes membrane that phosphorylate spectrin, band-3 protein, and phospholipids were compared in patients with myotonic muscular dystrophy and normal age- and sex-matched controls. These activities tended to be lower in the patients, but the differences were not statistically significant. In contrast, the temperature responses (the increase in activity in response to an increase in temperature from 30 degrees C to 37 degrees C) of the spectrin and band-3 protein kinase activities were significantly lower in the patients. Although they do not eliminate an alteration of one of the substrates, these results are consistent with the proposal that differences in erythrocytes from myotonic muscular dystrophy (MyD) patients are due to a membrane lipid change. Cholesterol is unlikely to be the altered lipid, as no difference in membrane cholesterol content was found.