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Effects of ATP analogues and basic fibroblast growth factor on astroglial cell differentiation in primary cultures of rat striatum
Int. J. Devl Neuroscience, Vol. 13, No. 7, pp. 685-693.1995
Pergamon
0736-5748(95)00064-X
Elsevier ScienceLtd Copyright © 1995ISDN Printed in Great Britain. All rights reserved 0736-5748/95$9.50+0.00
EFFECTS OF ATP A N A L O G U E S A N D BASIC FIBROBLAST GROWTH FACTOR ON ASTROGLIAL CELL DIFFERENTIATION IN PRIMARY CULTURES OF RAT STRIATUM M. P. A B B R A C C H I O , * S. C E R U T I , * R. L A N G F E L D E R , * F. C A T T A B E N I , * M. J. S A F F R E Y t ~ t and G. B U R N S T O C K t § *Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy: ?Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, U.K. (Received 21 February 1995; revised 1 June 1995;accepted 1 June 1995)
Abstraet--We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and a[3-methylene-ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose-dependent. The effect of e~13-methylene-ATPwas antagonized by the trypanoside suramin and mimicked by 2-methyl-thio-ATP, suggesting the involvement of a suramin-sensitive P2-purinoceptor. Neither an additive nor a synergistic effect between al3-methylene-ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways. It is concluded that, like basic fibroblast growth factor, ATP can promote the maturation of astrocytes towards a more differentiated phenotype characterized by the presence of longer astrocytic processes. These findings might have interesting implications for astroglial cell differentiation during brain development and for ischemia- and trauma-associated hypergliosis. Keywords: astroglial cells, ATP, reactive astrogliosis.
Glial cells m a k e up a substantial p r o p o r t i o n of the central n e r v o u s system. Within glia, astrocytes represent the m o s t n u m e r o u s cell type and play key roles in n o r m a l brain function, 4 d e v e l o p m e n t 3,22 and in the p a t h o l o g y o f the n e r v o u s system. 7 A s t r o c y t e s react to various types of injury with astrogliosis, a reaction characterized by b o t h increased proliferation and cellular h y p e r t r o p h y . 7,1° P r o m i n e n t reactive astrogliosis is seen after acute traumatic brain injury, i n f l a m m a t o r y demyelinating disease, A I D S d e m e n t i a and o t h e r viral infections, and such n e u r o d e g e n e r a t i v e diseases as A l z h e i m e r ' s disease. 7 Characterization of the e n d o g e n o u s factors which regulate astroglial cell function has t h e r e f o r e i m p o r t a n t implications in n e r v o u s system pathophysiology. G r o w i n g evidence supports a role for purines ( n a m e l y A T P and adenosine) as trophic factors and e n d o g e n o u s regulators of cell g r o w t h a n d differentiation in b o t h d e v e l o p m e n t and adulthood. ~ Purines are involved in egg fertilization, 8 e m b r y o n i c m o r p h o g e n e s i s 13 and organogenesis. 11,26 Via activation o f specific p u r i n o c e p t o r subtypes, purines regulate n e u r o n a l function and neurotransmission in b o t h central a n d peripheral n e r v o u s systems, 5,6,14,25 A role in m o d u l a t i o n of astroglial cell function has b e e n m o r e recently suggested. 2,3,16,23 W e have previously d e m o n s t r a t e d that analogues of b o t h adenosine and A T P can regulate the n u m b e r of astroglial cells in culture p r o b a b l y by influencing their proliferation rate. 2 H o w e v e r , besides cell proliferation, a k e y role for astrocytic h y p e r t r o p h y in the initiation and m a i n t e n a n c e of astrogliosis has b e e n e m p h a s i z e d recentlyJ ° Increases o f cellular size, increased staining for glial fibrillary acidic p r o t e i n ( G F A P ) and the presence of l o n g e r and thicker astrocytic processes are taken as an index of the astroglial cell differentiation k n o w n to occur in every type of astrogliosis, even in the absence of astrocytic proliferation (e.g. fetal anoxia). 1° §To whom all correspondence should be addressed. ~Present address: Department of Biology, The Open University, Walton Hall, Milton Keynes, MK7 6AA, U.K. Abbreviations: a[3-meATP, c~13-methylene-ATP;bFGF, basic fibroblast growth factor; FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; HBSS, Hank's balanced sale solution; 2meS-ATP, 2-methylthio-ATP; PBS, phosphate-buffered saline. 685
686
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On these bases, the present study was aimed at evaluating the effects of purine analogues on the elongation of GFAP-positive astrocytic processes in the same experimental model (neuron-glia primary cultures from neonatal rat c. striatum) which has previously been utilized for the proliferative studies. 2 Based on preliminary observations suggesting induction of 'stellation' and increased astrocytic branching after application of the relatively hydrolysis-resistant ATP analogue cx[3-methylene-ATP (al3-meATP), this study focussed on ATP derivatives. Moreover, since increased GFAP expression has been demonstrated to occur following exposure to a number of growth factors such as, for example, basic fibroblast growth factor (bFGF), 15 the morphological effects induced by purine analogues on cultured astrocytes have been compared with those induced by bFGF.
EXPERIMENTAL PROCEDURES
Cultures Cultures were prepared by a slight modification of the previously described method. 2 Briefly, after rapid decapitation, the brains of three or four seven-day-old Sprague-Dawley rats were removed under sterile conditions, the corpus striata were transferred to Hank's balanced salt solution (HBSS) supplemented with 0.6% glucose and gently chopped into small pieces using a sterile razor blade; pieces were then washed three times in HBSS without calcium and magnesium (buffer 1). Ten milliliters of a solution of 0.125% trypsin in buffer 1 containing 10 ~g/ml DNAase I were added to the washed tissue, which was incubated at 37°C for 45 min. After four washes with 5 ml of buffer 1 supplemented with 8 mM MgCI 2, 10% fetal calf serum (FCS) and 10 wg/ml DNAase I, the tissue was mechanically dissociated into single cells in 5 ml of the same solution containing 20 i~g/ml DNAase I, using a sterile pipette. Dissociated cells were centrifuged at 800 rpm for 10 rain and the pellet was resuspended in 2 ml of medium 199 supplemented with 10% FCS and 5 mg/ml glucose. The total number of cells was determined in a Burke chamber using the Trypan Blue dye exclusion test. The cell suspension was diluted with medium 199+10% FCS and 5 mg/mi glucose to a final concentration of 1.9×105 viable cells/mL Three hundred and sixty microliters of the cell suspension were inoculated into each well of 24-well dishes (each containing a poly-L-lysine-coated glass coverslip), followed by 360 ~1 of medium 199 + 10% FCS + 5 mg/ml glucose and 80 ~1 of buffer 1 containing either ot[3-meATP, 2-methylthio-ATP (2meS-ATP) or bFGF at various concentrations, or different combinations of the various agents as indicated in the legends to the figures and tables. A final volume of 800 p,l/well was used both in control cultures and in cultures treated with two agents together by increasing or reducing, respectively, the volume of medium added to the well. Cultures were then maintained in a 5% CO 2 incubator at 37°C. This first incubation with FCS was done to favour attachment of dissociated cells to the dishes. After 24 h, the serum-supplemented medium was replaced with a chemically defined serum-free medium, containing 2×10 -8 M progesterone, 0.12 U.I./ml insulin, 3×10 -8 M sodium selenite, 5 wg/ml transferrin, 100 I~M putrescine, 0.08% bovine serum albumin (BSA) and 5 mg/ml glucose, in the absence (control cultures) or presence of the various agents. In selected experiments the action of the trypanoside suramin (an antagonist of P2 purinoceptors), alone or in combination with ot[3-meATP or bFGF, was tested.
Immunofluorescence staining and analysis of GFAP-positive processes. After 72 h in culture, cells were fixed with methanol at -20°C for 10 min, and then washed three times (10 min each) with phosphate-buffered saline (PBS). For evaluation of astroglial process formation, astrocytes were labelled by indirect immunofluorescence using rabbit anti-GFAP immunoglobulins (1:500; overnight incubation at room temperature). Cells were then washed three times (10 min each) with PBS and incubated with biotinylated donkey anti-rabbit secondary antibody (1:250; 1 h at room temperature), followed by streptavidin-fluorescein (1:100; 1 h at room temperature). All antibodies were diluted in antibody diluting solution containing 0.1% Triton X-100, 0.1% sodium azide and 0.01% bovine serum albumin in PBS. After three final washes with PBS, coverslips were mounted in Immumount and labelled cells examined using a Zeiss fluorescence microscope equipped with a fluorescein filter. The length and number of GFAP-positive
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astrocytic processes was measured using an image video system consisting of a Macintosh computer (equipped with NIH Image 1.47 software) and a video camera connected to the microscope. Values for the total number of GFAP-positive processes/cell, the total length of processes/cell and the mean length/process were obtained from 50 cells/condition. Statistical analysis was performed on the data obtained from two different experiments (100 cells/condition) with unpaired Student's t-test.
Materials Sprague-Dawley rats were obtained from Charles River (Italy). HBSS, HBSS without calcium and magnesium, FCS, Medium 199, PBS tablets and 20% glucose solution were purchased from Gibco BRL, Life Technologies (European Division). Trypsin, DNAase 1, od3-meATP, 2meS-ATP, human recombinant bFGF, progesterone, insulin, sodium selenite, transferrin, putrescine, BSA, sodium azide and paraformaldehyde were obtained from Sigma Chemicals Co. (U.S.A.), while suramin was obtained from Bayer (Germany). Cultures were grown in 24-well dishes obtained from Nunc. Rabbit primary antibody against GFAP was purchased from D A K O A/S (Denmark); biotinylated donkey anti-rabbit secondary antibody and streptavidin-fluorescein were purchased from Amersham, Life Science (U.K). Coverslips were mounted in Immumount, purchased from Shandon Italscientifica (Italy).
RESULTS
Effects of bFGFand A TP analogues on the elongation of GFAP-positive astrocytic processes Both bFGF and a[3-meATP induced dramatic morphological changes of astrocytes, characterized by the presence of long and thick astrocytic processes which stained strongly for GFAP (Fig. 1). The quantitative analysis of these effects is shown in Tables 1 and 2. Exposure of cells to the growth factor produced a marked and dose-dependent increase of the mean length of processes/cell with maximal effects at a concentration of 1 ng/ml. No differences were detected in the mean number of processes/cell with respect to control cultures (Table 1). Table 2 shows the same parameters in cultures treated with al3-meATP. Similarly to bFGF, the ATP analogue induced a dose-dependent increase of the mean length of processes/cell, without affecting the number of GFAP-positive processes/cell. Effects were maximal at 10 -5 M, still maintained at higher concentrations and quantitatively comparable with the maximal effect obtained with bFGF. Details of the effects induced by bFGF and c~13-meATP on the expression of GFAP-positive astrocytic processes are shown in Figs 2 and 3. In these figures, the frequency distribution of GFAP-positive cells with different mean process length is shown. It is evident that in control cultures the majority of cells belonged to sub-populations with mean length of processes up to 150 ixm. Exposure to increasing bFGF concentrations induced a dose-dependent shift of cell distribution towards populations with longer processes (Fig. 2). For example, in control cultures, about 70 cells/100 had a mean length of processes between 0 and 150 ~m, whereas only 30 cells/100 had these characteristics in cultures exposed to 1 ng/ml bFGF. Conversely, almost no control cell had processes longer than 300 p.m, whereas a progressively higher percentage of bFGF-treated cells displayed processes in the 301-500 Ixm ranges. Comparable results were obtained with al3-meATP, which induced a similar shift towards cell populations characterized by higher astrocytic lengths (Fig. 3). For the purine analogue, maximal effects were obtained at 10 -5 M, whereas at 5 × 10 -5 M a slight reduction of effect was evident. In order to verify if the simultaneous exposure of cultures to bFGF and oL[3-meATP could synergistically affect astroglial cell differentiation, we grew cells in the presence of both agents. An example of these experiments is shown in Fig. 4 for the combination of the three concentrations of a[3-meATP with 0.5 ng/ml bFGF. Neither synergism nor additivity between oLI3-meATP and bFGF were detected; on the contrary, a reduction of process length was evident in comparison with the two agents alone. Similar results were also obtained for all the other possible combinations of a[3-meATP and bFGF (data not shown). In order to determine if other purine analogues could modulate the elongation of GFAP-positive processes in our system, in another set of experiments we grew cells in the presence of 2meS-ATP.
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Fig. 1. Immunofluorescence micrographs showing GFAP-positive cells in control cultures (A) and in cultures grown in the presence of 1 ng/ml bFGF (B) or in the presence of 10 -5 M c~13-meATP (C). Note the dramatic morphological changes (extension of longer and thicker processes) induced by the two agents. Scale bar=50 p.m.
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Table 1. Effect of bFGF on GFAP-positive astrocytic processes in rat striatal cultures
Control cells
bFGF bFGF bFGF bFGF bFGF
(0.01 ng/ml) (0.I ng/ml) (0.5 ng/ml) (1 ng/ml) (10 ng/ml)
Mean length of processes/cell
Mean number of processes/cell
(p~m-+S.E.)
(No.-+S.E.)
131.63_+7.47 171.22-+8.62" 184.98_+ 17.05~ 196.12-+9.92~: 199.95-+ 10.43~: 189.53_+ 10.36~
1.98_+0.09 2.03_+0.08 1.93-+0.08 2.04_+0.10 2.15-+0.10 1.98_+0.10
Rat striatal primary cultures were grown for 24 h in complete medium and then for an additional 48 h in chemically defined serum-free medium, in the absence (control) or in the presence of the indicated concentrations of bFGF. At day 3 in culture, cells were fixed, immunostained for GFAP and the length and number of astrocytic processes were measured using an image video analysis system (see Experimental Procedures for details). Results are the mean-+S.E, of 100 cells/condition, obtained from two different experiments. Similar results were obtained in three other experiments. *P<0.002; tP<0.006; $P<0.001 with respect to control cells, Student's t-test.
Table 2. Effect of ctl3-meATP on GFAP-positive astrocytic processes in rat striatal cultures
Control cells
al3-meATP (5× 10 -6 M) al3-meATP (10 -5 M) c~13-meATP (5 x 10-ZM)
Mean length of processes/cell
Mean number of processes/cell
(p,m-+S.E.)
(No._+S.E.)
131.63---7.47 154.26-+7.12" 189.84-+9.51t 177.26-+ 9.30t
1.98-+0.09 2.06-+0.09 2.01-+0.09 1.92_ + 0.07
Rat striatal primary cultures were grown for 24 h in complete medium and then for an additional 48 h in chemically defined serum-free medium, in the absence (control) or in the presence of the indicated concentrations of al3-meATP. At day 3 in culture, cells were fixed, immunostained for GFAP and the length and number of astrocytic processes were measured using an image video analysis system (see Experimental Procedures for details). Results are the mean-+ S.E. of 100 cells/condition, obtained from two different experiments. Similar results were obtained in three other experiments. *P<0.03 tP<0.001 with respect to control cells, Student's t-test.
This molecule is less resistant to hydrolysis than al3-meATP but is considered a more selective agonist at P2Y-purinoceptors with respect to a cxl3meATP. 9 Exposure of cultures to 10 -5 M 2meS-ATP (the maximally effective concentration for otl3-meATP) resulted in a significant increase of the mean length of processes, although this increase seemed lower with respect to that induced by af3-meATP (a 24.5_+7% increase with respect to the 44_+7% increase induced by the same concentration of etl3-meATP, mean of three experiments run in triplicate, P<0.02 with respect to control cells). As with the other agents tested, no difference in the mean number of processes/cell was observed in the 2meS-ATP-treated cultures with respect to control cells (data not shown).
Effect of suramin on ot[5-meA TP- and bFGF-treated cultures In order to verify whether the effects induced by txl3-meATP could be related to the activation of extracellular receptors, we exposed cells to both the purine analogue and the trypanoside suramin, which is known to act as an antagonist at both P2X and P2Y purinoceptors. 12 Suramin alone (10 -5 M) did not significantly affect the length of processes (Table 3), while, in the same set of experiments, 10 -5 M oLI3-meATP produced an increase of length of about 35% with respect to control cultures. Exposure to both agents completely prevented the action of the ATP analogue (Table 3), thus suggesting the involvement of a suramin-sensitive P2 purinoceptor. The possibility that suramin may interfere with the action of bFGF has been reported by other authors. 27 We therefore tested this possibility in our system. As expected, exposure of cultures to bFGF induced a 74% increase of process length with respect to control cultures (Table 4); suramin did not affect either the length of processes p e r s e or block the action of the growth factor when the two were added in combination, therefore ruling out any interaction of suramin with the activities of bFGF under our experimental conditions.
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40
• control W bFGFj1 ng/ml 30 0
20
'a ,,Q E ,.,¢ z
10
0
o
~
0
0
0
°
o
g
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N
0
0
o
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N
V~
Fig. 2. Effects of bFGF on astroglial cells in culture. Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium in the absence (control) or presence of the indicated concentration of bFGF. At day 3, cells were fixed, immunostained for GFAP and the total length of processes/cell was evaluated using an image video analysis system. Cells were then grouped according to mean length of processes in the 0-100, 101-150, 151-200, etc. ranges as indicated on the x axis. The distribution of cell populations for control cultures (black histograms) and for cultures exposed to 1 ng/ml bFGF are shown; intermediate effects were obtained using lower (0.01 and 0.1 ng/ml) concentrations of the growth factor. For data shown, S.E. was, in all cases, lower than 10%. Data refer to two experiments which were analysed in detail by utilizing the above-described method; however, a similar pattern of distribution for cell populations was obtained in three other independent experiments.
40
W @ O
• control [ ] (zJ3-meATP 10 -5 M
30
"6 .Q E
20
z
10
0
Fig. 3. Effects of cq3-meATP on astroglial cells in culture. Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium in the absence (control) or presence of the indicated concentration of al3-meATP. At day 3, cells were fixed, immunostained for GFAP and the total length of processes/cell was evaluated using an image video analysis system. Cells were then grouped according to mean length of processes in the 0-100, 101-150, 151-200, etc. ranges as indicated on the x axis. The distribution of cell populations for control cultures (black histograms) and for cultures exposed to 10 -5 M al3-meATP are shown; proportional effects were obtained by using other aI3-meATP concentrations. For data shown, S.E. was, in all cases, lower than 10%. Two experiments were analysed in detail by utilizing the above-described method; however, a similar pattern of distribution for cell populations was obtained in three other independent experiments. DISCUSSION Adenosine and ATP have previously been shown to affect the proliferative response of astrocytes i n c u l t u r e . 2,23 I n t h i s s t u d y , w e h a v e i n v e s t i g a t e d w h e t h e r p u r i n e a n a l o g u e s c a n a l s o m o d u l a t e another important parameter of astrocytic activation: the formation of GFAP-positive astrocytic
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160-140 120
100 80 v
60 ..d 40 20
Control
al~-rneATP 10-5 M
bFGF + ctl~-meATP
bFGF
10-5 M ct~-meATP
ct[3-meATP
5 x 10-6 M
5 x 10-5 M
bFGF ÷ II~-meATP 5x IO-6M
bFGF + al~-meATP 5xlO-SM
Fig. 4. Effect of the exposure to both al3-meATP and bFGF on the length of astrocytic processes in striatal cultures. Cells were grown in the presence of al3-meATP at the indicated concentrations alone or in combination with bFGF (0.5 ng/ml). After 72 h in culture, cells were fixed, immunostained for GFAP and the mean length of processes/ceU (reported in the y axis as percentage of control) was calculated using an image video analysis system. **P<0.02; ***P<0.004 with respect to bFGF alone, Student's t-test.
Table 3. Effect of suramin on c~13-meATPinduced elongation of astrocytic processes in rat striatal cultures Mean length of processes/cell (% of control_+S.E.) Suramin (10-5 M) c~13-meATP(10-~M) al3-meATP (10-5 M)+ Suramin (10-5 M)
111.5_+8.7 152.3_+9" 113.3_+9.6
Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium, in the absence (control) or presence of 10-5 M suramin and 10-5 M ~13meATP alone or in combination as indicated. At day 3 in culture, cells were fixed, immunostained for GFAP and the length of processes/cells were calculated using an image video analysis system (see Experimental Procedures for details). Results are the mean-S.E, of 50 cells and similar results were obtained in two other experiments. *P<0.02 with respect to control cells, Student's t-test. processes. This p a r a m e t e r c a n b e t a k e n as a m a r k e r of the astrocytic h y p e r t r o p h y a n d f u n c t i o n a l d i f f e r e n t i a t i o n which is k n o w n to occur in reactive astrogliosis. 7 T h e p r e s e n t results show that, like k n o w n r e g u l a t o r s of astroglial cell f u n c t i o n such as b F G F , b o t h a l 3 - m e A T P a n d 2 m e S - A T P can i n d u c e e l o n g a t i o n of astrocytic processes. F o r etl3-meATP, the effect was d o s e - d e p e n d e n t a n d q u a n t i t a t i v e l y c o m p a r a b l e with that i n d u c e d by the g r o w t h factor. M o r e o v e r , the effect was r e l a t e d to the a c t i v a t i o n of specific e x t r a c e l l u l a r P2 p u r i n o c e p t o r s , since it could b e p r e v e n t e d b y the p u r i n o c e p t o r a n t a g o n i s t s u r a m i n , which, conversely, did n o t affect b F G F - i n d u c e d e l o n g a t i o n of processes. T h e e l o n g a t i o n of G F A P - p o s i t i v e astrocytic processes i n d u c e d b y A T P a n a l o g u e s indicates t h a t G F A P e x p r e s s i o n is likely to b e i n c r e a s e d in these cells. This is in a g r e e m e n t with p r e v i o u s studies o n G F A P e x p r e s s i o n in rat c e r e b r a l cortical astrocytes. N e a r y a n d c o - w o r k e r s 17 s h o w e d t h a t b o t h A T P a n d A D P could e v o k e i n t r a c e l l u l a r calcium m o b i l i z a t i o n , a n d i n c r e a s e d
692
M.P. Abbracchio et al. Table 4. Effect of suramin on bFGF-induced elongation of astrocytic processes in rat striatal cultures Mean length of processes/cell (% of control-+S.E.) Suramin (10 -5 M) bFGF (1 ng/ml) bFGF (1 ng/ml.)+ Suramin (10-~M)
110.7_+8.2 174.1±9.4" 160.6+_8.3 *
Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium, in the absence (control) or presence of 10 -5 M suramin and 1 ng/ml bFGF alone or in combination as indicated. At day 3 in culture, cells were fixed, immunostalned for GFAP and the length of processes/cell were calculated using an image video analysis system (see Experimental Procedures for details). Results are the mean+_S.E, of 50 cells and similar results were obtained in two other experiments. *P<0.0001 with respect to control cells, Student's t-test.
phosphorylation of two astrocytic protein bands, one of which (the 53 kDa protein) co-migrated with GFAP. More recently, these authors confirmed these data by demonstrating that GFAP content was increased by 35-40% in astrocytes exposed to ATP. 18 Increased GFAP expression represents the prototypic marker of astrocytic activation and differentiation in reactive astrogliosis. 7 From a functional point of view, it is conceivable that this event may produce changes of astrocytic morphology assisting wound repair by stabilizing the tissue surrounding neural injuries, leading to a walling off of areas of tissue necrosis and filling in the space resulting from neuronal loss.7 In further support of the idea that GFAP increases in post-traumatic recovery are functionally important, suppression of GFAP expression by antisense mRNA has been found to obliterate the ability of astroglial cells to support neurite regrowth in vitro 10 and to abolish the formation of stable astrocytic processes in response to neuronal signals. 28 Interestingly, when etl3-meATP was added together with bFGF, neither an additive nor synergistic effect between the two agents on the elongation of GFAP-positive processes was found. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded. A quite different interaction between bFGF and ATP was reported by Neary and co-workers 19 on astrocytic D N A synthesis. These authors indeed showed a synergistic activation of astrocytic proliferation by a combination of these two agents. Such qualitatively different results may be related to the use of different experimental protocols in the two studies. A serum-containing medium was used in Neary's study while a chemically defined serum-free medium was used in the present study, which promotes cell differentiation more than proliferation. It is not surprising that, when used in combination, the growth factor and the purine analogue could exert different effects on such different cellular programs. Moreover, three to four-week-old astrocytic cultures were utilized in Neary's study, 19 whereas three-day-old primary cultures were used in the present study. Cell passaging and time in culture have been shown to profoundly affect astrocytic gene expression; 21 differences in culture age may therefore also contribute to different responses to purines and bFGF. At present we do not have an explanation for the inhibitory effect on process outgrowth detected when o~[3-meATP and bFGF were added in combination. Preliminary data from our laboratory suggest that both the growth factor and the purine analogue rapidly induce the expression of the c-fos primary response gene product. This would suggest that both ATP and the growth factor may act as endogenous regulators of the elongation of astrocytic processes by utilizing common intracellular mechanisms. It might be hypothesized that feed-back inhibitory mechanisms are activated when both agents are present, and that such mechanisms are responsible for a reduction of effect. In this respect, we are currently evaluating whether purinoceptors can modulate the synthesis and/or the release of bFGF by astroglial cells, playing a role in a form of autocrine regulation of astrocytic differentiation by this growth factor. Studies on astrocytoma cell lines transfected with sense and antisense mRNA for bFGF 24 are now in progress in our laboratory to test this hypothesis.
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In conclusion, these results suggest that purines can not only regulate astrocytic proliferation 2 but also promote the maturation of astrocytes towards a more differentiated phenotype. Purines can thus act as endogenous regulators of astroglial cell function like growth factors, cytokines and myelin basic proteins, 2° which might have intriguing implications in astroglial cell maturation during development and in reactive astrogliosis. Acknowledgements--Authors wish to thank Dr Pietro Marini for help in image analysis and Dr D. Christie for editorial help. M. J. Saffrey was supported by the Wellcome Trust.
REFERENCES 1. Abbracchio M. P., Ceruti S., Langfelder R., Cattabeni F., Saffrey M. J. and Burnstock G. (1994) Trophic actions of purines. J. Neurochem. 63 (1), $40. 2. Abbracchio M. P., Saffrey M J., Hopker V. and Burnstock G. (1994) Modulation of astroglial cell proliferation by analogues of adenosine and ATP in primary cultures of rat striatum. Neuroscience 59, 67-76. 3. Abbracchio M. P., Ceruti S., Burnstock G. and Cattabeni F. (1994) Purinoceptors on glial cells of the central nervous system: functional and pathological implications. In Adenosine and Adenine Nucleotides: from Molecular Biology to Integrative Physiology (eds Belardinelli L. and Pelleg A.), pp. 271-280. Kluwer Academic, Norwell, MA. 4. Barres B. A. (1991) New roles for gila. J. Neurosci. Res. 11, 3685-3694. 5. Burnstock G. (1990) Co-transmission. The Fifth Heymans Lecture--Ghent, February 17,1990. Arch. Int. Pharmacodyn. Then. 304, 7-33. 6. Burnstock G. (1993) Physiological and pathological roles of purines: an update. Drug Dev. Res. 28, 195-206. 7. Eddleston M. and Mucke L. (1993) Molecular profile of reactive astrocytes--implications for their role in neurologic diseases. Neuroscience 54, 15-36. 8. Foresta C., Rossato M. and Di Virgilio F. (1992) Extracellular ATP is a trigger for the acrosome reaction in human spermatozoa. J. biol. Chem. 257, 19,443-19,447. 9. Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W., Harden K., Jacobson K. A., Left P. and Williams M. (1994) Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46, 143-156. 10. Hatten M. E., Liem R. K. H., Shelanski M. L. and Mason C. A. (1991) Astroglia in CNS injury. Glia 4, 233-243. 11. Hiller S. R., Palmatier B. Y. and Fithian E. M. (1977) Precocious evagination of the embryonic chick thyroid in ATP-containing medium. J. EmbryoL exp. Morphol. 42, 163-175. 12. Hoyle C. H. V., Knight G. E. and Burnstock G. (1990) Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinatory bladder and Taenia coil Br. J. Pharmacol. 99, 617-621. 13. Knudsen T. B. and Elmer W. A. (1987) Evidence for negative control of growth by adenosine in the mammalian embryo: induction of HmX/+ mutant limb outgrowth by adenosine deaminase. Differentiation 33, 270-279. 14. Linden J., Jacobson M. A., Hutchins C. and Williams M. (1994) Adenosine receptors. In Handbook of Receptors and Channels (ed. Peroutka S. J.), pp. 29-43. CRC Press, Boca Raton. 15. Logan A. and Berry M. (1993) Transforming growth factor-131 and basic fibroblast growth factor in the injured CNS. TiPS 14, 337-343. 16. Neary J. T. and Norenberg M. D. (1992) Signalling by extracellular ATP: physiological and pathological considerations in neuronal-astrocytic interactions. Prog. Brain Res. 94, 145-151. 17. Neary J. T., Laskey R., van Breemen C., Blicharska J., Norenberg L. O. B. and Norenberg M. D. (1991) ATP-evoked calcium signal stimulates protein phosphorylation/dephosphorylation in astrocytes. Brain Res. 566, 89-94, 18. Neary J. T., Baker L., Jorgensen S. L. and Norenberg M. D. (1993) Extracellular ATP induces steUation and increases glial fibrillary acidic protein content and DNA synthesis in primary astrocytic cultures. Acta Neuropathol. Berlin 87, 8-13. 19. Neary J. T., Whittemore S. R., Zhu Q. and Norenberg M. D. (1994) Synergistic activation of DNA synthesis in astrocytes by fibroblast growth factors and extracellular ATP. J. Neurochem. 63, 490-494. 20. Norton W. T., Aquino D. A., Hozumi I., Chiu F.-C. and Brosnan C. F. (1992) Quantitative aspects of reactive gliosis: a review. Neurochem. Res. 17, 877-885. 21. Passaquin A.-C., Schreier W. A. and De Vellis J. (1994) Gene expression in astrocytes is affected by subculture. Int. J. Devl Neurosci. 12, 363-372. 22. Rakic P. (1991) Glial cells in development. Ann. N.Y. Acad. Sci. 633, 96-99. 23. Rathbone M. P., DeForge S., DeLuca B., Gabel B., Laurenssen C., Middlemiss P. and Parkinson S. (1992) Purinergic stimulation of cell division and differentiation: mechanisms and pharmacological implications. Med. Hypoth. 37, 213-219. 24. Redekop G. J. and Naus C. C. G. (1995) Transfection with bFGF sense and antisense cDNA resulting in modification of malignant glioma growth. J. Neurosurg. 82, 83-90. 25. Rudolphi K. A., Schubert P., Parkinson F. E. and Fredholm B. B. (1992) Adenosine and brain ischemia. Cerebrovasc. Brain Metab. Rev. 4, 346-369. 26. Smuts M. S. (1981). Rapid nasal pit formation in mouse embryos stimulated by ATP-containing medium. J. exp. Zool. 216, 409-414. 27. Voogd T. E., Vansterkenburg E. L. M., Wilting J. and Janssen L. H. M. (1993) Recent research on the biological activity of suramin. Pharmacol. Rev. 45, 177-203. 28. Weinstein D. E., Shelanski M. L. and Liem R. (1991) Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons. J. Cell Biol. 112, 1205-1213. DN 13-7-B
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EFFECTS OF ATP A N A L O G U E S A N D BASIC FIBROBLAST GROWTH FACTOR ON ASTROGLIAL CELL DIFFERENTIATION IN PRIMARY CULTURES OF RAT STRIATUM M. P. A B B R A C C H I O , * S. C E R U T I , * R. L A N G F E L D E R , * F. C A T T A B E N I , * M. J. S A F F R E Y t ~ t and G. B U R N S T O C K t § *Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy: ?Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, U.K. (Received 21 February 1995; revised 1 June 1995;accepted 1 June 1995)
Abstraet--We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and a[3-methylene-ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose-dependent. The effect of e~13-methylene-ATPwas antagonized by the trypanoside suramin and mimicked by 2-methyl-thio-ATP, suggesting the involvement of a suramin-sensitive P2-purinoceptor. Neither an additive nor a synergistic effect between al3-methylene-ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways. It is concluded that, like basic fibroblast growth factor, ATP can promote the maturation of astrocytes towards a more differentiated phenotype characterized by the presence of longer astrocytic processes. These findings might have interesting implications for astroglial cell differentiation during brain development and for ischemia- and trauma-associated hypergliosis. Keywords: astroglial cells, ATP, reactive astrogliosis.
Glial cells m a k e up a substantial p r o p o r t i o n of the central n e r v o u s system. Within glia, astrocytes represent the m o s t n u m e r o u s cell type and play key roles in n o r m a l brain function, 4 d e v e l o p m e n t 3,22 and in the p a t h o l o g y o f the n e r v o u s system. 7 A s t r o c y t e s react to various types of injury with astrogliosis, a reaction characterized by b o t h increased proliferation and cellular h y p e r t r o p h y . 7,1° P r o m i n e n t reactive astrogliosis is seen after acute traumatic brain injury, i n f l a m m a t o r y demyelinating disease, A I D S d e m e n t i a and o t h e r viral infections, and such n e u r o d e g e n e r a t i v e diseases as A l z h e i m e r ' s disease. 7 Characterization of the e n d o g e n o u s factors which regulate astroglial cell function has t h e r e f o r e i m p o r t a n t implications in n e r v o u s system pathophysiology. G r o w i n g evidence supports a role for purines ( n a m e l y A T P and adenosine) as trophic factors and e n d o g e n o u s regulators of cell g r o w t h a n d differentiation in b o t h d e v e l o p m e n t and adulthood. ~ Purines are involved in egg fertilization, 8 e m b r y o n i c m o r p h o g e n e s i s 13 and organogenesis. 11,26 Via activation o f specific p u r i n o c e p t o r subtypes, purines regulate n e u r o n a l function and neurotransmission in b o t h central a n d peripheral n e r v o u s systems, 5,6,14,25 A role in m o d u l a t i o n of astroglial cell function has b e e n m o r e recently suggested. 2,3,16,23 W e have previously d e m o n s t r a t e d that analogues of b o t h adenosine and A T P can regulate the n u m b e r of astroglial cells in culture p r o b a b l y by influencing their proliferation rate. 2 H o w e v e r , besides cell proliferation, a k e y role for astrocytic h y p e r t r o p h y in the initiation and m a i n t e n a n c e of astrogliosis has b e e n e m p h a s i z e d recentlyJ ° Increases o f cellular size, increased staining for glial fibrillary acidic p r o t e i n ( G F A P ) and the presence of l o n g e r and thicker astrocytic processes are taken as an index of the astroglial cell differentiation k n o w n to occur in every type of astrogliosis, even in the absence of astrocytic proliferation (e.g. fetal anoxia). 1° §To whom all correspondence should be addressed. ~Present address: Department of Biology, The Open University, Walton Hall, Milton Keynes, MK7 6AA, U.K. Abbreviations: a[3-meATP, c~13-methylene-ATP;bFGF, basic fibroblast growth factor; FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; HBSS, Hank's balanced sale solution; 2meS-ATP, 2-methylthio-ATP; PBS, phosphate-buffered saline. 685
686
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On these bases, the present study was aimed at evaluating the effects of purine analogues on the elongation of GFAP-positive astrocytic processes in the same experimental model (neuron-glia primary cultures from neonatal rat c. striatum) which has previously been utilized for the proliferative studies. 2 Based on preliminary observations suggesting induction of 'stellation' and increased astrocytic branching after application of the relatively hydrolysis-resistant ATP analogue cx[3-methylene-ATP (al3-meATP), this study focussed on ATP derivatives. Moreover, since increased GFAP expression has been demonstrated to occur following exposure to a number of growth factors such as, for example, basic fibroblast growth factor (bFGF), 15 the morphological effects induced by purine analogues on cultured astrocytes have been compared with those induced by bFGF.
EXPERIMENTAL PROCEDURES
Cultures Cultures were prepared by a slight modification of the previously described method. 2 Briefly, after rapid decapitation, the brains of three or four seven-day-old Sprague-Dawley rats were removed under sterile conditions, the corpus striata were transferred to Hank's balanced salt solution (HBSS) supplemented with 0.6% glucose and gently chopped into small pieces using a sterile razor blade; pieces were then washed three times in HBSS without calcium and magnesium (buffer 1). Ten milliliters of a solution of 0.125% trypsin in buffer 1 containing 10 ~g/ml DNAase I were added to the washed tissue, which was incubated at 37°C for 45 min. After four washes with 5 ml of buffer 1 supplemented with 8 mM MgCI 2, 10% fetal calf serum (FCS) and 10 wg/ml DNAase I, the tissue was mechanically dissociated into single cells in 5 ml of the same solution containing 20 i~g/ml DNAase I, using a sterile pipette. Dissociated cells were centrifuged at 800 rpm for 10 rain and the pellet was resuspended in 2 ml of medium 199 supplemented with 10% FCS and 5 mg/ml glucose. The total number of cells was determined in a Burke chamber using the Trypan Blue dye exclusion test. The cell suspension was diluted with medium 199+10% FCS and 5 mg/mi glucose to a final concentration of 1.9×105 viable cells/mL Three hundred and sixty microliters of the cell suspension were inoculated into each well of 24-well dishes (each containing a poly-L-lysine-coated glass coverslip), followed by 360 ~1 of medium 199 + 10% FCS + 5 mg/ml glucose and 80 ~1 of buffer 1 containing either ot[3-meATP, 2-methylthio-ATP (2meS-ATP) or bFGF at various concentrations, or different combinations of the various agents as indicated in the legends to the figures and tables. A final volume of 800 p,l/well was used both in control cultures and in cultures treated with two agents together by increasing or reducing, respectively, the volume of medium added to the well. Cultures were then maintained in a 5% CO 2 incubator at 37°C. This first incubation with FCS was done to favour attachment of dissociated cells to the dishes. After 24 h, the serum-supplemented medium was replaced with a chemically defined serum-free medium, containing 2×10 -8 M progesterone, 0.12 U.I./ml insulin, 3×10 -8 M sodium selenite, 5 wg/ml transferrin, 100 I~M putrescine, 0.08% bovine serum albumin (BSA) and 5 mg/ml glucose, in the absence (control cultures) or presence of the various agents. In selected experiments the action of the trypanoside suramin (an antagonist of P2 purinoceptors), alone or in combination with ot[3-meATP or bFGF, was tested.
Immunofluorescence staining and analysis of GFAP-positive processes. After 72 h in culture, cells were fixed with methanol at -20°C for 10 min, and then washed three times (10 min each) with phosphate-buffered saline (PBS). For evaluation of astroglial process formation, astrocytes were labelled by indirect immunofluorescence using rabbit anti-GFAP immunoglobulins (1:500; overnight incubation at room temperature). Cells were then washed three times (10 min each) with PBS and incubated with biotinylated donkey anti-rabbit secondary antibody (1:250; 1 h at room temperature), followed by streptavidin-fluorescein (1:100; 1 h at room temperature). All antibodies were diluted in antibody diluting solution containing 0.1% Triton X-100, 0.1% sodium azide and 0.01% bovine serum albumin in PBS. After three final washes with PBS, coverslips were mounted in Immumount and labelled cells examined using a Zeiss fluorescence microscope equipped with a fluorescein filter. The length and number of GFAP-positive
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astrocytic processes was measured using an image video system consisting of a Macintosh computer (equipped with NIH Image 1.47 software) and a video camera connected to the microscope. Values for the total number of GFAP-positive processes/cell, the total length of processes/cell and the mean length/process were obtained from 50 cells/condition. Statistical analysis was performed on the data obtained from two different experiments (100 cells/condition) with unpaired Student's t-test.
Materials Sprague-Dawley rats were obtained from Charles River (Italy). HBSS, HBSS without calcium and magnesium, FCS, Medium 199, PBS tablets and 20% glucose solution were purchased from Gibco BRL, Life Technologies (European Division). Trypsin, DNAase 1, od3-meATP, 2meS-ATP, human recombinant bFGF, progesterone, insulin, sodium selenite, transferrin, putrescine, BSA, sodium azide and paraformaldehyde were obtained from Sigma Chemicals Co. (U.S.A.), while suramin was obtained from Bayer (Germany). Cultures were grown in 24-well dishes obtained from Nunc. Rabbit primary antibody against GFAP was purchased from D A K O A/S (Denmark); biotinylated donkey anti-rabbit secondary antibody and streptavidin-fluorescein were purchased from Amersham, Life Science (U.K). Coverslips were mounted in Immumount, purchased from Shandon Italscientifica (Italy).
RESULTS
Effects of bFGFand A TP analogues on the elongation of GFAP-positive astrocytic processes Both bFGF and a[3-meATP induced dramatic morphological changes of astrocytes, characterized by the presence of long and thick astrocytic processes which stained strongly for GFAP (Fig. 1). The quantitative analysis of these effects is shown in Tables 1 and 2. Exposure of cells to the growth factor produced a marked and dose-dependent increase of the mean length of processes/cell with maximal effects at a concentration of 1 ng/ml. No differences were detected in the mean number of processes/cell with respect to control cultures (Table 1). Table 2 shows the same parameters in cultures treated with al3-meATP. Similarly to bFGF, the ATP analogue induced a dose-dependent increase of the mean length of processes/cell, without affecting the number of GFAP-positive processes/cell. Effects were maximal at 10 -5 M, still maintained at higher concentrations and quantitatively comparable with the maximal effect obtained with bFGF. Details of the effects induced by bFGF and c~13-meATP on the expression of GFAP-positive astrocytic processes are shown in Figs 2 and 3. In these figures, the frequency distribution of GFAP-positive cells with different mean process length is shown. It is evident that in control cultures the majority of cells belonged to sub-populations with mean length of processes up to 150 ixm. Exposure to increasing bFGF concentrations induced a dose-dependent shift of cell distribution towards populations with longer processes (Fig. 2). For example, in control cultures, about 70 cells/100 had a mean length of processes between 0 and 150 ~m, whereas only 30 cells/100 had these characteristics in cultures exposed to 1 ng/ml bFGF. Conversely, almost no control cell had processes longer than 300 p.m, whereas a progressively higher percentage of bFGF-treated cells displayed processes in the 301-500 Ixm ranges. Comparable results were obtained with al3-meATP, which induced a similar shift towards cell populations characterized by higher astrocytic lengths (Fig. 3). For the purine analogue, maximal effects were obtained at 10 -5 M, whereas at 5 × 10 -5 M a slight reduction of effect was evident. In order to verify if the simultaneous exposure of cultures to bFGF and oL[3-meATP could synergistically affect astroglial cell differentiation, we grew cells in the presence of both agents. An example of these experiments is shown in Fig. 4 for the combination of the three concentrations of a[3-meATP with 0.5 ng/ml bFGF. Neither synergism nor additivity between oLI3-meATP and bFGF were detected; on the contrary, a reduction of process length was evident in comparison with the two agents alone. Similar results were also obtained for all the other possible combinations of a[3-meATP and bFGF (data not shown). In order to determine if other purine analogues could modulate the elongation of GFAP-positive processes in our system, in another set of experiments we grew cells in the presence of 2meS-ATP.
688
M . P . A b b r a c c h i o et al.
Fig. 1. Immunofluorescence micrographs showing GFAP-positive cells in control cultures (A) and in cultures grown in the presence of 1 ng/ml bFGF (B) or in the presence of 10 -5 M c~13-meATP (C). Note the dramatic morphological changes (extension of longer and thicker processes) induced by the two agents. Scale bar=50 p.m.
ATP and astroglial cell differentiation
689
Table 1. Effect of bFGF on GFAP-positive astrocytic processes in rat striatal cultures
Control cells
bFGF bFGF bFGF bFGF bFGF
(0.01 ng/ml) (0.I ng/ml) (0.5 ng/ml) (1 ng/ml) (10 ng/ml)
Mean length of processes/cell
Mean number of processes/cell
(p~m-+S.E.)
(No.-+S.E.)
131.63_+7.47 171.22-+8.62" 184.98_+ 17.05~ 196.12-+9.92~: 199.95-+ 10.43~: 189.53_+ 10.36~
1.98_+0.09 2.03_+0.08 1.93-+0.08 2.04_+0.10 2.15-+0.10 1.98_+0.10
Rat striatal primary cultures were grown for 24 h in complete medium and then for an additional 48 h in chemically defined serum-free medium, in the absence (control) or in the presence of the indicated concentrations of bFGF. At day 3 in culture, cells were fixed, immunostained for GFAP and the length and number of astrocytic processes were measured using an image video analysis system (see Experimental Procedures for details). Results are the mean-+S.E, of 100 cells/condition, obtained from two different experiments. Similar results were obtained in three other experiments. *P<0.002; tP<0.006; $P<0.001 with respect to control cells, Student's t-test.
Table 2. Effect of ctl3-meATP on GFAP-positive astrocytic processes in rat striatal cultures
Control cells
al3-meATP (5× 10 -6 M) al3-meATP (10 -5 M) c~13-meATP (5 x 10-ZM)
Mean length of processes/cell
Mean number of processes/cell
(p,m-+S.E.)
(No._+S.E.)
131.63---7.47 154.26-+7.12" 189.84-+9.51t 177.26-+ 9.30t
1.98-+0.09 2.06-+0.09 2.01-+0.09 1.92_ + 0.07
Rat striatal primary cultures were grown for 24 h in complete medium and then for an additional 48 h in chemically defined serum-free medium, in the absence (control) or in the presence of the indicated concentrations of al3-meATP. At day 3 in culture, cells were fixed, immunostained for GFAP and the length and number of astrocytic processes were measured using an image video analysis system (see Experimental Procedures for details). Results are the mean-+ S.E. of 100 cells/condition, obtained from two different experiments. Similar results were obtained in three other experiments. *P<0.03 tP<0.001 with respect to control cells, Student's t-test.
This molecule is less resistant to hydrolysis than al3-meATP but is considered a more selective agonist at P2Y-purinoceptors with respect to a cxl3meATP. 9 Exposure of cultures to 10 -5 M 2meS-ATP (the maximally effective concentration for otl3-meATP) resulted in a significant increase of the mean length of processes, although this increase seemed lower with respect to that induced by af3-meATP (a 24.5_+7% increase with respect to the 44_+7% increase induced by the same concentration of etl3-meATP, mean of three experiments run in triplicate, P<0.02 with respect to control cells). As with the other agents tested, no difference in the mean number of processes/cell was observed in the 2meS-ATP-treated cultures with respect to control cells (data not shown).
Effect of suramin on ot[5-meA TP- and bFGF-treated cultures In order to verify whether the effects induced by txl3-meATP could be related to the activation of extracellular receptors, we exposed cells to both the purine analogue and the trypanoside suramin, which is known to act as an antagonist at both P2X and P2Y purinoceptors. 12 Suramin alone (10 -5 M) did not significantly affect the length of processes (Table 3), while, in the same set of experiments, 10 -5 M oLI3-meATP produced an increase of length of about 35% with respect to control cultures. Exposure to both agents completely prevented the action of the ATP analogue (Table 3), thus suggesting the involvement of a suramin-sensitive P2 purinoceptor. The possibility that suramin may interfere with the action of bFGF has been reported by other authors. 27 We therefore tested this possibility in our system. As expected, exposure of cultures to bFGF induced a 74% increase of process length with respect to control cultures (Table 4); suramin did not affect either the length of processes p e r s e or block the action of the growth factor when the two were added in combination, therefore ruling out any interaction of suramin with the activities of bFGF under our experimental conditions.
690
M . P . A b b r a c c h i o et al.
40
• control W bFGFj1 ng/ml 30 0
20
'a ,,Q E ,.,¢ z
10
0
o
~
0
0
0
°
o
g
~
N
0
0
o
0
~
N
V~
Fig. 2. Effects of bFGF on astroglial cells in culture. Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium in the absence (control) or presence of the indicated concentration of bFGF. At day 3, cells were fixed, immunostained for GFAP and the total length of processes/cell was evaluated using an image video analysis system. Cells were then grouped according to mean length of processes in the 0-100, 101-150, 151-200, etc. ranges as indicated on the x axis. The distribution of cell populations for control cultures (black histograms) and for cultures exposed to 1 ng/ml bFGF are shown; intermediate effects were obtained using lower (0.01 and 0.1 ng/ml) concentrations of the growth factor. For data shown, S.E. was, in all cases, lower than 10%. Data refer to two experiments which were analysed in detail by utilizing the above-described method; however, a similar pattern of distribution for cell populations was obtained in three other independent experiments.
40
W @ O
• control [ ] (zJ3-meATP 10 -5 M
30
"6 .Q E
20
z
10
0
Fig. 3. Effects of cq3-meATP on astroglial cells in culture. Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium in the absence (control) or presence of the indicated concentration of al3-meATP. At day 3, cells were fixed, immunostained for GFAP and the total length of processes/cell was evaluated using an image video analysis system. Cells were then grouped according to mean length of processes in the 0-100, 101-150, 151-200, etc. ranges as indicated on the x axis. The distribution of cell populations for control cultures (black histograms) and for cultures exposed to 10 -5 M al3-meATP are shown; proportional effects were obtained by using other aI3-meATP concentrations. For data shown, S.E. was, in all cases, lower than 10%. Two experiments were analysed in detail by utilizing the above-described method; however, a similar pattern of distribution for cell populations was obtained in three other independent experiments. DISCUSSION Adenosine and ATP have previously been shown to affect the proliferative response of astrocytes i n c u l t u r e . 2,23 I n t h i s s t u d y , w e h a v e i n v e s t i g a t e d w h e t h e r p u r i n e a n a l o g u e s c a n a l s o m o d u l a t e another important parameter of astrocytic activation: the formation of GFAP-positive astrocytic
A T P and astroglial cell differentiation
691
160-140 120
100 80 v
60 ..d 40 20
Control
al~-rneATP 10-5 M
bFGF + ctl~-meATP
bFGF
10-5 M ct~-meATP
ct[3-meATP
5 x 10-6 M
5 x 10-5 M
bFGF ÷ II~-meATP 5x IO-6M
bFGF + al~-meATP 5xlO-SM
Fig. 4. Effect of the exposure to both al3-meATP and bFGF on the length of astrocytic processes in striatal cultures. Cells were grown in the presence of al3-meATP at the indicated concentrations alone or in combination with bFGF (0.5 ng/ml). After 72 h in culture, cells were fixed, immunostained for GFAP and the mean length of processes/ceU (reported in the y axis as percentage of control) was calculated using an image video analysis system. **P<0.02; ***P<0.004 with respect to bFGF alone, Student's t-test.
Table 3. Effect of suramin on c~13-meATPinduced elongation of astrocytic processes in rat striatal cultures Mean length of processes/cell (% of control_+S.E.) Suramin (10-5 M) c~13-meATP(10-~M) al3-meATP (10-5 M)+ Suramin (10-5 M)
111.5_+8.7 152.3_+9" 113.3_+9.6
Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium, in the absence (control) or presence of 10-5 M suramin and 10-5 M ~13meATP alone or in combination as indicated. At day 3 in culture, cells were fixed, immunostained for GFAP and the length of processes/cells were calculated using an image video analysis system (see Experimental Procedures for details). Results are the mean-S.E, of 50 cells and similar results were obtained in two other experiments. *P<0.02 with respect to control cells, Student's t-test. processes. This p a r a m e t e r c a n b e t a k e n as a m a r k e r of the astrocytic h y p e r t r o p h y a n d f u n c t i o n a l d i f f e r e n t i a t i o n which is k n o w n to occur in reactive astrogliosis. 7 T h e p r e s e n t results show that, like k n o w n r e g u l a t o r s of astroglial cell f u n c t i o n such as b F G F , b o t h a l 3 - m e A T P a n d 2 m e S - A T P can i n d u c e e l o n g a t i o n of astrocytic processes. F o r etl3-meATP, the effect was d o s e - d e p e n d e n t a n d q u a n t i t a t i v e l y c o m p a r a b l e with that i n d u c e d by the g r o w t h factor. M o r e o v e r , the effect was r e l a t e d to the a c t i v a t i o n of specific e x t r a c e l l u l a r P2 p u r i n o c e p t o r s , since it could b e p r e v e n t e d b y the p u r i n o c e p t o r a n t a g o n i s t s u r a m i n , which, conversely, did n o t affect b F G F - i n d u c e d e l o n g a t i o n of processes. T h e e l o n g a t i o n of G F A P - p o s i t i v e astrocytic processes i n d u c e d b y A T P a n a l o g u e s indicates t h a t G F A P e x p r e s s i o n is likely to b e i n c r e a s e d in these cells. This is in a g r e e m e n t with p r e v i o u s studies o n G F A P e x p r e s s i o n in rat c e r e b r a l cortical astrocytes. N e a r y a n d c o - w o r k e r s 17 s h o w e d t h a t b o t h A T P a n d A D P could e v o k e i n t r a c e l l u l a r calcium m o b i l i z a t i o n , a n d i n c r e a s e d
692
M.P. Abbracchio et al. Table 4. Effect of suramin on bFGF-induced elongation of astrocytic processes in rat striatal cultures Mean length of processes/cell (% of control-+S.E.) Suramin (10 -5 M) bFGF (1 ng/ml) bFGF (1 ng/ml.)+ Suramin (10-~M)
110.7_+8.2 174.1±9.4" 160.6+_8.3 *
Cells were grown for 24 h in complete medium and then for an additional 48 h in serum-free medium, in the absence (control) or presence of 10 -5 M suramin and 1 ng/ml bFGF alone or in combination as indicated. At day 3 in culture, cells were fixed, immunostalned for GFAP and the length of processes/cell were calculated using an image video analysis system (see Experimental Procedures for details). Results are the mean+_S.E, of 50 cells and similar results were obtained in two other experiments. *P<0.0001 with respect to control cells, Student's t-test.
phosphorylation of two astrocytic protein bands, one of which (the 53 kDa protein) co-migrated with GFAP. More recently, these authors confirmed these data by demonstrating that GFAP content was increased by 35-40% in astrocytes exposed to ATP. 18 Increased GFAP expression represents the prototypic marker of astrocytic activation and differentiation in reactive astrogliosis. 7 From a functional point of view, it is conceivable that this event may produce changes of astrocytic morphology assisting wound repair by stabilizing the tissue surrounding neural injuries, leading to a walling off of areas of tissue necrosis and filling in the space resulting from neuronal loss.7 In further support of the idea that GFAP increases in post-traumatic recovery are functionally important, suppression of GFAP expression by antisense mRNA has been found to obliterate the ability of astroglial cells to support neurite regrowth in vitro 10 and to abolish the formation of stable astrocytic processes in response to neuronal signals. 28 Interestingly, when etl3-meATP was added together with bFGF, neither an additive nor synergistic effect between the two agents on the elongation of GFAP-positive processes was found. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded. A quite different interaction between bFGF and ATP was reported by Neary and co-workers 19 on astrocytic D N A synthesis. These authors indeed showed a synergistic activation of astrocytic proliferation by a combination of these two agents. Such qualitatively different results may be related to the use of different experimental protocols in the two studies. A serum-containing medium was used in Neary's study while a chemically defined serum-free medium was used in the present study, which promotes cell differentiation more than proliferation. It is not surprising that, when used in combination, the growth factor and the purine analogue could exert different effects on such different cellular programs. Moreover, three to four-week-old astrocytic cultures were utilized in Neary's study, 19 whereas three-day-old primary cultures were used in the present study. Cell passaging and time in culture have been shown to profoundly affect astrocytic gene expression; 21 differences in culture age may therefore also contribute to different responses to purines and bFGF. At present we do not have an explanation for the inhibitory effect on process outgrowth detected when o~[3-meATP and bFGF were added in combination. Preliminary data from our laboratory suggest that both the growth factor and the purine analogue rapidly induce the expression of the c-fos primary response gene product. This would suggest that both ATP and the growth factor may act as endogenous regulators of the elongation of astrocytic processes by utilizing common intracellular mechanisms. It might be hypothesized that feed-back inhibitory mechanisms are activated when both agents are present, and that such mechanisms are responsible for a reduction of effect. In this respect, we are currently evaluating whether purinoceptors can modulate the synthesis and/or the release of bFGF by astroglial cells, playing a role in a form of autocrine regulation of astrocytic differentiation by this growth factor. Studies on astrocytoma cell lines transfected with sense and antisense mRNA for bFGF 24 are now in progress in our laboratory to test this hypothesis.
ATP and astroglial cell differentiation
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In conclusion, these results suggest that purines can not only regulate astrocytic proliferation 2 but also promote the maturation of astrocytes towards a more differentiated phenotype. Purines can thus act as endogenous regulators of astroglial cell function like growth factors, cytokines and myelin basic proteins, 2° which might have intriguing implications in astroglial cell maturation during development and in reactive astrogliosis. Acknowledgements--Authors wish to thank Dr Pietro Marini for help in image analysis and Dr D. Christie for editorial help. M. J. Saffrey was supported by the Wellcome Trust.
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