Extracellular guanosine 5' triphosphate enhances nerve growth factor-induced neurite outgrowth via increases in intracellular calcium.

Extracellular guanosine 5' triphosphate (GTP) enhances nerve growth factor-dependent neurite outgrowth from rat pheochromocytoma (PC12) cells; cultures of PC12 cells exposed to GTP and nerve growth factor together contain significantly more neurite-bearing cells than do those exposed to either nerve growth factor or GTP alone [Gysbers J. W. and Rathbone M. P. (1996) Int. J. devl Neurosci. 14, 19-34]. PC12 cells contain specific cell surface binding sites for extracellular GTP, which do not bind ATP or uridine 5' triphosphate. Exposure of PC12 cells to extracellular GTP (300microM) produced a robust and sustained increase in intracellular Ca(2+) ([Ca(2+)](i)), different from the transient response to the addition of ATP. The GTP-induced [Ca(2+)](i) increase was blocked by the L-type calcium channel inhibitor, nifedipine. The L-type Ca(2+) channel inhibitors, nifedipine or verapamil, also inhibited the enhancement of neurite outgrowth by GTP, but did not affect neurite outgrowth stimulated by nerve growth factor alone. Pre-treatment of PC12 cells with ryanodine (0.5-50microM) depleted calcium from internal stores and prevented the further release of calcium by GTP. Similarly, pre-treatment of PC12 cells with thapsigargin (an inhibitor of internal store Ca(2+)/ATPase) or dantrolene (which blocks Ca(2+) release from some of these stores) also reduced the enhancement of neurite outgrowth by GTP. Therefore, Ca(2+)-induced Ca(2+) release from specific stores, present in PC12 cells, is involved in the enhancement of nerve growth factor-induced neurite outgrowth by GTP, possibly acting at specific binding sites on the cell surface. GTP is proving to be an important extracellular trophic modulator in the central nervous system. These studies show that the neuritogenic actions of GTP involve moderate but sustained increases in intracellular Ca(2+) which are likely due to activation of L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from intracellular stores. These effects of extracellular GTP are likely mediated at the cell surface and may be related to specific GTP binding sites which are distinct from G-proteins and from hitherto described purine nucleotide (P2) receptors. These data indicate a mechanism whereby the neuritogenic effects of GTP are mediated and emphasize the importance of considering GTP as a neurotrophic mediator.

Trophic effects of purines in neurons and glial cells.

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.

Nitric oxide donors enhance neurotrophin-induced neurite outgrowth through a cGMP-dependent mechanism.

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 microM) 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 microM-10mM) or sodium nitroprusside (100 nM-1 micro M) 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 imply that NO may act alone or with other growth factors to regulate synapse formation and maintenance by stimulating neurite outgrowth.

Neurite outgrowth in PC12 cells is enhanced by guanosine through both cAMP-dependent and -independent mechanisms.

Extracellular guanosine, guanosine triphosphate (GTP), and 5'-N'-ethylcarboxamidoadenosine (NECA), each significantly enhanced the proportion of nerve growth factor (NGF)-treated rat pheochromocytoma (PC12) cells which had neurites, greater than that in cultures exposed to NGF alone. Guanosine and NECA, but not GTP, increased intracellular cAMP concentrations. An adenylate cyclase inhibitor, SQ22536, completely blocked the cAMP increase induced by both guanosine and 0.1 microM NECA. However, SQ22536 only partially blocked guanosine enhanced neurite outgrowth, although it completely blocked the neuritogenic effect of NECA. Therefore guanosine-enhanced neurite outgrowth through both cAMP-dependent and -independent mechanisms, while the effect of GTP was cAMP-independent.

GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells.

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 microM extracellular GTP or 300 microM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20-35% of PC12 cells had neurites after 48 hr, and the addition of 300 microM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40-65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics. GTP probably did not exert its effects via the P2X or P2Y purinoceptors because the adenine nucleotides ATP, ATP gamma S, ADP beta S, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP >> ATP, does not fit the profile of any known P2Y, P2X or P2U receptor. The poorly hydrolyzable GTP analogues, GTP gamma S and GDP beta s were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms. The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P1 receptors, because the A2 receptor antagonists, 1,3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the A1 receptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the P1 purinoceptors. The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.

Extracellular guanosine and guanosine-5'-triphosphate increase: NGF synthesis and release from cultured mouse neopallial astrocytes.

Cultures of neonatal mouse cortical astrocytes synthesized NGF mRNA and released immunoreactive NGF (ir-NGF) into the culture medium. Addition of 10 microM guanosine or GTP to the cultures increased ir-NGF release by 6 and 2 fold, respectively, after 24 h, and increased NGF mRNA 6 fold after 4 h and 2-3 fold after 24 h. In contrast, neither adenosine nor ATP (each 1-100 microM) affected either NGF mRNA synthesis or ir-NGF release.

Guanosine enhances NGF-stimulated neurite outgrowth in PC12 cells.

Guanosine at 30 and 300 microM elicited the de novo extension of neurites from PC12 cells. With saturating concentrations of NGF, guanosine acted in a synergistic manner to enhance neuritogenesis. Adenosine alone also stimulated neurite outgrowth, but did not enhance NGF-induced neuritogenesis. 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog and A1/A2 receptor agonist, also alone had neuritogenic effects. It enhanced NGF-induced neuritic outgrowth but not to the same extent as guanosine. However, when NECA was added together with guanosine in the presence of NGF, these compounds elicited a greatly enhanced neuritogenic response. This suggested that the mechanisms through which NECA modulates the neuritogenic effects may be different from those of guanosine and NGF.

Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types.

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.

Extracellular purine nucleosides stimulate cell division and morphogenesis: pathological and physiological implications.

Extracellular purine nucleosides and nucleotides are ubiquitous, phylogenetically ancient, intercellular signals. Purines are released from hypoxic, damaged or dying cells. Purine nucleosides and nucleotides are potent mitogens for several types of cells such as fibroblasts, endothelial cells and neuroglia. They also induce other cell types to differentiate. For example, they act synergistically with nerve growth factor to stimulate neurite outgrowth from a pheochromocytoma cell line (PC12). We propose that after injury to tissues, including the central nervous system, purine nucleosides and nucleotides interact synergistically with other growth factors. They stimulate proliferation and morphological changes in the various cell types involved in the wound healing response. In the central nervous system this response includes glial proliferation, capillary endothelial cell proliferation, and sprouting of nerve axons. Since many actions of extracellular purines are mediated through specific cell surface receptors, this hypothesis has broad pharmacological implications.

Adenosine and its nucleotides stimulate proliferation of chick astrocytes and human astrocytoma cells.

Aqueous extracts of the brains of 18-day-old white Leghorn chicken embryos contain several substances that stimulate proliferation of primary cultures of chick brain astrocytes. Most of the mitogens are peptides. Purification of one mitogenic fraction was obtained by centrifugation, passage through Amicon Diaflo membranes of nominal molecular weight cutoffs 30, 1 and 0.5 kDa, ion exchange chromatography and reverse phase high performance liquid chromatography (HPLC) using a Deltapak C18 column. The mitogenic fraction contained no amino acids. On the basis of its behaviour on thin layer chromatography, its ultraviolet absorption spectrum, its 1H and 31P nuclear magnetic resonance spectra and its behaviour on positive and negative ion fast atom bombardment mass spectrometry, the mitogenic material was identified as adenosine-5'-monophosphate (AMP). Other adenosine compounds including adenosine, ADP and ATP also stimulated proliferation of and [3H]leucine incorporation into primary cultures of astrocytes. Nitrobenzylthyioinosine (NBTI), an inhibitor of nucleoside transport, did not prevent the stimulation of [3H]leucine incorporation into cultured astrocytes. Polyadenylic acid (Poly A), that mimics the effect of adenosine at adenosine receptors, also stimulated proliferation of the astrocytes. The effects of adenosine and Poly A were not inhibited by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) but were inhibited by 1,3-dipropyl-7-methyl-xanthine (DPMX), indicating that adenosine and Poly A acted at the cell surface, likely through adenosine A2 receptors. The stimulatory effect of ATP was biphasic. The proliferative effect of low, but not of high, concentrations of ATP were abolished by DPMX. The purinergic P2 receptor agonist 2-methylthioATP and, at higher concentrations, the P2y agonist, alpha,beta-methyleneATP also stimulated incorporation of [3H]thymidine. These data indicate that high concentrations of ATP stimulate cell proliferation through at a P2, possibly a P2y receptor. These results have considerable biological significance. After brain injury, or when cells in brain die or become hypoxic, nucleotides and nucleosides are released from the cells. Their extracellular concentrations can exceed those required to stimulate astrocyte proliferation in vitro. Therefore they may be partly responsible for gliotic changes following cell death in brain.