355
Brain Research, 554 (1991) 355-357 (~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391247522
BRES 24752
Effect of nitric oxide on ,-[all]glutamate binding to rat brain synaptic membranes Hiroyuki Fujimori and Hidemitsu Pan-Hou Department of Analytical Chemistry in Hygiene, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka (Japan)
(Accepted 16 April 1991) Key words: Nitric oxide; L-Glutamate binding; Neurotransmission; Synaptic membrane receptor; Sodium nitroprusside; Regulation of nitric oxide
Nitric oxide (NO), which is spontaneously generated from sodium nitroprusside, was shown to inhibit L-[3H]glutamatebinding to rat brain synaptic membranes in a concentration-dependent manner. The L-glutamate binding inhibited by NO, was largely recoverable by the addition of hemoglobin, a scavenger of NO, to the assay medium. These results suggest that NO may play an important role in the modulation of excitatory neurotransmission through direct interaction of L-glutamate binding to its physiological synaptic membrane receptors. L-Glutamate is believed to be the major excitatory transmitter in the mammalian central nervous systems and there are at least 3 separate subtypes of receptor, which are characterized by selective interaction with N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) 3'23. Activation of these receptors triggers a variety of transmembrane biochemical processes including stimulation of calcium influx 24, activation of inositol phospholipid turnover 17, and accumulation of cellular cGMP 2'5'18 in neural cells. The elevation of cGMP levels induced by L-glutamate and its structural analogs in the cells prepared from rat cerebellum has been shown to be mediated by Ca2÷-dependent formation of nitric oxide (NO) from L-arginine 1'5-7'1°. The biosynthesis of NO from the terminal guanidino nitrogen atoms of L-arginine was originally demonstrated in vascular endothelial cells and now has been identified in many cells and tissues 8"1°'11'19. Accumulating evidence suggests that NO may play an important role as a messenger molecule in the regulation of cell function and cellular communication in many biological systems 4' 9,10,15. However, at present, there is little direct evidence that the short-lived diffusible NO can directly participate in the neurotransmission in the central nervous systems, particularly in the primary interaction of ligands with its specific physiological membrane receptors. The functional consequences of the released NO induced by the activation of excitatory amino acids in neural tissue are largely unknown. One L-glutamate binding molecule isolated from rat brain synaptic membranes was found to be a metallo-
protein whose activity is dependent on the integrity of its iron-sulfur center at the recognition site of glutamate binding 13'14. The biological action of NO is generally considered to be mediated through direct nitrosation of the transition metals of active molecules 8'9'12'22. In view of these findings, it is of interest to know whether NO can directly modulate L-glutamate binding to its physiological membrane receptors. In the present article, we provide the first evidence that NO, spontaneously produced from sodium nitroprusside directly regulates L-glutamate binding to its synaptic membrane receptors. A possible role for NO in the maintenance of homeostasis of glutamatergic transmission is suggested. Crude brain synaptic membranes were prepared from whole brain of male albino Wistar rats weighing 180-220 g according to the procedures described by Zukin et al. 25. Extensively washed and frozen rat brain synaptic membranes were thawed at room temperature and washed twice with 20 vols. of 50 mM Tris-acetate buffer (pH 7.4). The washed synaptic membranes (approximately 10 mg protein) were incubated in 30 ml of the Tris-acetate buffer (pH 7.4) containing 0.08% (v/v) Triton X-100 at 2 °C. After 10 min of incubation, the treated membranes were washed twice with 20 vols. of the chilled Tris-acetate buffer before use 2°'21. For L-[3H]glutamate binding, the Triton X-100-treated synaptic membranes (0.2 mg protein) were suspended in 0.5 ml of 50 mM Tris-acetate buffer (pH 7.4) containing varying concentrations of sodium nitroprusside and preincubated at 37 °C for 15 min. After cooling on ice, the assay was initiated by the addition of 10 nM
Correspondence: H. Pan-Hou, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-01, Japan.
356
l--lNone F ~ H b (20 aM)
100
o ~j
% 75
H
5O0 4--: C O : NONe
/\ /~1,
%
i. ,i
I:!~
i : 8 1 u ( 1 . 0 mr,l) • :NMDA(O. 1 in,~1) : g.,'~ •. 3 ,,t.~)
a4,
~: 25o 50
2: i. ,4 io l
1:4 I:il I.;4
r~
25 re5
J
0
I
I I I I 20 30 Time (min) Fig. 2. Effect of sodium nitroprusside (SNP), L-glutamate (Glu) and NMDA on dissociation of L-[3H]glutamate binding to the Triton X-100-treated synaptic membranes. Triton X-100-treated synaptic membranes were incubated with 10 nM L-[3H]glutamate in 0.5 ml of 50 mM Tris-acetate buffer (pH 7.4) at 2 °C. At the time indicated by the arrow, SNP, Glu or NMDA was added to the incubation mixture and the incubation was continued at 2 °C. Data are the mean + S.E.M. of 5 independent determinations. 0
10 30 SNP (JJM)
100
300
Fig. 1. Effect of sodium nitroprusside (SNP) on the NMDA-specific L-[3H]glutamate binding to the Triton X-t00-treated synaptic membranes prepared from rat brain in the absence or presence of 20/~M hemoglobin (Hb). Data are the mean + S.E.M. of 5 independent determinations. L-[3H]glutamic acid (spec. act. 54.7 Ci/mmol; New England Nuclear), in the absence or presence of 20/~M of hemoglobin (Sigma). After incubation at 2 °C for 15 min, the reaction was terminated by the addition of 5 ml of the chilled Tris-acetate buffer and filtration through a Whatman GF/B glass microfiber filter pretreated with 0.3% polyethylenimine. After washing the filter 4 times with 5 ml of the chilled Tris-acetate buffer, the radioactivity trapped on the filter was measured by a liquid scintillation spectrometer (Aloka LSC-900) using 7 ml of modified Triton-toluene scintillant at a counting efficiency of 38-42% 20. Non-specific and N-methyl-Daspartate (NMDA)-specific L-[3H]glutamate bindings were obtained from the radioactivity displaced by 1 mM L-glutamic acid and 0.1 mM N M D A , respectively 2°'el. The assay was carried out in 5 independent determinations with a variation of less than 10%. Effect of sodium nitroprusside on L-[3H]glutamate binding to the Triton X-100-treated synaptic membranes is shown in Fig. 1. Under conditions employed in the present study, approximately 90% of the specific L[3H]glutamate binding observed is displaced by 0.1 mM N M D A . This result is in agreement with that reported by Monahan and Michel 16. This NMDA-specific L - [ 3 H ] glutamate binding was markedly inhibited by sodium nitroprusside in a concentration-dependent manner. The binding activity inhibited by sodium nitroprusside, was largely recovered by the addition of 20/~M hemoglobin in the assay medium. The recovery is explained by the ability of hemoglobin to bind NO through an interaction with the iron atom in the heme. These findings clearly
I 10
1
indicate that NO, spontaneously generated from sodium nitroprusside, has an ability to inhibit L-glutamate binding to its physiological membrane receptors. As shown in Fig. 2, L-Jail]glutamate binding to the Triton X-100-treated synaptic membranes progressively increased in proportion to the incubation time and almost reached an equilibrium within 10 min at 2 °C. Addition of non-radioactive L-glutamate (1 mM) or N M D A (0.1 mM) to the assay medium at 15 min after the initiation of incubation at 2 °C induced a prompt dissociation of the binding. Sodium nitroprusside (0.3 mM) also exerted a prompt dissociation of the L-glutamate binding. The result obtained suggests that N O generated from sodium nitroprusside, has an ability not only to inhibit Lglutamate binding to its synaptic membrane receptors, but also to be capable of inducing dissociation of the ligand from its recognition site of the physiological receptors. The N M D A - r e c e p t o r complex is prominently involved in excitatory transmission in the mammalian central nervous system. It has been shown that activation of NMDA-receptors in rat cerebellum induced a large increase in c G M P levels, an effect which is mediated by the Ca2+-dependent formation of N O from L-arginine and subsequent activation of soluble guanylate cyclase 1' 5-7,10. The excitatory amino acid induced elevation of cerebellar cGMP has been used as a biochemical index to study signal transduction following application of agonist(s) and antagonist(s) 2'18. However, the functional consequences of increase in c G M P levels and the precise modulatory role of c G M P in the neurotransmission in the
357 c e r e b e l l u m are still a m a t t e r of controversy. The biosynthesis of N O from L-arginine is now known to be a wide-spread mechanism for regulation of cell function and cellular communication in several biological systems 4'9'1°'15. N O is a n o n - p o l a r molecule like O2, and it can readily diffuse across cell m e m b r a n e s . All the biological effects of N O are m e d i a t e d through an interaction with i r o n sulfur center of m e t a l l o p r o t e i n in target cells8'9'12'22. The g l u t a m a t e binding protein, one of the c o m p o n e n t s of the g l u t a m a t e receptors, isolated from rat brain synaptic m e m b r a n e was found to be a h e m o p r o t e i n containing 2 g atoms of F e - S p e r mole of the purified protein 13"14. The results o b t a i n e d in the present study d e m o n s t r a t e that exogenous N O is capable of modulating the primary interaction of L-glutamate with its physiological synaptic receptors most likely via direct modification of F e - S center of the ligand binding molecules. Taking together 1 Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamatelinked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9030-9033. 2 Ferrendelli, J.A., Chang, N.M. and Kinscherf, D.A., Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids, J. Neurochem., 22 (1974) 535-540. 3 Foster, A. and Fagg, G., Acidic amino acid binding sites in mammalian neuronal membranes; their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 4 Gaily, J.A., Montague, ER., Reeke Jr., G.N. and Edelman, G.M., The NO hypothesis: possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 3547-3551. 5 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. 6 Garthwaite, J., Garthwaite, G., Palmer, R.M.J. and Moncada, S., NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices, Eur. J. Pharmacol., 172 (1989) 413-416. 7 Garthwaite, J., Southam, E. and Anderton, M., A kainate receptor linked to nitric oxide synthesis from arginine, J. Neurochem., 53 (1989) 1952-1954. 8 Hibbs Jr., J.B., Taintor, R.R., Vavrin, Z. and Rachlin, E.M., Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157 (1988) 87-94. 9 Ignarro, L.J., Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling, Pharmacol. Toxicol., 67 (1990) 1-7. 10 Kowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5159-5162. 11 Mccall, T.B., Boughton-Smith, N.K., Palmer, R.M.J., Whittle, B.J.R. and Moncada, S., Synthesis of nitric oxide from Larginine by neutrophils. Release and interaction with superoxide anion, Biochem. J., 261 (1989) 293-296. 12 Meyer, J., Comparison of carbon monoxide, nitric oxide and nitrite as inhibitors of the nitrogenase from Clostridium pasteurianum, Arch. Biochem. Biophys., 210 (1981) 246-256. 13 Michaelis, E.K., The glutamate receptor-like protein of brain synaptic membranes is metalloprotein, Biochem. Biophys. Res.
this finding with the previous observations as r e p o r t e d so far, we speculate that the C a 2 ÷ - d e p e n d e n t intracellular generation of N O induced by activation of N M D A receptors m a y be a negative f e e d b a c k mechanism to turn off the excessive or p r o l o n g e d activation of the N M D A receptors by the ligands, when the N O p r o d u c e d diffuses across the m e m b r a n e barriers and subsequently acts on the binding sites. T h e r e f o r e , the physiological implication of Ca2÷-triggered N O formation m a y be involved in the maintenance of the homeostasis of the r e c e p t o r activity rather than via mechanisms involving cGMP. T h e dynamic regulation of N O formation requires further study since large amounts of N O may elicit cytotoxic action via destruction of the Fe-S center in bioactive molecules. This work was supported in part by a grant from the Central Research Laboratory, Osaka Institute of Technology. Commun., 87 (1979) 106-113. 14 Michaelis, E.K., Michaelis, M.L., Chang, H.H., Grubbs, R.D. and Kuonen, D.R., Molecular characteristics of glutamate receptors in the mammalian brain, Mol. Cell. Biochem., 38 (1981) 163-179. 15 Moncada, S., Palmer, R.M.J. and Higgs, E.A., Biosynthesis of nitric oxide from L-arginine; a pathway for the regulation of cell function and communication, Biochem. Pharmacol., 38 (1989) 1709-1715. 16 Monahan, J.B. and Michel, J., Identification and characterization of N-methyl-D-aspartate specific L-[3H]glutamate recognition site in synaptic plasma membranes, J. Neurochem., 48 (1987) 1699-1708. 17 Nicoleni, F., Wroblewski, J.T., Novelli, A., Alho, H., Guidoni, A. and Costa, E., The activation of inositol phospholipid metabolism as a signal transducing system for dicarboxylic excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1986) 1905-1911. 18 Novelli, A., Nicoletti, E, Wroblewski, J.T., Alho, H., Costa, E. and Guidotti, A., Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells, J. Neurosci., 7 (1987) 40-47. 19 Palmer, R.M.J., Ashton, D.S. and Moncada, S., Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature, 333 (1988) 664-666. 20 Pan-Hou, H., Fujimori, H. and Suda, Y., Effect of porphyrins on [3H]-Lglutamate binding to rat brain synaptic membranes, Chem. Express, 6 (1991) 33-36. 21 Pan-Hou, H., Suda, Y., Ohe, Y., Sumi, M. and Yoshioka, M., Effect of aspartame on N-methyl-D-aspartate sensitive L[3H]glutamate binding sites in rat brain synaptic membranes, Brain Research, 520 (1990) 351-353. 22 Reddy, D., Lancaster Jr., J.R. and Cornforth, D.P., Nitrite inhibition of Clostridium botulinum: electron spin resonance detection of iron-nitric oxide complexes, Science, 221 (1983) 769-770. 23 Watkins, J.C., Excitatory amino acid and central synaptic transmission, Trends Pharmacol., 5 (1984) 373-376. 24 Wroblewski, J.T., Nicoletti, E and Costa, E., Different coupling of excitatory amino acid receptors with C a 2+ channels in primary cultures of cerebellar granule cells, Neuropharmacology, 24 (1985) 919-921. 25 Zukin, S.R., Young, A.B. and Snyder, S.H., Gamma-aminobutyric acid binding to receptor sites in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 4802-4807.
Brain Research, 554 (1991) 355-357 (~) 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391247522
BRES 24752
Effect of nitric oxide on ,-[all]glutamate binding to rat brain synaptic membranes Hiroyuki Fujimori and Hidemitsu Pan-Hou Department of Analytical Chemistry in Hygiene, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka (Japan)
(Accepted 16 April 1991) Key words: Nitric oxide; L-Glutamate binding; Neurotransmission; Synaptic membrane receptor; Sodium nitroprusside; Regulation of nitric oxide
Nitric oxide (NO), which is spontaneously generated from sodium nitroprusside, was shown to inhibit L-[3H]glutamatebinding to rat brain synaptic membranes in a concentration-dependent manner. The L-glutamate binding inhibited by NO, was largely recoverable by the addition of hemoglobin, a scavenger of NO, to the assay medium. These results suggest that NO may play an important role in the modulation of excitatory neurotransmission through direct interaction of L-glutamate binding to its physiological synaptic membrane receptors. L-Glutamate is believed to be the major excitatory transmitter in the mammalian central nervous systems and there are at least 3 separate subtypes of receptor, which are characterized by selective interaction with N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) 3'23. Activation of these receptors triggers a variety of transmembrane biochemical processes including stimulation of calcium influx 24, activation of inositol phospholipid turnover 17, and accumulation of cellular cGMP 2'5'18 in neural cells. The elevation of cGMP levels induced by L-glutamate and its structural analogs in the cells prepared from rat cerebellum has been shown to be mediated by Ca2÷-dependent formation of nitric oxide (NO) from L-arginine 1'5-7'1°. The biosynthesis of NO from the terminal guanidino nitrogen atoms of L-arginine was originally demonstrated in vascular endothelial cells and now has been identified in many cells and tissues 8"1°'11'19. Accumulating evidence suggests that NO may play an important role as a messenger molecule in the regulation of cell function and cellular communication in many biological systems 4' 9,10,15. However, at present, there is little direct evidence that the short-lived diffusible NO can directly participate in the neurotransmission in the central nervous systems, particularly in the primary interaction of ligands with its specific physiological membrane receptors. The functional consequences of the released NO induced by the activation of excitatory amino acids in neural tissue are largely unknown. One L-glutamate binding molecule isolated from rat brain synaptic membranes was found to be a metallo-
protein whose activity is dependent on the integrity of its iron-sulfur center at the recognition site of glutamate binding 13'14. The biological action of NO is generally considered to be mediated through direct nitrosation of the transition metals of active molecules 8'9'12'22. In view of these findings, it is of interest to know whether NO can directly modulate L-glutamate binding to its physiological membrane receptors. In the present article, we provide the first evidence that NO, spontaneously produced from sodium nitroprusside directly regulates L-glutamate binding to its synaptic membrane receptors. A possible role for NO in the maintenance of homeostasis of glutamatergic transmission is suggested. Crude brain synaptic membranes were prepared from whole brain of male albino Wistar rats weighing 180-220 g according to the procedures described by Zukin et al. 25. Extensively washed and frozen rat brain synaptic membranes were thawed at room temperature and washed twice with 20 vols. of 50 mM Tris-acetate buffer (pH 7.4). The washed synaptic membranes (approximately 10 mg protein) were incubated in 30 ml of the Tris-acetate buffer (pH 7.4) containing 0.08% (v/v) Triton X-100 at 2 °C. After 10 min of incubation, the treated membranes were washed twice with 20 vols. of the chilled Tris-acetate buffer before use 2°'21. For L-[3H]glutamate binding, the Triton X-100-treated synaptic membranes (0.2 mg protein) were suspended in 0.5 ml of 50 mM Tris-acetate buffer (pH 7.4) containing varying concentrations of sodium nitroprusside and preincubated at 37 °C for 15 min. After cooling on ice, the assay was initiated by the addition of 10 nM
Correspondence: H. Pan-Hou, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-01, Japan.
356
l--lNone F ~ H b (20 aM)
100
o ~j
% 75
H
5O0 4--: C O : NONe
/\ /~1,
%
i. ,i
I:!~
i : 8 1 u ( 1 . 0 mr,l) • :NMDA(O. 1 in,~1) : g.,'~ •. 3 ,,t.~)
a4,
~: 25o 50
2: i. ,4 io l
1:4 I:il I.;4
r~
25 re5
J
0
I
I I I I 20 30 Time (min) Fig. 2. Effect of sodium nitroprusside (SNP), L-glutamate (Glu) and NMDA on dissociation of L-[3H]glutamate binding to the Triton X-100-treated synaptic membranes. Triton X-100-treated synaptic membranes were incubated with 10 nM L-[3H]glutamate in 0.5 ml of 50 mM Tris-acetate buffer (pH 7.4) at 2 °C. At the time indicated by the arrow, SNP, Glu or NMDA was added to the incubation mixture and the incubation was continued at 2 °C. Data are the mean + S.E.M. of 5 independent determinations. 0
10 30 SNP (JJM)
100
300
Fig. 1. Effect of sodium nitroprusside (SNP) on the NMDA-specific L-[3H]glutamate binding to the Triton X-t00-treated synaptic membranes prepared from rat brain in the absence or presence of 20/~M hemoglobin (Hb). Data are the mean + S.E.M. of 5 independent determinations. L-[3H]glutamic acid (spec. act. 54.7 Ci/mmol; New England Nuclear), in the absence or presence of 20/~M of hemoglobin (Sigma). After incubation at 2 °C for 15 min, the reaction was terminated by the addition of 5 ml of the chilled Tris-acetate buffer and filtration through a Whatman GF/B glass microfiber filter pretreated with 0.3% polyethylenimine. After washing the filter 4 times with 5 ml of the chilled Tris-acetate buffer, the radioactivity trapped on the filter was measured by a liquid scintillation spectrometer (Aloka LSC-900) using 7 ml of modified Triton-toluene scintillant at a counting efficiency of 38-42% 20. Non-specific and N-methyl-Daspartate (NMDA)-specific L-[3H]glutamate bindings were obtained from the radioactivity displaced by 1 mM L-glutamic acid and 0.1 mM N M D A , respectively 2°'el. The assay was carried out in 5 independent determinations with a variation of less than 10%. Effect of sodium nitroprusside on L-[3H]glutamate binding to the Triton X-100-treated synaptic membranes is shown in Fig. 1. Under conditions employed in the present study, approximately 90% of the specific L[3H]glutamate binding observed is displaced by 0.1 mM N M D A . This result is in agreement with that reported by Monahan and Michel 16. This NMDA-specific L - [ 3 H ] glutamate binding was markedly inhibited by sodium nitroprusside in a concentration-dependent manner. The binding activity inhibited by sodium nitroprusside, was largely recovered by the addition of 20/~M hemoglobin in the assay medium. The recovery is explained by the ability of hemoglobin to bind NO through an interaction with the iron atom in the heme. These findings clearly
I 10
1
indicate that NO, spontaneously generated from sodium nitroprusside, has an ability to inhibit L-glutamate binding to its physiological membrane receptors. As shown in Fig. 2, L-Jail]glutamate binding to the Triton X-100-treated synaptic membranes progressively increased in proportion to the incubation time and almost reached an equilibrium within 10 min at 2 °C. Addition of non-radioactive L-glutamate (1 mM) or N M D A (0.1 mM) to the assay medium at 15 min after the initiation of incubation at 2 °C induced a prompt dissociation of the binding. Sodium nitroprusside (0.3 mM) also exerted a prompt dissociation of the L-glutamate binding. The result obtained suggests that N O generated from sodium nitroprusside, has an ability not only to inhibit Lglutamate binding to its synaptic membrane receptors, but also to be capable of inducing dissociation of the ligand from its recognition site of the physiological receptors. The N M D A - r e c e p t o r complex is prominently involved in excitatory transmission in the mammalian central nervous system. It has been shown that activation of NMDA-receptors in rat cerebellum induced a large increase in c G M P levels, an effect which is mediated by the Ca2+-dependent formation of N O from L-arginine and subsequent activation of soluble guanylate cyclase 1' 5-7,10. The excitatory amino acid induced elevation of cerebellar cGMP has been used as a biochemical index to study signal transduction following application of agonist(s) and antagonist(s) 2'18. However, the functional consequences of increase in c G M P levels and the precise modulatory role of c G M P in the neurotransmission in the
357 c e r e b e l l u m are still a m a t t e r of controversy. The biosynthesis of N O from L-arginine is now known to be a wide-spread mechanism for regulation of cell function and cellular communication in several biological systems 4'9'1°'15. N O is a n o n - p o l a r molecule like O2, and it can readily diffuse across cell m e m b r a n e s . All the biological effects of N O are m e d i a t e d through an interaction with i r o n sulfur center of m e t a l l o p r o t e i n in target cells8'9'12'22. The g l u t a m a t e binding protein, one of the c o m p o n e n t s of the g l u t a m a t e receptors, isolated from rat brain synaptic m e m b r a n e was found to be a h e m o p r o t e i n containing 2 g atoms of F e - S p e r mole of the purified protein 13"14. The results o b t a i n e d in the present study d e m o n s t r a t e that exogenous N O is capable of modulating the primary interaction of L-glutamate with its physiological synaptic receptors most likely via direct modification of F e - S center of the ligand binding molecules. Taking together 1 Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamatelinked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9030-9033. 2 Ferrendelli, J.A., Chang, N.M. and Kinscherf, D.A., Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids, J. Neurochem., 22 (1974) 535-540. 3 Foster, A. and Fagg, G., Acidic amino acid binding sites in mammalian neuronal membranes; their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 4 Gaily, J.A., Montague, ER., Reeke Jr., G.N. and Edelman, G.M., The NO hypothesis: possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 3547-3551. 5 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. 6 Garthwaite, J., Garthwaite, G., Palmer, R.M.J. and Moncada, S., NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices, Eur. J. Pharmacol., 172 (1989) 413-416. 7 Garthwaite, J., Southam, E. and Anderton, M., A kainate receptor linked to nitric oxide synthesis from arginine, J. Neurochem., 53 (1989) 1952-1954. 8 Hibbs Jr., J.B., Taintor, R.R., Vavrin, Z. and Rachlin, E.M., Nitric oxide: a cytotoxic activated macrophage effector molecule, Biochem. Biophys. Res. Commun., 157 (1988) 87-94. 9 Ignarro, L.J., Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling, Pharmacol. Toxicol., 67 (1990) 1-7. 10 Kowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5159-5162. 11 Mccall, T.B., Boughton-Smith, N.K., Palmer, R.M.J., Whittle, B.J.R. and Moncada, S., Synthesis of nitric oxide from Larginine by neutrophils. Release and interaction with superoxide anion, Biochem. J., 261 (1989) 293-296. 12 Meyer, J., Comparison of carbon monoxide, nitric oxide and nitrite as inhibitors of the nitrogenase from Clostridium pasteurianum, Arch. Biochem. Biophys., 210 (1981) 246-256. 13 Michaelis, E.K., The glutamate receptor-like protein of brain synaptic membranes is metalloprotein, Biochem. Biophys. Res.
this finding with the previous observations as r e p o r t e d so far, we speculate that the C a 2 ÷ - d e p e n d e n t intracellular generation of N O induced by activation of N M D A receptors m a y be a negative f e e d b a c k mechanism to turn off the excessive or p r o l o n g e d activation of the N M D A receptors by the ligands, when the N O p r o d u c e d diffuses across the m e m b r a n e barriers and subsequently acts on the binding sites. T h e r e f o r e , the physiological implication of Ca2÷-triggered N O formation m a y be involved in the maintenance of the homeostasis of the r e c e p t o r activity rather than via mechanisms involving cGMP. T h e dynamic regulation of N O formation requires further study since large amounts of N O may elicit cytotoxic action via destruction of the Fe-S center in bioactive molecules. This work was supported in part by a grant from the Central Research Laboratory, Osaka Institute of Technology. Commun., 87 (1979) 106-113. 14 Michaelis, E.K., Michaelis, M.L., Chang, H.H., Grubbs, R.D. and Kuonen, D.R., Molecular characteristics of glutamate receptors in the mammalian brain, Mol. Cell. Biochem., 38 (1981) 163-179. 15 Moncada, S., Palmer, R.M.J. and Higgs, E.A., Biosynthesis of nitric oxide from L-arginine; a pathway for the regulation of cell function and communication, Biochem. Pharmacol., 38 (1989) 1709-1715. 16 Monahan, J.B. and Michel, J., Identification and characterization of N-methyl-D-aspartate specific L-[3H]glutamate recognition site in synaptic plasma membranes, J. Neurochem., 48 (1987) 1699-1708. 17 Nicoleni, F., Wroblewski, J.T., Novelli, A., Alho, H., Guidoni, A. and Costa, E., The activation of inositol phospholipid metabolism as a signal transducing system for dicarboxylic excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1986) 1905-1911. 18 Novelli, A., Nicoletti, E, Wroblewski, J.T., Alho, H., Costa, E. and Guidotti, A., Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells, J. Neurosci., 7 (1987) 40-47. 19 Palmer, R.M.J., Ashton, D.S. and Moncada, S., Vascular endothelial cells synthesize nitric oxide from L-arginine, Nature, 333 (1988) 664-666. 20 Pan-Hou, H., Fujimori, H. and Suda, Y., Effect of porphyrins on [3H]-Lglutamate binding to rat brain synaptic membranes, Chem. Express, 6 (1991) 33-36. 21 Pan-Hou, H., Suda, Y., Ohe, Y., Sumi, M. and Yoshioka, M., Effect of aspartame on N-methyl-D-aspartate sensitive L[3H]glutamate binding sites in rat brain synaptic membranes, Brain Research, 520 (1990) 351-353. 22 Reddy, D., Lancaster Jr., J.R. and Cornforth, D.P., Nitrite inhibition of Clostridium botulinum: electron spin resonance detection of iron-nitric oxide complexes, Science, 221 (1983) 769-770. 23 Watkins, J.C., Excitatory amino acid and central synaptic transmission, Trends Pharmacol., 5 (1984) 373-376. 24 Wroblewski, J.T., Nicoletti, E and Costa, E., Different coupling of excitatory amino acid receptors with C a 2+ channels in primary cultures of cerebellar granule cells, Neuropharmacology, 24 (1985) 919-921. 25 Zukin, S.R., Young, A.B. and Snyder, S.H., Gamma-aminobutyric acid binding to receptor sites in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 4802-4807.
