Introduction 1 2 3 4 6 7 9 3 3 5 10 in vitro 10 11 in vivo 12 13 14 3 15 16 17 20 16 16 19 13 21 3 15 15 22 23 24 22 22 22 15 25 26 l l 2+ 27 28 29 31 in vitro 32 33 32 34 34 35 38 36 39 40 42 35 36 43 Together, these data led us to question whether guanosine enhanced NGF-dependent neurite outgrowth is through a mechanism involving cGMP, and if so, whether it was attributable to the stimulation of either NO or CO synthesis. Materials and methods Cell culture and treatments Tissue culture supplies were from Life Technologies. All other supplies were obtained from Sigma RBI unless otherwise stated. 2.5S NGF was a generous gift from Dr. M. Coughlin, Department of Medicine, McMaster University, 6-(phenylamino)-5,8-quinolinedione (LY83583) was obtained from (Calbiochem), copper protoporphyrin from (Porphyrin Products), and zinc protoporphyrin IX from (Research Biochemical). 15 2 Neurite outgrowth assay 15 d,l 4 N ω l l 2 44 Western immunoblot analysis 6 Determination of cyclic GMP 5 Statistical analysis Statistical analysis was carried out using a two-way ANCOVA, when applicable, or a two-way ANOVA followed by Fischer's LSD test for multiple comparisons. Results Guanosine enhances NGF-dependent neurite outgrowth via activation of soluble guanylate cyclase 23 26 45 46 47 1 P 1 48 Figure 1 P P  49 46 Inhibition of nitric oxide synthase (NOS) has no effect on guanosine-enhanced NGF-dependent neurite outgrowth 25 l l l N ω l l 50 25 51 l l 25 51 l 2a 2b 2c Figure 2 1 l 1 P l d,l  Inhibition of heme oxygenase (HO) attenuates guanosine-enhanced NGF-dependent neurite outgrowth 52 52 53 3 3 54 Figure 3 1 P P  Guanosine induces heme oxygenase-1 (HO-1) expression 55 36 4b P P 4 56 57 P 4 P 4 P P 4 P Figure 4 d,l 2b P P P P  36 5b Figure 5 d,l 2b  Guanosine increases intracellular cGMP concentrations in PC12 cells during guanosine-enhanced NGF-dependent neurite outgrowth 36 P P P P Inhibition of heme oxygenase (HO) attenuates intracellular cGMP concentrations in PC12 cells during guanosine enhanced NGF-dependent neurite outgrowth 3 4 6 58 59 3 P P 7 P P Figure 6 d,l 2b P P P P  Figure 7 d,l 2b P P P  Discussion 15 22 23 22 25 26 26 35 45 60 32 26 33 l 61 62 Rather than NO, our data indicated that CO might activate sGC in response to guanosine in undifferentiated PC12 cells. The heme oxygenase inhibitor, ZnPP attenuated both the ability of guanosine to enhance NGF-dependent neurite outgrowth, and its ability to increase intracellular cGMP, alone or in the presence of NGF. This led us to conclude that guanosine either activated constitutive HO-2 or induced the expression of either HO-1 and/or the HO-2 isoforms of this enzyme. 55 63 64 63 64 63 64 56 57 63 64 36 65 66 67 68 42 69 36 65 23 70 71 i 5 70 5 72 73 i 74 76 22 24 63 64 Guanosine plus NGF in combination induce HO-1 expression to a similar extent than NGF alone at 6, 12 and 24 h. At 48 h, however the effect of guanosine plus NGF on HO-1 expression is comparable to that of control and lower than NGF alone. Since the mechanisms that induce HO-1 expression are complex, the roles played by guanosine and NGF, and the time course of these effects are unclear. We have shown, however that guanosine alone is insufficient to cause major neurite outgrowth. It does however enhance the NGF-induced neuritogenesis. Neuritogenesis is not solely due to HO-1 expression. Either HO-1 or HO-2 could be responsible for these early events since ZnPP, a non-selective HO inhibitor reduces the concentration of cGMP elicited by guanosine or guanosine plus NGF by 12 h. 18 20 18 20 2+ 36 36 77 78 57 79 80 Since HO-1 expression in the guanosine treated cells was not observed until 24 h, and since neither iNOS nor nNOS was detectable at these times, our data is most parsimoniously explained by activation of HO-2. Although we have not determined HO-2 activity, we have shown, using Western immunoblot analysis that this isozyme is expressed in PC12 cells constitutively, throughout the time course of the experiment (from 0 to 48 h) during all treatment conditions. 35 38 81 82 81 82 83 84 85 37 38 86 i 86 i