Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Abstract

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). [Formula: see text]-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.

Figure 1

Methylene blue attenuates guanosine-enhanced nerve growth factor-dependent neurite outgrowth in PC12 cells. 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 (P < 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 (**P < 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.

Figure 2

(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. PC12 cells were cultured with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) as described in Figure 1. Cultures were treated with the general nitric oxide synthase inhibitor, l-NAME (0.1–10 mM) for 48 h and the number of cells bearing one or more neurites were determined as described in Figure 1. Open bars: NGF treatment, closed bars: NGF plus guanosine treatment. Neurite outgrowth in cultures treated with guanosine plus NGF was significantly (P < 0.01, two-way independent ANOVA) greater than in cultures treated with NGF alone. l-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-d,l-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.

Figure 3

Inhibition of heme oxygenase attenuates guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured with NGF (40 ng/ml) or NGF (40 ng/ml) plus guanosine (300 µM) as described in Figure 1. 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 (P < 0.01 two-way independent ANOVA) greater proportion of neurite-bearing cells than cultures treated with NGF alone. Zinc protoporphyrin-IX significantly decreased (**P < 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.

Figure 4

Guanosine induces the expression of heme oxygenase-1 protein in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-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 2b. The expression of heme oxygenase-1 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 ANCOVA followed by Fischer's LSD post-hoc comparison test (○ P < 0.05 compared with control time point 0); (○○ P < 0.01 compared with time point 0); (* P < 0.05 compared with control at same time point); (** P < 0.01 compared with control at same time point). (a) Data represent the mean optical density ± SEM obtained in six independent experiments. (b) Results are representative Western immunoblots obtained in these experiments.

Figure 5

Guanosine has no effect on heme-oxygenase-2 protein expression in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-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 2b. 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.

Figure 6

Guanosine increases cGMP concentrations during guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-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 2b. 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 (○ P < 0.05, compared to time point 0); (* P < 0.05, relative to control); (** P < 0.01, relative to control); (Δ P < 0.05, relative to NGF). Data presented represent the mean relative optical density T SEM obtained in six independent experiments.

Figure 7

Inhibition of heme oxygenase attenuates cGMP concentrations during guanosine enhanced NGF-dependent neurite outgrowth in PC12 cells. PC12 cells were cultured on plates coated with poly-d,l-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 2b. The selective inhibitor of heme oxygenase, zinc protoporphyrin-IX (100 nM), was added to some cultures prior to the addition of guanosine, or NGF, or guanosine plus NGF. Cells were lysed after 12 h and cGMP concentrations were determined by a competitive enzyme immunoassay. Open bars: no zinc protoporphyrin-IX added, closed bars: zinc protoporphyrin-IX added. Statistical analysis was performed using a one-way ANOVA followed by Fischer's LSD post-hoc comparison test (** P < 0.01, relative to control); (ΔΔ P < 0.01, relative to NGF); (○ P < 0.05, relative to treatment without zinc protoporphyrin-IX). Data presented represent the mean relative optical density T SEM obtained in six independent experiments.