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 (approximately 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 x 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 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) 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.

1 Extracellular guanosine has diverse e ects 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 e ects of extracellular guanosine are compatible with an action on G-protein linked cell membrane receptors. 4 This site was speci®c for guanosine, and the order of potency in displacing 50 nM [ 3 H]-guanosine was: guanosine=6-thio-guanosine4inosine46-thio-guanine4guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine ca eine, theophylline, GDP, GMP and ATP were unable to signi®cantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well-characterized receptors for adenosine and purines. 5 The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein.

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 microM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 microM) 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 microM) or propentophylline (100 microM) 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 A(1) or A(2) adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A(3) 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 A(3) adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A(3) adenosine receptors.

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3 ¶,5 ¶-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). N ! -Nitro-L-arginine methyl ester (L-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.

By, using adenosine as a substrate, we detected the presence of enzyme activity in a crude extract of tobacco tissue that converts this cytokinin into adenosine. The extract also contains strong hydrolase activity that converts adenosine to adenine. N6-(A'-Isopentenyl)adenosine and its derivatives N6-(A'-isopentenyl)-2-methylthioadenosine, N-(4-hydroxy-3-methylbut-2-trans-enyl)-adenosine, andN-(4-hydroxy-3-methylbut-2-enyl)- Chromatography. Whatman No. 1 paper was used. Solvents: A: ethanol-0.1 M ammonium borate, pH 9.0 (1:9, v/v); B: isopropanol-concentrated ammonium hydroxide-water (7:1:2, v/v); C: 1-butanol-concentrated ammonium hydroxide-water (86:5:14, v/v). Merck Silica Gel H was used for preparative thin layer chromatography. Plates were developed in a 15% (v/v) methanol in methylene chloride mixture.Detection of Radioactivity. For the assays of enzyme activity 3.8-cm wide strips from the developed paper chromatograms were cut out and scanned in a Nuclear Chicago Actigraph III instrument. The areas on the chart paper corresponding to the peaks of radioactivity were cut out and weighed. The calculation of the relative amounts of radioactive compounds was based on the weight of the paper cut-outs.For accurate measurement of radioactivity in the synthetic procedures a liquid scintillation counter was used.Preparation of [8-"C]-N6-(Av2-Isopentenyl)adenosine. Two syntheses of N"-(zX'-isopentenyl)adenosine have been reported. In the first route, A-isopentenylamine is condensed with 6. chloro-(9-8-D-ribofuranosyl)purine (12). In the second, adenosine is alkylated with A'-isopentenylbromide (5, 9, 12). For preparation of a radioactively labeled compound, the latter approach offers greater versatility because of the commercial availability of different forms of labeled adenosine.We synthesized [8-l"C]-N'-(A'-isopentenyl)adenosine, following the route of Martin and Reese (9). The small scale of the reaction presented difficulties because of potential side reactions; further, the product is highly labile and consequently must be purified and stored in a manner different from that used for larger scale synthesis of an unlabeled sample.An aqueous solution of [8-"C]-adenosine (100 Muc; 0.4854 mg; 1.82 Mumole; in 10 ml) was evaporated to dryness in vacuo at 35 C. The residue was dried from absolute ethanol (2 X 2 ml) and placed in a desiccator over phosphorus pentoxide for 16 hr. Adenosine (4.85 mg, 18.2 Mmoles) was dried and added to the radioactive material. A suspension of adenosine (5.335 mg, 20 Mumoles), A-isopentenyl bromide (5.2 mg, 4 ,u; 35 Mmoles) in dimethylformamide (70 pl) was stirred at room temperature. After 24 hr, aqueous dimethylamine (70 ,ul) and methanol (100 p1) were added, and the reaction mixture was stirred for a further 16 hr. The reaction mixture was then evaporated to dryness in vacuo; the residue re-evaporated with 95% ethanol (2 X 200 ul) and finally with water (2 X 200 pi).The solid residue was triturated with 95% ethanol (2 X 2 ml) 775 www.plantphysiol.org on May 11, 2018 -Publish...

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.

Like adenine-based purines, extracellular nonadenine-based purines have a multitude of trophic effects on the growth, differentiation, and survival of target cells. The nonadenine-based purines, which include guanosine, inosine, and GTP, apparently exert their trophic effects by interacting with both intercellular targets as well as those on the cell surface. Specifically, guanosine and inosine target the protein kinase N-kinase, in promoting remarkable nerve process extension, even in long tracts of the central nervous system after injury. In contrast, GTP may exert its effects via a cell surface receptor coupled to the release of calcium from internal stores. In other cases trophic effects may be mediated by the enhancement of release of adenine-based purines by guanosine. Additionally, evidence is presented for the existence of a high-affinity binding site for guanosine with receptor-like characteristics on the plasma membranes of astrocytes and brain tissue. This site may be G-protein-coupled and exert its effects through activation of the MAP kinase cascade. One effect apparently mediated through this mechanism is the production and release by astrocytes of trophic protein growth factors such as NGF and TGFβ. These have substantial neuroprotective effects. Additionally, this pathway is apparently involved in modulating the expression of P2Y 1 and P2Y 2 receptors in response to extracellular guanosine. Extracellular nonadenine-based purines can interact with other growth factors, but these interactions are not always synergistic. For example, combinations of guanosine and FGF are antagonistic and reduce the growth of microvascular cells in vitro. Some of the properties of the nonadenine-based purines likely derive from their unique intracellular metabolism in which conversion of guanine to xanthine is the final catabolic step. This step is catalyzed by guanase, the activity of which varies markedly in different brain regions, raising the possibility that guanine or guanosine are involved in neurotransmission. Together these data suggest several potentially useful pharmacological approaches involving nonadenine-based purines to modulate trophic effects in the central nervous system. Drug Dev. Res. 52:303-315, 2001.

The sarcoplasmic reticulum (SR) Ca2+ pump in membranes isolated from arterial smooth muscle is damaged by reactive oxygen species (ROS). Because angiotensin II (ANG II) contracts arterial smooth muscle by mobilizing intracellular Ca2+ concentrations ([Ca2+])i, we determined the effects of ROS pretreatment on ANG II-induced contractions in coronary artery rings and [Ca2+]i transients in smooth muscle cells (SMC) cultured from them. This experimental design eliminates direct ROS interference in assay solutions, thus monitoring only the tissue damage. Pretreating the arteries with peroxide inhibited the ANG II contractions with the concentration for half-maximal activation (K0.5) = 74 +/- 5 microM. Peroxide (250 microM) inhibited the contractions to ANG II and cyclopiazonic acid (CPA, SR Ca(2+)-pump inhibitor) by 78.3 +/- 5.1 and 67.4 +/- 6.3%, respectively, but did not significantly affect the contractions by 60 mM KCl. Pretreating SMC with peroxide inhibited the ANG II-induced increase in [Ca2+]i with K0.5 = 24 +/- 3 microM for peroxide. Peroxide (100 microM) inhibited the increase in [Ca2+]i in response to ANG II and CPA by 78.9 +/- 5.1 and 38.3 +/- 4.9%, respectively. The SR Ca(2+)-pump activity was also measured as the Ca(2+)-dependent formation of 115-kDa acylphosphate. Pretreating SMC with 100 microM peroxide inhibited the acylphosphate levels by 36.3 +/- 3.2%. Peroxide (100 microM) pretreatment of SMC did not significantly affect their ANG II binding.(ABSTRACT TRUNCATED AT 250 WORDS)