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

BRAIN RESEARCH ELSEVIER

Brain Research 677 (1995) 152-156

Short communication

Extracellular guanosine and guanosine-5'-triphosphate increase: NGF synthesis and release from cultured mouse neopallial astrocytes Pamela J. Middlemiss a,*, John W. Gysbers b, Michel P. Rathbone a,b a

Department of Biomedical Sciences, McMaster University, Health Science Centre, 4N25, 1200 Main Street West, Hamilton, Ont. L8N 3Z5, Canada Department of Medicine, McMaster University, Hamilton, Ont., Canada

Accepted 24 January 1995

Abstract

Cultures of neonatal mouse cortical astrocytes synthesized NGF mRNA and released immunoreactive NGF (ir-NGF) into the culture medium. Addition of 10 /zM 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/zM) affected either NGF mRNA synthesis or ir-NGF release. Keywords: NGF synthesis; NGF mRNA; Astrocyte; Guanosine; GTP; Neurotrophin; ATP; Adenosine; cAMP

Extracellular purine nucleosides, e.g. guanosine (Guo) and adenosine (Ado), or nucleotides, e.g. guanosine-5'-triphosphate (GTP) and adenosine-5'-triphosphate (ATP), stimulate proliferation of astrocytes [1,18,23] and microglia [28] in vitro. The effects of Guo and G T P were greater than those of A d o and A T P [29,30]. Extracellular purines also stimulated reactive gliosis in vivo. [15]. Astroglial reaction after central nervous system (CNS) injury was previously considered largely deleterious [31,32]. However, astrocytes that become 'reactive' after brain injury or in pathological conditions may synthesize neurotrophins such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) [3]. In contrast, in u n d a m a g e d adult brain, neurotrophins are largely confined to neurons [33]. Therefore after CNS injury, production of neurotrophins by astrocytes enhance neuronal sprouting, formation of collateral circuits and hence functional recovery [33]. Astrocytes in culture, like reactive astrocytes in vivo, can synthesize neurotrophins (for review see [33]). Secretion of N G F by astrocytes in culture is not constant [4,16,42] but depends on culture conditions [5]. Hence

* Corresponding author. Fax: (1) (905) 547-6892. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00156-5

results are not always consistent among investigators. Nevertheless, serum, several growth factors and cytokines all stimulate neurotrophin synthesis in astrocytes [36,43]. Recently Zafra et al. [44] reported that cytokines enhanced the synthesis of N G F m R N A by astrocytes while norepinephrine and glutamate receptor agonists did not [36,44]. However, Pechan et al. [25] reported that glutamate induced N G F gene expression in astrocytes. The role of cAMP in the stimulation of N G F synthesis in astrocytes is also controversial. Whereas some investigators have reported that activation of adenylate cyclase increased N G F m R N A synthesis in rat cortical astrocytes [35], others did not [43,44]. The effects of adenylate cyclase activation on N G F m R N A synthesis were sensitive to culture conditions [44]. Other factors, including F G F - 2 [36,42] and hydrogen peroxide ( H 2 0 2 ) , a cytotoxic molecule, have been also reported to increase astrocyte N G F m R N A [17,22]. Since Guo, Ado, G T P or A T P stimulated astrocyte proliferation we questioned whether they also affected the synthesis a n d / o r release of N G F by astrocytes. Moreover Guo, and to a lesser extent Ado, increased intracellular adenosine-3',5'-monophosphate (cAMP) in astrocytes whereas G T P and A T P had no effect [27]. As c A M P may stimulate N G F synthesis in astrocytes [35], this was further impetus to test the effects of extracellular purines on N G F synthesis.

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Astrocytes from the cerebral cortex of newborn N I H Swiss mice (Harlan) were isolated according to Hertz et al. [14] and maintained in modified Dulbecco's medium [13] containing 10% horse serum (HS). After 2 weeks in culture the astrocytes formed a confluent monolayer. The cultures used in the assays consisted of > 95% glial fibrillary acidic protein positive, flat epithelial-like cells [13] with < 5% macrophages and approximately 1% oligodendrocytes of the total cells. The isolation method, the age of animals used, low seeding density and frequent medium changes kept the percentage of oligodendrocytic cell precursors and other cell types to a minimum. Oligodendrocytes can express N G F [2]. However, since oligodendrocytes represent such a small proportion of the total cell population, their contribution to the total level of N G F in the astrocyte conditioned medium (ACM) would be minimal. Thus the neurotrophins produced in the ACM would be almost entirely derived from the astrocytes. N G F release into astrocyte culture medium was determined using cultures, in logarithmic growth phase. Cultures were treated with Guo, Ado, G T P or ATP dissolved in phosphate buffered saline (PBS) or, as a control, PBS alone. After 24 h the medium was removed from the cultures and centrifuged. The supernatant, ACM, was then tested in a two-site, sandwich type ELISA [19]. Samples of ACM (100 /xl) or 2.5 S HPLC-purified mouse submandibular salivary gland N G F (Cedarlane) as the standard, were used in the assay and read using a Microfluor ELISA reader (exci-

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Fig. 1. The effect of Guo and Ado on the production of ir-NGF by mouse cortical astrocytes. Confluent monolayers of astrocytes were trypsinized and replated into 24 well plates (5×104 cells/well). After 2 h the medium was then replaced (DMEM containing 10% HS) and Guo or Ado or PBS was added to the cells. 24 h later the medium was removed and the ir-NGF level (expressed as pg/well) in the ACM was measured by ELISA. Bars represent the mean _+S.E.M. 10 /~M Guo significantly increased ir-NGF level in ACM above ir-NGF level for control cultures: A N O V A (* P < 0.001), Tukey's HSD.

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Fig. 2. The effect of GTP and Guo on the production of ir-NGF by mouse cortical astrocytes. The same experimental design was used as described in Fig. 1. Bars represent the mean_+ S.E.M. GTP significantly increased ir-NGF level in the ACM above ir-NGF level from control cultures: A N O V A (* P < 0.001, * * P < 0.0001), Tukey's HSD.

tation 360 nm; emission 450 nm). The sensitivity of this assay was 10 pg N G F / w e l l . The data obtained was analyzed by a one-way analysis of variance (ANOVA) followed by Tukey's HSD for significance. N G F m R N A was quantified by slot blot analysis using a random primed 32p-labelled 0.995 kb cDNA N G F probe in plasmid p G E M . N G F ( + ) . Northern blot analysis confirmed that this probe hybridized a 1.3 kb band of m R N A identical in size to N G F m R N A [34]. To ensure the film response was linear with respect to increasing amounts of 32p, various concentrations of total R N A was used. Autoradiography was performed using Hyperfilm-MP ( A m e r s h a m / U S B ) and intensifying screens. The data obtained was analyzed by a one-way A N O V A followed by Tukey's HSD for significance. Both Guo (Fig. 1) and G T P (Fig. 2) increased the immunoreactive-NGF (ir-NGF) level in ACM. The amount of ir-NGF in the ACM varied with the concentration of Guo or G T P added to the cultures. For both Guo and G T P the greatest increase in ir-NGF was observed at 10/~M. However, although Guo increased ir-NGF to 97,108 pg/well (Fig. 1), the increase due to G T P was only 33,571 pg/well (Fig. 2). In contrast, neither Ado (1-100 /~M) (Fig. 1) nor ATP ( 1 - 1 0 0 / x M ) (Fig. 2) increased the ir-NGF level in ACM above ir-NGF levels from control cultures. The N G F antibody used in the ELISAs recognizes NGF, B D N F and NT-3 [20]. The reaction of N G F antibody with NT-3 was slightly less than with N G F and about 100 fold greater than with BDNF (Dr. M. Coughlin, personal communication). Astrocytes produce NGF, NT-3 and B D N F [33]. These neurotrophins share 40-65% amino acid sequence homology and contain six conserved cysteine residues [24]. Therefore,

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Fig. 3. The effect of Guo (10 /zM) and GTP (10 /xM) on NGF mRNA levels in astrocytes 4 and 24 h after treatment. Total RNA, from harvested astrocyteswas extracted using TRIzol reagent (protocol Gibco BRL/Life TechnologiesInc.). Total RNA 0.25-4/xg, were spotted onto Hybond-Nfilters (Amersham) [40] and then hybridized. The resultant autoradiographs were densitometrically scanned (MCID Image Analysis) and the relative density per microgram of total RNA (ROD//xg RNA) spotted was determined for each sample. Bars represent the mean + S.E.M. By 4 and 24 h Guo and GTP significantly increased the ROD//xg RNA level above the ROD//xg RNA level from control cultures: ANOVA (* P < 0.001, * * P < 0.0001), Tukey's HSD.

it is likely that the N G F antibody reacts with a similar epitope exposed on the surface of each neurotrophin. The amino acid homology is greater between NT-3 and N G F than between B D N F and N G F [24]. This accords with their immunological cross-reactivity. To confirm that the astrocytes were indeed synthesizing N G F in response to Guo or GTP, we measured the relative amounts of N G F m R N A after treatment with these purines. Total m R N A was extracted from astrocytes 4 or 24 h after Guo, G T P or PBS (vehicle control) was added to their culture medium. Northern blot analysis revealed a single band of m R N A at 1.3 kb when probed with N G F (data not shown). Slot blot analysis showed that the amount of N G F m R N A produced in cells 4 h after addition of Guo or GTP was approximately 6 fold greater than that found in the PBS controls (Fig. 3). 24 h after Guo or GTP was added to the cultures, the amount of N G F m R N A in treated cultures was less than at 4 h, yet still 2 - 3 times greater than in the PBS controls (Fig. 3). The increase in N G F m R N A expression in astrocytes after treatment with Guo or GTP was similar to the 6 - 7 fold increase observed 4 - 6 h after exposure to H z O 2 [22,25] or glutamate [26]. In those cases, N G F m R N A levels increased 6 - 7 fold over untreated cultures 4 - 6 h after treatment. These data indicate that Guo or GTP, but not Ado

or ATP, stimulate N G F synthesis by astrocytes. Interestingly, Guo [10] and GTP [9], but not Ado or ATP, also stimulate neurite outgrowth from PC-12 cells in culture [11]. This raises the possibility that Guo and GTP, to a much greater extent than Ado and ATP, may stimulate neurotrophin synthesis in several cell types. N G F secretion by astrocytes is related to the rate of cell growth and decreases after serum deprivation [5]. Since extracellular purine nucleosides and nucleotides stimulate astrocyte proliferation [1,23,29,30] might this underlie their ability to increase N G F synthesis? We consider this unlikely; whereas all four purines stimulate astrocyte proliferation [1,23,29,30], only Guo and, to a lesser extent GTP, enhance N G F synthesis and release. GTP can be converted to Guo by a series of ectoenzymes which includes a nucleoside triphosphatase, nucleoside diphosphate phosphohydrolase and a 5'nucleotidase [7,8,21,37]. The ectoenzymes involved have complex kinetics and therefore would be unlikely to convert GTP stoichiometrically to Guo alone. Since both Guo and GTP were maximally effective at 10 p.M, it is therefore unlikely that GTP acted simply as a reservoir from which Guo was synthesized. Substances that increase intracellular cAMP may increase N G F synthesis by astrocytes [35]. Guo increases intracellular cAMP in astrocytes [27]. Therefore, Guo may employ cAMP in the signal transduction cascade that stimulates N G F synthesis. This cannot be true for GTP as it does not increase cAMP in astrocytes [27]. Therefore, Guo and GTP may enhance N G F synthesis through different signalling pathways. After brain injuries such as stroke, the extracellular concentration of Guo is elevated over 1 week [12,39]. Therefore, astrocytes are exposed to levels of extracellular Guo that may substantially exceed 10/zM [45], for prolonged periods. Our data indicate that at these concentrations either Guo or GTP is capable of stimulating N G F synthesis in astrocytes. In contrast to Guo, extracellular Ado concentrations may be elevated more transiently following tissue injury [12,39]. Extracellular Ado is rapidly removed by a number of processes, including re-uptake and the activity of ectoenzymes such as adenosine deaminase [6,38,41]. Thus extracellular Guo, rather than Ado, may well be particularly suited to providing a sustained influence to stimulate synthesis of neurotrophins by astrocytes after CNS injury. Experiments are under way to test whether Guo produces this effect in vivo as well as in vitro.

Acknowledgements We thank Drs. M. Fahnestock for providing the N G F c D N A probe and for advice, J. Staniz for assis-

P.J. Middlemiss et al. /Brain Research 677 (1995) 152-156

t a n c e w i t h t h e E L I S A , M. C o u g h l i n f o r p r o v i d i n g t h e NGF and anti-NGF and for advice, and M.P. Elliott for e d i t o r i a l c o m m e n t s . T h i s w o r k w a s s u p p o r t e d by grants from The Hospital For Sick Children Foundation, Toronto;, Advanced ImmunoTherapeutics, Tustin, C A ; a n d G. C a l d e r . J . W . G . h e l d a N e u r o - O n c o l o g y Foundation Post-Doctoral Fellowship.

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