Journal of Neurochemislry Raven Press, L a . , New York 0 1992 International Society for Neurochemistry

Rapid Communication

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices Valerie Boss and P. Jeffrey Conn Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, U.S.A.

Abstract: Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein-coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R-amino-l,3~yclopentanedicarboxylic acid (ACPD) and 1S,3S-ACPD, but not the inactive isomer, I R,3S-ACPD, induce a concentration-dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1 S,3R-ACPD-induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR-iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L-2-Amino3-phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis-coupled mGluRs. Key Words: I-Amino- 1,3-cyclopentanedicarboxylic acid-Metabotropic glutamate receptor-~-2-Amino-3-phosphonopropionic acidHippocampus-Phosphatidylcholine-Phospholipase D. Boss V. and Conn P.J. Metabotropic excitatory amino acid receptor activation stimulates phospholipase D in hippocampal slices. J. Neurochem. 59, 2340-2343 (1992).

Excitatory amino acids (EAAs), such as glutamate, exert many of their effects in the CNS by activating a family of metabotropic glutamate receptors (mGluRs) that are coupled to effector systems through G proteins (for reviews, see Schoepp et al., 1990a; Conn and Desai, 1991). At least five mGluRs have been cloned from rat brain (Houamed et al., 1991; Masu et al., 1991; Abe et al., 1992; Tanabe et al., 1992); however, the signal transduction mechanisms associated with these receptors have not been fully characterized. Resubmitted manuscript received September 8, 1992; accepted September I I , 1992. Address correspondence and reprint requests to Dr. P. J. Conn at Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. Abbreviulions used: ACPD, I-aminocyclopentane- 1,3-dicarboxacid; DAPV, ylic acid; L - A P ~L-2-amino-3-phosphonopropionic ,

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A common signal transduction mechanism involves the receptor-mediated breakdown of membrane phospholipids. The most well-known pathway to utilize such a mechanism includes the hydrolysis of phosphoinositides by phospholipase C. However, recent experiments have determined that many receptors are coupled to the hydrolysis of phosphatidylcholine (PC). Both phospholipase C and phospholipase D (PLD) can hydrolyze PC, but the PLD pathway probably plays the predominant role in signal transduction (for reviews, see Loffelholz, 1990; Thompson et al., 1991). This pathway converts PC into choline and phosphatidic acid (PA), which can then be converted to diacylglycerol by phosphatidate phosphohydrolase. trans- 1-Amino- 1,3-cyclopentanedicarboxylicacid (transACPD) selectively activates mGluRs (Palmer et al., 1989; Desai and Conn, 1990;Winder and Conn, 1992a). Previous studies have suggested that the regional distribution of trans-ACPD-induced diacylglycerol formation within the hippocampus (Hwang et al., 1990)differs from that of transACPD-induced inositol phosphate formation (Boss et al., 1992). Because PC is the major lipid constituent of the lipid bilayer, the production of diacylglycerol from PC is often quantitatively much greater than that from the less abundant phosphoinositides. If trans-ACPD activates receptors coupled to PLD, the bulk of diacylglycerol could be formed by PLD-catalyzed PC hydrolysis. We therefore examined the possibility that metabotropic EAA receptors exist in the hippocampus that are coupled to the activation of PLD.

EXPERIMENTAL PROCEDURES PLD activity was assayed by measuring the transphosphatidylation of phospholipids in the presence of ethanol, which results in the formation of phosphatidylethanol (Pet). Agonist-induced formation of Pet is generally considered to be diagnostic of agonist-induced PLD activation. PLD nor~2-amino-5-phosphonovalericacid; CNQX, 6cyano-2,3-dihydroxy-7-nitroquinoxaline;EAA, excitatory amino acid; iGluR, ionotropic glutamate receptor; KRB, Krebs-bicarbonate buffer; mGluR, metabotropic glutamate receptor; NMDA, N-methyl+ aspartate; PA, phosphatidic acid; PC, phosphatidylcholine; Pet, phosphatidylethanol; PLD, phospholipase D.

ACPD-STIMULATED PLD ACTIVATION mally catalyzes the hydrolysis of PC into choline and PA. However, in the presence of an exogenous primary alcohol, PLD catalyzes a transphosphatidylation reaction that transfers the alcohol (instead of water) to the phosphatidyl moiety, producing Pet (instead of PA) (for review, see Thompson et al., 1990). Cross-chopped hippocampal slices (350 X 350 pm) were incubated in Krebs-bicarbonate buffer (KRB) for 30 min, as described previously (Desai and Conn, 1990). PLD activity was assayed according to the protocol of Llahi and Fain (1 992). In brief, the slices were rinsed with warmed KRB and then incubated for 3 h in the presence of [',PIorthophosphate (60-80 pCi/ml) in KRB. The slices were washed extensively with KRB, and then 25-pl aliquots of gravity-packed slices were added to tubes containing solutions of EAA receptor antagonists in KRB (or KRB alone as controls) and incubated for 15 min. EAA receptor agonists (or KRB) and ethanol (final concentration, 170 mM) were then added to the slices in a final volume of 250 pl, and they were incubated for an additional 30 min. Control tubes in which the ethanol was omitted were always included in each assay. The reaction was stopped with 1.2 ml of chloroform/ methanol (1:2 vol/vol). All incubations were camed out under an atmosphere of 95% OJ5% C 0 2 at 37°C in a shaking water bath. Lipids were extracted by addition of 500 p1 each of0.25 M HCI and chloroform. The tubes were mixed vigorously, and then the phases were separated by low-speed centrifugation for at least 5 min. Aliquots (500 p l ) of the organic (lower) layer were dried under N, for 30 min. The samples were resuspended in 10 pl of chloroform/methanol(9: 1 vol/vol), and the entire volume was immediately spotted onto silica gel-HL plates. Standard solutions of PA, PC, and Pet (in chloroform) were also spotted onto the plates. A solvent system of chloroform/methanol/acetic acid (65: 15:2 by volume) was used to separate Pet from other lipids. The plates were analyzed using autoradiography. The plates were then exposed to iodine so that the positions of the phospholipid standards could be detected. All of the radioactivity was located in spots corresponding to the positions of PA, PC, and Pet. These spots were scraped off into scintillation vials, and their radioactivity was measured using liquid scintillation counting. The radioactivity corresponding to [32P]Pet in control samples in which ethanol was omitted was subtracted as background. Data were analyzed as the percentage of total radioactivity incorporated into lipids that was converted to ["PIPet ([32P]Pet/32Pin total lipid X 100). Statistical comparisons were always made between percent conversion values, using Student's t test.

Materials [32P]Orthophosphoricacid (carrier-free; 8,800 Ci/mmol) was obtained from DuPont-New England Nuclear. Silica gel-HL plates were supplied by Analtech. Phospholipid standards were purchased from Avanti Polar Lipids. 1S,3RACPD, IS,3S-ACPD, ~2-amino-5-phosphonovalericacid (D-APV), L-2-amino-3-phosphonopropionicacid (L-AP3), and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline(CNQX) were obtained from Tocris Neuramin (U.K.) 1R,3S-ACPD was a gift from Dr. James A. Monn (Lilly Research Laboratories). All other reagents were purchased from Sigma.

RESULTS AND DISCUSSION Consistent with a previous report (Llahi and Fain, 1992), addition of ethanol to the incubation medium resulted in

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incorporation of 32Pinto a lipid extracted from brain slices that comigrated with the Pet standard. This [32P]Petwas never seen in the absence of added ethanol (data not shown), suggesting that [32P]Petwas formed by the PLD-catalyzed transphosphatidylation of a labeled endogenous phospholipid. Both 1S,3R-ACPD and lS,3S-ACPD induced concentration-dependent increases in [32P]Petformation (Fig. 1). The maximal response to either isomer was approximately twice the basal value, with an EC, of -30 pMfor lS,3R-ACPD and 60 M f o r 1 S.3S-ACPD. 1R.3S-ACPD caused a smaller increase in PC hydrolysis, but this response was highly variable and failed to reach statistical significance (p > 0.05) at any of the concentrations tested. This stereoselectivity is virtually identical to that obtained in previous studies of mGluRs that are coupled to phosphoinositide hydrolysis (Irving et al., 1990; Schoepp et al., 199 1 ;Desai et al., 1992), inhibition of adenylate cyclase (Schoepp et al., 1992), increased cyclic AMP accumulation (Winder and Conn, 1992a,b), and various physiological responses (Desai et al., 1992). The effects of ACPD on PLD activity were compared with those of norepinephrine and carbachol. In the presence of 50 pit4 pargyline, norepinephrine, at a concentration (100 pA4) that elicits an almost maximal PLD activation in rat brain (Llahi and Fain, 1992), induced an increase in Pet formation of 3.1 k 0.2 times the basal value (n = 14 experiments in duplicate). Thus, norepinephrine was slightly more efficacious than the active isomers of ACPD in stimulating PLD activity in rat hippocampus. Carbachol(1 mM) had no effect on PLD activity (n = 3 experiments in triplicate). Although the active isomers of ACPD are generally held to be selective agonists of mGluRs, because ionotropic glutamate receptor (iGluR) antagonists were not included in the above experiments, it was necessary to address the possibility that the PLD response was mediated by cross-activation of iGluRs. Therefore, IS,3R-ACPD was applied to hippocampal slices in the presence of DAPV ( 100 p M ) , a selective N-methyl-Daspartate (NMDA) receptor antagonist, and CNQX (50 pA4), a selective cr-amino-3-hydroxy-5methylisoxazole-4-propionicacid (AMPA) antagonist. Neither of these compounds blocked l S, 3R-ACPD-induced ["PIPet formation at concentrations that completely block

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FIG. 1. Effect of ACPD isomers on PLD-catalyzed formation of [=P]Pet in hippocampalslices. Tissue was incubated for 30 min at 37°C with or without ACPD. Data are expressed as percentages of the basal (without ACPD) incorporation of radioactivity into [32P]Petand are mean f SEM (bars) values from four experiments for 1S.3R- and lS,S-ACPD and six experiments for lR,S-ACPD. All experiments were performed in duplicate.

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V. BOSS AND P. J. CONN

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iGluR function (Fig. 2), suggesting that APCD-induced PLD activation is not iGluR mediated. The selective iGluR agonists NMDA and kainate [ 1 mM, in the presence of CNQX (50 p M ) and DAPV (100 pM)], did not increase [32P]Petformation (Fig. 3). Indeed, 1 mM NMDA induced a small but significant decrease in [32P]Pet formation. Further experiments revealed that the inhibition of basal Pet formation by 1 mM NMDA was similar whether NMDA was applied alone, with 100 mM D-APV, or with 500 m M DAPV (data not shown). This result suggests that DAPV-insensitive NMDA receptors may mediate an inhibition of PLD, although the mechanism underlying this inhibition is unknown. The nonselective iGluR-mGluR agonists ibotenate (100 p M ) and quisqualate (100 p M ) (both in the presence of iGluR antagonists) (Schoepp et al., 1 9 9 0 ~Conn ; and Desai, 1991 ) caused significant increases in [32P]Pet formation (Fig. 3). However, it is interesting that the putative endogenous agonist glutamate (at 5 1 mM) did not elicit a detectable increase in [32P]Petformation (Fig. 3). Previous studies have shown that metabotropic EAA receptor-mediated responses to glutamate are generally large in systems where there is no glutamate uptake, but relatively small in systems where glutamate uptake is intact (for review, see Conn and Desai, 1991). It is possible that the ineffectiveness ofglutamate in stimulating [32P]Pet formation was due to glutamate uptake. Alternatively, the receptor coupled to PLD activity may use another EAA as an endogenous agonist. Previous studies have shown that L - A P ~blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, where it acts as a weak partial agonist (Irving et al., 1990; Schoepp et al., 1990b). In addition, LAP3 inhibits 1S,3R-ACPD-induced cyclic AMP accumulation in hippocampal slices (Winder and Conn, 1 9 9 2 ~ ) . When measuring PLD activation, we found that 500 p M L - A P ~produced almost as great a response as the maximal response to 1S,3R-ACPD. Thus, the receptor that mediates this response may be pharmacologically distinct from some previously characterized mGluRs. However, it is interesting to note that L - A P is ~ a full agonist at mGluRs that are negatively coupled to adenylate cyclase (Schoepp and Johnson, 1992). These experiments demonstrate that activation of metabotropic EAA receptors can lead to a significant increase in PLD activity in hippocampus. It is likely that PLD catalyzes

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FIG. 2. Effects of iGluR antagonists and L - A P on ~ the formation of [32P]Pet.Hippocampal slices were incubated with CNQX (50 &f), D-APV (100 pA4). or L-AP3 (500 p M ) for 15 min before addition of 100 pM 1S,3R-ACPD (or buffer in samples without 1S.3RACPD) for a further incubation of 30 min at 37°C. Data are expressed as percentages of total radioactivity incorporated into the lipid phase that was converted to r3'P]Pet ([32P]Pet/32Pin total lipid X 100) and are mean k SEM (bars) values from three experiments performed in triplicate. 'Significantly different from no ACPD control at p < 0.01.

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FIG. 3. Effect of mGluR and iGluR agonists on PLD-catalyzed formation of [=P]Pet in hippocampalslices. Tissue was incubated for 30 min at 37°C with glutamate (glu), NMDA, or kainate (kain) at 1 mM; quisqualate (quis). ibotenate (ibo), or lS.3R-ACPD (ACPD) at 100 p M ; or KRB in control tubes (cont). Data are mean k SEM (bars) values from three experiments performed in triplicate. ' p 4 0.01, significantly less than the control; **p < 0.02, "'p < 0.005.significantly greater than the control.

the breakdown of membrane PC (for reviews, see Lijffelholz, 1990; Thompson et al., 1991); however, it is possible that PLD could hydrolyze other phospholipids. Because PC is the most abundant phospholipid in the cell membrane, metabotropic EAA receptor-mediated PLD activation is likely to play an important role in mediating physiological responses elicited by EAAs in hippocampal neurons. Further experiments will be necessary to determine whether PLD is directly coupled to a distinct metabotropic EAA receptor, or whether an increase in PLD activity is secondary to the release of a second messenger. However, the data obtained using L-AP-3 suggest that the receptor that is associated with PLD activation is different from mGluRs in the hippocampus that are coupled to phosphatidylinositol-specific phospholipase C or to increased accumulation of cyclic AMP. Acknowledgment: We thank Dr. James A. Monn for generously supplying 1R, 3S-ACPD. This work was supported by grant NS28405 from the National Institutes of Health.

REFERENCES Abe T., Sugihara H., Nawa H., Shigemoto R., Mizuno N., and

Nakanishi S. (1992) Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca'+ signal transduction. J. Bid. Chem. 267, 1336 1-13368. Boss V.. Desai M. A., Smith T. S., and Conn P. J. (1992) trunsACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation. Bruin Res. (in press). Conn P. J. and Desai M. A. (199 1) Pharmacology and physiology of metabotropic glutamate receptors in mammalian central nervous system. Drug. Dev. Res. 24,207-229. Desai M. A. and Conn P. J. (1990) Selective activation of phosphoinositide hydrolysis by a rigid analogue of glutamate. Neurosci. Lett. 109, 157-162. Desai M. A. and Conn P. J. (1991) Excitatory effects of ACPD receptor activation in the hippocampus are mediated by direct effects on pyramidal cells and blockade of synaptic inhibition. J. Neiirophysiol. 66, 40-52. Desai M. A,, Smith T. S., and Conn P. J. (1992) Multiple metabotropic glutamate receptors regulate hippocampal function. Synapse (in press). Houamed K. M., Kuijper J. L., Gilbert T. L., Haldeman B. A,, OHara P. J., Mulvihill E. R., Almers W., and Hagen F. S. (1991) Cloning, expression, and gene structure of a G protein-

ACPD-STIMULATED PLD ACTIVATION coupled glutamate receptor from rat brain. Science 252, I3 181321. Hwang P. M., Bredt D. S., and Snyder S. S. (1990) Autoradiographic imaging of phosphoinositide turnover in the brain. Science 249,802-804. Irving A. J., Schofeld J. G., Watkins J. C., Sunter D. C., and Collingridge G. L. (1990) IS.3R-ACPD stimulates and L - A P blocks ~ Ca2+mobilization in rat cerebellar neurons. Eur. J. PharmaCOI. 186, 363-365. Llahi S. and Fain J. N. (1992) q-Adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex. J. Biol. Chem. 267, 3679-3685. Loffelholz K. (1990) Receptors linked to hydrolysis ofcholine phospholipids: the role of phospholipase D in a putative mechanism of signal transduction, in Current Aspecfs offhe Neurosciences (Osborne N. N., ed), pp. 49-76. Macmillan, London. Masu M., Tanabe Y., Tsuchida K., Shigemoto R., and Nakanishi S. (1991) Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765. Manzoni O., Prezaeu L., Sladeczek F., and Bockaert J. (1 992)fransACPD inhibits CAMPformation via a pertussis toxin-sensitive G-protein. Eur. J. Pharmacol. Mol. Pharmacol. 225,357-358. Palmer E., Monaghan D. T., and Cotman C. W. (1989) transACPD, a selective agonist of the phosphoinositide-hydrolysiscoupled excitatory amino acid receptor. Eur. J. Pharmacol. 166,585-587. Schoepp D. D. and Johnson B. G. (1992) Pharmacology ofmetabotropic glutamate receptor inhibition of CAMP formation in.the adult rat hippocampus. Neurochem. Inf. (in press).

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) Schoepp D. D., Bockaert J., and Sladeczek F. ( 1 9 9 0 ~Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors. Trends Pharmacol. Sci. 11, 508-515. Schoepp D. D., Johnson B. G., Smith E. C. R., and McQuaid L. A. (19906) Stereoselectivity and mode of inhibition of phosphoinositide-coupled excitatory amino acid receptor by 2amino-3-phosphonopropionicacid. Mol. Pharmacol. 38,222228. Schoepp D. D., Johnson B. G., True R. A., and Monn J. A. (199 1 ) Comparison of ( IS,3R)- 1-aminocyclopentanedicarboxylic acid ( I S,3R-ACPD)- and 1R.3S-ACPD-stimulated brain phosphoinositide hydrolysis. Eur. J. Phannacol. Mol. Pharmacol. 207,351-353. Schoepp D. D., Johnson B. G., and Monn J. A. (1 992) Inhibition of cyclic AMP formation by a selective metabotropic glutamate receptor agonist. J. Neurochem. 58, 1 184-1 186. Tanabe Y., Masu M., Ishii T., Shigemoto R., and Nakanishi S. (1992) A family of metabotropic glutamate receptors. Neuron 8, 1-20. Thompson N. T., Bonser R. W., and Lawrence G. G. (1991) R a p tor-coupled phospholipase D and its inhibition. Trends Pharmacol. Sci. 12, 404-408. Winder D. G. and Conn P-J. (1992~)Activation of metabotropic glutamate receptors in the hippocanipuskeases cyclic AMP accumulation. J. Neurochem. 59, 375-378. Winder D. G. and Conn P. J. (19926) Activation of metabotropic glutamate receptors increases cyclic AMP accumulation in hippocampus by potentiating responses to endogenous adenosine. J. Neurosci. (in press).

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