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Nitric oxide (NO), a diffusible and unstable gas, has been implicated in inter‐ and intra‐cellular communication in the nervous system. NO also plays a role in neural development, plasticity and alterations of synaptic function such as long‐term potentiation and long‐term depression (Gally et al.: Proc NY Acad Sci, 87:354–355, 1990; Zhuo et al.: Science 260:1946–1950, 1993; Schuman and Madison.: Science 254:1503–1506, 1991; Bruhwyler et al.: Neurosci Biobehav Rev 17:373–384, 1993) some of which likely involve growth and remodelling of neurites. Some actions of NO are mediated directly by protein modification (e.g., nitrosylation) and others by activation of soluble guanylyl cyclase (soluble GC), which increases intracellular levels of guanosine 3′,5′‐cyclic monophosphate (cGMP). NO is synthesized by the enzyme nitric oxide synthase (NOS), which is induced by treatment of CNS neurons (Holtzman et al.: Neurobiol Disease 1:51–60, 1994) or pheochromocytoma PC12 cells (Hirsch et al.: Curr Biol 3:749–754, 1993) with NGF. NO has been proposed to mediate some of the effects of NGF on PC12 cells by inhibiting cell division (Peunova and Enikolopov: Nature 374:68–73, 1995). In addition, NO can substitute for NGF by delaying the death of trophic factor‐deprived PC12 cells through a mechanism that does not involve a cytostatic action (Farinelli et al.: J Neurosci 16:2325–2334, 1996). We investigated whether NO stimulated neurite outgrowth from hippocampal neurons and PC12 cells. Primary cultures of E17 mouse hippocampal neurons co‐cultured with neopallial astrocytes were exposed to the NO donors sodium nitrite (100 μM) or sodium nitroprusside (100 nM). After 48 hr, NO donor‐treated cultures contained a greater proportion of cells bearing neurites and neurites that were much longer than those found in control cultures. In cultures of PC12 cells, NO donors also enhanced the neuritogenic effects of NGF. The proportion of PC12 cells with neurites 48 hr after exposure to NO donors sodium nitrite (100 μM–10 mM) or sodium nitroprusside (100 nM–1 μM) plus 2.5S nerve growth factor (NGF) was approximately twice the proportion of cells with neurites in sister cultures grown in NGF alone. Neither of the NO donors elicited neurites from the PC12 cells in the absence of NGF. The effects of the NO donors were likely mediated by release of NO since their effects were antagonized by addition of hemoglobin, which avidly binds NO, to the culture medium. The enhancement by NO of NGF‐mediated neurite outgrowth in PC12 cells appeared to occur through a cGMP‐dependent mechanism. The NO donors stimulated a prompt increase in intracellular cGMP in PC12 cells. Moreover their action was mimicked by addition of the membrane‐permeant cGMP analogs 8‐Bromo‐cGMP (8‐Br‐cGMP) and para (chlorophenylthio)‐cGMP (pCPT‐cGMP) to the culture medium and by atrial natriuretic factor which stimulates particulate guanylyl cyclase. The neuritogenic activity of the NO donors was inhibited by LY83583 and methylene blue, inhibitors of guanylyl cyclase. These data i...

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). N ! -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.

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Presumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [3H]thymidine at a low rate. [3H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A2 receptor antagonist, but not by 1,3-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A1 antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P2 receptor agonists, alpha, beta-methyleneATP and 2-methylthioATP, also stimulated [3H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A2 receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.

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Like adenine-based purines, extracellular nonadenine-based purines have a multitude of trophic effects on the growth, differentiation, and survival of target cells. The nonadenine-based purines, which include guanosine, inosine, and GTP, apparently exert their trophic effects by interacting with both intercellular targets as well as those on the cell surface. Specifically, guanosine and inosine target the protein kinase N-kinase, in promoting remarkable nerve process extension, even in long tracts of the central nervous system after injury. In contrast, GTP may exert its effects via a cell surface receptor coupled to the release of calcium from internal stores. In other cases trophic effects may be mediated by the enhancement of release of adenine-based purines by guanosine. Additionally, evidence is presented for the existence of a high-affinity binding site for guanosine with receptor-like characteristics on the plasma membranes of astrocytes and brain tissue. This site may be G-protein-coupled and exert its effects through activation of the MAP kinase cascade. One effect apparently mediated through this mechanism is the production and release by astrocytes of trophic protein growth factors such as NGF and TGFβ. These have substantial neuroprotective effects. Additionally, this pathway is apparently involved in modulating the expression of P2Y 1 and P2Y 2 receptors in response to extracellular guanosine. Extracellular nonadenine-based purines can interact with other growth factors, but these interactions are not always synergistic. For example, combinations of guanosine and FGF are antagonistic and reduce the growth of microvascular cells in vitro. Some of the properties of the nonadenine-based purines likely derive from their unique intracellular metabolism in which conversion of guanine to xanthine is the final catabolic step. This step is catalyzed by guanase, the activity of which varies markedly in different brain regions, raising the possibility that guanine or guanosine are involved in neurotransmission. Together these data suggest several potentially useful pharmacological approaches involving nonadenine-based purines to modulate trophic effects in the central nervous system. Drug Dev. Res. 52:303-315, 2001.

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