Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Inhibition of [3H]MK-801 Binding by Ferrous (II) but NotFerric (III) Ions in a Manner Different from That by Sodium

Nitroprusside (II) in Rat Brain Synaptic Membranes

Makoto Shuto, Kiyokazu Ogita, Takao Minami, Hiroko Maeda, and Yukio Yoneda

Department of Pharmacology, Setsunan University, Osaka, Japan

Abstract: The addition of sodium nitroprusside (SNP)significantly inhibited binding of (+ ) -5-[~HI methyl-i 0,li -dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine([3H]MK-801) to an ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of >1 biM in ratbrain synaptic membranes not extensively washed. How-ever, neither S-nitroso- N-acetylpenicillamine nor S-ni-troso-L-glutathione inhibited binding even at 100 biM. Ofthe two compounds structurally related to SNP (Il), simi-larly potent inhibition was induced by potassium ferrocya-nide (Il) but not by potassium ferricyanide (Ill). In addi-tion, ferrous chloride (Il) induced much more potent inhi-bition of binding than ferric chloride (III), at a similarconcentration range. In contrast, iron chelators preventedthe inhibition by ferrous chloride (Il) without markedlyaffecting that by SNP (Il) and potassium ferrocyanide (Il).Pretreatment with ferrous chloride (Il) also led to potentinhibition of [3H]MK-801binding in a manner insensitiveto subsequent addition of the iron chelators. Pretreatmentwith Triton X-100 resulted in significant potentiation ofthe ability of ferrous chloride (Il) to inhibit [3H]MK-801binding irrespective of the addition of agonists, moreover,although binding of other radioligands to the non-NMDAreceptors was unaltered after pretreatment first with Tri-ton X-100 and then with ferrous chloride (II). These re-sults suggest that ferrous ions (Il) may interfere selec-tively with opening processes of the NMDA channelthrough mechanisms entirely different from those under-lying the inhibition by both SNP (Il) and potassium ferro-cyanide (Il) in rat brain. Key Words: [3H]MK-801bind-ing —Sodium nitroprusside— Potassium ferrocyanide—Ferrous chloride— Iron chelators— NMDA receptor.J. Neurochem. 69, 744—752 (1997).

Recent molecular biological studies have success-fully cloned complementary DNAs encoding each sub-class of brain excitatory amino acid (EAA) receptors,including metabotropic and ionotropic receptors (Holl-mann and Heinemann, 1994; Nakanishi and Masu,1994). An ionotropic subclass sensitive to the exoge-nous agonist N-methyl-D-aspartic acid (NMDA) is areceptor ionophore complex consisting of at least the

following four different domains: (1) an ion channeldomain permeable to Ca2~ions, (2) an NMDA recog-nition domain with high affinity for the endogenousagonist L-glutamic acid (L-Glu), (3) a glycine (Gly)recognition domain insensitive to strychnine, and (4)a polyamine recognition domain with several indefiniteproperties. lonotropic subclasses insensitive to NMDAare also distinguishable by preference to other exoge-nous agonists such as DL-a--amino-3-hydroxy-5-meth-ylisoxazole-4-propionic (AMPA) and kainic (KA)acids.

Among these ionotropic subclasses, the NMDA re-ceptor is supposed to play a key role in mechanismsunderlying neurotoxicity by EAA through generationof nitric oxide (NO) radicals in particular situations.For example, cytotoxicity by NMDA is prevented inprimary cortical cultures by the inhibitor of NO syn-thase (NOS), NG~nitro~L~arginine(L-NA), which hasno significant effect on either binding of ( + )-5- [3H] -

methyl- 10,1 1-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) to the NMDA channel orNMDA currents under patch-clamp conditions (Daw-son et al., 1991). In cultured CHP 100 neuroblastomacells, cell death by NMDA is significantly preventedby the potent NOS inhibitor L-NA methyl ester and theNO scavenger hemoglobin, in addition to protectionby the NMDA antagonist MK-801 (Corasaniti et al.,1992)~Similar attenuation by NO inhibitors is also

Received January 2, 1997; revised manuscript received March 17,1997; accepted March 18, 1997.

Address correspondence and reprint requests to Dr. Y. Yoneda atDepartment of Pharmacology, Faculty of Pharmaceutical Sciences,Setsunan University, 45-l Nagaotoge-cho, Hirakata, Osaka 573-01,Japan.

Abbreviations used: AMPA, DL-a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; CGP 39653, DL- (E) -2-amino-4-propyl-5-phosphono-3-pentenoic acid; DCKA, 5,7-dichiorokynurenic acid;EAA, excitatory amino acid; Glu, glutamic acid; Gly, glycine; KA,kainic acid; MK-80 1, 5-methyl-10,11 -dihydro-5H-dibenzo [ad] -

cyclohepten-5, I 0-imine; L-NA, N°-nitro-L-arginine; NMDA, N-methyl-D-aspartic acid; NO, nitric oxide; NOS, nitric oxide synthase;SNAP, S-nitroso-N-acetylpenicillamine; SNG, S-nitroso-L-glutathi-one; SNP, sodium nitroprusside.

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INHIBITION BY FERROUS IONS OF NMDA CHANNEL 745

shown with rat hippocampal slices (Izumi et al.,1992). The flavoprotein inhibitor diphenyleneiodo-nium protects neuronal cells significantly against cyto-toxicity by NMDA (Dawson et al., 1993) at concentra-tions effective at inhibiting NOS (Stuehr et al., 1991).Moreover, sodium nitroprusside (SNP), which is ableto liberate NO radicals from its molecule, mimics neu-rotoxicity by NMDA in a manner sensitive to preven-tion by radical scavengers in primary brain cultures(Dawson et al., 1991, 1993).

There is emerging evidence in the literature, how-ever, against possible involvement of NO radicals inNMDA neurotoxicity. Indeed, blockade of NO forma-tion does not always prevent neurotoxicity by NMDAand by L-Glu (Demerle-Pallardy et al., 1991; Lerner-Natoli et al., 1992; Pauwels and Leysen, 1992). Inprimary cultures of rat and mouse cerebral cortex, L-NA markedly exacerbates the stimulation by NMDAof formation of cyclic GMP without affecting cellulardamage by NMDA (Hewett et al., 1993). Several dif-ferent NO donors including SNP protect cortical neu-rons against neurotoxicity by NMDA (Lei et al.,1992). Far from it, NO radicals are not involved inprotection by SNP against NMDA neurotoxicity incul-tured cerebellar granule cells (Kiedrowski et al.,1991). Furthermore, SNP modulates the influxof Ca2~ions across the NMDA channel in cultured forebrainneurons (Hoyt et al., 1992) and cerebellar granule cells(Kiedrowski et al., 1992). Although NMDA neurotox-icity is attenuated by several antagonists of calmodulin,which is responsible for activation by Ca2~ions ofNOS (Dawson et al., 1991, 1993), we have previouslydemonstrated that [3HJMK-801 binding is markedlyinhibited in a concentration-dependent manner in ratbrain synaptic membranes by these calmodulin antago-nists, including calmidazolium, chlorpromazine, pre-nylamine, trifluoperazine, N- (6-aminohexyl ) -1 -naph-thalenesulfonamide, and N- (6-aminohexyl ) -5-chioro-l-naphthalenesulfonamide (Ogita et al., 1992).

In this article, therefore, we have attempted to evalu-ate the effects of SNP, often used as an NO donor, on[3HIMK-801 binding to anion channel associated withthe native NMDA receptor in rat brain synaptic mem-branes, by using ligand binding techniques.

MATERIALS AND METHODS

Materials[3H]MK-80l([3-3H]MK-801, 740 GBq/mmol), [3H]DL-

(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) ([propyl-2,3-3H] CGP 39653, 1.26 TBq/mmol),5,7- [3HI dichiorokynurenic acid ([3H] DCKA) ([3- 3H] -

DCKA, 603 GBq/mmol), [3HI AMPA ([5-methyl- 3H] -

AMPA, 2.22 TBq/mmol), and [3H]KA ([vinylidene-3H]-KA, 2.15 TBqImmol) were purchased from NEN—Du Pont(Boston, MA, U.S.A.). Both [3H]Glu([L-3,4-3H]Glu, 1.48TBq/mmol) and [3H]Gly ([2-3H]Gly, 1.11 TBq/mmol)were supplied by American Radiolabeled Chemicals(St. Louis, MO, U.S.A.). S-Nitroso-N-acetylpenicillamine(SNAP) (Dojindo, Kumamoto, Japan), S-nitroso-L-glutathi-

one (SNG) (Cayman Chemical, Ann Arbor, MI, U.S.A.),and o-phenanthroline hydrochloride (Wako, Osaka, Japan)were all provided by local suppliers. Both SNP and deferoxa-mine mesylate were obtained from Sigma Chemicals (St.Louis, MO, U.S.A.). Other chemicalsused were of the high-est purity commercially available. All solutions were freshlyprepared immediately beforeeach use. Some lipophilic com-pounds such as SNAP and SNG were dissolved in dimethylsulfoxide, which did not significantly affect [3H]MK-80 1binding, at a final concentration of up to 2%.

Membrane preparationCrude synaptic membrane fractions obtained from whole

brains (including cerebellum) of male Wistar rats weighing200—250 g were washed once by suspension in 40 volumesof 50 mM Tris-acetate buffer (pH 7.4), using a Physcotronhomogenizer at setting 6 for 2 min at 4°C, followed bycentrifugation at 50,000 g for 30 mm, as described previously(Enomoto et al., 1992). Buffers andany other solutions usedin the present study were all sterilized each time before useby filtration through a nitrocellulose membrane filter with apore size of 450 nm to avoid possible microbial contamina-tion (Yoneda and Ogita, 1989). Resultant pellets were sus-pended in 8 volumes of 0.32 M sucrose and store‘d at —80°Cuntil used. On the day of the experiment, these frozen sus-pensions were thawed at room temperature and used directlyfor binding assays, described below as “nonwashed“ mem-brane preparations. In addition, resultant pellets were sub-jected to procedures for preparation of “Triton-treated“membranes, as needed, to deplete endogenous agonists asmuch as possible, as described elsewhere (Ogita andYoneda, 1988).

[3H]MK-801 bindingAn aliquot (~-~0.15 mg of protein) of nonwashed mem-

branes was incubated with 5 nM [3H]MK-80l in 0.5 ml 50mM Tris-acetate buffer (pH 7.4) at 30°Cfor 30 min unlessindicated otherwise (Enomoto et al., 1992). Incubation wasterminated by the addition of 3 ml of cold buffer at 2°Candsubsequent filtration through a Whatman GF/B glass—fiberfilter under constant vacuum of 15 mm Hg. The filter wasrinsed with 3 ml of cold buffer at 2°Cfour times within 10s, and radioactivity retained on the filter was measured by aliquid scintillation spectrometer, using 5 ml of modified Tri-ton-toluene scintillant at a counting efficiency of 40—42%(Ogita et al., 1986). Nonspecific binding of [3H]MK-801wasdefined by theaddition of both o-2-amino-5-phosphono-valeric and 7-chlorokynurenic acids at 0.1 mM to avoidpossible interference with binding by adsorption of the li-gand to membrane phospholipids (Suzuki et al., 1993). Inour preliminary experiments, [3H]MK-801 binding was notaffected significantly by 5 mM Tris-acetate buffer (pH 7.4)under all experimental conditions used, in contrast to theprevious report in which 5 mM Tris-HC1 buffer (pH 7.4)was used (Wong et al., 1988). Therefore, 50 mM Tris-acetate buffer (pH 7.4) was used throughout binding assaysreported here, to determine binding of all radioligands relatedto ionotropic EAA receptors simultaneously, under similarconditions.

Binding of other radioligandsAn aliquot (~-~0.15 mg of protein) of Triton-treated mem-

branes was incubated with 10 nM [3HTIAMPA(Ogita et al.,1994), [3H]KA (Ogita et al., 1994), [3H]Gly(Ogita et al.,1989), [3H]DCKA (Yoneda et al., 1993), [3H]Glu(Ogita

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s

s

746 M. SHUTO ET AL.

ited [3H]MK-801 binding at equilibrium, in a concen-tration-dependent manner, at a concentration of >1‚uM in the absence of added spermidine (IC

50 13.9± 2.7 ‚uM). In contrast, SNG induced slight but statis-tically significant potentiation of binding at 100 ‚uM,but SNAP did not affect binding significantly at theconcentrations used. Further addition of spermidine,however, attenuated the inhibition by SNP (IC50 51.1± 7.5 ‚uM) without markedly altering binding in thepresence of SNAP or SNG (Fig. 1, right). In the pres-ence of added spermidine, however, SNG was not ableto stimulate [

3H]MK-801 binding significantly at thehighest concentration.

FIG. 1. Effects of in vitro addition of three different NO donorson [3HJMK-801 binding. Incubation was performed in buffer con-taining at least four different concentrations of the NO donorindicated in either the presence or the absence of 1 mM spermi-dine. Values are from four separate experiments. °°p< 0.01,significantly different from each control value obtained in theabsence of.any test drugs added. Control binding (fmol/mg ofprotein): none, 318.0 ± 13.6; spermidine, 683.0 ±26.5. SPD,spermidine.

and Yoneda, 1988), and 2 nM [3H]CGP 39653 (Zuo et al.,1993) under the respectively appropriate equilibrium condi-tions. Incubation was terminated by the rapid filtrationmethod described above. Unlabeled compounds used at 0.1mM to define each nonspecific binding were as follows:[3H]AMPA,Glu; [3H]KA, Glu; [3H]Gly, D-serine; [3H]-DCKA, Gly; [3H]Glu, NMDA; and [3H]CGP 39653, Glu.Binding assays were all performed one by one at intervalsof20 sin triplicate with variations of <10%. Protein contentwas determined by the method of Lowry et al. (1951).

Data analysisThe concentration of a test compound to inhibit binding

by 50% (IC50) was calculated according to the Hill plot

analysis by the computer program LOTUS-l-2-3 for theNEC PC. Results are expressed as mean ± SE values, andthe statistical significance was determined by two-tailed Stu-dent‘s t test or one-way analysis of variance.

NO donors

RESULTS

In nonwashed membranes of rat whole brain,[3HJMK-801 binding reached equilibrium within 30

min at 30°C,due to the abundance of endogenous ago-nists such as Glu and Gly. Further addition of eitherGlu or Gly at 10 ‚uM did not markedly potentiate bind-ing, in fact, whereas binding was more than doubledby the addition of spermidine at 1 mM as shown pre-viously (Ogita et al., 1992; Yoneda et al., 1995).Therefore, subsequent screening experiments weresimilarly performed, using these nonwashed mem-branes in either the presence or the absence of 1 mMspermidine.

As shown in Fig. 1 (left), SNP significantly inhib-

Iron chelatorsAn attempt was next made to determine whether

binding was modulated by the in vitro addition of sev-eral compounds related to SNP (II). These includedSNP (II), potassium ferrocyanide (II), potassium fer-ricyanide (III), and iron ions (II and III) (Fig. 2). Ofthe two compounds structurally related to SNP (II),moreover, potassium ferrocyanide (II) (IC

50 27.9± 4.2 ‚uM) (lower middle panel) was much more po-tent for inhibiting binding in a concentration-depen-dent manner, at a concentration range of 1—100 ‚uM,than potassium ferricyanide (III) (IC50 >100 ‚uM)(lower right panel). In a similar manner, ferrous chlo-ride (II) (IC50 23.4 ±1.2 ‚uM) (upper left panel) wasmuch more potent for inhibitingbinding at a concentra-tion range of 1—100 ‚uM than ferne chloride (III) (IC50>100 ‚uM) (upper right panel). Finally among the testcompounds related to SNP, SNP (II) was most potentfor inhibiting binding, with progressively less potentinhibition by ferrous chloride (II), potassium ferrocya-nide (II), ferric chloride (III), and potassium ferricya-nide (III).

To evaluate the possible participation of iron ions,the effects of addition of two different iron chelators,on the inhibition of [

3HJMK-801 binding by SNP-related compounds, were examined. For this purpose,incubation was performed in buffer containing threedifferent concentrations of an SNP-related compoundin either the presence or the absence of iron chelatorssuch as o-phenanthroline and deferoxamine (Fig. 2).Neither o-phenanthroline nor deferoxamine affectedbinding significantly at the concentration range used[(fmol/mg of protein): none, 291.0 ± 7.1; 10 ‚uM o-phenanthroline, 304.8 ± 24.5; 100 ‚uM o-phenanthro-line, 284.9 ±23.5; 10 ‚uMdeferoxamine, 287.7 ± 19.2;100 ‚uM deferoxamine, 278.0 ± 15.2]. o-Phenanthro-line was effective for preventing the inhibition by fer-rous chloride (II) at a concentration range of 10—100‚uM (Fig. 2a, upper left panel) [IC

50(1.tM): none, 21.7± 4.2; 10 ‚uM, 45.3 ± 10.3; 100 ‚uM, 62.4 ±2.9 (p< 0.01)], without markedly affecting that by otherSNP-related compounds including fernic chloride (III)(upper right panel) [IC50(1.tM): none, >100; 10 ‚uM,>100; 100 ‚uM, >1001, SNP (II) (lower left panel)[IC50(p~M):none, 14.4 ± 2.0; 10p~M,11.0 ± 1.6; 100

J. Neurochem., Vol. 69, No. 2, 1997

INHIBITION BY FERROUS IONS OF NMDA CHANNEL 747

FIG. 2. Effects of in vitro addition of iron chelators on inhibition by iron-containing compounds of [3H)MK-801 binding. Incubationwas performed in buffer containing at least three different concentrations of each test compound in either the presence or the absenceof o-phenanthroline (a) or deferoxamine (b) at 10 or 100 ~sM.Values are from four separate experiments. °p< 0.05; **p < 0.01,significantly different from each control value obtained in the absence of any iron-containing compounds. Control binding (fmol/mgof protein): none, 291.0 ±7.1; 10

1iM o-phenanthroline, 304.8 ±24.5; 100 1iM o-phenanthroline, 284.9 ±23.5; 10 1iM deferoxamine,287.7 ±19.2; 100 1~Mdeferoxamine, 278.0 ±15.2.

‚uM, 10.1 ± 0.81, potassium ferrocyanide (II) (lowermiddle panel) [IC50(,uM): none, 14.5 ± 2.3; 10 ‚uM,18.2 ± 2.7; 100 ‚uM, 12.5 ± 2.31, and potassium fer-ricyanide (III) (lower right panel) [IC50(,uM): none,>100; 10 ‚uM, >100; 100 ‚uM, >1001 (Fig. 2a). In asimilar manner, another iron chelaton, deferoxamine,markedly prevented the inhibition by ferrous chloride(II) in a concentration-dependent fashion at 10—100‚uM(Fig. 2b, upper left panel) [IC50(~tM):none, 21.7±4.2; 10 ‚uM, 39.5 ±7.8; 100 ‚uM, >100], withoutmarkedly altering that by ferne chloride (III) [IC50(1.tM): none, >100; 10 ‚uM, >100; 100 ‚uM, >100],SNP (II) [IC50(1~M):none, 14.4 ± 2.0; 10 ‚uM, 12.3± 1.7; 100 ‚uM, 11.7 ±1.41, potassium ferrocyanide(II) [IC50([1M): none, 14.5 ±2.3; 10 ‚uM, 15.4 ±2.1;100 ‚uM, 14.7 ±1.4], and potassium fernicyanide (III)[IC50(tiM): none, >100; 10 ‚uM, >100; 100 ‚uM,>100] (Fig. 2b). In the presence of deferoxamine at100 ‚uM, however, ferne chloride (III) did not inhibitbinding at all. In contrast, potassium ferricyanide (III)significantly inhibited binding in the presence of defer-oxamine at 10—100 ‚uM.

Pretreatment with ferrous chlorideTo evaluate reversibility of the inhibition by iron-

containing substances, synaptic membranes were atfirst incubated with ferrous chloride (II) or potassiumferrocyanide (II) at different concentrations from I to100 ‚uM, followed by two cycles of centrifugation andsuspension for extensive washing. These washing pro-cedures markedlyattenuated [

3H]MK-801 binding dueto removal from membrane preparations of endoge-nous agonists such as Glu and Gly (346.8 ± 7.1 vs.87.7 ± 24.1 fmol/mg of protein). As shown in Fig.3a (left panel), more potent inhibition was induced

after such pretreatment with ferrous chloride (II) thanafter the in vitro addition [IC

50(j.tM): addition, 23.4± 1.2; pretreatment, 6.0 ± 1.6 (p < 0.01 )]. In particu-lar, complete abolition was seen with [

3H]MK-801binding in membranes pretreated with ferrous chloride(II) at 100 ‚uM. In a similar manner, pretreatment withpotassium ferrocyanide (II) caused more potent inhibi-tion of binding compared with its addition (Fig. 3a,right panel) [IC

50(~.tM):addition, 27.9 ±4.2; pretreat-ment, 16.7 ±4.71.An attempt was next made to determine whether iron

chelators still affect binding in membranes subjected topretreatment with ferrous chloride (II) and subsequentwashing procedures. Nonwashed membranes were in-cubated with ferrous chloride (II) at different concen-trations, followed by two cycles of washing proceduresas shown in the right margin of Fig. 3b. Consequently,binding was determined in either the presence or theabsence of each iron chelator at 100 ‚uM. However,both chelators did not markedly modulate the inhibi-tion of binding by pretreatment with ferrous chloride(II) at concentrations up to 100 ‚uM (Fig. 3b) [IC50([1M): none, 2.9 ± 0.8; o-phenanthroline, 3.3 ± 1.2;deferoxamine, 5.0 ± 1.5].

Pretreatment with Triton X-100Pretreatment with Triton X-100 at a low concentra-

tion resulted in almost complete abolition of [3H]MK-

801 binding without affecting any binding profilesthemselves, due to depletion from membrane prepara-tions of endogenous agonists such as Glu and Gly(Enomoto et al., 1992; Ogita et al., 1992). In contrastto nonwashed membranes, in fact; the addition of Glualone markedly potentiated binding in these Triton-treated membranes. In addition, Gly additionally en-

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748 M. SHUTO ET AL.

FIG. 3. Effects of pretreatment with ferrous chloride (Il) on [3H]MK-801binding, a: The first incubation was done with ferrous chloride(Il) or potassium ferrocyanide (II) at different concentrations from 1 to 100

1iM, followed by two cycles of washing procedures andsubsequent determination of [

3H]MK-801 binding under the routine conditions. In addition to these membranes, nonwashed membraneswere also incubated under routine conditions in buffer containing at least three different concentrations of ferrous chloride (Il) orpotassium ferrocyanide (Il). b: An aliquot of nonwashed membranes was pretreated with ferrous chloride (Il) as described above,followed by determination of [3HJMK-801binding in either the presence or the absence of o-phenanthroline or deferoxamine at 100

1iM. Values are from four separate determinations. *p < 0.05; “p < 0.01, significantly different from each control value obtained inmembranes not treated with ferrous chloride (Il). Control binding (fmol/mg of protein): none, 126.2 ±24.8; o-phenanthroline, 113.2±22.0; deferoxamine, 152.5 ±27.9.

hanced binding in the presence of added Glu alone,and further addition of spermidine doubled binding inthe presence of both Glu and Gly. Therefore, non-washed membranes were first subjected to incubationin either the presence or the absence of Triton X-100and subsequent washing procedures. Each membranepreparation was then treated with ferrous chloride(II),followed by extensive washing and subsequent deter-mination of binding in the presence of three differenttypes of agonists for the NMDA receptor complex. Asshown in Fig. 4, the addition of these agonists pre-vented significantly the inhibition by ferrous chloride(II) in membranes not treated with Triton X-100 [IC50([1M): none, 3.5 ±0.6; Glu, 5.5 ± 1.2; Glu/Gly, 10.9±0.8 (p < 0.01); Glu/Gly/spermidine, 15.2 ± 1.5 (p< 0.01), different from the control value determinedin the absence of any added agonistsj. In addition,pretreatment with Triton X-100 led to potentiation ofthe inhibition by ferrous chloride (II), irrespective ofthe addition of those NMDA agonists [IC50 (,uM):none, not detectable; Glu, 2.0 ±0.6 (p < 0.05); Glu!Gly, 3.1 ± 1.0 (p < 0.05); Glu!Gly/spermidine, 3.5± 0.3 (p < 0.05), different from each control valueobtained in membranes not treated withTriton X-100 j.

To test the specificity of ferrous chloride (II) for[3H]MK-801 binding to the NMDA channel, binding

of other radioligands was examined by using thesemembranes treated at first with Triton X-100 and sub-sequently with ferrous chloride (II). Pretreatment withTriton X-100 did not markedly affect binding of [3H1-AMPA (163.8 ± 9.4 vs. 169.7 ±10.6 fmol!mg ofprotein) and [3HIKA (199.5 ± 7.9 vs. 172.8 ±21.4)to the non-NMDA receptors. In contrast, pretreatment

with Triton X-100 resulted in marked potentiation ofbinding of [3H]Glu (148.8 ± 17.7 vs. 345.9 ± 75.8)and II3HICGP 39653 (135.0 ±32.3 vs. 201.2 ±64.8)to the NMDA recognition domain, in addition to [3H1-Gly (52.1 ± 5.6 vs. 92.3 ± 12.7) and [3H]DCKA(283.7 ± 15.9 vs. 512.5 ±38.2) to the Gly recognitiondomain. However, ferrous chloride (II) did not altersignificantly [3H]AMPA and [3H]KA binding to thenon-NMDA receptors, independent of pretreatmentwithTriton X-100 at the concentration range used (Fig.5a). Ferrous chloride (II) was effective for inhibitingsignificantly binding of [3H]Glu and [3H]CGP 39653to the NMDA recognition domain in membranes nottreated with Triton X-100 but rather ineffective inmembranes treated with Triton X-100 (Fig. 5b). Fur-thermore, ferrous chloride (II) inhibited significantlybinding of [3HIDCKA, but not of [3H]Gly, to theGly recognition domain in membranes not treated withTriton X-100 (Fig. 5c). In membranes treated withTriton X-100, ferrous chloride (II) was rather ineffec-tive for inhibiting [3HJDCKA binding [10 ‚uM, 82.6± 2.6 vs. 111.8 ± 10.6 (p < 0.05); 100 ‚uM, 35.6±3.5 vs. 51.9 ± 3.7 (p < 0.05)].

DISCUSSION

The essential importance of the present findings isthat ferrous ions (II) could inhibit [3HJMK-801 bind-ing to the native NMDA channel through peculiarmechanisms that are completely different from thoseunderlying the inhibition by SNP (II) and potassiumferrocyanide (II) in rat brain synaptic membranes.Namely, both o-phenanthroline and deferoxamine

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INHIBITION BY FERROUS IONS OF NMDA CHANNEL 749

FIG. 4. Effects of pretreatment with Triton X-100 on inhibitionby ferrous chloride (Il) of [3H]MK-801binding. An aliquot ofnonwashed membranes was incubated with Triton X-100 as de-scribed previously (Ogita and Yoneda, 1988), followed by twocycles of washing procedures. Membranes were similarly incu-bated in the absence of Triton X-100, followed by the samewashing procedures. These membrane preparations were againincubated with ferrous chloride (II) at three different concentra-tions, accompanied by two extra cycles of washing proceduresand subsequent determination of [3HIMK-801binding in eitherthe presence or the absence of 10

1iM Glu, 10 pM Gly, and 1mM spermidine. Values are from four independent determina-tions. **p < 0.01, significantly different from each control valueobtained in membranes not treated with ferrous chloride (II).Control binding (fmol/mg of protein): none: Glu, 282.6 ±20.2;Glu/Gly, 320.3 ±17.3; Glu/Gly/spermidine, 579.3 ±30.5; Tri-ton: Glu, 121.2 ±10.9; Glu/Gly, 164.7 ±4.4; Glu/Gly/spermi-dine, 381.9 ±4.6.

markedly prevented the inhibition by ferrous chloride(II), without affecting that by SNP (II) and potassiumferrocyanide (II). These data clearly challenge the hy-pothesis that ferrous ions (II) may play a critical rolein mechanisms underlying the inhibition by SNP (II)and potassium ferrocyanide (II) of [

3H]MK-801 bind-ing to the NMDA channel. In cqntrast, ferrous ions(II) are shown to be responsible for the inhibition bySNP (II) and potassium ferrocyanide (II) of theNMDA-induced elevation of intracellular Ca2~ions incultured cerebellar granule cells (Oh and McCaslin,1995). In the latter report, however, the authors didnot attempt to examine the effects of iron chelators onthe inhibition by SNP (II), potassium ferrocyanide(II), and ferrous sulfate (II) (Oh and McCaslin,1995). Iron chelators are quite useful for differentiat-ing mechanisms underlying the inhibition by SNP (II),potassium ferrocyanide (II), and ferrous chloride (II)as demonstrated here. The present study has, for thefirst time, provided the evidence against the prevailingview that ferrous ions (II) at least in part participatein the modulation of the NMDA channel by SNP (II)and potassium ferrocyanide (II). The data from experi-ments usingother radioligands also suggest that ferrousions (II) may selectively modulate opening processesof the native NMDA channel without affecting those

FIG. 5. Effects of pretreatment with ferrous chloride (Il) on bind-ing to non-NMDA receptors (a), NMDA recognition domain (b),and Gly recognition domain (c). An aliquot of nonwashed mem-branes was treated or not treated with Triton X-100 first, andwashed twice thereafter. These membranes were incubated withferrous chloride (Il) at different concentrations, followed bywashing and subsequent determination of binding of each ra-dioligand under appropriate routine conditions. Values are fromfour separate experiments. °p< 0.05; **p < 0.01, significantlydifferent from each control binding obtained in membranes nottreated with ferrous chloride (Il). Control binding (fmol/mg ofprotein): [3HIAMPA:Triton (—)‘ 163.8 ±9.4; Triton (+)‘ 169.7±10.6; [3H]KA:Triton(—),199.5 ±7.9;Triton(+), 172.8 ±21.4;[3H]Glu:Triton (—)‘ 148.8 ± 17.7; TritDn (+)‘ 345.9 ± 75.8;[3H]CGP39653: Triton (—)‘ 135.0 ± 32.3; Triton (+)‘ 201.2±64.8; 13H]Gly: Triton (—)‘ 52.1 ±5.6; Triton (+)‘ 92.3 ±12.7;[3HIDCKA:Triton (—)‘ 283.7 ±15.9; Triton (+)‘ 512.5 ±38.2.

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750 M. SHUTO ET AL.

of other ionotropic EAA channels such as AMPA andKA receptors.Furthermore, ferrous chloride (II) induced irrevers-

ible inhibition of binding after extensive washing ofmembrane preparations. Iron chelators did not restore[3HIMK-801 binding in these membranes previouslytreated with ferrous chloride (II). These resultsstrongly suggest the absence of ferrous chloride (II),at a concentration effective for inhibiting binding, fromthose membranes that were previously treated withfer-rous chloride (II), accompaniedby extensive washing.Therefore, it is unlikely that ferrous ions (II) them-selves are involved directly in the inhibition of[3H1MK-801 binding in membranes treated with fer-rous chloride (II). Nonetheless, iron ions are shownto lead to particular neurodegenerative disorders in as-sociation with several active radical species (Gerlachet al., 1994). For example, lipid peroxidation is sup-posed to be responsible for mediating neuronal injuriesassociated with iron ions (Floyd and Carney, 1991;Halliwell, 1992; Hall and Braughler, 1993). Indeed,ferrous ions (II) markedly facilitate formation of lipidhydroperoxides, including free fatty acid hydroperox-ide, phosphatidylcholine hydroperoxide, and phospha-tidylethanolamine hydroperoxide, with a concomitantloss of the cellular viability in cultured spinal neurons(Zhang et al., 1996). Lipid peroxidation by iron ionscould ignite cascade processes crucial for the crisis of avariety of neurological disorders associated with aging(Youdim et al., 1993), which is shown to induce in-creased accumulation of iron ions in particular regionsof rat brain (Benkovic and Connor, 1993). These pre-vious findings all suggest that lipid peroxidation mayat least in part participate in mechanisms underlyingthe inhibition by ferrous chloride (II) of binding dem-onstrated here. From this point of view, it is notewor-thy that pretreatment of membranes with phospholi-pase A

2 or C leads to marked prevention of the inhibi-tion by ferrous chloride (II) of [

3H]MK-801 bindingunder similar experimental conditions (our unpub-lished data).

The exact mechanisms underlying the potentiationby pretreatment with Triton X-100, of the ability offerrous chloride (II) to inhibit [3H]MK-801 binding,irrespective of the addition of three different types ofNMDA agonists, are not clear at present. One possibleexplanation for the potentiation is that Triton treatmentmay deplete from membranes different types of endog-enous NMDA agonists that all have abilities to coun-teract the inhibition by ferrous chloride (II). In mem-branes not treated with Triton X-100, in fact, the IC

50value of ferrous chloride (II), in the presence of Glualone, was doubled by the addition of Gly and tripledby the further addition of spermidine. However, threedifferent NMDA agonists invariably failed to affectsignificantly the potencies of ferrous chloride (II) toinhibit binding in membranes treated with Triton X-100. If the potentiation by Triton X-100 should bemerely due to removal of endogenous agonists as de-

scribed above, the addition of different NMDA ago-nists would also have attenuated the ability of ferrouschloride (II) to inhibit [

3HIMK-801 binding in mem-branes treated with Triton X-100, as seen in mem-branes not treated withTriton X-100. Thus, the potenti-ation by Triton X-100 could be brought about throughparticular unidentified mechanisms other than thedepletion of endogenous agonists from membranepreparations. Triton treatment would get rid of certainmembranous constituents crucial for the action againstthe inhibition by ferrous chloride (II) in rat brain syn-aptic membranes, without affecting biochemical andpharmacological profiles of [3HIMK-801 binding tothe native NMDA channel. Similar mechanisms mightunderlie the differential effects of Triton treatment onbinding of radiolabeled agonists and antagonists to theNMDA and Gly recognition domains. However, theapparent contradiction that Triton treatment markedlypotentiated binding of radioligands to the NMDA re-ceptor without affecting that to the non-NMDA recep-tors is explained by considering differential affinitiesof the endogenous agonist L-Glu for these ionotropicreceptors. In other words, L-Glu has >10-fold higheraffinity for the NMDA recognition domain than forthe non-NMDA receptors (Ogita and Yoneda, 1988;Ogita et al., 1994).

In contrast, however, the data cited above suggestthat SNP may directly interfere with the opening pro-cesses of the NMDA channel, to lead to blockade ofthe influx of Ca2* ions, which are responsible for acti-vation by calmodulin of NOS in certain situations. Thisinterference does not appear to be associated with lib-eration of an NO radical from an SNP molecule. Forexample, binding was not inhibited by the addition ofother different NO donors with abilities to liberate NOradicals from their molecules more effectively thanSNP. In addition to generation of NO radicals from itsmolecule, nevertheless, SNP is shown to be highlyreactive with thiol groups on the NMDA receptor com-plex (Lipton and Stamler, 1994), to result in protectionof neuronal cells against toxicity by NMDA (Liptonet al., 1993). Our results presented in this study sup-port the previous findings on neuroprotective actionsof SNP against NMDA toxicity through inhibition ofthe opening processes of the NMDA channel. On thecontrary, the present study may challenge the hypothe-sis that the NO radical is a messenger of neurotoxicityby NMDA, as previously advocated by Dawson et al.(1991, 1993). Furthermore, binding of [3H]MK-801to the NMDA channel is sensitive to inhibition by avariety ofcompounds usually used for proving possibleparticipation of NO radicals in NMDA neurotoxicity,i.e., the NO donor SNP, the NOS inhibitors diphenylio-donium and diphenyleneiodonium (Shuto et al., 1997),and calmodulin antagonists (Ogita et al., 1992). Ac-cordingly, these positive compounds could all modu-late the influx of Ca2~ions across the NMDA channel,to affect neurotoxicity of NMDA in a manner indepen-dent of generation of NO radicals. Diphenyliodonium

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INHIBITION BY FERROUS IONS OF NMDA CHANNEL 751

indeed prevents the elevation of intracellular Ca2~ionselicited by NMDA in cultured rat spinal neurons (Na-.kamura et al., 1997). Nevertheless, NO radicals andrelated nitroso compounds could have dual actions, asreported by Lipton et al. (1993), and, depending onexperimental conditions, a protective or neurotoxic ef-fect may be exposed.

Moreover, SNP (II) could be degraded to ferrocya-nide (IT), ferricyanide (III), iron ions (II and III), andcyanide under certain conditions, after liberating NOradicals (Butler and Glidewell, 1987). In particular,both ferrocyanide (II) and ferricyanide (III) are highlysimilar structurally to SNP (II). Moreover, SNP (II)is highly reactive with divalent cations, often crucialfor the functions of different constitutional proteins, aswell as several active moieties on biological constit-uents, which result in critical functional disorders.Therefore, NO radicals would not always participatein mechanisms underlying the diversity of pharmaco-logical actions of SNP (II). Neuroprotection by SNPis supposed to be mediated by a direct interaction ofthe nitroferricyanide (II) molecule itself with the redoxsite on the NMDA receptor complex, in fact, whereasneurotoxicity by SNP (II) is attributable to the peroxy-nitrite anion (OONO ~)‘ which is formed from a reac-tion between nitric oxide (NO) and superoxide anion(O2~) radicals (Lipton and Stamler, 1994). Becausesuperoxide anion radicals do not appear to evolve un-der the in vitro conditions used here, the aforemen-tioned supposition supports that the selective inhibitionby SNP (II) demonstrated here involves mechanismsother than liberation of NO radicals from its molecule.

One possible interpretation is that SNP (II) mayinhibit ~3H]MK-8Ol binding to the NMDA channelthrough liberation of ferrocyanide (II) during incuba-tion, as mentioned above. In fact, potassium ferrocya-nide (II) inhibited binding with potencies similar tothose of SNP (II). The addition of spermidine is alsoeffective for preventing the inhibition by potassiumferrocyanide (II) as well as that by SNP (II) (ourunpublished data). These data suggest that ferrocya-nide (II) may play a key role in the inhibition by SNP(II) of [3HIMK-801 binding to the NMDA channel.Similar participation of breakdown products includingferrocyanide (II) is seen in the inhibition by SNP (II)of phosphatidylinositide hydrolysis induced by excit-atory amino acids in cultured cerebellar granule cells(Yu and Chuang, 1996). However, it is still unclearhow ferrocyanide (II) could fully explain mechanismsunderlying the interference by SNP (II) with the open-ing processes of the NMDA channel.

It thus appears that ferrous ions (II) may interferewith the opening processes of the NMDA channelthrough mechanisms different from those for the inhi-bition by SNP (II), in a manner independent of libera-tion of NO radicals. The modulatory mechanisms byferrous ions (Il) may be operative in vivo under partic-ular pathological and/or etiological conditions associ-ated with abnormalities of the NMDA receptor corn-

plex in the brain. Unfortunately, binding experimentsare certainly not the best examples of NMDA research,and some simple electrophysiological evidence couldstrengthen the concepts put forth in future studies.

Acknowledgment: This study was supported in part byGrants-in-Aid for Scientific Research to Y.Y. from the Min-istry of Education, Science, Sports, and Culture, Japan.

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