Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP Undifferentiated rat pheochromocytoma -LRB- PC12 -RRB- cells extend neurites when cultured in the presence of nerve growth factor -LRB- NGF -RRB- . 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 -LRB- cGMP -RRB- within the first 24 h whereas addition of soluble guanylate cyclase -LRB- sGC -RRB- inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth . sGC may be activated either by nitric oxide -LRB- NO -RRB- or by carbon monoxide -LRB- CO -RRB- . - Nitro-l-arginine methyl ester -LRB- l-NAME -RRB- , a non-isozyme selective inhibitor of nitric oxide synthase -LRB- NOS -RRB- , had no effect on neurite outgrowth induced by guanosine . Neither nNOS -LRB- the constitutive isoform -RRB- , nor iNOS -LRB- the inducible isoform -RRB- 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 -LRB- HO -RRB- , 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 . Introduction The roles of extracellular purine nucleosides and nucleotides as neurotransmitters and modulators are well documented -LSB- 1 -RSB- . Extracellular purines also exert trophic effects on cells ; influencing growth , division -LSB- 2 , 3 -RSB- differentiation , and even apoptosis -LSB- 4 -- 6 -RSB- . Purines are released from cells under physiological conditions , acting as neurotransmitters and neuromodulators -LSB- 7 -- 9 -RSB- . Trauma and other insults to the central nervous system are also potent stimuli that cause the release of purines -LSB- 3 -RSB- , most of which are converted by ectoenzymes to adenosine and guanosine -LSB- 3 , 5 -RSB- . After insults to cells , more guanine-based than adenine-based purines are released -LSB- 10 -RSB- . Furthermore , the extracellular concentration of guanosine remains elevated for prolonged periods in vitro -LSB- 10 -RSB- and for up to a week after central nervous system -LRB- CNS -RRB- injury -LSB- 11 -RSB- , implying that extracellular guanosine may exert trophic effects in vivo . Sprouting of neurites , which later become axons and dendrites , is an important change associated with neural development and differentiation -LSB- 12 , 13 -RSB- . Neurite sprouting through regeneration or collateral sprouting also plays an important role in the functional recovery following injury to the central or peripheral nervous systems -LSB- 14 -RSB- . Amongst its trophic effects , guanosine stimulates neurite outgrowth and enhances NGF-dependent neurite outgrowth -LSB- 3 , 15 -RSB- . PC12 cells serve as a useful model for studying cell signaling -LSB- 16 -RSB- . They respond to many growth factors , neurotrophins and hormones , which initiate multiple signaling pathways -LSB- 17 -- 20 -RSB- . The specific cellular targets of these signaling pathways mediate the distinct responses of differentiation , proliferation and survival , all of which can be assessed -LSB- 16 -RSB- . The addition of NGF to PC12 cells causes a sustained activation of ERK , a mitogen-activated protein kinase , through activation of the TrkA receptor -LSB- 16 , 19 -RSB- . This leads to the development of many phenotypic characteristics in PC12 cells , which are also associated with mature sympathetic neurons -LSB- 13 , 21 -RSB- . Extracellular guanosine not only stimulates neurite outgrowth from primary cultures of rat hippocampal neurons -LSB- 3 -RSB- , and PC12 cells -LSB- 15 -RSB- but also enhances the neuritogenic effects of NGF on PC12 cells -LSB- 15 , 22 , 23 -RSB- . Both in astrocytes -LSB- 24 -RSB- and in PC12 cells -LSB- 22 -RSB- , guanosine increases intracellular adenosine 3 ′ ,5 ′ - cyclic monophosphate -LRB- cAMP -RRB- . Some of the neuritogenic effects of guanosine in PC12 cells appeared to be mediated through this mechanism -LSB- 22 -RSB- . However , guanosine also has cAMP-independent effects , synergistic with NGF , because its effects are not abolished by an adenylate cyclase inhibitor -LSB- 22 -RSB- and are synergistic with substances that increase intracellular cAMP -LSB- 15 -RSB- . The cAMP-independent component of the signal transduction mechanism through which guanosine synergistically enhances NGF-dependent neurite outgrowth is unknown . However , several lines of evidence indicate a role for guanosine 3 ′ ,5 ′ - cyclic monophosphate -LRB- cGMP -RRB- . First , extracellular guanosine increases intracellular cGMP in rat mesenteric artery -LSB- 25 -RSB- . Second , nitric oxide -LRB- NO -RRB- donors enhance NGF-dependent PC12 cell neurite outgrowth via a cGMP-dependent mechanism ; and thirdly , the neuritogenic effects of the NO donors are abolished by guanylyl cyclase inhibitors and mimicked by cGMP analogs -LSB- 26 -RSB- . The mechanism by which cGMP enhances neurite outgrowth is currently unclear . Nitric oxide synthases are a family of enzymes , which synthesize NO through the catalytic conversion of l-arginine to l-citrulline . The constitutively expressed forms are the endothelial -LRB- eNOS -RRB- or neuronal -LRB- nNOS -RRB- , which are regulated by the cytosolic concentration of Ca2 + -LSB- 27 -RSB- . The inducible isoform -LRB- iNOS -RRB- is widely distributed , and becomes active only hours after an inducing event -LSB- 28 -RSB- . NO is involved in cell communication and signal transduction in many systems including the CNS -LSB- 29 -- 31 -RSB- . NGF induced different isoforms of NOS in PC12 cells after 4 days in vitro -LSB- 32 , 33 -RSB- . Peunova and Enikolopov -LSB- 32 -RSB- reported that the NO produced preceding the development of the differentiated phenotype is due predominantly to iNOS . The diffusible gas carbon monoxide -LRB- CO -RRB- is a putative neurotransmitter -LSB- 34 -RSB- . Heme oxygenase -LRB- HO -RRB- synthesizes CO from the biologically active substrate biliverdin , which is rapidly reduced to bilirubin , and iron from intracellular heme -LSB- 34 -RSB- . CO can modulate activities attributed to cGMP in the nervous system -LSB- 35 -- 38 -RSB- . It has been proposed that CO production is responsible for baseline cGMP levels in the hippocampus -LSB- 36 -RSB- . In cerebellar granule cell cultures , CO produced by HO affects intracellular cGMP concentrations by modulating the NO-soluble guanylate cyclase signaling system -LSB- 39 -RSB- . Conversely , NO , synthesized by NOS may induce the expression of HO-1 -LSB- 40 -- 42 -RSB- , indicating a close and reciprocal interaction between the NOS-NO and the HO-CO signaling systems -LSB- 35 , 36 -RSB- . Based on these findings it has been proposed that a possible role for HO-1 is to counteract NO toxicity -LSB- 43 -RSB- . 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 . 2.5 S NGF was a generous gift from Dr. M. Coughlin , Department of Medicine , McMaster University , 6 - -LRB- phenylamino -RRB- -5,8-quinolinedione -LRB- LY83583 -RRB- was obtained from -LRB- Calbiochem -RRB- , copper protoporphyrin from -LRB- Porphyrin Products -RRB- , and zinc protoporphyrin IX from -LRB- Research Biochemical -RRB- . PC12 cells were maintained in either RPMI 1640 medium supplemented with 5 % heat-inactivated -LRB- HI -RRB- fetal calf serum -LRB- FCS -RRB- , 5 % HI-horse serum -LRB- HS -RRB- and 1 % antibiotic-antimycotic -LRB- Anti-Anti ; 10,000 units of penicillin , 10,000 µg of streptomycin , 25 µg amphotericin B/ml in 0.85 % saline -RRB- -LSB- 15 -RSB- or F-12K -LRB- Kaighn 's Modification -RRB- medium supplemented with 15 % HI-HS , 2.5 % HI-FCS , and 1 % Anti-Anti at 37 °C in a 5 % CO2 environment . Neurite outgrowth assay To evaluate the effect of test compounds on neurite outgrowth in PC12 cells , they were added to cultures for 48 h as previously described -LSB- 15 -RSB- . Briefly , PC12 cells were plated onto poly-d , l-ornithine -LRB- PORN -RRB- - coated 24-well plates at a density of 2.5 × 104 cells/well . Cells were cultured in RPMI 1640 supplemented with 1.5 % HI-HS , 1.5 % HI-FCS and 1 % Anti-Anti . Guanosine was dissolved in 10 % sodium hydroxide -LRB- 1N NaOH -RRB- and when added to the culture medium the final concentration was 0.01 % sodium hydroxide . In experiments in which guanosine -LRB- 300 µM -RRB- was added to each well first , followed within 15 min by the addition of 2.5 S NGF -LRB- 40 ng/ml -RRB- . Methylene blue , LY83583 , hemoglobin , Nω-nitro-l-arginine methyl ester -LRB- l-NAME -RRB- , copper protoporphyrin-IX -LRB- CuPP -RRB- and zinc protoporphyrin-IX -LRB- ZnPP -RRB- , were added 15 min prior to the addition of guanosine . All test substances remained in the medium throughout the 48-h test period . Cells were maintained at 37 °C in a humidified atmosphere of 95 % air , 5 % CO2 . After 48 h , cells were fixed with 10 % formalin in phosphate buffered saline -LRB- PBS -RRB- , pH 7.4 for 10 min and stored at 4 °C in PBS -LRB- pH 7.4 -RRB- containing sodium azide -LRB- 0.1 % , w/v -RRB- until counted -LRB- usually within 2 weeks -RRB- . Total cell number was determined by counting two randomly selected areas in each well . The observer was blind to the treatments the cells had received . At least 50 cells per well were counted , and the number of cells bearing one or more neurites determined using a NIKON Diaphot microscope equipped with phase contrast optics . Neurites were defined as processes extending at least one cell body diameter from the cell with growth cones at their tips -LSB- 44 -RSB- . Western immunoblot analysis The expression of HO-1 , HO-2 , iNOS , nNOS , and β-actin in PC12 cells was determined by Western immunoblot analysis . PC12 cells were seeded onto PORN-coated plates at a density of 1 × 106 cells/plate and maintained at 37 ° for 48 h prior to treatments . Cells were placed in a medium containing 3 % HI-FCS and 3 % HI-HS 12 h before the start of treatments . Cells were then treated with various agents for 0 , 6 , 12 , 24 , or 48 h . At the end of the treatment period cells were washed once with PBS and then harvested at 4 °C using a lysis buffer -LRB- 25 µM Tris/HCl pH 7.4 , 10 µM NaCl , 10 µM EDTA , 100 µl/10 ml Tween 20 , 10 µM sodium pyrophosphate decahydrate , 10 mM sodium orthovanadate , 5 µg/ml leupeptin , 10 mM glycerophosphate -RRB- . Cells were disrupted by sonication and aliquots -LRB- 25 µl -RRB- were removed for the determination of protein concentrations using the bicinchoninic acid -LRB- BCA -RRB- protein assay -LRB- Pierce , Illinois , USA -RRB- . Cells lysates were separated on 12 % SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes -LRB- PALL , Michigan , USA -RRB- . Membranes were incubated with a specific primary antibody overnight at 4 °C then were exposed to a secondary antibody for 1 h at room temperature . Positive control peptides for nNOS and iNOS -LRB- obtained from Cayman Chemical , Ann Arbor . MI -RRB- were used as electrophoresis standard . The following antibodies were used : monoclonal antibody to brain NOS -LRB- Sigma , dilution : 1:1,000 -RRB- , monoclonal antibody to the inducible NOS -LRB- Sigma , dilution : 1:1,000 -RRB- , anti-iNOS/bNOS , goat anti-mouse IgG-HRP -LRB- StressGen Biotechnologies , dilution 1:100,000 -RRB- , monoclonal antibody to β-actin -LRB- abCAM , dilution : 1:20,000 -RRB- , monoclonal anti-β-actin , goat anti-mouse IgG-HRP -LRB- Novus Biological , dilution 1:100,000 -RRB- , monoclonal antibody to HO-1 -LRB- StressGen Biotechnologies , dilution 1:2,000 -RRB- , anti-HO-1 , goat anti-mouse IgG-HRP -LRB- StressGen Biotechnologies , dilution 1:200,000 -RRB- , rabbit polyclonal antibody to HO-2 / HO-1 -LRB- StressGen Biotechnologies , dilution 1:1,000 -RRB- , anti HO-2 / HO-1 , goat anti-rabbit IgG-HRP -LRB- StressGen Biotechnologies , dilution 1:200,000 -RRB- . Immunocomplexes were then visualized using a chemiluminescence substrate -LRB- Sigma-Aldrich -RRB- . Immunoblots were quantified by densitometric analyses , using the Northern Eclipse program -LRB- EPIX -RRB- . All bands on the immunoblots were normalized to their corresponding β-actin bands prior to statistical analysis . Determination of cyclic GMP PC12 cells were seeded onto PORN-coated 12-well plates at a density of 5 × 105 cells/well and maintained at 37 °C for 48 h prior to treatments . Cells were placed in a medium containing 3 % HI-FCS and 3 % HI-HS 12 h before the start of treatments . Cells were then treated with various agents for different times -LRB- 0 , 6 , 12 , 24 , or 48 h -RRB- . At the end of the treatment period cGMP was extracted and analyzed using a cGMP enzyme immunoassay -LRB- EIA -RRB- kit -LRB- Amersham Biosciences -RRB- . The assay is based on a competition between unlabeled cGMP in the sample or -LRB- standard -RRB- and a fixed quantity of peroxidase-labeled cGMP for a limited number of binding sites on a cGMP specific antibody . With fixed amounts of antibody and cGMP-peroxidase conjugate , the amount of bound cGMP-peroxidase conjugate is inversely proportional to the concentration of unlabeled cGMP . 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 As we have reported previously , we have evaluated the effect of guanosine on NGF-dependent neurite outgrowth during the first 48 h , as guanosine most likely exerted its effects within the first 1 -- 2 h after treatment -LSB- 23 -RSB- . Therefore , all experiments reported in this manuscript were performed during the first 48 h of PC12 cell treatment with various agents . PC12 cells contain both soluble , and particulate isoforms of guanylate cyclase -LRB- GC -RRB- -LSB- 26 , 45 -RSB- . To determine whether activation of GC was necessary for the neuritogenic effect of guanosine , inhibitors of GC were added for 48 h to PC12 cells treated with NGF plus guanosine . Methylene blue at concentrations ranging from 0.1 to 1 mM was added to PC12 cell cultures that contained NGF alone -LRB- 40 ng/ml -RRB- , or guanosine -LRB- 300 mM -RRB- plus NGF -LRB- 40 ng/ml -RRB- . Cultures were then evaluated for neurite outgrowth . At the concentrations used in these experiments , methylene blue inhibits sGC but not particulate GC -LSB- 46 , 47 -RSB- . Methylene blue had no effect on the outgrowth of neurites induced by NGF alone -LRB- Figure 1 -RRB- . However , in cultures treated with both guanosine and NGF , the addition of methylene blue reduced significantly -LRB- P < 0.01 -RRB- the proportion of neurite-bearing cells in a concentration-dependent manner -LRB- Figure 1 -RRB- . LY83583 inhibits both , particulate GC and sGC -LSB- 48 -RSB- . When LY83583 -LRB- 10 nM -RRB- was added to PC12 cell cultures , it inhibited neurite outgrowth elicited by guanosine plus NGF but had no effect on neurite outgrowth in cultures treated with NGF alone -LRB- data not shown -RRB- . These data support the hypothesis that cGMP plays a role in enhancing the effect of guanosine on NGF-mediated neurite outgrowth . Moreover , since methylene blue at the concentrations used inhibits soluble but not particulate GC , these data imply that guanosine activates sGC . 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 -LRB- 40 ng/ml -RRB- or NGF -LRB- 40 ng/ml -RRB- plus guanosine -LRB- 300 µM -RRB- and with increasing concentrations of methylene blue -LRB- 0 -- 1 µM -RRB- . 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 -LRB- P < 0.01 , two-way independent ANOVA -RRB- greater proportion of neurite-bearing cells than those treated with NGF alone . Methylene blue -LRB- 0.1 to 1.0 µM -RRB- 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 -LRB- ** P < 0.01 , two-way independent ANOVA -RRB- 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 may activate sGC either directly , or indirectly by stimulating the formation of NO , CO or hydroxyl radicals , which are all physiological activators of sGC -LSB- 49 -RSB- . To distinguish between these possibilities we added hemoglobin -LRB- 100 nM -RRB- to cultures of PC12 cells . Extracellular hemoglobin directly scavenges NO and CO because these substances avidly bind to the heme moiety , thereby inhibiting their GC activating activity -LSB- 46 -RSB- . Hemoglobin reduced the synergistic effects of guanosine on NGF-mediated neurite outgrowth but had no effect on neurite outgrowth elicited by NGF alone -LRB- data not shown -RRB- . Although these data imply that guanosine stimulates the synthesis of a sGC-activating factor , they do not show whether this factor is NO , CO or hydroxyl radical . Inhibition of nitric oxide synthase -LRB- NOS -RRB- has no effect on guanosine-enhanced NGF-dependent neurite outgrowth In the rat mesenteric artery , guanosine stimulates NO formation -LSB- 25 -RSB- . Therefore , we investigated if guanosine could also stimulate NO synthesis in PC12 cells . To examine whether NO was involved in the signal transduction pathway that mediated the enhancement of neurite outgrowth by guanosine through sGC , we inhibited the enzyme NOS , which catalyzes the conversion of l-arginine to l-citrulline plus NO . The l-arginine analog , Nω-nitro-l-arginine methyl ester hydrochloride -LRB- l-NAME -RRB- -LSB- 50 -RSB- inhibits NOS competitively -LSB- 25 , 51 -RSB- . In this experiment , we reduced the concentration of l-arginine in the culture medium from 1 mM to 80 µM , thus permitting more effective competition of the NOS inhibitor with l-arginine . Under these conditions , L-NAME inhibits NOS at 10 µM -LSB- 25 , 51 -RSB- . When added to the culture medium of PC12 cells at 10 µM l-NAME did not inhibit NGF-dependent neurite outgrowth , and had no effect on the guanosine-mediated enhancement of NGF-dependent neurite outgrowth -LRB- Figure 2a -RRB- . To confirm that NO was not involved in the signal transduction pathway that mediates the enhancement of neurite outgrowth by guanosine , we determined the expression of two subtypes of NOS isoenzymes : iNOS , the inducible isoform -LRB- Figure 2b -RRB- , and nNOS , the constitutive isoform -LRB- Figure 2c -RRB- . PC12 cultures were exposed to guanosine alone -LRB- 300 µM -RRB- , or NGF alone -LRB- 40 ng/ml -RRB- , or to the combination of guanosine plus NGF for 48 h , and NOS expression was determined by Western immunoblot analysis . We analyzed various protein concentrations -LRB- 5 -- 50 µg/ml -RRB- and determined β-actin expression on the same Western immunoblots . Using protein concentrations as high as 50 µg/ml neither nNOS nor iNOS expression could be detected in PC12 cells treated with guanosine , or NGF , or the combination of the two agents . These data indicate that since NOS is not expressed during the first 48 h of treatment , thus NO can not mediate the effect of guanosine on neurite outgrowth . Figure 2 -LRB- a -RRB- 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 -LRB- 40 ng/ml -RRB- or NGF -LRB- 40 ng/ml -RRB- plus guanosine -LRB- 300 µM -RRB- as described in Figure 1 . Cultures were treated with the general nitric oxide synthase inhibitor , l-NAME -LRB- 0.1 -- 10 mM -RRB- 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 -LRB- P < 0.01 , two-way independent ANOVA -RRB- greater than in cultures treated with NGF alone . l-NAME -LRB- 0.1 -- 10 µM -RRB- 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 . -LRB- b -RRB- 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 -LRB- 3 % heat-inactivated fetal calf serum and 3 % heat-inactivated horse serum -RRB- for 12 h , and were treated with guanosine -LRB- G , 300 µM -RRB- or NGF -LRB- N , 40 ng/ml -RRB- or guanosine -LRB- 300 µM -RRB- plus NGF -LRB- 40 ng/ml -RRB- -LRB- G + N -RRB- , or with no added treatments -LRB- C -RRB- . The expression of inducible nitric oxide synthase was determined at various time points -LRB- 6 , 12 , 24 and 48 h -RRB- by Western immunoblot analysis . Recombinant inducible nitric oxide synthase protein -LRB- 50 ng -RRB- was used as a positive control -LRB- P -RRB- . 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 . -LRB- c -RRB- 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 -LRB- b -RRB- , and the expression of neuronal nitric oxide synthase was determined at various time points -LRB- 6 , 12 , 24 and 48 h -RRB- by Western immunoblot analysis . Recombinant neuronal nitric oxide synthase protein -LRB- 50 ng -RRB- was used as a positive control -LRB- P -RRB- . 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 . Inhibition of heme oxygenase -LRB- HO -RRB- attenuates guanosine-enhanced NGF-dependent neurite outgrowth Since NO apparently did not mediate the effects of guanosine , we examined the possibility that guanosine stimulated CO synthesis . Heme oxygenase -LRB- HO -RRB- , the enzyme which synthesizes CO , has been detected in rat adrenal glands -LSB- 52 -RSB- . Zinc protoporphyrin-IX -LRB- ZnPP -RRB- inhibits both the constitutive HO -LRB- HO-2 -RRB- and the inducible HO -LRB- HO-1 -RRB- isoforms of this enzyme -LSB- 52 , 53 -RSB- . We , therefore , tested the effect of increasing concentrations of ZnPP on the ability of NGF or , NGF plus guanosine to induce neurite outgrowth . Addition of ZnPP -LRB- 0.01 , 0.1 , 1 µM -RRB- to PC12 cell cultures did not significantly reduce outgrowth of neurites induced by NGF -LRB- Figure 3 -RRB- , when compared to control cultures . In contrast , ZnPP attenuated the ability of guanosine to enhance NGF-stimulated neurite outgrowth -LRB- Figure 3 -RRB- . As a control for non-specific effects of metalloporphyrins we used copper protoporphyrin-IX -LRB- CuPP -RRB- , which does not inhibit heme oxygenase -LSB- 54 -RSB- . The addition of CuPP to PC12 cells did not inhibit either NGF-dependent neurite outgrowth or the ability of guanosine to enhance NGF-dependent neurite outgrowth -LRB- data not shown -RRB- . Figure 3 Inhibition of heme oxygenase attenuates guanosine-enhanced NGF-dependent neurite outgrowth in PC12 cells . PC12 cells were cultured with NGF -LRB- 40 ng/ml -RRB- or NGF -LRB- 40 ng/ml -RRB- plus guanosine -LRB- 300 µM -RRB- as described in Figure 1 . Some cultures were treated with the selective inhibitor of heme oxygenase zinc protoporphyrin-IX -LRB- 0.01 Y1 µM -RRB- 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 -LRB- P < 0.01 two-way independent ANOVA -RRB- greater proportion of neurite-bearing cells than cultures treated with NGF alone . Zinc protoporphyrin-IX significantly decreased -LRB- ** P < 0.01 -RRB- 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 . Guanosine induces heme oxygenase-1 -LRB- HO-1 -RRB- expression PC12 cells have been previously shown to express both HO-1 and HO-2 proteins under basal conditions -LSB- 55 -RSB- . Since the addition of ZnPP to PC12 cultures decreased NGF-dependent neurite outgrowth , we attempted to establish whether guanosine treatment had any effect on the expression of the HO-1 protein during the first 48 h of this process . We treated PC12 cultures with guanosine alone -LRB- 300 µM -RRB- , or with NGF alone -LRB- 40 ng/ml -RRB- , or with a combination of guanosine plus NGF for 48 h , and determined HO-1 expression by Western immunoblot analysis . We used the monoclonal antibody directed against rat HO-1 , which recognizes a single protein band , at 32 kDa , the reported molecular weight for this enzyme -LSB- 36 -RSB- -LRB- Figure 4b -RRB- . Western immunoblots were quantified and normalized against their corresponding β-actin band . Statistical analysis revealed no differences in β-actin expression for any of the treatments at the different time points -LRB- data not shown -RRB- ; therefore , this was used as a control for normalizing HO-1 protein expression . There was no significant change in HO-1 expression in untreated PC12 cells during the 48-h period . In cells exposed to guanosine alone HO-1 expression , unexpectedly , was significantly reduced at 6 h compared to untreated cells at 0 time -LRB- P < 0.05 -RRB- , and at 6 h -LRB- P < 0.01 -RRB- -LRB- Figure 4 -RRB- . A possible explanation for this result may stem from the observation that HO-1 gene expression is suppressed by elevated intracellular calcium concentrations -LSB- 56 -RSB- and guanosine has been shown to increase intracellular calcium concentrations in astrocytes -LSB- 57 -RSB- . Since increases in calcium are rapid and transient , this may account for the reduced HO-1 expression at 6 h . The addition of NGF , however , is sufficient to override this effect and so at later time points HO-1 expression is up-regulated by the combination of NGF and guanosine . The addition of guanosine plus NGF significantly increased HO-1 expression at 12 h -LRB- P < 0.05 -RRB- , compared to that of control cells at 0 time -LRB- Figure 4 -RRB- . After 24 h all three treatments -LRB- guanosine alone , or NGF alone or guanosine plus NGF -RRB- led to a significant increase in HO-1 expression compared to control cells at 0 time -LRB- P < 0.01 -RRB- -LRB- Figure 4 -RRB- . In cells exposed to guanosine alone the enhanced HO-1 expression at 24 h was also significantly different from that detected in untreated cells at 24 h -LRB- P < 0.05 -RRB- . Whereas in cells treated with NGF alone , or with the combination of NGF plus guanosine this difference was significant at P < 0.01 -LRB- Figure 4 -RRB- . After 48 h , HO-1 expression was elevated significantly only in cells exposed to NGF alone when compared to untreated cells at 0 time , and at 48 h -LRB- P < 0.05 -RRB- for both comparisons . In cells exposed to guanosine alone or to NGF plus guanosine HO-1 expression by 48 h declined and was similar to untreated control values at 0 time and at 48 h. 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 -LRB- 3 % heat-inactivated fetal calf serum and 3 % heat-inactivated horse serum -RRB- for 12 h , and were treated with guanosine -LRB- G , 300 µM -RRB- or NGF -LRB- N , 40 ng/ml -RRB- or guanosine -LRB- 300 µM -RRB- plus NGF -LRB- 40 ng/ml -RRB- -LRB- G + N -RRB- , or with no added treatments -LRB- C -RRB- as described in Figure 2b . The expression of heme oxygenase-1 was determined at various time points -LRB- 6 , 12 , 24 and 48 h -RRB- 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 -LRB- C -RRB- , closed bars : guanosine plus NGF -LRB- G + N -RRB- treatment , stippled bars : NGF -LRB- N -RRB- treatment , hatched bars : guanosine -LRB- G -RRB- treatment . Statistical analysis was performed using a two-way ANCOVA followed by Fischer 's LSD post-hoc comparison test -LRB- ○ P < 0.05 compared with control time point 0 -RRB- ; -LRB- ○ ○ P < 0.01 compared with time point 0 -RRB- ; -LRB- * P < 0.05 compared with control at same time point -RRB- ; -LRB- ** P < 0.01 compared with control at same time point -RRB- . -LRB- a -RRB- Data represent the mean optical density ± SEM obtained in six independent experiments . -LRB- b -RRB- Results are representative Western immunoblots obtained in these experiments . In parallel experiments , we determined whether guanosine had an effect on the expression of the constitutive isoform , HO-2 in these cells . We treated PC12 cultures with guanosine alone -LRB- 300 µM -RRB- , or NGF alone -LRB- 40 ng/ml -RRB- , or in combination guanosine plus NGF for 48 h and determined HO-2 expression by Western immunoblot analysis . Using the monoclonal antibody directed against rat HO-2 we detected a single protein band , of molecular weight 36 kDa , as described for HO-1 -LSB- 36 -RSB- -LRB- Figure 5b -RRB- . Western immunoblots were quantified and normalized against their corresponding β-actin band . Again , statistical analysis revealed no differences in β-actin expression for any of the treatments at the different time points -LRB- data not shown -RRB- , therefore , this was used as a control for normalizing HO-2 protein bands . HO-2 expression was detectable at 0 time in untreated PC12 cells , but none of the treatments had any significant effect on its expression at any time point determined . In the guanosine-treated cells HO-2 expression was elevated slightly at 12 h , but this was not statistically significant . 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 -LRB- 3 % heat-inactivated fetal calf serum and 3 % heat-inactivated horse serum -RRB- for 12 h , and were treated with guanosine -LRB- G , 300 µM -RRB- or NGF -LRB- N , 40 ng/ml -RRB- or guanosine -LRB- 300 µM -RRB- plus NGF -LRB- 40 ng/ml -RRB- -LRB- G + N -RRB- , or with no added treatments -LRB- C -RRB- as described in Figure 2b . The expression of heme oxygenase-2 was determined at various time points -LRB- 6 , 12 , 24 and 48 h -RRB- 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 -LRB- C -RRB- , closed bars : guanosine plus NGF -LRB- G + N -RRB- treatment , stippled bars : NGF -LRB- N -RRB- treatment , hatched bars : guanosine -LRB- G -RRB- treatment . Statistical analysis was performed using a two-way ANOVA . -LRB- a -RRB- Data represent the mean optical density T SEM obtained in three independent experiments . -LRB- b -RRB- Results are representative Western immunoblots obtained in these experiments . Guanosine increases intracellular cGMP concentrations in PC12 cells during guanosine-enhanced NGF-dependent neurite outgrowth CO is a known activator of sGC -LSB- 36 -RSB- . Since we have shown that HO-1 expression is elevated in PC12 cells exposed to guanosine , we next investigated whether this increased HO-1 expression is accompanied by elevation in intracellular cGMP concentrations . PC12 cell cultures were treated with guanosine alone -LRB- 300 µM -RRB- , or NGF alone -LRB- 40 ng/ml -RRB- , or in combination guanosine plus NGF for 48 h. Intracellular cGMP concentrations were determined after 6 , 12 , 24 and 48 h of treatment . In cells exposed to guanosine alone , cGMP concentrations increased significantly after 6 h compared to untreated cells at this time point -LRB- P < 0.05 -RRB- . In PC12 cells treated with guanosine plus NGF , cGMP concentrations increased significantly after 6 h compared to untreated cells at 0 time -LRB- P < 0.05 -RRB- , remained significantly elevated at 12 h -LRB- P < 0.05 -RRB- and at 24 h -LRB- P < 0.05 -RRB- , and declined to values comparable to control by 48 h. Inhibition of heme oxygenase -LRB- HO -RRB- attenuates intracellular cGMP concentrations in PC12 cells during guanosine enhanced NGF-dependent neurite outgrowth Previous experiments have demonstrated that the HO inhibitor , ZnPP , blocked guanosine enhanced NGF-dependent neurite outgrowth of PC12 cells -LRB- Figure 3 -RRB- . We have also shown that HO-1 expression and cGMP concentrations were elevated during the first 12 -- 24 h of this neurite outgrowth process -LRB- Figures 4 and 6 , respectively -RRB- . We therefore examined whether pre-treatment of cells with the HO inhibitor ZnPP had any effect on cGMP concentrations . Although metalloporphrins , such as ZnPP-IX have been used widely to demonstrate the role for this enzyme in numerous physiological situations , their selectivity for HO has been questioned ; as they may also inhibit haemoproteins such sGC and NOS -LSB- 58 -RSB- . However , metalloporphrins used at a concentration below 10 µM are more selective for HO -LSB- 59 -RSB- . Since we found that ZnPP , at 100 nM concentration significantly attenuated guanosine-enhanced neurite outgrowth -LRB- Figure 3 -RRB- , we used this concentration of ZnPP to test its effect on intracellular cGMP concentration . PC12 cell cultures were exposed to guanosine alone -LRB- 300 µM -RRB- , or NGF alone -LRB- 40 ng/ml -RRB- , or in combination guanosine plus NGF for 12 h. ZnPP -LRB- 100 nM -RRB- was added to some cultures prior to the addition of guanosine , or NGF , or NGF plus guanosine , and intracellular cGMP concentrations were determined . In cells exposed to guanosine alone , or to guanosine plus NGF , cGMP concentrations were significantly elevated compared to untreated cells -LRB- P < 0.01 -RRB- and to cells treated with NGF alone -LRB- P < 0.01 -RRB- -LRB- Figure 7 -RRB- . In cultures pretreated with ZnPP , cGMP concentrations were significantly reduced in cells exposed to guanosine alone -LRB- P < 0.05 -RRB- , or guanosine plus NGF -LRB- P < 0.05 -RRB- compared to the corresponding treatments without the inhibitor . Treatment of PC12 cells with NGF did not increase intracellular cGMP concentrations , and the addition of ZnPP had no effect on cGMP concentrations . 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 -LRB- 3 % heat-inactivated fetal calf serum and 3 % heat-inactivated horse serum -RRB- for 12 h , and were treated with guanosine -LRB- G , 300 µM -RRB- or NGF -LRB- N , 40 ng/ml -RRB- or guanosine -LRB- 300 µM -RRB- plus NGF -LRB- 40 ng/ml -RRB- -LRB- G + N -RRB- , or with no added treatments -LRB- C -RRB- 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 -LRB- C -RRB- , closed bars : guanosine plus NGF -LRB- G + N -RRB- treatment , stippled bars : NGF -LRB- N -RRB- treatment , hatched bars : guanosine -LRB- G -RRB- treatment . Statistical analysis was performed using a two-way ANCOVA followed by Fischer 's LSD post-hoc comparison test -LRB- ○ P < 0.05 , compared to time point 0 -RRB- ; -LRB- * P < 0.05 , relative to control -RRB- ; -LRB- ** P < 0.01 , relative to control -RRB- ; -LRB- Δ P < 0.05 , relative to NGF -RRB- . 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 -LRB- 3 % heat-inactivated -RRB- fetal calf serum and 3 % heat-inactivated horse serum -RRB- for 12 h , and were treated with guanosine -LRB- G , 300 µM -RRB- or NGF -LRB- N , 40 ng/ml -RRB- or guanosine -LRB- 300 µM -RRB- plus NGF -LRB- 40 ng/ml -RRB- -LRB- G + N -RRB- , or with no added treatments -LRB- C -RRB- as described in Figure 2b . The selective inhibitor of heme oxygenase , zinc protoporphyrin-IX -LRB- 100 nM -RRB- , 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 -LRB- ** P < 0.01 , relative to control -RRB- ; -LRB- ΔΔ P < 0.01 , relative to NGF -RRB- ; -LRB- ○ P < 0.05 , relative to treatment without zinc protoporphyrin-IX -RRB- . Data presented represent the mean relative optical density T SEM obtained in six independent experiments . Discussion Addition of guanosine to cultures of undifferentiated , NGF-naïve PC12 cells modestly increases the proportion of cells with neurites after 48 h and , in cultures treated with maximally effective concentrations of NGF , addition of guanosine produces a significant further increase in the proportion of neurite-bearing cells -LSB- 15 -RSB- . This implied that the effects of NGF and guanosine are mediated through distinct signaling systems . We reported previously that , whereas some of the neuritogenic effects of guanosine were mediated by increases in intracellular cAMP -LSB- 22 , 23 -RSB- , there was also a cAMP-independent component -LSB- 22 -RSB- . Several points led us to consider that a signaling pathway involving cGMP may be responsible for the cAMP-independent component . Vuorinen et al. -LSB- 25 -RSB- had shown that extracellular guanosine increases intracellular cGMP in rat mesenteric artery through a NO-dependent mechanism ; nitric oxide donors enhance NGF-dependent PC12 cell neurite outgrowth via a cGMP-dependent mechanism -LSB- 26 -RSB- ; and the neuritogenic effects of the NO donors are abolished by guanylyl cyclase inhibitors and mimicked by cGMP analogs -LSB- 26 -RSB- . Our data indicated that indeed cGMP is involved in the neuritogenic effects of guanosine in PC12 cells , but , surprisingly , that NO is not . Increases in intracellular cGMP are often due to activation of GC by NO -LSB- 35 , 45 -RSB- . Schulick et al. -LSB- 60 -RSB- demonstrated that activation of GC by NO donors or atrial natriuretic peptide enhanced NGF-dependent neurite outgrowth in PC12 cells . Further , in PC12 cells NGF upregulates NOS expression , and NO plays an important role in their cellular differentiation -LSB- 32 -RSB- . However , NGF-induced differentiation of undifferentiated NGF-naïve PC12 cells could not be abolished by inhibitors of sGC , and NO alone is not sufficient to induce PC12 cell differentiation -LSB- 26 , 33 -RSB- . In the experiments reported here , pre-treating undifferentiated , NGF-naïve PC12 cells with the NOS inhibitor l-NAME , had no effect on the guanosine-enhanced NGF-induced neurite outgrowth . Moreover , like others -LSB- 61 , 62 -RSB- , we found that neither nNOS nor iNOS isoforms were expressed during the first 48 h of cellular differentiation of PC12 cells . Therefore , NO can not mediate the neuritogenic effects of guanosine on PC 12 cells during the first 48 h. 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 . Our data indicated that , as others have reported -LSB- 55 -RSB- , both HO-1 and HO-2 were expressed constitutively in PC12 cells under basal cell culture conditions . Guanosine induced a significant increase in the expression of HO-1 protein at 24 h in undifferentiated PC12 cells , but had no significant effect on HO-2 expression . Guanosine , in the presence of NGF , produced an even more rapid and pronounced enhancement of HO-1 expression , which was detectable after 12 h. Treatment of PC12 cells with NGF alone also increased the expression of HO-1 . Several authors reported recently that NGF promotes HO-1 gene transcription in PC12 cells -LSB- 63 , 64 -RSB- via activation of the MEK pathway . HO-1 mediates the anti-oxidative and antiapoptotic effects of NGF in serum-deprived PC12 cells -LSB- 63 -RSB- . In PC12 cells exposed to oxidative stress induced by 6-hydroxy dopamine , NGF promotes HO-1 upregulation , in this case via the PI3K/Akt/PKB pathway -LSB- 64 -RSB- . Others -LSB- 63 , 64 -RSB- have reported increased HO-1 protein by 6 -- 9 h after exposure to NGF , whereas in our study we observed no change in HO-1 protein concentration until 24 h after NGF addition . This discrepancy may be due to a difference in the PC12 cell line , or to the experimental conditions to which the cells were exposed . We did not expect to find inhibition of HO-1 protein expression by guanosine at 6 h . It has been reported however , that the HO-1 gene expression is suppressed by the elevation of intracellular calcium concentration -LSB- 56 -RSB- . Since we have found that guanosine increases intracellular calcium concentrations in astrocytes -LSB- 57 -RSB- , similar mechanisms may downregulate HO-1 expression in PC12 cells at the 6-h time point . As the guanosine-stimulated calcium increases are rapid and transient , at later time points -LRB- 12 -- 24 h -RRB- other intracellular pathways , such as the MAPK and PI-3 kinase pathways are activated , and these are known to promote HO-1 gene expression . Most likely , by 48 h these pathways are no longer activated . In contrast to HO-1 , neither guanosine nor NGF , alone or in combination with guanosine , affected HO-2 expression . This confirms the results of others who have found that HO-2 is not regulated by NGF -LSB- 63 , 64 -RSB- . The HO-1 isozyme is induced by diverse intracellular signals -LRB- reviews : Maines -LSB- 36 -RSB- , Immenshchuh and Ramadori -LSB- 65 -RSB- -RRB- , that include some of the pathways which activate protein kinases , such as PKA -LSB- 66 -RSB- , or PKC -LSB- 67 , 68 -RSB- , or the second messenger cGMP -LSB- 42 -RSB- most likely via PKG -LSB- 69 -RSB- . These pathways likely converge on nuclear effectors such as the nuclear factor-κB -LRB- NF-κB -RRB- , activator protein-1 -LRB- AP-1 -RRB- and heat shock factor , to enhance HO-1 gene transcription by interacting with the regulatory elements in the promoter region the gene -LSB- 36 , 65 -RSB- . We reported previously that inhibition of transport of guanosine into PC12 cells did not affect its neuritogenic activity -LSB- 23 -RSB- , raising the possibility that its effects are mediated by activation of a cell surface , membrane-bound receptor . Indeed , recently we have identified and pharmacologically characterized a specific binding site for guanosine in rat brain membranes -LSB- 70 -RSB- , in cultured astrocytes -LSB- 71 -RSB- and in PC12 cells -LRB- F. Caciagli et al. , unpublished observations -RRB- , with characteristics of a Gi-protein coupled receptor -LSB- 5 , 70 -RSB- . Occupation of this putative receptor by guanosine activates the MAPK pathway -LSB- 5 -RSB- and the PI3K/Akt/PKB pathway -LSB- 72 , 73 -RSB- . These effects are most likely mediated by the β,γ-subunit of the Gi-protein , coupled to the guanosine receptor -LSB- 74 -- 76 -RSB- . Guanosine has also been shown to increase intracellular cAMP concentrations in several cell types -LSB- 22 -- 24 -RSB- and phosphorylate the cAMP binding protein , CREB -LRB- F. Caciagli et al. , unpublished observations -RRB- . In PC12 cells NGF-promoted induction of HO-1 expression requires the activation of the MEK/ERK pathway -LSB- 63 -RSB- and the PI3K/Akt/PKB pathway -LSB- 64 -RSB- . Since guanosine , via its putative receptor may also stimulate these intracellular pathways , HO-1 expression in PC12 cells may be regulated by similar mechanisms . 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. NGF activates a number of signalling pathways in PC12 cells , including the MAP kinase and PI3-kinase pathway -LSB- 18 -RSB- and the c-JUN N-terminal kinases -LRB- JNKs . -RRB- -LSB- 20 -RSB- . Induction of the HO-1 gene by these pathways seems to override the inhibitory effect of guanosine on HO-1 expression . HO-1 expression at 12 and 24 h is upregulated to a similar extent by guanosine alone , or by NGF alone or by guanosine plus NGF , so there is no additive effect of the combination treatment . As the mechanisms involved in HO-1 gene induction are complex and not clearly defined , our results suggest that the activation of these processes by the three treatments is comparable . At 48 h HO-1 expression in cells treated with guanosine alone , or guanosine plus NGF is similar to control . In the NGF-treated cells , however HO-1 expression is still significantly upregulated compared to control . We attribute this to a difference in the time course of HO-1 gene activation by guanosine , and NGF . Since NGF-activated intracellular signalling pathways are complex and manifold -LSB- 18 , 20 -RSB- these effects may persist for a longer time . These results are different from those we obtained at 6 h . At this time point , the only significant effect on HO-1 expression is its inhibition by guanosine alone , and this may be due to downregulation of HO-1 expression by guanoisne-stimulated elevations in intracellular Ca2 + concentrations as described above . In several types of cells , HO-1 expression is regulated by the NO-cGMP pathway -LRB- reviewed by Maines -LSB- 36 -RSB- -RRB- . In the case of guanosine this mechanism is not likely to play a significant role , as neither iNOS , nor nNOS was detected in NGF-treated PC12 cells during the first 48 h of guanosine-promoted neurite outgrowth . In contrast , upregulation of HO-1 expression by activation of the constitutive HO-2 via the transcription factor NF-κB , is a very plausible alternative mechanism -LSB- 36 -RSB- . HO-2 may be activated by PKC , which in turn is stimulated by increased intracellular calcium concentrations -LSB- 77 -RSB- . PKC may also activate sGC directly , by phosphorylation , thus providing a receptor-mediated elevation of cGMP concentrations in PC12 cells -LSB- 78 -RSB- . We have shown that guanosine increases intracellular calcium concentrations under certain conditions -LSB- 57 -RSB- , and this in turn may activate PKC . Thus , guanosine may activate HO-2 in a calcium-dependent manner . The time course of intracellular cGMP elevation elicited by guanosine , or by guanosine plus NGF , is consistent with this hypothesis . In PC12 cells exposed to guanosine alone , or to guanosine and NGF , cGMP concentrations were significantly elevated by 6 h and until 12 h in cells exposed to guanosine plus NGF . Although the addition of NGF to PC12 cells induces HO-1 expression at 24 and 48 h , this treatment does not increase cGMP concentrations . Initially we were also puzzled by these data , as we expected a parallel increase in the cGMP concentrations with this treatment . It has been reported by Liu et al. -LSB- 79 -RSB- that NGF treatment of PC12 cells rapidly decreases sGC mRNA and protein in a transcription - and translation-dependent manner . So our interpretation of these results is that although HO-1 protein is upregulated by NGF , there is a concurrent suppression of sGC expression , so no increase in cGMP is detected under these conditions . In addition , NGF may also inhibit the activities of some phosphodiesterase isoforms , and thus decrease intracellular cGMP concentrations -LSB- 80 -RSB- . 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 -LRB- from 0 to 48 h -RRB- during all treatment conditions . CO synthesized by HO-1 and HO-2 will bind and activate sGC , increasing intracellular cGMP -LSB- 35 -- 38 -RSB- and this in turn may activate protein kinase G -LRB- PKG -RRB- -LSB- 81 , 82 -RSB- and regulate phosphodiesterases -LRB- PDE -RRB- -LSB- 81 , 82 -RSB- . Our data are compatible with the neuritogenic effects of guanosine being mediated by cGMP , possibly from early activation of HO-2 , and later the induction of HO-1 , leading to CO generation , and activation of sGC . But how these molecular events enhance neuritogenesis is unknown . CO itself may directly activate several intracellular signaling pathways , including the MAPK pathway -LSB- 83 -RSB- and p38 MAPK -LSB- 84 -RSB- , the transcription factor NF-κB -LSB- 85 -RSB- and induce the transcription of immediate early genes -LSB- 37 , 38 -RSB- . Recently , Xiang et al. -LSB- 86 -RSB- proposed that navigation of growing axons in response to guidance cues , is mediated by Gi protein coupled receptors , via elevation of cytosolic cGMP -LSB- 86 -RSB- . This is another mechanism by which guanosine may promote differentiation of PC12 cells . In conclusion , we have demonstrated the importance of HO , CO and cGMP in mediating the neuritogenic effect of guanosine . The relationship of this signaling mechanism to the putative Gi-protein-linked receptor for guanosine is currently the subject of active investigation in our laboratories .

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O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O O O B I O O B O O O O B O B O O O O O O O O O O O O O O O O O B O B O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O

carbon monoxide
cyclic gmp
guanosine
heme oxygenase
neurite outgrowth
ngf
nitric oxide synthase
pc12 cells