Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms.

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.

Non-adenine based purines accelerate wound healing.

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5-2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8-3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.

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 (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.

Rat brain guanosine binding site. Biological studies and pseudo-receptor construction.

Rat brain guanosine binding sites were studied by (i). a pharmacological approach to confirm the hypothesis of the existence of specific G-coupled receptors for guanosine (1) and, for the first time, delineate a structure-activity relationship for a series of guanosine derivatives; (ii). a molecular modelling approach to design a pseudo-receptor construction. GTP and its non-hydrolysable analogue Gpp[NH]p decreased [3H]-guanosine binding to rat brain membranes. Gpp[NH]p 30 and 100 microM induced a dose-dependent decrease in [3H]-guanosine affinity and PTX pretreatment of rat brain membranes caused a 50% reduction in binding. In slices from rat brain cortex, guanosine induced a dose-dependent increase in intracellular cAMP. This increase is specific for guanosine, since neither the pretreatment with adenosine deaminase nor the A(1) and A(2) adenosine receptor antagonists were able to modify the guanosine-induced cAMP accumulation. The structure-activity relationship showed that the potency order of the best substances able to displace 50 nM [3H]-guanosine was guanosine (1)=6-thioguanosine (3)>8-bromoguanosine (4)>inosine (10)>7-methylguanosine (6)=3'-deoxyguanosine (9)>2'-deoxyguanosine (8)=guanine (11)=6-thioguanine (12)>>N(2)-methylguanosine (5). The competition studies confirmed that [3H]-guanosine site was distinct from the well characterized ATP and adenosine binding sites. The present results are rationalized in terms of a putative pseudo-receptor construct which includes all the relevant physicochemical interaction between guanosine analogues and their putative binding sites. This construct will be useful for the in silico screening of compound libraries in search for new potent and structurally diverse pharmacological tools.

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 (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.

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 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.

Specific [(3)H]-guanosine binding sites in rat brain membranes.

1. 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. 2. Specific binding sites for [(3)H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [(3)H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122 x 10(7) M(-1) min(-1) and 1.69 min(-1), respectively. A single high affinity binding site for [(3)H]-guanosine with a K(D) of 95.4 +/- 11.9 nM and B(max) 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 [(3)H]-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. 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. 6. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.