Glutamate stimulates the phosphorylation of glial fibrillary acidic protein in slices of immature rat hippocampus via a metabotropic receptor

Neurochem. Int. Vol. 24, No. 6, pp. 517 523, 1994

~

Pergamon

0197-0186(94)E0036-S

Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0197-0186/94 $7.00+0.00

GLUTAMATE STIMULATES THE PHOSPHORYLATION OF GLIAL FIBRILLARY ACIDIC PROTEIN IN SLICES OF IMMATURE RAT HIPPOCAMPUS VIA A METABOTROPIC RECEPTOR* SUSANA T. WOFCHUK a n d RICHARD RODNIGHT t Departamento de Bioquimica, Instituto de Biociencias UFRGS (Centro), 90.050 Porto Alegre, RS, Brazil (Received 9 November 1993 ; accepted 2 February 1994)

Abstract--Phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats (10-16 days postnatal) was strongly stimulated by glutamate in the presence of Ca 2÷. This effect apparently occurred via a metabotropic receptor since the specific agonist of metabotropic glutamate receptors, IS,3R-l-aminocyclopentane-l,3-dicarboxylicacid (IS,3R-ACPD), stimulated GFAP phosphorylation by 173% whilst the mixed agonists, ibotenate and quisqualate, stimulated to a lesser extent. Ionotropic agonists were mainly ineffective. The action of 1S,3R-ACPD was blocked by L(+)-2-amino-3-phosphonopropionic acid (L-AP3) a specific antagonist of the metabotropic glutamate receptor coupled to the hydrolysis of phosphoinositides and was reduced by 70% by preincubation of the slices with pertussis toxin. In contrast to these results with immature animals glutamate had little or no effect on the phosphorylation of GFAP in hippocampal slices from adult rats.

A protein phosphorylation system characteristically active in incubated slices of the rat hippocampus has as its substrate a detergent-insoluble phosphoprotein originally designated ppH-47 (Gongalves et al., 1990; Rodnight and Leal, 1990; Rodnight et al., 1991) and recently identified as a highly phosphorylated form of the astrocyte cell marker glial fibrillary acidic protein (GFAP) (Gongalves and Rodnight, 1992). To maintain continuity we will use the term G F A P / p p H - 4 7 when referring to this protein. Phosphorylation of G F A P / p p H - 4 7 was found to be strongly stimulated by glutamate in hippocampal slices from immature rats in the age range 11-16 days

postnatal in a reaction apparently dependent on external Ca 2+ (Wofchuk and Rodnight, 1990). This observation was of particular interest in view of the now considerable literature on the presence of glutamate receptors in astrocytes (Pearce and Murphy, 1988; Backus et al., 1989 ; Teichberg, 1991 ; Blankenfeld and Kettenmann, 1991) and on the action of glutamate and its agonists in regulating Ca-entry and mobilization from internal stores in cultured astrocytes (Pearce et al., 1986; Enkvist et al., 1989; Glaum et al., 1990 ; Ahmed et al., 1990 ; Cornell-Bell et al., 1990 ; Jensen and Chiu, 1990, 1991 ; Barres, 1991 ; Usowicz et al., 1991; Inagaki et al., 1991; Cornell-Bell and Finkbeiner, 1991 ; Charles et al., 1991 ; Van den Pol et al., 1992). In this paper we examine the nature of stimulation of the phosphorylation of GFAP/ppH-47 by glutamate and show that this apparently occurs via a metabotropic receptor.

*Part of this work was presented at the Excitatory Amino Acid Neurotransmission meeting, Marseilles, France, 29 August-2 September 1993, organized by Max R6casens. tAuthor to whom all correspondence should be addressed : Richard Rodnight, Departamento de Bioquimica, Instituto de Biociencias, UFRGS (Centro), Rua Sarmento Leite 500, 90.050 Porto Alegre, RS, Brazil. Abbreviations : GFAP, glial fibrillary acidic protein ; NMDA, N-methyl-D-aspartic acid ; 1S,3R-ACPD, (1S,3 R) - 1-aminocyclo-pentane-l,3-dicarboxylicacid; AMPA, (RS)-ct- Radiochemicals and compounds amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ; [32P]Na2HPO4 was purchased from CNEN, Sao Paulo, PLC, phospholipase C ; DNQX, 6,7-dinitroquinoxaline- Brazil. Acrylamide (product A8887), bis-acrylamide, L-glu2,3-dione. tamic acid, N-methyl-D-aspartate (NMDA), kainate and 517

EXPERIMENTAP LROCEDURES

518

~USANA T. WOFCHUK and RICHARD RODNIGHT

ibotenate were obtained from Sigma (St Louis, MO, U.S.A.). Quisqualate, (RS)-~-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), (IS,3R)-I-aminocyclopentane1,3-carboxylic acid (IS.3R-ACPD) and l(+)-2-amino-3phosphono-propionic acid (L-AP3), were purchased from Tocris Neuramin (Bristol, U.K.). Pertussis toxin came from List Biological Laboratories (Campbell, CA. U.S.A.). The toxin was activated immediately before use by incubation with 50 mM dithiothreitol (DTT) for 20 min at 37 C accordingloBerti Mattera el a/. (1992). 4 nimal.~ Wistar rats of either sex in the age range 11 16 days postnatal ("immature") or 60 90 days C'adult '') came from the local breeding colony. Preparation and labellinq slices Rats were killed by decapitation and the hippocampus dissected on ice within 3 rain. Slices (0,4 mm) were cut with a McIIwain chopper, trimmed to identical sizes and labelled with 32p essentially as previously described (Rodnight et al., 1988: Rodnight and Leal, 1990). Adjacent slices were used for test and control situations. In most experiments 16 slices were prepared and were incubated and analysed in the following order, where C = control and T = test: CTTCCTTCCTTCCTTC. The basic medium contained 124 mM NaCI, 4 mM KCI, 1.2 mM MgSO4, 25 mM Na HEPES (pH 7.4), 12 mM glucose, 1 mM CaC12 and was gassed with 02. Slices were pre-incubated in 100 pl of basic medium at 25 C tk)r 45 min, which was then replaced with 50 #1 of medium containing 40/tCi of [32]phosphate with or without additions. In some experiments agents were added to both pre-incubation and incubation media. The labelling reaction was normally allowed to proceed for 1 h at 30 C and stopped with I ml of 10% trichloroacetic acid. After a minimum of 10 min in ice, slices were washed twice by decantation with 4% trichloroacetic acid to remove excess radioactivity, briefly with water to remove acid and then immediately dissolved in first dimension electrophoresis buffer as described previously (Rodnight et al., 1988). Quisqualate and ibotenate were dissolved in dimethylsulphoxide and diluted to the appropriate concentration with medium. L-AP3 and pertussis toxin were added to the pre-incubation and incubation media. Whenever dimethylsulphoxide was used to dissolve reagents an equivalent concentration was included in the control tubes. Media lacking Ca "-~ contained t mM EGTA. Two-dimensional eh, ctrophoresis The procedure described previously was employed (Rodnight et al., 1988). Nonequilibrium pH gradient electrophoreis (NEPHGE) was used for the first dimension and 8% (T;C,2.5%) polyacrylamide slab gels for the second dimension. These were prepared in batches of 8 in a casting box. Two first dimension rod-gels--one control and one test were mounted as mirror images with the positive end in the centre on one second dimension slab gel. This procedure was adopted to minimize inter-gel variation during electrophoresis. A typical separation is shown in Fig. I. Quan t!lication of GFA P/ppH-47 phosphorylation After drying gels were exposed to X-ray film (X-Omat XKI) at - 7 0 ' C with intensifying screens. Assessment was made by densitometric scanning of the autoradiographs and

measuring peak heights. No advantage was gained by measuring peak areas, as GFAP/ppH-47 always migrated as a dense round spot (see Fig. 1). Variation in peak height between the 8 controls in a typical experiment was less than :+ 10% of the mean. On each gel, containing one control and one test sample, the peak heights of the control spots were normalized to 100% and the percentage difference from the test spot calculated. Data were analysed statistically by a paired t-test, values from each get being treated as one pair. Only films from sample pairs showing equal overall labelling were scanned; on the average t in 10 films were discarded for this reason,

RESULTS

Slice preservation u n d e r the micro-conditions of incubation was excellent as shown by A T P levels ( 1.25 itmol/g/tissue after 30 min incubation-- see R o d n i g h t et al., 1988) a n d by the satisfactory protein phosp h o r y l a t i o n p a t t e r n s o b t a i n e d in slices from b o t h i m m a t u r e and adult animals. Thus m a j o r kinase substrates such as synapsin 1, M A R C K S , B - 5 0 / G A P 43 a n d D A R P P - 3 2 as well as n u m e r o u s unidentified substrates were labelled at b o t h ages. I n c o r p o r a t i o n of 32p into G F A P / p p H - 4 7 increased markedly with age presumably due to increased synthesis of the substrate molecule. To investigate the nature of the effect o f glutamate in stimulating the p h o s p h o r y l a t i o n of G F A P / p p H - 4 7 nearly 3-fold in i m m a t u r e h i p p o c a m p a l slices (Wofchuk a n d Rodnight, 1990) we tested k n o w n agonists of glutamate receptors, initially at a c o n c e n t r a t i o n of 1 m M . N M D A a n d A M P A h a d no effect on G F A P / p p H - 4 7 p h o s p h o r y l a t i o n , kainate gave a small stimulation which just reached statistical significance, the mixed agonist ibotenate stimulated 1.84old and I S,3R-ACPD, a selective agonist of glutamatergic m e t a b o t r o p i c receptors, stimulated the system to the same extent as that given previously by glutamate (Table, 1 ; Fig. 1). Quisqualate, a n o t h e r mixed agonist, also stimulated G F A P / p p H - 4 7 p h o s p h o r y l a t i o n , but only at a c o n c e n t r a t i o n of 50/~M, suggesting that the toxicity o f 1 m M quisqualate prevented the response. H a l f maximal stimulation by 1S,3R-ACPD required a c o n c e n t r a t i o n of approx. 20 # M (Fig. 2). The effect of 100 /~M 1S,3R-ACPD was completely blocked by p r e - i n c u b a t i o n o f slices with 1 m M L-AP3, an inhibitor of glutamatergic m e t a b o t r o p i c receptors a n d was inhibited by p r e - i n c u b a t i o n with activated pertussis toxin (Table 2). D N Q X , a n a n t a g o n i s t of ionotropic n o n - N M D A glutamate receptors, did not inhibit the stimulation given by glutamate (data not shown). We considered the possibility t h a t the effect of glutamate and I S , 3 R - A C P D was due to the action of

519

Glutamate stimulation of GFAP phosphorylation Table 1. Effect of glutamate agonists on the phosphorylation of GFAP/ppH-47 in immature hippocampal slices

Agonist Glutamate*, 1 mM (10) NMDA, 1 mM (5) AMPA, 1 mM (5) Kainate, 1 mM (9) Quisqualate, 1 mM (6) Quisqualate, 50/JM (6) lbotenate, l mM (8) IS, 3R-ACPD, 1 mM (9) IS, 3R-ACPD, 100 #M (9)

Mean normalized peak heights (Control = 100)

Mean differences (percent)

P value

280+ 37 80+ 13 115-t-14 139_+14 91 _+16 251 + 38 183_+33 273_+34 268_+20

+ 180 -20 + 15 +39 -9 + 15t +83 + 173 + 168

<0.001 NS NS <0.03 NS <0.011 <0.004 <0.001 <0.001

*Value from Wofchuk and Rodnight (1990). Number of observations given in parentheses. Significancewas determined by a paired t-test.

n o r a d r e n a l i n e released t h r o u g h the activation o f presynaptic g l u t a m a t e receptors. However the inclusion o f the fl-adrenergic a n t a g o n i s t p r o p r a n o l o l (100 ~ M ) in the m e d i u m h a d no effect o n the stimulation o f G F A P by 100 ~ M 1 S , 3 R - A C P D (data n o t shown). We previously reported t h a t the stimulation o f G F A P / p p H - 4 7 p h o s p h o r y l a t i o n by glutamate in slices from i m m a t u r e animals is d e p e n d e n t o n the presence o f Ca 2+ in the i n c u b a t i o n m e d i u m ( W o f c h u k a n d R o d n i g h t 1990). In later work we f o u n d t h a t this

kDa

a p p a r e n t dependence o f the glutamate effect o n Ca 2+ was related to the fact t h a t in the absence o f Ca 2+ in the m e d i u m i n c o r p o r a t i o n o f n p into G F A P / p p H 47 was increased to approximately the same level as o b t a i n e d with glutamate in the presence of C a 2+ as s h o w n in Fig. 3. The small increase in p h o s p h o r y l a t i o n given by glutamate in m e d i u m lacking Ca 2÷ did not reach statistical significance a n d the effects o f glut a m a t e a n d C a 2+ lack were n o t additive. Confirming o u r previous o b s e r v a t i o n ( W o f c h u k

(A)

(B)

(c)

(D)

80

20

50--

Fig. 1. Autoradiographs showing stimulation of GFAP/ppH-47 phosphorylation by 1 mM glutamate (A,B) and 100 #M IS,3R-ACPD (C,D), where A and C are controls and B and D are tests. Hippocampal slices from 15-day-old rats were incubated and analysed by NEPHGE in the first dimension and electrophoresis in 8% gels in the second dimension. Arrows point to GFAP/ppH-47.

520

SUSANA T. WOFCHUK a n d RICHARD RODNIGHT

300 --

~

400

/

N +Ca N+ca+glo .o

9

300

8

- " ~¢~

~-Ca

E??::~t

[ ] -Ca+gl~

"..",.,~I

"-" 200

200 - -

°~,.N

.-,:,;~, e~ x~

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2::,.-,,,-

100

'-,"'3{

too-

1

I

I

I

0

1

2

3

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Fig. 2. Sensitivity of G F A P / p p H - 4 7 p h o s p h o r y l a t i o n to I S , 3 R - A C P D in slices from i m m a t u r e animals. E a c h p o i n t is the m e a n of 6 - 9 observations. Values significantly different from control ( P < 0 . 0 1 ) are m a r k e d w i t h an asterisk.

and Rodnight, 1990) Fig. 3 also shows that in adult animals glutamate only had little or no effect on the phosphorylation of ppH-47/GFAP. DISCUSSION

In this paper we show that the phosphorylation of GFAP in slices of immature hippocampus is regulated by a glutamatergic receptor. Since the distribution of GFAP is restricted to astrocytes it is very probable that the receptor is located on this cell type. We cannot rigorously exclude the possibility that the effect is secondary to a glutamate-mediated release from neurones of another transmitter which then acts on a non-glutamatergic astrocytic receptor. However, the failure of the fl-adrenergic antagonist propranolol to inhibit the stimulation given by ACPD shows that the putative secondary transmitter cannot act via a fl-

2",/',.y Immature Adult Fig. 3. C o m p a r i s o n of the effect of g l u t a m a t e a n d C a 2÷ on G F A P / p p H - 4 7 p h o s p h o r y l a t i o n in h i p p o c a m p a l slices from i m m a t u r e and adult tissue. In the i m m a t u r e block the three c o l u m n s m a r k e d by an asterisk are significantly different from the 100% control, but do not differ significantly one from another. Each b l o c k is the m e a n of 6 - 9 observations.

adrenergic receptor. This observation eliminates the possibility that noradrenaline, which has been shown to stimulate GFAP phosphorylation in a C-6 glioma cell culture (Browning and Ruina, 1984), is responsible for the effect of glutamate agonists. Moreover, astrocytes in culture are known to express both metabotropic and, with the exception of the NMDA type, ionotropic glutamatergic receptors (Pearce and Murphy, 1988 ; Teichberg, 1991). The evidence for a metabotropic receptor comes from the effect of the rigid glutamate analogue 1S,3RACPD, which stimulated the phosphorylation of GFAP/ppH-47 to approximately the same extent as glutamate. 1S,3R-ACPD has a high selectivity for metabotropic, compared to ionotropic, glutamatergic receptors as assessed by phosphoinositide hydrolysis in rat hippocampus (Schoepp et al, 1991), receptor

Table 2. Inhibition of the I S,3R-ACPD-stimulated phosphorylation of GFAP/ppH-47 in immature hippocampal slices

Addition IS,3R-ACPD (8) IS,3R-ACPD+L-AP3 (4) 1S,3 R-ACPD + toxin (7)

Mean normalized peak heights (Control = 100)

Mean differences (percent)

P value

268 ___22 108+ 19 130 _+14

+ 168 +8 + 30

< 0.001 NS NS

Concentrations of agonist and antagonists were as follows: IS,3R-ACPD: 100/zM; L-AP3:1 mM; pertussis toxin: 5 ug/ml. L-AP3 and toxin were included in the pre-incubation medium; when added only to the incubation L-AP3 was ineffective~ Number of observations given in parentheses. Significance was determined by a paired t-test.

Glutamate stimulation of GFAP phosphorylation binding studies (Schoepp and True, 1992) and mobilization of Ca 2÷ from internal stores in cultured cerebellar neurons (Irving et al., 1990). It is reasonable to conclude therefore that the main effect of the physiological agonist glutamate on this phosphorylation system is mediated by a metabotropic receptor. The lower stimulation given by the mixed agonist ibotenate also points to the involvement of a metabotropic receptor. The partial inhibition resulting from pre-incubation of slices with pertussis toxin indicates the involvement of a GTP binding protein, probably of the category Go (Rosenthal et al., 1990). The failure of 1 mM quisqualate to stimulate GFAP phosphorylation was presumably due to the high toxicity of this agonist for neurons in immature hippocampal slices (Garthwaite and Garthwaite, 1989). Although astrocytes are reported to be relatively resistant to the toxicity of excitatory amino acids (Choi et al., 1987), it is possible that the extensive swelling of neurons that occurs on exposure of immature slices to a high concentration of quisqualate may have restricted access of the agonist to the astrocytic receptor sites. The small statistically significant stimulation of GFAP phosphorylation by kainate suggests that ionotropic receptor activation may contribute minimally to the stimulatory effect of glutamate. Kainate receptors are expressed in glia, for example in the Bergmann glia of the cerebellum (Teichberg, 1991) and in Type2 cortical astrocytes in culture (Usowicz et al., 1989 ; Jensen and Chiu, 1991). However, in the absence of an effect of the pure ionotropic agonist AMPA, it is more likely that the effect of kainate is secondary to the release of glutamate by the activation of presynaptic kainate receptors (Pastuszko et al., 1984). The precise linkage between metabotropic receptor stimulation and ppH-47/GFAP phosphorylation remains obscure, but most likely involves Ca 2÷. Thus in preliminary work we have found that labelling of GFAP/ppH-47 by [32P]ATP in cytoskeletal preparations prepared from immature hippocampus was more than 98°,/o dependent on added Ca 2÷ and calmodulin indicating that the intermediate filaments possess associated Ca:÷~ calmodulin-dependent kinase activity. This appears to be the same bound enzyme activity described in a cytoskeletal fraction prepared from astrocyte cultures by Harrison and Mobley (1992) and is very probably the activity responsible for the phosphorylation of GFAP/ppH47 in slices in the present study. It is also relevant to note that astrocytes in culture express protein kinase activity stimulated by Ca 2÷ and calmodulin (BabcockAnderson et al., 1989). However, the precise role of

521

external Ca 2+ is unclear. In particular it is not known whether the sites in ppH-47/GFAP whose phosphorylation is stimulated through activation of the metabotropic receptor are identical to those whose phosphorylation is inhibited by the presence of Ca 2÷ in the medium. If they are identical, glutamate/ACPD would appear to be acting by neutralizing the Ca :÷induced inhibition. We have suggested (Wofchuk and Rodnight, 1993) that this might be occurring by inhibition of the entry of Ca 2+ through a G-protein linked mechanism and a decrease in the activity of a putative Ca 2÷-dependent protein phosphatase event directly or indirectly involved in the dephosphorylation of ppH47/GFAP. If, on the other hand, site analysis shows that the sites phosphorylated under the two conditions are different, then the glutamate effect may be related to the liberation of Ca 2÷ from internal stores consequent on the generation ofinositol trisphosphate from PLC-mediated hydrolysis of phosphoinositides. We consider this last interpretation less likely since if it were the case the effects of glutamate and Ca 2+ lack should be additive. Besides, site analysis identification of the subtype of metabotropic glutamate receptor involved in the response should help to clarify the problem. While present evidence is insufficient on this point the inhibition of the system by L-AP3, an antagonist of metabotropic receptors linked to the hydrolysis of phosphoinositides (Schoepp et al., 1990), suggests that the receptor is coupled to a phospholipase C and this conclusion is supported by observations showing that in cultured astrocytes glutamate increases phosphoinositide hydrolysis (Pearce and Murphy, 1988) and releases Ca 2÷ from internal stores (Ahmed et al., 1990). However mRNA expression data show that mGluR3, which is negatively linked to adenylate cyclase, is the predominant metabotropic glutamate receptor in glia (Shigemoto, 1993a,b). Further work using more selective agonists and antagonists is needed to reach a conclusion. It is of interest to note that the period during which the astrocytic metabotropic receptor is expressed (1016 days postnatal) corresponds to the onset of massive synaptogenesis in the rat brain (Aghajanian and Bloom, 1967). Interactions between neurons and astrocytes are well documented (Barres, 1991) and it is probable that during development astrocytes play a dynamic role in the formation of new synapses (Meshul et al., 1987; Arenander and de Vellis, 1992) and glutamate liberated by developing neurons may conceivably be involved in this phenomenon. Finally it is relevant to note that site-specific phosphorylation of intermediate filament proteins, includ-

522

SUSANAT. WOFCHUKand RICHARD RODNIGHT

ing G F A P , has been shown to be a necessary step in the disassembly of the filament structure during mitosis (lnagaki et al., 1987, 1990; C h o u et al., 1989; Nishizawa et al., 1991 ; M a t s u o k a et al., 1992 ; N a k a mura et al., 1992). It is tempting to speculate therefore that glutamate liberated by developing n e u r o n s is signalling an increase in the p h o s p h o r y l a t i o n state of G F A P and a consequent increase in the n u m b e r of mitotic astrocytes. A n i m p o r t a n t step in investigating this hypothesis will be to determine the specific phosphorylation sites in G F A P modified by m e t a b o t r o p i c receptor stimulation. work was supported by the Brazilian funding agencies CNPq, FINEP and FAPERGS

Acknowledgement--This

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