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Trans-ACPD inhibits cAMP formation via a pertussis toxin-sensitive G-protein
European Journal of Pharmacology - Molecular Pharmacology Section. 225 (1992) 357-358 q~ 1992Elsevier SciencePublishers~.V. All rights reserved 0922-4106/92/$05.00
357
EJPMOL 0110R
Rapid communication
Trans-ACPD inhibits cAMP formation via a pertussis toxin-sensitive G-protein Oiivier M a n z o n i , L a u r e n t Prezeau, Fritz Sladeczek a n d JoEl Bockaert Centre CNRS-INSEIL~I de Pharmacologie-Endoerinologie, 34094 Montpellier, France
Received 25 Februa~ 1992,accepted 5 March 1992
In prima~ cultured striatal neurons we found that (±)-trans-l-amino-cyclopentyt-t,3-dicarboxylate (trans-ACPD) could inhibit forskolin-induced cAMP formation in a dose-dependent manner (ECs0 156±38 p~M, n =5, maximal inhibition 37.8 _~:1.2, n = 37). The t;ans-ACPD-induced inhibition was totally abolished in neurons preincubated with Bordetella pertussis toxin (1 ~g/ml), demonstrating the involvement of z G-protein. This is the first report in intact neurons of a glutamate metabotropic receptor negatively coupled to cAMP formation. Metabotropie receptor; cAMP formation; Striatal neurons
In the central nervous system, a glutamate (Glu) receptor (Qp receptor) is coupled to phospholipase C via a G-protein (Schoepp et al., 1990). We bare previously shown that ( + )-trans-l-amino-cyclopentyl-l,3-dicarboxylate (trans-ACPD) was a specific agonist at the Qp receptor in striatal neurons in primary culture and in rat brain mRNA injected X e n o p u s oocytes (Manzoni et al., 1990). The Qp receptor has been cloned (Masu et al.. 1991) and the investigators recently described a family of metabotropic glutamate receptors (Tanabe et al., 1992). When transfected, several members of this family could inhibit cAMP formation (Tanabe et at., 1992). Moreover, Miller recently pointed out that Glu metabotropic Qp receptors and muscarinic receptors share several transduetion mechanism systems, in addition to phospholipase C activation (Miller, 1991). We therefore tested the putative effects of trans-ACPD on the forskolin-induced cAMP formation in primary cultured striatal neurons. Primary cultures of striatal neurons were prepared in serum-free medium, as previously described (Manzoni et al., 1991). The cAMP content of cells was measured using the prelabelling technique described by Weiss et al. (1985). On the sixth day, the cells were washed and incubated at 37°C (5% COz/95% air mixture) with 2 / z C i / m l of [3H]adenine (24 Ci/mol). After 2 h, the cultures were washed and incubated with 0.75 mM isobutylmethylxanthine and testing agents in 1 ml
Correspondence to: Olivier Manzoni, Centre CNRS-INSERM de Pharmacologie-Endocrinologie,Rue de la Cardonille, 34094 Montpellier Cedex 5, France.Tel. 67-14.29.38;Fax 67-54.24.32.
of HEPES buffer saline (composition: NaCI 146 raM, KCI 4.2 mM, MgC12 0.5 mM, glucose 0.1%, HEPES 20 m M / p H 7.2), for I0 rain at 37°C in the presence of forskolin (10/zM). In the incubation buffer, Ca 2+ was omitted and tetrodotoxin (TTX; 3 # M ) was added, to avoid effects which could be mediated either by cell depolarization or Ca z+ infltLxes. The reaction was stopped by aspiration of the media and addition of t ml of ice-cold 5% trichloroacetic acid. Cells were loosened with a rubber scraper and 100/zl of 5 mM ATP and 5 mM cAMP were added to the mixture. Cellular protein was centrifuged at 5 0 0 0 × g . [3H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex and alumina columns, cAMP formation is expressed as percentage conversion: [3H]ATP to [3H]cAMP : [3H]cAMP × 100/[3H]cAMP + [3H]ATP, To avoid any inhibitory effects of -/-aminobutyric acid (GABA) released upon Glu stimulation during cAMP formation measurements, we performed all our experiments on 6 DIV striatal neurons (at that development stage, no GABA release could be detected, Weiss et al., 1986). To reduce the effects of ionotropic Gtu receptors on cAMP production, experiments were performed in the presence of 10 /xM MK 801 (a non-competitive NMDA receptor antagonist) and 30 ~ M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (a specific com?~etitive antagonist of non-NMDA receptors). Fig. 1A shows that under these conditions transACPD inhibited forskolin- (10 /zM) induced cAMP formation in a dose-dependent manner. The maximal inhibition obtained was 37.8 __+1.2 (n = 37) and the ECs0 of the trans-ACPD effect was 156 +_38/~M (n = 5). The observation that trans-ACPD could inhibit
358
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Q control F1 +l/ag/ml PTX
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maximal effect was totally abolished, d e m o n s t r a t i n g the involvement o f a toxin-sensitive G - p r o t e i n (fig. 1B). U s i n g n e u r o n a l cultures, in which n e u r o t r a n s m i t t e r s are not released, s u p p r e s s i n g extracellular Ca 2+, a n d blocking d e p o l a r i z a t i o n with T T X , we w e r e able to r e d u c e t h e possibility that t r a n s - A C P D - i n d u c e d inhibition o f forskolin-stimulated c A M P p r o d u c t i o n could b e indirect. T h e s e data d e m o n s t r a t e a novel action of t r a n s - A C P D : t h e inhibition o f c A M P f o r m a t i o n may be, via a new Glu r e c e p t o r , negatively c o u p l e d to an adenylyl cyclase. A l t h o u g h the exact m e c h a n i s m o f t h e coupling r e m a i n s to b e d e m o n s t r a t e d , a G - p r o t e i n sensitive to P T X is implicated. In preliminary e x p e r i m e n t s we have f o u n d that t h e p h a r m a c o l o g y o f this putative n e w Glu r e c e p t o r is d i f f e r e n t to that o f t h e m e t a b o t r o p i c Q p r e c e p t o r coupled to p h o s p h o l i p a s e C. T h e d e t a i l e d p h a r m a c o l o g i c a l a n d functional c h a r a c t e r i z a t i o n o f this r e c e p t o r is und e r c u r r e n t investigation.
Acknowledgements IO~M Forsk . lmM tmns-ACPD
Fig. 1. Effects of trans-ACPD on forskolin-induced cAMP formation in striatal neurons. (A) Concentration-dependent effect of transACPD. Neurons were exposed to increasing concentrations of agonist in the presence of 10/zM forskolin. In the presence of forskolin alone, the conversion of [3H]ATP to [3H]cAMP was 2.25:0.18% (n = 11) in this representative experiment which was repeated 4 times. Each value is the mean ± standard error of triplicate determinations. (B) Blockade of the inhibitory effect of trans-ACPD by PTX. In the presence of forsko!i, (!0 /~,M), the conversion of [3H]ATP to [3H}cAMP was i.8+0.2% (n = 11). Neurons were incubated with 1 ~g/ml PTX overnight. Each value is the mean ±S.E.M. of three independent experiments. A Student's t-test was performed between the cAMP productions obtained in neurons treated with or without PTX. P < 0.001. c A M P formation in the p r e s e n c e o f M K 801, C N Q X , T T X and in the p r e s e n c e of low external Ca 2+, suggested that a n o n - i o n o t r o p i c Glu r e c e p t o r was involved. T h e next step was to test the involvement o f a Bordetella pertussis toxin (PTX)-sensitive G - p r o t e i n . W h e n the n e u r o n s were p r e i n c u b a t e d with P T X (1 / z g / m l , dose necessary to achieve a c o m p l e t e A D P ribosylation, not shown) overnight, the t r a n s - A C P D
We are grateful to Angle Turner-Madeuf for skillful secretarial assistance. Supported by grants from CNRS, INSERM, Bayer France/Troponwerke (Germany), and DRET (contract No. 88/163).
References Manzoni, O., L. Fagni, J.-P. Pin, F. Rassendren, F. Poulat, F. Sladeczek and J. Bockaert, 1990, ACPD stimulates q, isqualate phosphoinositide-coupled receptors but not ionotropic glutamate receptors in striatal neurons and Xenopusoocytes, Mol. Pharmacol. 38, I. Masu, M., Y. Tanabe, K. Tsuchida, R. Shigemoto and S. Nakanishi, 1991, Sequence and expression of a metabotropic glutamate receptor, Nature 349, 760. Miller, R.J., 1991, Metabotropic excitatory arf,ino acid receptors reveal their true colors, Trends Pharmaco]. Sci. 12, 365. Schoepp, D.D., J. Bockaert and F. Sladeczek, 1990, Metabotropic excitatory amino acid receptors: pharmacological and funciioiial characteristics, Trends Pharmacol. Sci. 11,508. Tanabe, Y., Masu, M., Ishii, T., Shigemoto, R. and S. Nakanishi, 1992, A family of metabotropic receptors, Neuron 8, 169. Weiss, S., M. Sebben, A. Garcia-Sainz and J. Bockaert, 1985, D2dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture, Mol. Pharmacol. 27, 595. Weiss, S., J.P. Pin, M. Sebben, D.E. Kemp, F. Sladeczek, J. Gabrion and J. Bockaert, 1986, Synaptogenesisof cultured striatal neurons in serum-free medium: a morphological and biochemical study, Proc. Natl. Acad. Sci. USA 83, 2238.
357
EJPMOL 0110R
Rapid communication
Trans-ACPD inhibits cAMP formation via a pertussis toxin-sensitive G-protein Oiivier M a n z o n i , L a u r e n t Prezeau, Fritz Sladeczek a n d JoEl Bockaert Centre CNRS-INSEIL~I de Pharmacologie-Endoerinologie, 34094 Montpellier, France
Received 25 Februa~ 1992,accepted 5 March 1992
In prima~ cultured striatal neurons we found that (±)-trans-l-amino-cyclopentyt-t,3-dicarboxylate (trans-ACPD) could inhibit forskolin-induced cAMP formation in a dose-dependent manner (ECs0 156±38 p~M, n =5, maximal inhibition 37.8 _~:1.2, n = 37). The t;ans-ACPD-induced inhibition was totally abolished in neurons preincubated with Bordetella pertussis toxin (1 ~g/ml), demonstrating the involvement of z G-protein. This is the first report in intact neurons of a glutamate metabotropic receptor negatively coupled to cAMP formation. Metabotropie receptor; cAMP formation; Striatal neurons
In the central nervous system, a glutamate (Glu) receptor (Qp receptor) is coupled to phospholipase C via a G-protein (Schoepp et al., 1990). We bare previously shown that ( + )-trans-l-amino-cyclopentyl-l,3-dicarboxylate (trans-ACPD) was a specific agonist at the Qp receptor in striatal neurons in primary culture and in rat brain mRNA injected X e n o p u s oocytes (Manzoni et al., 1990). The Qp receptor has been cloned (Masu et al.. 1991) and the investigators recently described a family of metabotropic glutamate receptors (Tanabe et al., 1992). When transfected, several members of this family could inhibit cAMP formation (Tanabe et at., 1992). Moreover, Miller recently pointed out that Glu metabotropic Qp receptors and muscarinic receptors share several transduetion mechanism systems, in addition to phospholipase C activation (Miller, 1991). We therefore tested the putative effects of trans-ACPD on the forskolin-induced cAMP formation in primary cultured striatal neurons. Primary cultures of striatal neurons were prepared in serum-free medium, as previously described (Manzoni et al., 1991). The cAMP content of cells was measured using the prelabelling technique described by Weiss et al. (1985). On the sixth day, the cells were washed and incubated at 37°C (5% COz/95% air mixture) with 2 / z C i / m l of [3H]adenine (24 Ci/mol). After 2 h, the cultures were washed and incubated with 0.75 mM isobutylmethylxanthine and testing agents in 1 ml
Correspondence to: Olivier Manzoni, Centre CNRS-INSERM de Pharmacologie-Endocrinologie,Rue de la Cardonille, 34094 Montpellier Cedex 5, France.Tel. 67-14.29.38;Fax 67-54.24.32.
of HEPES buffer saline (composition: NaCI 146 raM, KCI 4.2 mM, MgC12 0.5 mM, glucose 0.1%, HEPES 20 m M / p H 7.2), for I0 rain at 37°C in the presence of forskolin (10/zM). In the incubation buffer, Ca 2+ was omitted and tetrodotoxin (TTX; 3 # M ) was added, to avoid effects which could be mediated either by cell depolarization or Ca z+ infltLxes. The reaction was stopped by aspiration of the media and addition of t ml of ice-cold 5% trichloroacetic acid. Cells were loosened with a rubber scraper and 100/zl of 5 mM ATP and 5 mM cAMP were added to the mixture. Cellular protein was centrifuged at 5 0 0 0 × g . [3H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex and alumina columns, cAMP formation is expressed as percentage conversion: [3H]ATP to [3H]cAMP : [3H]cAMP × 100/[3H]cAMP + [3H]ATP, To avoid any inhibitory effects of -/-aminobutyric acid (GABA) released upon Glu stimulation during cAMP formation measurements, we performed all our experiments on 6 DIV striatal neurons (at that development stage, no GABA release could be detected, Weiss et al., 1986). To reduce the effects of ionotropic Gtu receptors on cAMP production, experiments were performed in the presence of 10 /xM MK 801 (a non-competitive NMDA receptor antagonist) and 30 ~ M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (a specific com?~etitive antagonist of non-NMDA receptors). Fig. 1A shows that under these conditions transACPD inhibited forskolin- (10 /zM) induced cAMP formation in a dose-dependent manner. The maximal inhibition obtained was 37.8 __+1.2 (n = 37) and the ECs0 of the trans-ACPD effect was 156 +_38/~M (n = 5). The observation that trans-ACPD could inhibit
358
A
1:3 ¢P o
100
'o ¢ ~90 "T "
¢i
o =80 =¢'o L. O ~,.
ito ~ 7 0
~
~0 ~1///
: m
i
!
i
-6
-5
-4
-Log
[trans-ACPDJ
Q control F1 +l/ag/ml PTX
•
i
i
-3
-2
B
maximal effect was totally abolished, d e m o n s t r a t i n g the involvement o f a toxin-sensitive G - p r o t e i n (fig. 1B). U s i n g n e u r o n a l cultures, in which n e u r o t r a n s m i t t e r s are not released, s u p p r e s s i n g extracellular Ca 2+, a n d blocking d e p o l a r i z a t i o n with T T X , we w e r e able to r e d u c e t h e possibility that t r a n s - A C P D - i n d u c e d inhibition o f forskolin-stimulated c A M P p r o d u c t i o n could b e indirect. T h e s e data d e m o n s t r a t e a novel action of t r a n s - A C P D : t h e inhibition o f c A M P f o r m a t i o n may be, via a new Glu r e c e p t o r , negatively c o u p l e d to an adenylyl cyclase. A l t h o u g h the exact m e c h a n i s m o f t h e coupling r e m a i n s to b e d e m o n s t r a t e d , a G - p r o t e i n sensitive to P T X is implicated. In preliminary e x p e r i m e n t s we have f o u n d that t h e p h a r m a c o l o g y o f this putative n e w Glu r e c e p t o r is d i f f e r e n t to that o f t h e m e t a b o t r o p i c Q p r e c e p t o r coupled to p h o s p h o l i p a s e C. T h e d e t a i l e d p h a r m a c o l o g i c a l a n d functional c h a r a c t e r i z a t i o n o f this r e c e p t o r is und e r c u r r e n t investigation.
Acknowledgements IO~M Forsk . lmM tmns-ACPD
Fig. 1. Effects of trans-ACPD on forskolin-induced cAMP formation in striatal neurons. (A) Concentration-dependent effect of transACPD. Neurons were exposed to increasing concentrations of agonist in the presence of 10/zM forskolin. In the presence of forskolin alone, the conversion of [3H]ATP to [3H]cAMP was 2.25:0.18% (n = 11) in this representative experiment which was repeated 4 times. Each value is the mean ± standard error of triplicate determinations. (B) Blockade of the inhibitory effect of trans-ACPD by PTX. In the presence of forsko!i, (!0 /~,M), the conversion of [3H]ATP to [3H}cAMP was i.8+0.2% (n = 11). Neurons were incubated with 1 ~g/ml PTX overnight. Each value is the mean ±S.E.M. of three independent experiments. A Student's t-test was performed between the cAMP productions obtained in neurons treated with or without PTX. P < 0.001. c A M P formation in the p r e s e n c e o f M K 801, C N Q X , T T X and in the p r e s e n c e of low external Ca 2+, suggested that a n o n - i o n o t r o p i c Glu r e c e p t o r was involved. T h e next step was to test the involvement o f a Bordetella pertussis toxin (PTX)-sensitive G - p r o t e i n . W h e n the n e u r o n s were p r e i n c u b a t e d with P T X (1 / z g / m l , dose necessary to achieve a c o m p l e t e A D P ribosylation, not shown) overnight, the t r a n s - A C P D
We are grateful to Angle Turner-Madeuf for skillful secretarial assistance. Supported by grants from CNRS, INSERM, Bayer France/Troponwerke (Germany), and DRET (contract No. 88/163).
References Manzoni, O., L. Fagni, J.-P. Pin, F. Rassendren, F. Poulat, F. Sladeczek and J. Bockaert, 1990, ACPD stimulates q, isqualate phosphoinositide-coupled receptors but not ionotropic glutamate receptors in striatal neurons and Xenopusoocytes, Mol. Pharmacol. 38, I. Masu, M., Y. Tanabe, K. Tsuchida, R. Shigemoto and S. Nakanishi, 1991, Sequence and expression of a metabotropic glutamate receptor, Nature 349, 760. Miller, R.J., 1991, Metabotropic excitatory arf,ino acid receptors reveal their true colors, Trends Pharmaco]. Sci. 12, 365. Schoepp, D.D., J. Bockaert and F. Sladeczek, 1990, Metabotropic excitatory amino acid receptors: pharmacological and funciioiial characteristics, Trends Pharmacol. Sci. 11,508. Tanabe, Y., Masu, M., Ishii, T., Shigemoto, R. and S. Nakanishi, 1992, A family of metabotropic receptors, Neuron 8, 169. Weiss, S., M. Sebben, A. Garcia-Sainz and J. Bockaert, 1985, D2dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture, Mol. Pharmacol. 27, 595. Weiss, S., J.P. Pin, M. Sebben, D.E. Kemp, F. Sladeczek, J. Gabrion and J. Bockaert, 1986, Synaptogenesisof cultured striatal neurons in serum-free medium: a morphological and biochemical study, Proc. Natl. Acad. Sci. USA 83, 2238.