Neuroscience Research, 10 (1991) 71-77 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50
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NEURES 00425
Possible heterogeneity of metabotropic glutamate receptors induced in Xenopus oocytes by rat brain mRNA Shigeru T a n a b e 1, Isao Ito 2 and Hiroyuki Sugiyama 3,, 2
..
1 Department of Biology, Faculty of Science, Okayama University, Okayama, Fujtgotemba Research Labs, Chugai Pharmaceutical Company, Gotemba and J Department of Cellular Physiology, National Institute for Physiological Sciences, Okazaki (Japan) (Received 1 October 1990; Accepted 24 October 1990)
Key words: Excitatory amino acid; Glutamate receptor; Metabotropic glutamate receptor; Xenopus oocytes
SUMMARY Pharmacological properties of metabotropic glutamate receptors were studied in Xenopus oocytes injected with rat brain mRNA. trans-l-Amino-cyclopentyl-l,3-dicarboxylic acid (t-ACPD), a conformationaily restricted analog of glutamate, induced oscillatory inward currents in mRNA-injected oocytes. These t-ACPD responses showed several characteristics identical to those of the other metabotropic responses including the metabotropic glutamate responses stimulated by quisqualate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) effectively suppressed the t-ACPD and ibotenate responses. However, quisqualate responses were not affected substantially by D,L-AP3. These findings suggest that the metabotropic glutamate receptors in the oocytes may be classified into at least two subtypes according to their pharmacological properties: one preferentially activated by quisqualate and insensitive to AP3, and the other activated by t-ACPD and ibotenate and antagonized by AP3.
Receptors for excitatory amino acids such as glutamate have been classified into two major categories: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) 23. The iGluRs, including N-methyl-D-aspartate (NMDA)-type, aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type and kainate (KA)-type receptors 24, are directly coupled to cation-specific ion channels 8. In contrast, the mGluRs were first demonstrated in Xenopus oocytes injected with rat brain mRNA to function through the activation of GTP-binding proteins (G-proteins) and stimulate inositol phospholipid (PI) metabolism 22. The pharmacological properties of mGluRs have been studied in the oocytes, and the results indicated that glutamate (Glu), quisqualate (QA), ibotenate (IB) and L-homocystein sulfinate (L-HS) could activate mGluRs 23 Excitatory amino-acid-stimulated PI breakdown has also been studied biochemically in brain slices, cultured brain cells, or synaptoneurosomes 11.15.20 The pharmacological * Present address: Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan. Correspondence: Dr. Hiroyuki Sugiyama, Department of Biology, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan.
72 properties of excitatory amino acid recognition sites analyzed in these systems are not, however, completely consistent with each other or with those of mGluRs studied in Xenopus oocytes. For instance, L-2-amino-4-phosphonobutyrate (L-AP4) and L-serine-Ophosphate (L-SOP) were reported to inhibit the QA-stimulated PI responses in some systems 5,~0,16.18, whereas neither of them is effective on QA-stimulation in other systems 1.15,23. These discrepancies raised the question of whether or not these analyses, especially electrophysiological analyses using Xenopus oocytes and biochemical analyses using brain slices, are really analysing the same reactions and characterizing the same receptors. Xenopus oocytes injected with rat brain mRNA, in which the metabotropic (or G-protein-coupled) glutamate receptor was first demonstrated unambiguously 22, have several advantages for the study of this receptor compared to biochemical analyses using brain slices. In this system, all responses are recorded under voltage-clamp conditions, so that indirect responses mediated by voltage-dependent reactions such as the activation of voltage-dependent channels can be avoided. From the patterns of current responses, the responses mediated by iGluR can also be distinguished and eliminated easily and clearly from the responses mediated by mGluR 23. The responses can be evoked and measured repeatedly. Using the mRNAs extracted from whole brains, it may be possible to detect a receptor which is localized in a specific subregion of the brain. Recently, Palmer and colleagues reported that, in hippocampal slices, trans-l-aminocyclopentyl-l,3-dicarboxylic acid (t-ACPD), a conformationally restricted analog of glutamate, could activate PI breakdown in hippocampal slices without significantly affecting the iGluR sites x4. Following this report, Schoepp and Hillman reported that D,L-AP3 completely inhibited t-ACPD-stimulated PI breakdown observed in cortical slices ~9. On the other hand, we have already reported that metabotropic QA responses in oocytes are not inhibited by D,L-AP3 23 In the present study, we tried to confirm the existence of t-ACPD receptors directly coupled to phosphoinositide hydrolysis in Xenopus oocytes injected with rat brain mRNA, and examined the effects of D,L-AP3 on the t-ACPD-stimulated responses in this system. Experiments were performed as described previously 6,7,22. Poly(A)+RNA isolated from adult (200-300 g) rat brains and size-fractionated by sucrose density gradient centrifugation was injected into oocytes. After cultivation for a few days, the oocytes were analyzed electrophysiologically under voltage-clamp conditions (holding potential - 6 0 mV) in normal Ringer solution (88 mM NaCI, 2 mM KC1, 2 mM CaC12, 2 mM MgC12, 10 mM Tris, pH 7.6). Chemicals were obtained from the following sources: L-HS, t-ACPD, D,L-AP3 and L-AP4 from Tocris Neuramin; QA, IB, acetylcholine and atropine from Sigma; all other reagents from Wako Pure Chemicals. Under voltage-clamp conditions, bath application of t-ACPD induced characteristic current responses in Xenopus oocytes injected with rat brain m R N A in a dose-dependent manner (Fig. 1). Such t-ACPD responses were not observed with native oocytes (no m R N A injected). Responses to t-ACPD were observed as oscillatory inward currents with delayed onsets, and were almost completely suppressed by intracellular injection of E G T A (Fig. 1A). Atropine (10 nM), a potent inhibitor of muscarinic acetylcholine receptors, did not affect 100 ~M t-ACPD responses (102 _+ 5.9% of control, mean _+ SEM, n = 4), but intensive stimulation of muscarinic acetylcholine receptors strongly suppressed t-ACPD responses (cross-desensitization) (Fig. 1A). All of these features of the t-ACPD responses were the common properties of the responses evoked by muscarinic acetylcho-
73
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Fig. 1. Typical current responses evoked by t-ACPD in Xenopusoocytes injected with rat brain mRNA. (A) Inhibition of t-ACPD responses by intracellular EGTA (upper trace) and 'cross-desensitization' between t-ACPD and acetylcholine (ACh) responses (lower trace). (B) Dose-response curve of t-ACPD-induced inward currents. In (A), upper trace, 5 min after measurement of the control response (t-ACPD, 300 #M), 250 pmol EGTA was injected into the oocyte (arrow) 6. Five minutes after the injection, t-ACPD responses were almost completely inhibited. In (A), lower trace, an oocyte was first stimulated with 300 #M t-ACPD. After 10 min of washing, the oocyte was exposed to 200 #M ACh. Under such conditions, a second application of t-ACPD produced no appreciable responses. Calibrations were 1 min, 100 nA for the upper trace and 2 min, 200 nA for the lower trace. In (B) oocytes were exposed to increasing concentrations of t-ACPD. Data were expressed as percentages of the maximal responses (mean + SEM, n = 9). line 6, serotonina2, neurotensin 6 and metabotropic Q A stimulations 22 in Xenopus oocytes. The ECs0 for t - A C P D was about 100 # M (Fig. 1B). The t - A C P D responses were inhibited by D,L-AP3 in a dose-dependent m a n n e r (Fig. 2). The ICs0 of D,L-AP3 was about 300/~M (against 100/~M t - A C P D which is close to the ECs0 of the agonist). The inhibition of t - A C P D responses b y D,L-AP3 was reversible, and the t - A C P D responses completely recovered from AP3 inhibition after 1 0 - 1 5 min of washing (Fig. 2A). In contrast, metabotropic Q A ( 1 - 3 ~tM) responses were m u c h less sensitive to and essentially unaffected by D,L-AP3 at concentrations of up to 1 m M (Figs. 2A and 2B). Responses to 3 # M Q A were inhibited significantly by D,L-AP3 only at concentrations of 3 m M or more (Fig. 2B). Responses induced b y 2 / ~ M of acetylcholine were not inhibited b y 3 m M D,L-AP3 (95 _+ 2.3% of control, mean _+ SEM, n = 3). A P 4 also suppressed t - A C P D (100 ttM) responses in a reversible manner, although the extent was significantly less than that by AP3 (the response in the presence of 1 m M L-AP4 was 66 + 2.5% of the control (mean _+ SEM, n = 3)). D,L-AP3 and L-AP4 did not induce any appreciable responses by themselves if they were applied at concentrations of 1 - 3 m M (Fig. 2A). 6-Cyano-7-nitroquinoxaline-2,3-dione ( C N Q X , 20 /xM) did not affect the t - A C P D (100 # M ) responses (122 _+ 22.6%, m e a n _4- SEM, n = 3). These pharmacological properties of t - A C P D responses induced in Xenopus oocytes were very similar to those reported for the receptor sites coupled to PI hydrolysis in brain slices 14,19 Finally, we c o m p a r e d the inhibitory effects of D,L-AP3 (1 m M ) on the m G l u R responses induced b y QA, t - A C P D , Glu, IB and L-HS. ECs0s for these agonists were estimated to be 3, 100, 40, 30 and 70/~M, respectively (Refs. 23 and 24, and the present study). The results are summarized in Figure 3. D,L-AP3 suppressed t - A C P D responses
74 A
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Fig. 2. Effects of I),L-AP3 on current responses evoked by t-ACPD and QA (A), and the dose-inhibition curves (B). In (A) D,L-AP3 did not induce any appreciable currents by itself, but inhibited t-ACPD responses. The inhibitory effects of D,L-AP3 were reversible, and the responses completely recovered after 10-15 rain washing~ QA responses were not affected by D,L-AP3. Concentrations used were: t-ACPD, 100 #M; QA, 1 #M; D,L-AP3. 1 mM. Calibrations were 1 rnin and 50 nA, In (B) responses were measured repeatedly at about 15-rain intervals. Oocytes were pretreated with increasing concentrations of D,L-AP3 for 1 rain prior to perfusion with the agonists and the same concentrations of D,L-AP3. Data were expressed as percentages of the control responses (mean ± SEM, n = 3-12). Concentrations used were: t-ACPD (open circles), 100 #M; QA (closed circles ), 3/LM.
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Fig. 3. Effects of 1 mM D,L-AP3 on current responses evoked by various mGluR agonists. Peak amplitudes of the inward currents were measured in the presence or absence of 1 mM D,L-AP3. Results were expressed as percentages of the control (mean ± SEM). Numbers in parentheses indicate number of experiments.
75 even at a nearly saturating concentration of the agonist (300/~M). In addition, D,L-AP3 suppressed responses evoked by IB at its ECs0 concentration. However, D,L-AP3 did not inhibit QA responses significantly. L-Glu and L-HS responses were inhibited to about 60% of the control. Under voltage-clamp conditions, bath applications of t-ACPD evoked current responses in Xenopus oocytes injected with rat brain mRNA. These responses showed typical features in common with the metabotropic (or G-protein-mediated) responses such as muscarinic acetylcholine, serotonin, neurotensin and QA responses induced in Xenopus oocytes 6.12,22: oscillatory inward current responses, inhibition by intracellular EGTA, and 'cross-desensitization' with muscarinic acetylcholine responses. From these results as well as the structural similarity between t-ACPD and glutamate, it was concluded that a certain type of m G l u R which can be activated by t-ACPD was induced in the oocytes by rat brain mRNA. We found that m G l u R responses induced by several agonists showed different sensitivities to D,L-AP3. These results suggest that m G l u R may be classified into at least two subtypes: one preferring QA and insensitive to AP3, and the other preferring IB and t-ACPD and sensitive to AP3. t-Glu and L-HS may be mixed agonists for these two subtypes. The hypothesis that glutamate receptors coupled to PI metabolism may be heterogeneous has been suggested 21, based on the comparison of different results by different groups of researchers using different systems. They include: (1) AP4 suppresses IB-stimulated PI hydrolysis in hippocampal slices 4,9,10,16,17, whereas it does not antagonize QA effects in striatal neurons 3,25, cerebellar slices 1, synaptoneurosomes 15, and Xenopus oocytes 23. (2) In brain slices from young animals, excitatory amino-acid-stimulated PI breakdown progressively declines during postnatal development, but the time courses of these reductions are different for different agonists 4,9,19 (3) AMPA-insensitive and QA-sensitive glutamate binding sites are mainly located in the cerebellar molecular layer 2, whereas IB- or t-ACPD-stimulated PI hydrolysis is extremely high in the hippocampus 4,9,21. However, most of these arguments are based on indirect comparison, and the interpretation could be very complicated. Our findings reported here provide very strong and straightforward evidence for the pharmacological heterogeneity of mGluR. An argument against our hypothesis may be the observation that, in hippocampal and cerebellar slices, the QA and IB responses were not additive 1.16 However, in hippocampal slices, it has also been reported that QA responses were suppressed by activation of N M D A receptors 13. In the experiments on brain slices, the additivity was examined using high concentrations of QA and IB (100-500 #M) 1.16, and the possibility that IB may have suppressed the QA responses via activation of N M D A receptors cannot be ruled out. We have also reported that, in Xenopus oocytes, QA and IB responses were not additive 23. One of the possible explanations for this may be that QA-preferring and IB-preferring subtypes of mGluRs could be coupled to the same G-proteins, so that full activation of one subtype of these receptors could result in the depletion of G-proteins for the other subtype. Whatever the mechanism may be, it may be difficult to provide conclusive arguments for the additivity of responses mediated by complicated reaction pathways. It has been reported that both IB and t-ACPD responses in brain slices showed very similar pharmacological properties 4,19. In our study as well, both IB and t-ACPD responses were inhibited to a similar extent by 1 mM of AP3 (Fig. 3). However, there is no direct evidence at present to show that IB and t-ACPD are acting at precisely the same receptor site. In this regard, it has been reported that the regional distribution and the
76 ontogeny of IB-stimulated PI resonses are not strictly consistent with those of t-ACPD 4.1,~ To clarify this point, further studies are required. ACKNOWLEDGEMENT
We are grateful to Professor Tsuneo Yamaguchi, O k a y a m a University, for his support. REFERENCES 1 Blackstone, C.D., Supattapone, S.S. and Snyder, S.H., Inositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fiber-Purkinje-cell synaptic transmission, Proc. Natl. Acad. Sci. USA, 86 (1989) 4316-4320. 2 Cha, J.J., Makowiec, R.L., Penney, J.B. and Young, A.B., L-[3HlGlutamate labels the metabotropic excitatory amino acid receptor in rodent brain, Neurosci. Lett., 113 (1990) 78-83. 3 Coble, A. and Perrier, M.L., Pharmacology of excitatory amino acid receptors coupled to inositol phosphate metabolism in neonatal rat striatum, Neurochem. Int., 15 (1989) 1-8. 4 Desai, M.A. and Conn, P.J., Selective activation of phosphoinositide hydrolysis by a rigid analogue of glutamate, Neurosci. Lett., 109 (1990) 157-162. 5 Godfrey, P.P. and Taghavi, Z., The effect of non-NMDA antagonists and phorbol esters on excitatory amino acid stimulated inositol phosphate formation in rat cerebral cortex, Neurochem. Int., 16 (1990) 65-72. 6 Hirono, C., Ito, I. and Sugiyama, H., Neurotensin and acetylcholine evoke common responses in frog oocytes injected with rat brain messenger ribonucleic acid, J. Physiol., 382 (1987) 523-535. 7 Ito, I., Hirono, C., Yamagishi, S., Nomura, Y., Kaneko, S. and Suglyama, H., Roles of protein kinases in neurotransmitter responses in Xenopus oocytes injected with rat brain mRNA, J. Cell. Physiol., 134 (1988) 155-160. 8 MacDermott, A.B. and Dale, N., Receptor, ion channels and synaptic potentials underlying the integrative actions of excitatory amino acids, Trends Neurosci., 10 (1987) 280-284. 9 Nicoletti, F., ladarola, M.J., Wroblewski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with al-adrenoceptors, Proc. Natl. Acad. Sci. USA, 83 (1986) 1931-1935. 10 Nicoletti, F., Meek, J.L., Iadarola, M.J., Chuang, D.M., Roth, B,L. and Costa, E., Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippoeampus, J. Neurochem., 46 (1986) 40-46. 11 Nicoletti, F., Wroblewski, J.T., Fadda, E. and Costa, E., Pertussis toxin inhibits signal transduction at a specific metabolotropic glutamate receptor in primary cultures of cerebellar granule cells, Neuropharmacology, 27 (1988) 551-556. 12 Nomura, Y., Kaneko, S., Kato, K., Yamagishi, S. and Sugiyama, H., Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA, 34ol. Brain Res., 2 (1987) 113-123. 13 Palmer, E., Monaghan, D.T. and Cotman, C.W., Glutamate receptors and phosphoinositide metabolism: stimulation via quisquaiate receptors is inhibited by N-methyl-D-aspartate receptor activation, Mol. Brain Res., 4 (1988) 161-165. 14 Palmer, E., Monaghan, D.T. and Cotman, C.W., Trans-ACPD, a selective agonist of the phosphoinositidecoupled excitatory amino acid receptor, Eur. J. PharmacoL, 166 (1989) 585-587. 15 Recasens, M., Guiramand, J. and Nourigat, A., A new quisqualate receptor subtype (sAA2) responsible for the glutamate-induced inositol phosphate formation in rat brain synaptoneurosomes, Neurochem. Int., 13 (1988) 463-467. 16 Schoepp, D.D. and Johnson, B.G., Excitatory amino acid agonist-antagonist interactions at 2-amino-4-phosphonobutyric acid-sensitive quisqualate receptors coupled to phosphoinositide hydrolysis in slices of rat hippocampus, J. Neurochem., 50 (1988) 1605-1613. 17 Schoepp, I~.~D. and Johnson, B.G., Comparison of excitatory amino acid-stimulated phosphoinositide hydrolysis and N-[3H]acetylaspartylglutamate binding in rat brain: selective inhibition of phosphoinositide hydrolysis by 2-amino-3-phosphonoproprionate, J. Neurochem., 53 (1989) 273-278. 18 Schoepp, D.D. and Johnson, B.G., Inhibition of excitatory amino acid-stimulated phosphoinositide hydrolysis in the neonatal rat hippoeampus by 2-amino-3-phosphonopropionate, J. Neurochem., 53 (1989) 1865-1870. 19 Schoepp, D.D. and Hillman, C.C., Developmental and pharmacological characterization of quisqnalate, ibotenate and trans-l-amino-l,3-cyciopentanedicarboxylic acid stimulations of phosphoinositide hydrolysis in rat cortical brain slices, Biogenic Amines, 7 (1990) 331-340.
77 20 Sladeczek, F., Pin, J., R&:asens, M., Bockaert, J. and Weiss, S., Glutamate stimulated inositol phosphate formation in striatal neurones, Nature, 317 (1985) 717-719. 21 Sladeczek, F., R&:asens, M. and Bockaert, J., A new mechanism for glutamate receptor action: phosphoinositide hydrolysis, Trends Neurosci., 11 (1988) 545-549. 22 Sugiyama, H., lto, 1. and Hirono, C., A new type of glutamate receptor linked to inositol phospholipid metabolism, Nature, 325 (1987) 531-533. 23 Sugiyama, H., lto, I. and Watanabe, M., Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA, Neuron, 3 (1989) 129-132. 24 Watkins, J.C., Krogsgaard-Larsen, P. and HonorS, T., Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists, Trends Physiol. Sci., 11 (1990) 25-33. 25 Weiss, S., Two distinct quisqualate receptor systems are present on striatal neurons, Brain Res., 491 (1989) 189-193.
"7!
NEURES 00425
Possible heterogeneity of metabotropic glutamate receptors induced in Xenopus oocytes by rat brain mRNA Shigeru T a n a b e 1, Isao Ito 2 and Hiroyuki Sugiyama 3,, 2
..
1 Department of Biology, Faculty of Science, Okayama University, Okayama, Fujtgotemba Research Labs, Chugai Pharmaceutical Company, Gotemba and J Department of Cellular Physiology, National Institute for Physiological Sciences, Okazaki (Japan) (Received 1 October 1990; Accepted 24 October 1990)
Key words: Excitatory amino acid; Glutamate receptor; Metabotropic glutamate receptor; Xenopus oocytes
SUMMARY Pharmacological properties of metabotropic glutamate receptors were studied in Xenopus oocytes injected with rat brain mRNA. trans-l-Amino-cyclopentyl-l,3-dicarboxylic acid (t-ACPD), a conformationaily restricted analog of glutamate, induced oscillatory inward currents in mRNA-injected oocytes. These t-ACPD responses showed several characteristics identical to those of the other metabotropic responses including the metabotropic glutamate responses stimulated by quisqualate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) effectively suppressed the t-ACPD and ibotenate responses. However, quisqualate responses were not affected substantially by D,L-AP3. These findings suggest that the metabotropic glutamate receptors in the oocytes may be classified into at least two subtypes according to their pharmacological properties: one preferentially activated by quisqualate and insensitive to AP3, and the other activated by t-ACPD and ibotenate and antagonized by AP3.
Receptors for excitatory amino acids such as glutamate have been classified into two major categories: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) 23. The iGluRs, including N-methyl-D-aspartate (NMDA)-type, aamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type and kainate (KA)-type receptors 24, are directly coupled to cation-specific ion channels 8. In contrast, the mGluRs were first demonstrated in Xenopus oocytes injected with rat brain mRNA to function through the activation of GTP-binding proteins (G-proteins) and stimulate inositol phospholipid (PI) metabolism 22. The pharmacological properties of mGluRs have been studied in the oocytes, and the results indicated that glutamate (Glu), quisqualate (QA), ibotenate (IB) and L-homocystein sulfinate (L-HS) could activate mGluRs 23 Excitatory amino-acid-stimulated PI breakdown has also been studied biochemically in brain slices, cultured brain cells, or synaptoneurosomes 11.15.20 The pharmacological * Present address: Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan. Correspondence: Dr. Hiroyuki Sugiyama, Department of Biology, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan.
72 properties of excitatory amino acid recognition sites analyzed in these systems are not, however, completely consistent with each other or with those of mGluRs studied in Xenopus oocytes. For instance, L-2-amino-4-phosphonobutyrate (L-AP4) and L-serine-Ophosphate (L-SOP) were reported to inhibit the QA-stimulated PI responses in some systems 5,~0,16.18, whereas neither of them is effective on QA-stimulation in other systems 1.15,23. These discrepancies raised the question of whether or not these analyses, especially electrophysiological analyses using Xenopus oocytes and biochemical analyses using brain slices, are really analysing the same reactions and characterizing the same receptors. Xenopus oocytes injected with rat brain mRNA, in which the metabotropic (or G-protein-coupled) glutamate receptor was first demonstrated unambiguously 22, have several advantages for the study of this receptor compared to biochemical analyses using brain slices. In this system, all responses are recorded under voltage-clamp conditions, so that indirect responses mediated by voltage-dependent reactions such as the activation of voltage-dependent channels can be avoided. From the patterns of current responses, the responses mediated by iGluR can also be distinguished and eliminated easily and clearly from the responses mediated by mGluR 23. The responses can be evoked and measured repeatedly. Using the mRNAs extracted from whole brains, it may be possible to detect a receptor which is localized in a specific subregion of the brain. Recently, Palmer and colleagues reported that, in hippocampal slices, trans-l-aminocyclopentyl-l,3-dicarboxylic acid (t-ACPD), a conformationally restricted analog of glutamate, could activate PI breakdown in hippocampal slices without significantly affecting the iGluR sites x4. Following this report, Schoepp and Hillman reported that D,L-AP3 completely inhibited t-ACPD-stimulated PI breakdown observed in cortical slices ~9. On the other hand, we have already reported that metabotropic QA responses in oocytes are not inhibited by D,L-AP3 23 In the present study, we tried to confirm the existence of t-ACPD receptors directly coupled to phosphoinositide hydrolysis in Xenopus oocytes injected with rat brain mRNA, and examined the effects of D,L-AP3 on the t-ACPD-stimulated responses in this system. Experiments were performed as described previously 6,7,22. Poly(A)+RNA isolated from adult (200-300 g) rat brains and size-fractionated by sucrose density gradient centrifugation was injected into oocytes. After cultivation for a few days, the oocytes were analyzed electrophysiologically under voltage-clamp conditions (holding potential - 6 0 mV) in normal Ringer solution (88 mM NaCI, 2 mM KC1, 2 mM CaC12, 2 mM MgC12, 10 mM Tris, pH 7.6). Chemicals were obtained from the following sources: L-HS, t-ACPD, D,L-AP3 and L-AP4 from Tocris Neuramin; QA, IB, acetylcholine and atropine from Sigma; all other reagents from Wako Pure Chemicals. Under voltage-clamp conditions, bath application of t-ACPD induced characteristic current responses in Xenopus oocytes injected with rat brain m R N A in a dose-dependent manner (Fig. 1). Such t-ACPD responses were not observed with native oocytes (no m R N A injected). Responses to t-ACPD were observed as oscillatory inward currents with delayed onsets, and were almost completely suppressed by intracellular injection of E G T A (Fig. 1A). Atropine (10 nM), a potent inhibitor of muscarinic acetylcholine receptors, did not affect 100 ~M t-ACPD responses (102 _+ 5.9% of control, mean _+ SEM, n = 4), but intensive stimulation of muscarinic acetylcholine receptors strongly suppressed t-ACPD responses (cross-desensitization) (Fig. 1A). All of these features of the t-ACPD responses were the common properties of the responses evoked by muscarinic acetylcho-
73
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Fig. 1. Typical current responses evoked by t-ACPD in Xenopusoocytes injected with rat brain mRNA. (A) Inhibition of t-ACPD responses by intracellular EGTA (upper trace) and 'cross-desensitization' between t-ACPD and acetylcholine (ACh) responses (lower trace). (B) Dose-response curve of t-ACPD-induced inward currents. In (A), upper trace, 5 min after measurement of the control response (t-ACPD, 300 #M), 250 pmol EGTA was injected into the oocyte (arrow) 6. Five minutes after the injection, t-ACPD responses were almost completely inhibited. In (A), lower trace, an oocyte was first stimulated with 300 #M t-ACPD. After 10 min of washing, the oocyte was exposed to 200 #M ACh. Under such conditions, a second application of t-ACPD produced no appreciable responses. Calibrations were 1 min, 100 nA for the upper trace and 2 min, 200 nA for the lower trace. In (B) oocytes were exposed to increasing concentrations of t-ACPD. Data were expressed as percentages of the maximal responses (mean + SEM, n = 9). line 6, serotonina2, neurotensin 6 and metabotropic Q A stimulations 22 in Xenopus oocytes. The ECs0 for t - A C P D was about 100 # M (Fig. 1B). The t - A C P D responses were inhibited by D,L-AP3 in a dose-dependent m a n n e r (Fig. 2). The ICs0 of D,L-AP3 was about 300/~M (against 100/~M t - A C P D which is close to the ECs0 of the agonist). The inhibition of t - A C P D responses b y D,L-AP3 was reversible, and the t - A C P D responses completely recovered from AP3 inhibition after 1 0 - 1 5 min of washing (Fig. 2A). In contrast, metabotropic Q A ( 1 - 3 ~tM) responses were m u c h less sensitive to and essentially unaffected by D,L-AP3 at concentrations of up to 1 m M (Figs. 2A and 2B). Responses to 3 # M Q A were inhibited significantly by D,L-AP3 only at concentrations of 3 m M or more (Fig. 2B). Responses induced b y 2 / ~ M of acetylcholine were not inhibited b y 3 m M D,L-AP3 (95 _+ 2.3% of control, mean _+ SEM, n = 3). A P 4 also suppressed t - A C P D (100 ttM) responses in a reversible manner, although the extent was significantly less than that by AP3 (the response in the presence of 1 m M L-AP4 was 66 + 2.5% of the control (mean _+ SEM, n = 3)). D,L-AP3 and L-AP4 did not induce any appreciable responses by themselves if they were applied at concentrations of 1 - 3 m M (Fig. 2A). 6-Cyano-7-nitroquinoxaline-2,3-dione ( C N Q X , 20 /xM) did not affect the t - A C P D (100 # M ) responses (122 _+ 22.6%, m e a n _4- SEM, n = 3). These pharmacological properties of t - A C P D responses induced in Xenopus oocytes were very similar to those reported for the receptor sites coupled to PI hydrolysis in brain slices 14,19 Finally, we c o m p a r e d the inhibitory effects of D,L-AP3 (1 m M ) on the m G l u R responses induced b y QA, t - A C P D , Glu, IB and L-HS. ECs0s for these agonists were estimated to be 3, 100, 40, 30 and 70/~M, respectively (Refs. 23 and 24, and the present study). The results are summarized in Figure 3. D,L-AP3 suppressed t - A C P D responses
74 A
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10
30 (raM)
Fig. 2. Effects of I),L-AP3 on current responses evoked by t-ACPD and QA (A), and the dose-inhibition curves (B). In (A) D,L-AP3 did not induce any appreciable currents by itself, but inhibited t-ACPD responses. The inhibitory effects of D,L-AP3 were reversible, and the responses completely recovered after 10-15 rain washing~ QA responses were not affected by D,L-AP3. Concentrations used were: t-ACPD, 100 #M; QA, 1 #M; D,L-AP3. 1 mM. Calibrations were 1 rnin and 50 nA, In (B) responses were measured repeatedly at about 15-rain intervals. Oocytes were pretreated with increasing concentrations of D,L-AP3 for 1 rain prior to perfusion with the agonists and the same concentrations of D,L-AP3. Data were expressed as percentages of the control responses (mean ± SEM, n = 3-12). Concentrations used were: t-ACPD (open circles), 100 #M; QA (closed circles ), 3/LM.
(8)
~Z
100
(1 o) T
~
80
(6) (7)
.- 60 E o)
>
40
c~
20
100,300 30
HS 10 70
QA 1, 3
(~M)
Fig. 3. Effects of 1 mM D,L-AP3 on current responses evoked by various mGluR agonists. Peak amplitudes of the inward currents were measured in the presence or absence of 1 mM D,L-AP3. Results were expressed as percentages of the control (mean ± SEM). Numbers in parentheses indicate number of experiments.
75 even at a nearly saturating concentration of the agonist (300/~M). In addition, D,L-AP3 suppressed responses evoked by IB at its ECs0 concentration. However, D,L-AP3 did not inhibit QA responses significantly. L-Glu and L-HS responses were inhibited to about 60% of the control. Under voltage-clamp conditions, bath applications of t-ACPD evoked current responses in Xenopus oocytes injected with rat brain mRNA. These responses showed typical features in common with the metabotropic (or G-protein-mediated) responses such as muscarinic acetylcholine, serotonin, neurotensin and QA responses induced in Xenopus oocytes 6.12,22: oscillatory inward current responses, inhibition by intracellular EGTA, and 'cross-desensitization' with muscarinic acetylcholine responses. From these results as well as the structural similarity between t-ACPD and glutamate, it was concluded that a certain type of m G l u R which can be activated by t-ACPD was induced in the oocytes by rat brain mRNA. We found that m G l u R responses induced by several agonists showed different sensitivities to D,L-AP3. These results suggest that m G l u R may be classified into at least two subtypes: one preferring QA and insensitive to AP3, and the other preferring IB and t-ACPD and sensitive to AP3. t-Glu and L-HS may be mixed agonists for these two subtypes. The hypothesis that glutamate receptors coupled to PI metabolism may be heterogeneous has been suggested 21, based on the comparison of different results by different groups of researchers using different systems. They include: (1) AP4 suppresses IB-stimulated PI hydrolysis in hippocampal slices 4,9,10,16,17, whereas it does not antagonize QA effects in striatal neurons 3,25, cerebellar slices 1, synaptoneurosomes 15, and Xenopus oocytes 23. (2) In brain slices from young animals, excitatory amino-acid-stimulated PI breakdown progressively declines during postnatal development, but the time courses of these reductions are different for different agonists 4,9,19 (3) AMPA-insensitive and QA-sensitive glutamate binding sites are mainly located in the cerebellar molecular layer 2, whereas IB- or t-ACPD-stimulated PI hydrolysis is extremely high in the hippocampus 4,9,21. However, most of these arguments are based on indirect comparison, and the interpretation could be very complicated. Our findings reported here provide very strong and straightforward evidence for the pharmacological heterogeneity of mGluR. An argument against our hypothesis may be the observation that, in hippocampal and cerebellar slices, the QA and IB responses were not additive 1.16 However, in hippocampal slices, it has also been reported that QA responses were suppressed by activation of N M D A receptors 13. In the experiments on brain slices, the additivity was examined using high concentrations of QA and IB (100-500 #M) 1.16, and the possibility that IB may have suppressed the QA responses via activation of N M D A receptors cannot be ruled out. We have also reported that, in Xenopus oocytes, QA and IB responses were not additive 23. One of the possible explanations for this may be that QA-preferring and IB-preferring subtypes of mGluRs could be coupled to the same G-proteins, so that full activation of one subtype of these receptors could result in the depletion of G-proteins for the other subtype. Whatever the mechanism may be, it may be difficult to provide conclusive arguments for the additivity of responses mediated by complicated reaction pathways. It has been reported that both IB and t-ACPD responses in brain slices showed very similar pharmacological properties 4,19. In our study as well, both IB and t-ACPD responses were inhibited to a similar extent by 1 mM of AP3 (Fig. 3). However, there is no direct evidence at present to show that IB and t-ACPD are acting at precisely the same receptor site. In this regard, it has been reported that the regional distribution and the
76 ontogeny of IB-stimulated PI resonses are not strictly consistent with those of t-ACPD 4.1,~ To clarify this point, further studies are required. ACKNOWLEDGEMENT
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