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′.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′.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

Inhibition of heme oxygenase attenuates guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells

<p><b>Copyright information:</b></p><p>Taken from "Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP"</p><p></p><p>Purinergic Signalling 2005;1(2):161-172.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096532.</p><p></p> PC12 cells were cultured with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) as described in Figure . Some cultures were treated with the selective inhibitor of heme oxygenase zinc protoporphyrin-IX (0.01Y1 µM) for 48 h and the number of cells bearing one or more neurites was determined as described in Figure 1. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Cultures treated with guanosine plus NGF had a significantly ( < 0.01 two-way independent ANOVA) greater proportion of neurite-bearing cells than cultures treated with NGF alone. Zinc protoporphyrin-IX significantly decreased (** < 0.01) the neurite growth from cultures treated with guanosine plus NGF, but had no significant effect on neurite outgrowth in cultures treated with NGF alone. Data represent the mean ± SEM of 12 determinations from two replicate experiments

Methylene blue attenuates guanosine-enhanced nerve growth factor-dependent neurite outgrowth in PC12 cells

<p><b>Copyright information:</b></p><p>Taken from "Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP"</p><p></p><p>Purinergic Signalling 2005;1(2):161-172.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096532.</p><p></p> PC12 cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum, 5% heat-inactivated horse serum and 1% antibiotic-antimycotic for 48 h. Cultures were then treated with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) and with increasing concentrations of methylene blue (0–1 µM). After 48 h, the total cell number and number of cells bearing one or more neurites were determined by counting two random areas in each well. The mean proportion of neurite-bearing cells in cultures treated with NGF was approximately 25%–35%. Because this value varied slightly between experiments, all experimental values are expressed relative to the NGF treated cultures, which was defined as 100%. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Cultures treated with guanosine plus NGF had a significantly ( < 0.01, two-way independent ANOVA) greater proportion of neurite-bearing cells than those treated with NGF alone. Methylene blue (0.1 to 1.0 µM) had no effect on the proportion of neurite-bearing cells in cultures treated with NGF alone, but at concentrations from 0.1 to 1.0 µM, it significantly (** < 0.01, two-way independent ANOVA) reduced the proportion of neurite-bearing cells in cultures treated with guanosine plus NGF. Data represent the mean ± SEM of 12 determinations from two replicate experiments

Guanosine increases cGMP concentrations during guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells

<p><b>Copyright information:</b></p><p>Taken from "Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP"</p><p></p><p>Purinergic Signalling 2005;1(2):161-172.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096532.</p><p></p> PC12 cells were cultured on plates coated with poly--ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C) as described in Figure . Cells were lysed at time points 0, 6, 12, 24, and 48 h and cGMP concentrations were determined by a competitive enzyme immunoassay. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test (○ < 0.05, compared to time point 0); (* < 0.05, relative to control); (** < 0.01, relative to control); (Δ < 0.05, relative to NGF). Data presented represent the mean relative optical density T SEM obtained in six independent experiments

Guanosine has no effect on heme-oxygenase-2 protein expression in PC12 cells

<p><b>Copyright information:</b></p><p>Taken from "Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP"</p><p></p><p>Purinergic Signalling 2005;1(2):161-172.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096532.</p><p></p> PC12 cells were cultured on plates coated with poly--ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C) as described in Figure . The expression of heme oxygenase-2 was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Open bars: untreated controls (C), closed bars: guanosine plus NGF (G + N) treatment, stippled bars: NGF (N) treatment, hatched bars: guanosine (G) treatment. Statistical analysis was performed using a two-way ANOVA. (a) Data represent the mean optical density T SEM obtained in three independent experiments. (b) Results are representative Western immunoblots obtained in these experiments

(a) Inhibition of nitric oxide synthase has no effect on the proportion of neurite-bearing PC12 cells cultured for 48 h in the presence of NGF, or NGF plus guanosine

<p><b>Copyright information:</b></p><p>Taken from "Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP"</p><p></p><p>Purinergic Signalling 2005;1(2):161-172.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096532.</p><p></p> PC12 cells were cultured with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) as described in Figure . Cultures were treated with the general nitric oxide synthase inhibitor, -NAME (0.1–10 mM) for 48 h and the number of cells bearing one or more neurites were determined as described in Figure . Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Neurite outgrowth in cultures treated with guanosine plus NGF was significantly ( < 0.01, two-way independent ANOVA) greater than in cultures treated with NGF alone. -NAME (0.1–10 µM) had no effect on the proportion of neurite bearing cells in cultures treated with NGF alone, or in combination with guanosine. Data represent the mean ± SEM of 12 determinations from two replicate experiments. (b) Inducible nitric oxide synthase is not expressed in PC12 cells cultured for 48 h in the presence of guanosine, or NGF, or guanosine plus NGF. PC12 cells were cultured on plates coated with poly--ornithine for 72 h. Cells were then grown in serum-reduced medium (3% heat-inactivated fetal calf serum and 3% heat-inactivated horse serum) for 12 h, and were treated with guanosine (G, 300 µM) or NGF (N, 40 ng/ml) or guanosine (300 µM) plus NGF (40 ng/ml) (G + N), or with no added treatments (C). The expression of inducible nitric oxide synthase was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Recombinant inducible nitric oxide synthase protein (50 ng) was used as a positive control (P). Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test. Data presented are representative of at least three independent experiment. (c) Neuronal nitric oxide synthase is not expressed in PC12 cells cultured for 48 h in the presence of guanosine, or NGF, or guanosine plus NGF. PC12 cells were cultured as described in panel (b), and the expression of neuronal nitric oxide synthase was determined at various time points (6, 12, 24 and 48 h) by Western immunoblot analysis. Recombinant neuronal nitric oxide synthase protein (50 ng) was used as a positive control (P). Immunoblots were quantified by densitometric analysis and were normalized to the corresponding β-actin bands as described in the Materials and methods. Statistical analysis was performed using a two-way ANCOVA followed by Fischer's LSD post-hoc comparison test. Data presented are representative of at least three independent experiment