Ca2+ AND H+ ANTAGONIZE THE DECREASE OF [3H]MK-801 BINDING INDUCED BY GLUTAMATE AND GLYCINE IN THE PRESENCE OF Mg2+

doi:10.1016/0197-0186(95)00102-6

Neurochemistry International

Volume 28, Issue 4, April 1996, Pages 401-415

https://doi.org/10.1016/0197-0186(95)00102-6 Get rights and content

Abstract

We have studied the effects of various cations on [³H]MK-801 binding to N-methyl-d-aspartate (NMDA) receptors in membrane preparations of the rat cerebral cortex. Low concentrations of Tris, K⁺, Na⁺, Mg²⁺, and Ca²⁺ enhanced submaximally stimulated [³H]MK-801 binding. At high concentrations, all compounds inhibited [³H]MK-801 binding, possibly by a direct competitive effect. H⁺ decreased the observed association rate of [³H]MK-801 binding observed as a decreased [³H]MK-801 binding under nonequilibrium conditions, apparently by decreasing the sensitivity of the glutamate and glycine effects on the association rate. In addition, Tris, Na⁺, Mg²⁺, and possibly K⁺ at very high concentrations, permitted glutamate and glycine to decrease [³H]MK-801 binding, probably reflecting a decreased affinity of [³H]MK-801 binding. In contrast, Ca²⁺ and H⁺ antagonized these glutamate- and glycine-induced decreases of [³H]MK-801 binding observed in the presence of Mg²⁺, possibly by a direct competitive action on the permissive Mg²⁺ effect. These Ca²⁺ and H⁺ -induced increases in [³H]MK-801 binding in the presence of Mg²⁺ may correspond to an increase in the potency of the Mg²⁺ block. Copyright © 1996 Elsevier Science Ltd.

Section snippets

Materials

The (+)-isomer of MK-801 was used, both tritium-labeled ([³H]MK-801; specific activity 26 Ci/mmol; New England Nuclear, U.S.A.) and unlabeled (Research Biochemicals Inc., U.S.A.). Tris, l-glutamate, and NMDA were obtained from Sigma Chemical Co., U.S.A., and glycine from Merck, Germany. All salts were of analytical grade and were obtained from Merck, Germany.

Animals and tissue preparation

Male Sprague-Dawley rats (body wt 200–250 g; B&K Universal, Sollentuna, Sweden) were kept under regular lighting conditions and had free

Effects of cations at pH 7.4

We first analyzed the effects of Tris, K⁺, Na⁺, Ca²⁺, and Mg²⁺ on [³H]MK-801 binding at pH 7.4. At low concentrations, Tris markedly increased the binding of [³H]MK-801 whether or not glutamate and glycine were added [Fig. 2(A)]. In the presence of 5 mM Tris-HCl, K⁺, Na⁺, Ca²⁺, and Mg²⁺ also increased the binding of [³H]MK-801 except in the presence of saturating concentrations of glutamate and glycine [Fig. 2(B–D) Fig. 3(B)]. The rank order of potency for this effect, which we tentatively name

Effects of cations at normal pH

The present results show that low concentrations of Tris, Na⁺, K⁺, Ca²⁺, and Mg²⁺ increases non-equilibrium [³H]MK-801 binding, possibly by sharing a common site of action, the Mg²⁺-like effect 1 (Fig. 8). This effect may also be shared by other cations, such as Ba²⁺, Co²⁺, Sr²⁺, Mn²⁺, and La³⁺ (Reynolds and Miller, 1988a; Rajdev and Reynolds, 1992) and by the arcaine-sensitive, high-affinity binding site for polyamines (Ransom and Stec, 1988; Reynolds, 1990; Sacaan and Johnson, 1990). It is

CONCLUSION

These findings suggest that Tris, Na⁺, Mg²⁺, and to some extent K⁺ have at least 3 separate effects on the NMDA receptor: one which increases non-equilibrium [³H]MK-801 binding; one which corresponds to a competitive action at the [³H]MK-801 binding site; and one which permits glutamate and glycine to decrease the affinity of the [³H]MK-801 binding site. Ca²⁺ shares the first two effects of these compounds but acts as an antagonist on the third effect. H⁺ has at least two major effects on the

Acknowledgements

This work was supported by the Swedish Medical Research Council (14X-10377), the Åke Wiberg, Magnus Bergvall and Lars Hierta foundations, the Swedish Society of Medicine, and funds from Karolinska Institutet.

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NMDA receptors are glutamate-regulated ion channels of critical importance for many neurophysiological and neuropathological processes. Mg²+ blocks the NMDA receptor by binding to the channel pore with an apparent affinity that depends on the membrane potential. We have investigated the effect of NMDA and the required co-agonist glycine on the affinity of the Mg²+ block site in NR1-1a/NR2A NMDA receptors expressed in Xenopus oocytes. We found that NMDA and glycine increase the IC₅₀ value of the Mg²+-block site at pH 7.4 and in the presence of physiological concentration of Ca²+. The increase the IC₅₀ value may correspond to a decrease in Mg²+-block affinity. This effect may result in an increased influx of Ca²+, and this influx may constitute up to a third of the total Ca²+ influx induced by NMDA. At high pH, or at low concentrations of Ca²+, NMDA and glycine have an opposite effect and instead decreased the IC₅₀ value of the Mg²+-block. These results indicate that glutamate and glycine can regulate the affinity of the Mg²+-block site. This effect may have implications for the understanding the role of NMDA receptors both under physiological and pathophysiological conditions.
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NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([³H]MK-801) to NMDA receptors in the presence of 1 mM Mg²⁺ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [³H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na⁺, K⁺, and Ca²⁺ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na⁺ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg²⁺ and 10 mM Ca²⁺ at pH 7.4, the cross-over values for Na⁺, K⁺, and Ca²⁺ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H⁺-sensitive and the less Ca²⁺-sensitive, glutamate-induced decreases in [³H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10μM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1μM) antagonized the glutamate-induced decrease in [³H]MK-801 binding observed in presence of Mg²⁺ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH.
These results suggest that in addition to a previously described high-affinity binding site for H⁺ and Ca²⁺ there exist a low-affinity binding site for H⁺, Ca²⁺, Na⁺, and K⁺ on NMDA receptors. The latter site may under physiological conditions be blocked by Na⁺ or K⁺, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [³H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg²⁺-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca²⁺ influx through activated NMDA receptor channels.
NMDA receptor complex blockade by oral administration of magnesium: Comparison with MK-801
1997, Pharmacology Biochemistry and Behavior
The ion channel of the N-methyl-D-aspartate (NMDA) receptor complex is subject to a voltage-dependent regulation by Mg²⁺ cations. Under physiological conditions, this channel is supposed to be blocked by a high concentration of magnesium in extracellular fluids. A single dose of magnesium organic salts (i.e., aspartate, pyroglutamate, and lactate) given orally to normal mice rapidly increases the plasma Mg²⁺ level and reveals a significant dose-dependent antagonist effect of magnesium on the latency of NMDA-induced convulsions; this effect is similar to that seen after administration of the dizocilpine (MK-801) channel blocker. An anticonvulsant effect of Mg²⁺ treatment is also observed with strychnine-induced convulsions but not with bicuculline-, picrotoxin-, or pentylenetetrazol-induced convulsions. In the forced swimming test, Mg²⁺ salts reduce the immobility time in a way similar to imipramine and thus resemble the antidepressant-like activity of MK-801. This activity is masked at high doses of magnesium by a myorelaxant effect that is comparable to MK-801-induced ataxia. Potentiation of yohimbine fatal toxicity is another test commonly used to evaluate putative antidepressant drugs. Administration of Mg²⁺ salts, like administration of imipramine, strongly potentiates yohimbine lethality, in contrast to MK-801, which is only poorly active in this test. Neither Mg²⁺ nor MK-801 treatment can prevent reserpine-induced hypothermia. These data demonstrate that oral administration of magnesium to normal animals can antagonize NMDA-mediated responses and lead to antidepressant-like effects that are comparable to those of MK-801. This important regulatory role of Mg²⁺ in the central nervous system needs further investigation to evaluate the potential therapeutic advantages of magnesium supplementation in psychiatric disorders.
Unique properties of [3H]MK-801 binding in membranes from the rat spinal cord
1997, Brain Research
In order to investigate possible differences between NMDA receptor-coupled ion channels in the spinal cord and in the cerebral cortex, we have characterized [ $^{3} H$ ]MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine] binding and its regulation by glutamate and glycine in membrane preparations of the rat spinal cord and cerebral cortex. The K_D value of [ $^{3} H$ ]MK-801 binding was higher in the spinal cord than in the cerebral cortex, mainly due to a lower association rate constant. When corrected for the concentrations of residual endogenous amino acids, the EC₅₀ values for glycine were lower at spinal NMDA receptors compared to those in the cerebral cortex, whereas the EC₅₀ values for glutamate were similar in both regions. The IC₅₀ values of d-((3)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (d-CPP) were significantly lower in the spinal cord in the presence of saturating concentrations of glutamate. The IC₅₀ values of 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324) were significantly lower in the spinal cord under all conditions. These results suggest that NMDA receptors in the spinal cord display low affinity for MK-801, which may correspond to a lower affinity of the voltage-dependent Mg²⁺ block. Furthermore, NMDA receptors in the spinal cord appear to display high sensitivity to glycine and to glutamate and glycine antagonists.
Pharmacological characterization of [3H]MK-801 binding in the rat spinal cord
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Using a receptor binding assay for [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine (MK-801) the pharmacology of spinal cord NMDA receptors was compared to that of NMDA receptors in the cerebral cortex. The affinities of glutamate site agonists l-glutamate, l-aspartate, ibotenic acid, NMDA and quinolinic acid for stimulation of [³H]MK-801 binding were 6–10 times lower in the spinal cord and the efficacy of quinolinic acid was 50% of that of the other agonists in this region. Also the affinities of glycine site agonists glycine, d-serine, d-alanine and l-serine were lower in the spinal cord as were the affinities of the non-competitive antagonists phencyclidine, (±)-cyclazocine and dextromethorphan. The divalent cations Zn²⁺, Mg²⁺ and Ca²⁺ had 4–8 times lower affinity for spinal NMDA receptors while the affinity of Co²⁺ was 50 times lower. The affinity of [³H]MK-801 was 2.5-fold lower in the spinal cord. These data show that spinal cord NMDA receptors show qualitative and quantitative differences compared to those in the cerebral cortex.
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Ca2+ AND H+ ANTAGONIZE THE DECREASE OF [3H]MK-801 BINDING INDUCED BY GLUTAMATE AND GLYCINE IN THE PRESENCE OF Mg2+

Abstract

Section snippets

Materials

Animals and tissue preparation

Effects of cations at pH 7.4

Effects of cations at normal pH

CONCLUSION

Acknowledgements

Eur. J. Pharmac.

Eur. J. Pharmac.

Eur. J. Pharmac.

Brain Res.

FEBS Lett.

Eur. J. Pharmac.

Eur. J. Pharmac.

Neuroscience

Eur. J. Pharmac.

Neurochem. Int.

Neurosci. Lett.

Eur. J. Pharmac.

Eur. J. Pharmac.

Effect of extracellular pH on the potency of N-methyl-d-aspartic acid receptor competitive antagonists

Molec. Pharmac.

N-methyl-d-aspartate receptor regulation of uncompetitive antagonist binding in rat brain membranes: kinetic analysis

Molec. Pharmac.

Modulation of glycine affinity for NMDA receptors by extracellular Ca2+ in trigeminal neurons

J. Neurosci.

Block of N-methyl-d-aspartate-activated current by the anticonvulsant MK-801 : selective binding to open channels

Proc. natn. Acad. Sci. U.S.A.

Glycine potentiates the NMDA response in cultured mouse brain neurons

Nature

Determination of the equilibrium dissociation constants and number of glycine binding sites in several areas of the rat central nervous system, using a sodium-independent system

J. Neurochem.

Kinetic characterization of the phencyclidine-N-methyl-d-aspartate receptor interaction : evidence for a steric blockade of the channel

Biochemistry

Properties of NMDA receptor channels in neurons acutely isolated from epileptic (kindled) rats

J. Neurosci.

Interaction of l-glutamate and magnesium with phencyclidine recognition sites in rat brain : evidence for multiple affinity states of the phencyclidine/N-methyl-d-aspartate receptor complex

Molec. Pharamac.

Ca²⁺ AND H⁺ ANTAGONIZE THE DECREASE OF [³H]MK-801 BINDING INDUCED BY GLUTAMATE AND GLYCINE IN THE PRESENCE OF Mg²⁺

Modulation of glycine affinity for NMDA receptors by extracellular Ca²⁺ in trigeminal neurons