N-Methyl-d-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release...

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 139±148 139

Journal of Neurochemistry, 2001, 76, 139±148

N-Methyl-d-aspartate receptors mediating hippocampal

noradrenaline and striatal dopamine release display differential

sensitivity to quinolinic acid, the HIV-1 envelope protein gp120,

external pH and protein kinase C inhibition

Anna Pittaluga, Roberto Pattarini,1 Marco Feligioni and Maurizio Raiteri

Dipartimento di Medicina Sperimentale, Sezione di Farmacologia e Tossicologia, UniversitaÁ di Genova, Italy

Abstract

NMDA receptors regulating hippocampal noradrenaline (NA)

and striatal dopamine (DA) release have been compared

using superfused synaptosomes prelabelled with the [3H]cate-

cholamines. Both receptors mediated release augmentation

when exposed to NMDA plus glycine. Quinolinic acid (100 mM

or 1 mM) plus glycine (1 mM)-elicited [3H]NA, but not [3H]DA

release. The NMDA (100 mM)-evoked release of [3H]NA and

[3H]DA was similar and concentration-dependently enhanced

by glycine or D-serine (0.1±1 mM); in contrast, the HIV-1 envel-

ope protein gp120 potently (30±100 pM) enhanced the NMDA-

evoked release of [3H]NA, but not that of [3H]DA. Gp120 also

potentiated quinolinate-evoked [3H]NA release. Ifenprodil (0.1±

0.5 mM) or CP-101,606 (0.1±10 mM) inhibited the NMDA plus

glycine-evoked release of both [3H]catecholamines. Zinc

(0.1±1 mM) was ineffective. Lowering external pH from 7.4 to

6.6 strongly inhibited the release of [3H]NA elicited by NMDA

plus glycine, whereas the release of [3H]DA was unaffected.

The protein kinase C inhibitors GF 109203X (0.1 mM) or H7

(10 mM) selectively prevented the effect of NMDA plus glycine

on the release of [3H]NA. GF 109203X also blocked the

release of [3H]NA induced by NMDA or quinolinate plus

gp120. It is concluded that the hippocampal NMDA receptor

and the striatal NMDA receptor are pharmacologically distinct

native subtypes, possibly containing NR2B subunits but

different splice variants of the NR1 subunit.

Keywords: gp120, NMDA receptor subtypes, NR1 subunit

variants, pH, protein kinase C, quinolinic acid.

J. Neurochem. (2001) 76, 139±148.

A number of neurological and neuropsychiatric symptoms,

including cognitive de®cits, are present in a high percentage

of AIDS patients. In some cases these symptoms are likely

to re¯ect overt neurodegenerative events; in others they

may be a consequence of more subtle effects caused by

exogenous compounds originated from the HIV-1 virus or

by endogenous molecules whose concentration becomes

abnormally elevated (Giulian et al. 1996; Lipton 1998;

Schroeder et al. 1998).

The HIV-1 glycoprotein gp120 is an envelope protein that

is shed by the virus and appears to exert a number of

deleterious actions, some probably leading to neuronal

death. The coat protein is thought to act in concert with

agonists at glutamate receptors of the NMDA type,

including glutamate itself, aspartate and quinolinic acid.

Quinolinate is a product of tryptophan catabolism, usually

present in the extracellular ¯uid of the mammalian CNS at

relatively low concentrations (Stone 1993 and references

therein), but the levels of which are reported to increase

greatly during pathological conditions, including AIDS

(Achim et al. 1993; Martin et al. 1993; Beagles et al. 1998).

In previous works we found that gp120 can imitate

glycine or d-serine as a glutamate coagonist at NMDA

Received May 24, 2000; revised manuscript received August 3, 2000;

accepted August 11, 2000.

Address correspondence and reprint requests to Dr Anna Pittaluga,

Dipartimento di Medicina Sperimentale, Sezione di Farmacologia

e Tossicologia, Viale Cembrano 4, 16148 Genova, Italy.

E-mail: [email protected]

Abbreviations used: CP-101,606, (1S,2S)-1-(4-hydroxyphenyl)-2-(4-

hydroxy-4-phenylpiperidino)-1-propanol; DA, dopamine; GF 109203X,

dihydrochloride-3-{1-[3-(dimethylamino)propyl]-1H-indol-3-yl}-4-(1H-

indol-3-yl)-1H-pyrrole-2,5-dione; H7, 1-(5-isoquinolinylsulphonyl)-2-

methylpiperazine; NA, noradrenaline; PKC, protein kinase C.1The present address of Dr Roberto Pattarini is Department of

Pharmacology, University of Manitoba, Faculty of Medicine, 753

McDermot Avenue, Winnipeg, MB R3E OT6, Canada.

receptors localized on noradrenergic neurones in the hippo-

campus and neocortex of rats (Pittaluga and Raiteri 1994;

Pattarini et al. 1998) and humans (Pittaluga et al. 1996).

These NMDA receptors are known to mediate the increase

of noradrenaline (NA) release (Fink et al. 1990; Pittaluga

and Raiteri 1990), and gp120, as well as its V3 loop peptide,

can mimic glycine with impressive potency, i.e. four orders

of magnitude higher than that of the natural glutamate

coagonist (Pattarini et al. 1998).

Quinolinic acid is an NMDA receptor agonist (Stone and

Perkins 1981; Stone 1993; Moroni 1999). The af®nity of

quinolinate for the receptor is much weaker than that of

NMDA; however, as an excitotoxin in the adult rat brain in

vivo, quinolinic acid was found to be as potent as NMDA

(Foster and Schwarcz 1988). Of note, quinolinate-induced

lesions differ qualitatively from those produced by NMDA

(see Guidetti and Schwarcz 1999 for references). One

possible reason is that quinolinic acid preferentially acti-

vates subtypes of the NMDA receptor (Monaghan and

Beaton 1991; Prado de Carvalho et al. 1996).

Release-regulating receptors of the NMDA type are

located not only on noradrenergic axon terminals in the

hippocampus, but also on striatal dopaminergic axon

terminals where they mediate enhancement of dopamine

(DA) release (Roberts and Anderson 1979; Krebs et al.

1991). The two presynaptic NMDA receptors regulating

catecholamine release have not been compared in terms of

functional properties and structural characteristics. In the

present work we studied the release of NA from rat hippo-

campal synaptosomes and of DA from striatal synaptosomes

and compared the effects of agonists at the glutamate

recognition site (including quinolinic acid) and at the

glycine site (including gp120). We found differences

between the two NMDA receptors and tried to clarify the

reasons for these differences by investigating the NR2

subunits present and the possible involvement of splice

variants of the NR1 subunit.

Materials and methods

Drugs

[3H]NA (speci®c activity 30 Ci/mmol) and [3H]DA (speci®c

activity 43 Ci/mmol) were purchased from Amersham Radio-

chemical Centre (Buckinghamshire, UK). Glycine, l-aspartate,

quinolinic acid, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine

(H7) and d-serine were obtained from Sigma Chemical Co.

(St Louis, MO, USA); NMDA was from Tocris Cookson (Bristol,

UK). Recombinant HIV-1 IIIB glycoprotein gp120 was obtained

from AMS Raggio Italgene (Milan, Italy). Sphingosine and

dihydrochloride-3-{1-[3-(dimethylamino)propyl]-1H-indol-3-yl}-4-

(1H-indol-3-yl)-1H-pyrrole-2,5-dione (GF 109203X) were pur-

chased from RBI (Sigma Aldrich, Milan, Italy). The following

substances were generous gifts from the indicated companies:

6-nitroquipazine maleate (Duphar, Amsterdam, the Netherlands)

(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-

propanol (CP-101,606; P®zer, Groton, CT, USA), memantine

(Merz Co., Frankfurt, Germany) and ifenprodil (Synthelabo

Recherche, Bagneaux, France).

Animals

The experimental procedures were approved by the Ethical

Committee of the Pharmacology and Toxicology Section, Depart-

ment of Experimental Medicine, in accordance with the European

legislation (European Communities Council Directive of 24

November 1986, 86/609/EEC). Adult male rats (Sprague±Dawley;

200±250 g) were housed at constant temperature (22 ^ 18C) and

relative humidity (50%) under a regular light±dark schedule (lights

on 7.00 a.m. to 7.00 p.m.). Food and water were freely available.

The animals were killed by decapitation, and hippocampi and

striata were rapidly dissected.

Preparation of synaptosomes

Crude synaptosomes were prepared as previously described (Raiteri

et al. 1984). Brie¯y, the tissue was homogenized in 40 volumes of

0.32 m sucrose buffered at pH 7.4 with phosphate (®nal concentra-

tion 0.04 m). The homogenate was centrifuged (5 min, 1000 g) to

remove nuclei and cellular debris, and synaptosomes were isolated

from the supernatant by centrifugation (12 000 g for 20 min). The

synaptosomal pellet was then resuspended in a physiological

medium having the following composition (mm): NaCl, 125; KCl,

3; MgSO4, 1.2; CaCl2, 1.2; NaH2PO4, 1; NaHCO3, 22; glucose, 10

(aeration with 95% O2 and 5% CO2 at 378C); pH 7.2±7.4.

Release experiments

Hippocampal synaptosomes were labelled with [3H]NA (®nal

concentration: 30 nm) in the presence of 0.1 mm 6-nitroquipazine to

avoid false labelling of serotonergic terminals. Striatal synapto-

somes were labelled with [3H]DA (®nal concentration: 23 nm).

Both preparations were incubated with the radioactive label at

378C, for 15 min, in a rotary water bath and in an atmosphere of

95% O2 and 5% CO2.

After the labelling period, identical portions of the synaptosomal

suspension were introduced in parallel superfusion chambers

maintained at 378C (Raiteri et al. 1974; Raiteri and Raiteri 2000).

Superfusion was then carried out at a rate of 0.5 mL/min, using

media aerated with 95% O2 and 5% CO2, for a total period of

48 min. Starting at t � 36 min, four 3-min fractions were collected.

At t � 20 min the medium was replaced with a physiological

solution from which Mg21 ions were omitted. Agonists were added

at t � 39 min, corresponding to the end of the ®rst fraction

collected, and were present till the end of superfusion. Antagonists

were added 8 min before agonists and maintained throughout the

superfusion period. Protein kinase C (PKC) inhibitors were added

at t � 39 min, together with agonists and were present till the end

of the superfusion.

In a set of experiments, performed to evaluate the effect of pH on

the evoked neurotransmitter release, the physiological medium

containing bicarbonate was replaced at t � 20 min by a medium

having the following composition (mm): NaCl, 140; KCl, 3; CaCl2,

1.2; NaH2PO4, 1; NaHCO3, 5; glucose, 10; HEPES, 10. The pH of

this medium was adjusted to the desired value (pH 6.6, pH 7.4 and

pH 8.0) with NaOH.

140 A. Pittaluga et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 139±148

Fractions collected and solubilized superfused synaptosomes

were counted for radioactivity by liquid scintillation.

Calculation and statistics

The amounts of [3H]NA and [3H]DA released into each superfusate

fraction were expressed as percentages of the total synaptosomal

content at the start of the fraction collected. The effect of the drugs

was evaluated by calculating the ratio between the percent ef¯ux in

the fraction corresponding to the maximal effect (the third fraction)

and the ef¯ux in the ®rst fraction collected. This ratio was

compared to the corresponding ratio obtained under control

conditions. When studying the effect of NMDA antagonists, results

are expressed as percentages of the NMDA plus glycine-evoked

release. A two-tailed Student's t-test was used to analyse the

signi®cance of the difference between two means; multiple

comparisons were made by applying the Dunnett's test.

Results

Effects of NMDA, l-aspartate and quinolinic acid on

the release of [3H]NA from hippocampal synaptosomes

and of [3H]DA from striatal synaptosomes

Rat hippocampal or striatal synaptosomes were prelabelled

with [3H]NA or [3H]DA, respectively, and exposed during

superfusion to NMDA, l-aspartate or quinolinic acid alone

or in the presence of glycine. l-Aspartate was used because

previous results showed that this endogenous aminoacid,

differently from l-glutamate, is a selective agonist at NMDA

receptors (Roberts and Anderson 1979). The results with

NMDA con®rm previous data obtained with cortical (Fink

et al. 1990) or hippocampal (Pittaluga and Raiteri 1990)

synaptosomes and with striatal synaptosomes (Krebs et al.

1991) showing that the release of [3H]NA and [3H]DA could

be enhanced by NMDA alone, possibly due to the presence

of glycine contamination in the solutions (see Paudice et al.

1998 for details) and, more effectively, by NMDA plus

glycine (Table 1). l-Aspartate (100 mm) behaved very

similarly to 100 mm NMDA. Quinolinic acid signi®cantly

enhanced the release of tritium from hippocampal synapto-

somes labelled with [3H]NA only when added at 1 mm; this

effect was doubled by 1 mm glycine. Quinolinate, at 100 mm,

elicited [3H]NA release only in presence of 1 mm glycine. In

contrast, 1 mm quinolinic acid, either alone or in presence of

glycine, failed to signi®cantly elevate tritium release from

striatal synaptosomes labelled with [3H]DA (Table 1).

NMDA-evoked [3H]NA and [3H]DA release:

effects of glycine, dd-serine or gp120

The results with quinolinic acid suggest possible differences

between the NMDA receptors under study. Considering the

reciprocal in¯uences occurring between coagonist sites of

the NMDA receptor, we then compared the sensitivity of the

hippocampal and the striatal NMDA receptors to glycine

and d-serine, the endogenous coagonists of glutamate and to

gp120, the HIV-1 envelope protein previously found to

potently mimic glycine/d-serine at the NMDA receptor

regulating NA release from hippocampal axon terminals

(Pattarini et al. 1998). As shown in Table 2, glycine and d-

serine, added at 0.1 or 1 mm, concentration-dependently

potentiated the effects of 100 mm NMDA on the release

of [3H]NA or [3H]DA. In contrast, gp120 (30±1000 pm)

selectively potentiated the NMDA-induced release of

[3H]NA, but was ineffective on the NMDA-induced release

of [3H]DA. The potency of gp120 at the noradrenergic

terminals was about four orders of magnitude higher than

that of glycine and d-serine, respectively, con®rming pre-

vious data from this laboratory (Pattarini et al. 1998).

Enhancement of the quinolinate effect on [3H]NA

release by gp120

It was important to test if the effect of quinolinic acid

also could be affected by gp120. Figure 1 shows that the

quinolinate-evoked release of tritium from hippocampal

Table 1 Effect of different glutamatergic agonists on the release of [3H]NA from hippocampal synaptosomes and of [3H]DA from striatal

synaptosomes

[3H]NA [3H]DA

Control Glycine (1 mM) Control Glycine (1 mM)

NMDA (100 mM) 37.6 �̂ 3.1 75.5 �^ 4.7** 14.3 �^ 1.9 41.1 �̂ 2.4**

L-aspartate (100 mM) 55.1 �^ 4.6 81.4 �^ 8.4* 17.9 �^ 1.5 44.2 �̂ 2.7**

Quinolinate (100 mM) 2.5 �^ 3.1 18.7 �^ 4.3* 1.3 �^ 2.7 3.2 �̂ 2.8

(1 mM) 15.3 �^ 1.5 34.5 �^ 2.7* 0.05 �^ 2.5 5.5 �̂ 1.8

Synaptosomes from the two rat brain areas were exposed to different glutamatergic agonists (concentration as indicated) alone or in presence of

glycine. Results are expressed as percentage increase over basal release. Data are means ^ SEM of ®ve to seven experiments run in triplicate

(three superfusion chambers for each experimental condition).

*p , 0.05 vs. respective control; **p , 0.01 vs. respective control.

Native NMDA receptor subtypes 141


synaptosomes prelabelled with [3H]NA was concentration-

dependently potentiated by 30 and 100 pm gp120. We

previously found that the NA release caused by NMDA plus

gp120 in rat (Pittaluga and Raiteri, 1994) and human

(Pittaluga et al. 1996) noradrenergic neurones could be

prevented by the NMDA open-channel blocker memantine.

As shown in Fig. 1, memantine also blocked the effect of

quinolinate plus gp120, con®rming the involvement of

NMDA receptors. The selectivity of quinolinic acid and

of gp120 for the receptor regulating NA release is con-

®rmed by the failure of quinolinate (1 mm) plus gp120

(1 nm) to enhance signi®cantly the release of tritium from

striatal synaptosomes prelabelled with [3H]DA (data not

shown).

Effects of zinc or ifenprodil on the release of [3H]NA

or [3H]DA elicited by NMDA receptor agonists

To better understand the possible differences between the

two NMDA receptors under study, we examined some of the

characteristics usually attributed to the presence of parti-

cular receptor subunits. Figure 2 shows that Zn21 ions,

known to inhibit with submicromolar af®nity the function of

NMDA receptors containing NR2A subunits (Paoletti et al.

1997), were unable to inhibit the release of [3H]NA or

[3H]DA provoked by NMDA plus glycine in hippocampal or

striatal synaptosomes, respectively. Ifenprodil, at very low

micromolar concentration, was shown to inhibit the function

of NMDA receptors containing NR2B subunits (Williams

et al. 1993) and a structural analogue of ifenprodil, CP-

101,606, was reported to behave similarly (Menniti et al.

1997). We here found that both ifenprodil and CP-101,606

concentration-dependently prevented the NMDA plus gly-

cine-evoked release of [3H]NA and [3H]DA. The quinolinic

acid plus gp120-evoked release of [3H]NA from hippocampal

synaptosomes also was prevented by ifenprodil (1 mm

quinolinic acid 1 100 pm gp120 � 38.5 ^ 5.4; 1 mm

quinolinic acid 1 100 pm gp120 1 1 mm ifenprodil �10.6 ^ 1.86, p , 0.01), or by CP-101,606 (1 mm quino-

linic acid 1 100 pm gp120 � 41.3 ^ 4.5; 1 mm quinolinic

acid 1 100 pm gp120 1 10 mm CP-101,606 � 7.8 ^ 3.4;

p , 0.01), but it was totally insensitive to Zn21 ions (1 mm

quinolinic acid 1 100 pm gp120 � 39.7 ^ 4.3; 1 mm qui-

nolinic acid 1 100 pm gp120 1 1 mm Zn21 � 38.5 ^ 6.3).

At the concentrations used, Zn21 ions, ifenprodil or

Table 2 Effect of glycine, D-serine and gp120 on the NMDA-evoked release of [3H]NA from hippocampal synaptosomes and of [3H]DA from striatal

synaptosomes

[3H]NA [3H]DA

Control (100 mM NMDA) 35.3 �̂ 3.1 12.3 �^ 2.0

NMDA 1 glycine (0�.1 mM) 50.3 �^ 3.0*(1 42.5) 18.3 �̂ 1.7*(48.8)

(1 mM) 73.6 �^ 3.3**(1 108.5) 43.5 �^ 4.4**(253.7)

NMDA 1 D-serine (0�.1 mM) 47.2 �̂ 13.8*(33.7) 16.8 �̂ 2.2*(36.6)

(1 mM) 76.8 �^ 7.2**(117.5) 32.7 �̂ 4.1**(165.9)

NMDA 1 gp120 (30 pM) 47.2 �^ 5.6*(33.7) n.d.

(100 pM) 69.7 �̂ 4.3**(110.2) 9.4 �̂ 1.7 (24.2)

(1000 pM) 75.2 �̂ 7.5**(124.1) 12.7 �̂ 2.4 (3.2)

Synaptosomes from the two brain areas were exposed to 100 mM NMDA alone or in presence of glycinergic agonists (concentration as indicated).

Results are expressed as percentage increase over basal release. Data are means ^ SEM of six to nine experiments run in triplicate (three

superfusion chambers for each experimental condition); percent increases with respect to the NMDA-induced release of both the neurotransmitters

are reported in brackets. n.d., not determined.


Fig. 1 Potentiation by gp120 and antagonism by memantine of the

quinolinate-induced release of [3H]NA from hippocampal synapto-

somes. Synaptosomes were exposed to quinolinate in absence or

in presence of the protein starting at the end of the ®rst fraction

collected. Memantine was added 8 min before agonists and was

present till the end of superfusion. Results are expressed as percen-

tage increase over basal release. Data are means ^ SEM of three

to ®ve experiments run in triplicate (three superfusion chambers for

each experimental condition). *p , 0.05 vs. quinolinate; #p , 0.05

vs. quinolinate 1 gp120.



CP-101,606 did not, on their own, affect the basal tritium

release (data not shown).

The hippocampal NMDA receptor is selectively

sensitive to pH decrease and PKC inhibition

We ®nally analysed some of the characteristics of the NR1

subunit which is known to exist in eight splice variants (see

Discussion). In particular, we investigated the pH sensitivity

and the protein kinase C modulation of the hippocampal and

the striatal NMDA receptors mediating catecholamine release.

The study of the pH sensitivity were performed in

HEPES-buffered medium. Preliminary experiments showed

that the basal release of tritium from hippocampal and

striatal synaptosomes prelabelled, respectively, with [3H]NA

and [3H]DA, did not differ signi®cantly during superfusion

with media buffered at pH 7.4 with bicarbonate vs. HEPES.

The tritium released spontaneously in the ®rst 3-min

superfusate fractions collected, expressed as percentage of

the total tissue content, amounted to 1.83 ^ 0.14 (n � 4) in

hippocampal synaptosomes and to 2.16 ^ 0.08 (n � 5) in

striatal synaptosomes, when superfused in bicarbonate

buffer. The spontaneous release from noradrenergic synap-

tosomes remained unchanged at pH 6.6 or at pH 8.0. Only a

slight increase of basal release was observed in dopaminer-

gic terminals exposed to medium buffered with HEPES at

pH 6.6 (from 2.06 ^ 0.05 to 2.54 ^ 0.06; n � 4). How-

ever, changing pH had a clear differential effect on the

release of the two catecholamines elicited by NMDA plus

glycine. As shown in Fig. 3, the release of [3H]NA was

signi®cantly inhibited when the pH of the superfusion

medium was lowered to 6.6. In contrast, the NMDA-evoked

release of [3H]DA remained unaffected. No signi®cant

alteration of the release of [3H]NA and [3H]DA could be

observed when the pH was increased to 8.0.

The modulation of the NMDA receptor function by PKC

was investigated by stimulating release of [3H]NA and

[3H]DA from synaptosomes with NMDA plus glycine in

presence of the PKC inhibitors GF 109203X, H7 or

sphingosine. The evoked release of [3H]NA was signi®-

cantly prevented by GF 109203X or H7, which failed to

affect the evoked release of [3H]DA (Table 3). Table 3 also

shows that sphingosine was ineffective on the evoked

release of both [3H]catecholamines. The release of [3H]DA-

elicited by NMDA plus glycine from striatal synaptosomes

had been found to be insensitive to the PKC inhibitors

staurosporine and chelerythrine (CheÂramy et al. 1996). At

the concentrations used, the PKC inhibitors did not affect,

on their own, the basal tritium release or the NMDA-evoked

tritium release (data not shown).

Table 4 shows that GF 109203X, but not sphingosine,

could prevent the release of [3H]NA elicited by NMDA plus

gp120 from hippocampal synaptosomes. The release of

[3H]NA evoked by quinolinic acid plus gp120 also was

sensitive to GF 109203X, but insensitive to sphingosine.

Discussion

Methodological aspects

The two NMDA receptors compared herein are localized on

noradrenergic and dopaminergic axon terminals, respec-

tively. The technique we used to monitor NA and DA

release (a very thin layer of synaptosomes plated on micro-

porous ®lters and up-down superfused) has often been

shown to prevent indirect effects. In particular, the changes

of release of one transmitter (NA or DA) caused by a

Fig. 2 Effects of Zn21, ifenprodil and CP-101,606 on the release of

[3H]NA (X) and [3H]DA (W) induced by NMDA in presence of glycine

from hippocampal and striatal synaptosomes, respectively. Antago-

nists were added 8 min before agonists and were present till the end

of the superfusion. Results are expressed as percentages of the

NMDA 1 glycine effects. Data are means ^ SEM from 18 experi-

ments run in triplicate (three superfusion chambers for each experi-

mental condition). *p , 0.05 vs. respective control; **p , 0.01 vs.

respective control.



compound can exclusively be attributed to a direct action of

the compound on the terminal (noradrenergic or dopami-

nergic) that releases that transmitter. Comparison between

receptors by functional neurochemistry using tools known to

modulate receptor activity seems appropriate, therefore, if

one considers that: (i) the NMDA receptors present on

noradrenergic and dopaminergic axon terminals represent

such a small percentage of the NMDA receptors present in a

synaptosomal preparation that radioligand binding tech-

niques can not be conveniently applied; and (ii) puri®cation

of these axon terminals is not technically feasible so that

the exploitation of molecular biology approaches able to

provide structural information is impossible.

Quinolinic acid and gp120 may act selectively at

NMDA receptor subtypes

Molecular studies have shown that NMDA receptors are

hetero-oligomeric proteins composed of NR1 and NR2

subunits (reviewed by Hollmann and Heinemann 1994). The

NR2 subunit exists in four isoforms termed NR2A to NR2D

which can originate heterogeneity of NMDA receptors

(Meguro et al. 1992; Monyer et al. 1992). Evidence for the

existence of native NMDA receptor subtypes, however, is

scarce.

Prior to these molecular studies, results from microionto-

phoresis (Perkins and Stone 1983) and binding (Monaghan

and Beaton 1991) experiments had suggested the existence

of at least two subtypes of NMDA receptor, one insensitive

to quinolinic acid, present in the cerebellum, and the other

sensitive to both NMDA and quinolinate. More recently,

using recombinant NMDA receptors, Prado de Carvalho et

al. (1996) found that quinolinic acid could not activate

heterodimeric receptors containing NR1 plus the NR2C sub-

unit, known to be present predominantly in the cerebellum.

The present results showing that quinolinic acid could

enhance, in a glycine-sensitive manner, the release of NA

Fig. 3 [H1] dependency of the [3H]NA and [3H]DA release induced

by NMDA in presence of glycine from hippocampal and striatal

synaptosomes, respectively. Superfusion was carried out as

described in the Materials and methods section. Results are

expressed as percentage increase over basal release. Data are

means ^ SEM of eight to 12 experiments run in triplicate. (three

superfusion chambers for each experimental condition). *p , 0.05

vs. respective control.

Table 3 Effect of PKC-inhibitors on the release of [3H]NA from

hippocampal synaptosomes and of [3H]DA from striatal synaptosomes

induced by NMDA in presence of glycine

NMDA (100 mM) 1 glycine (1 mM)

[3H]NA [3H]DA

Control 55.6 �^ 2.6 40.2 �^ 3.2

GF109203X (0�.1 mM) 32.9 �^ 5.6* 39.2 �^ 3.3

H7 (10 mM) 37.6 �̂ 4.5* 38.8 �^ 2.6

Sphingosine (10 mM) 61.3 �^ 5.8 35.4 �^ 6.2

Synaptosomes were exposed to PKC inhibitors together with agonists

at the end of the ®rst fraction collected and were present till the end of

the superfusion. Results are expressed as percentage increase over

basal release. Data are means ^ SEM of ®ve to seven experiments

run in triplicate (three superfusion chambers for each experimental

condition).

*p , 0.05 vs. NMDA 1 glycine.

Table 4 Effect of PKC-inhibitors on the [3H]NA release induced

by NMDA or quinolinate in presence of gp120 from hippocampal

synaptosomes

NMDA (100 mM)

gp120 (100 pM)

Quinolinate (1 mM)

gp120 (100 pM)

Control 68.2 �̂ 5.5 36.3 �̂ 4.2

GF 109203X (0�.1 mM) 37.7 �̂ 6.9** 15.5 �^ 5.9*

Sphingosine (10 mM) 61.2 �̂ 3.5 34.7 �̂ 3.5

PKC inhibitors were added together with agonists at the end of the ®rst

fraction collected and were present till the end of the superfusion.

Results are expressed as percentage increase over basal release.

Data are means ^ SEM of four to six experiments run in triplicate

(three superfusion chambers for each expertimental condition).




but not that of DA suggest possible structural differences

between the NMDA receptors present, respectively, on

hippocampal noradrenergic and striatal dopaminergic axon

terminals (see below). The insensitivity to quinolinate is

therefore not restricted to cerebellar NMDA receptors, but it

also seems to be a characteristic of NMDA receptors present

on striatal dopaminergic axon terminals. Subtype selectivity

of quinolinate would be especially signi®cant, since the

compound, although a low af®nity agent, is endogenous and

its action on subpopulations of the NMDA receptor could

be of clinical interest. Certainly quinolinate seems to be

an interesting experimental tool to investigate receptor

subtypes.

Glycine and d-serine are endogenous glutamate coagonists

at NMDA receptors. As shown previously, and here con-

®rmed (see Table 2), the NMDA-evoked release of NA from

hippocampal synaptosomes can be potentiated by the HIV-1

envelope protein gp120. The protein seems to act through

its V3-loop at the glycine coagonist site of the NMDA

receptors localized on noradrenergic terminals, mimicking

glycine with a potency of approximately four orders of

magnitude higher than that of the endogenous agonists

(Pattarini et al. 1998). In contrast, gp120 was unable to

enhance the effect of NMDA alone on the release of DA

from striatal synaptosomes, whereas this effect could be

trebled by glycine (cf. Tables 1 and 2). These results with

gp120 also suggest involvement of two NMDA receptor

subtypes (see below).

Gp120 has been implicated in various neurological and

neuropsychiatric effects associated with AIDS. The selec-

tivity of action of the protein as a possible exogenous

glutamate coagonist at some NMDA receptors may there-

fore be of pathological interest. Also noteworthy, gp120 has

been proposed to act in concert with quinolinate and

glutamate/aspartate in the brain of AIDS patients (Lipton

1998) and quinolinate levels were shown to be largely

increased during HIV-1 infection and CNS in¯ammation

(Achim et al. 1993; Martin et al. 1993; Beagles et al. 1998).

Interestingly, low picomolar concentrations of gp120 could

potentiate the NA release brought about by quinolinate, an

effect prevented by the NMDA open channel antagonist

memantine (see Fig. 1). Considering the involvement of NA

in cognitive processes (McGaugh 1989; Izquierdo et al.

1993), the selectivity of quinolinic acid and gp120 for the

NMDA receptor-mediating release of NA in the hippo-

campus may play important role in AIDS dementia.

NR2B subunits in the hippocampal and striatal

NMDA receptors

Recent evidence indicates that NR2 subunits contain the

glutamate recognition site (Laube et al. 1997). The selec-

tivity for quinolinate of the receptor mediating NA release

could therefore be due to the presence, in the hippocampal

receptor, of NR2 subunits different from those in the striatal

receptor. Zinc ions at submicromolar concentrations are

known to preferentially affect NMDA receptors containing

NR2A subunits (Paoletti et al. 1997). The failure of 100 nm

Zn21 to inhibit the NMDA-evoked release of NA and that of

DA suggests that neither receptor contains NR2A subunits.

Instead, both receptors appear to contain subunits of the

NR2B isoform, in as much as the effects of NMDA plus

glycine on NA and DA release could be blocked by

ifenprodil or CP-101,606, two preferential inhibitors of

NMDA receptors containing NR2B subunits (Williams et al.

1993; Menniti et al. 1997). All together our results seem not

consistent with binding data showing that homoquinolinic

and quinolinic acid are relatively selective ligands for native

NR2B-containing receptors (Brown et al. 1998). Actually,

there seem to exist NR2B-containing receptors that do not

bind quinolinate. We considered the possibility that the

differential binding of quinolinic acid may not exclusively

originate from different NR2 subunits and that the NR1

subunit might play a role.

The NR1 subunit, which contains the glycine binding

domain (Kuryatov et al. 1994), is expressed in eight distinct

splice variants throughout the central nervous system. The

gene encoding the subunit contains three exons (5, 21 and

22) that are alternatively spliced to form these eight variants.

Exon-5 encodes a splice cassette of 21 amino acids (termed

N1) inserted in the extracellular amino-terminus domain.

Exons 21 and 22 encode two cassettes of 37 amino acids

(C1) and 38 amino acids (C2), respectively, located in the

intracellular carboxy-terminus domain (Nakanishi et al.

1992; Durand et al. 1993; Hollmann and Heinemann 1994;

Zukin and Bennett 1995). Since the NR1 splice variant can

affect the pharmacological and physiological properties of

heteromeric NR1-NR2 receptors (Hollmann et al. 1993; see

review by Zukin and Bennett 1995), the possibility exists

that the differential effects of quinolinate, as well as of

gp120, originate from the presence of different splice

variants of the NR1 subunit in the two NR2B-containing

NMDA receptors under study.

Different NR1 splice variants are present in the

hippocampal and striatal receptors

Protons can directly inhibit NMDA receptor function

(Traynelis and Cull-Candy 1990). The NR1 subunit contains

the pH sensor. The effect of protons is reduced by NR1

variants containing the exon-5 insert N1 (Traynelis et al.

1995). Our results suggest that the proton-sensitive NMDA

receptor sited on hippocampal noradrenergic nerve terminals

contain an N1-lacking NR1 subunit; in contrast, the proton-

insensitive NMDA receptor sited on striatal dopaminergic

nerve terminals may comprise an NR1 variant containing

the exon-5 insert.

The involvement of an exon-5 N1 insert may represent

one explanation for the differential effect of gp120. In

fact, the presence of the 21 amino acid cassette encoded by



exon-5 in the NR1 subunit has been proposed to in¯uence

the downstream S1 and S2 domains that constitute the

glycine binding pocket (Masuko et al. 1999). These

conformational changes could selectively affect the binding

of gp120, a molecule much larger than glycine/d-serine, and

prevent its action at the NMDA receptor regulating DA

release.

NMDA receptors can mediate calcium in¯ux with

consequent elevations in cytosolic calcium concentration

(Grimwood et al. 1996; Schousboe et al. 1997) and

activation of various enzymes, including PKC. This enzyme

can positively modulate the function of NMDA receptors

(Ben-Ari et al. 1992; Zukin and Bennett 1995; Bi et al.

1998; for reviews). Studies with recombinant receptors show

that the PKC potentiation correlates with the presence or

absence of the C1 sequence in the NR1 subunit, where the

presence of C1 leads to decrease in PKC potentiation.

Neither the N1 nor C2 exons appear to participate in the

potentiation (Logan et al. 1999).

Our results show that the effects of NMDA plus glycine

on NA release, but not on DA release, were blocked by

GF 109203X or H7 (Table 3). The selectivity of the

PKC blockers towards the NA release would be compatible

with the presence of the exon-21 C1 cassette in the striatal

NMDA receptor regulating DA release, but not in the

hippocampal NMDA receptor regulating the release of NA.

The PKC involved may be a Ca21-dependent diacyl-

glycerol-independent form, in as much as the NMDA

responses on NA release were sensitive to compounds

inhibiting PKC at the ATP binding site, but not by

sphingosine, which blocks PKC at the diacylglycerol

binding site.

The effects of NMDA plus gp120 or of quinolinate plus

gp120 also were prevented by GF 109203X, but not by

sphingosine (Table 4). Interestingly, NMDA agonists plus

gp120 applied to primary neuronal cultures were found to

cause a rise in the intracellular calcium level followed by

translocation of PKC from the cytosol to the membrane and

consequent sensitization of NMDA receptors (Ushijima et al.

1993). This also occurred in C6 astrocytoma cells exposed

to gp120 (Wyss-Coray et al. 1996). These authors also

found that PKC activity was upmodulated in the CNS of

gp120 transgenic mice and that brain tissue from patients

with HIV-1 showed increased PKC immunoreactivity.

Conclusions

We have compared two NMDA receptors located on

noradrenergic axon terminals of the hippocampus, where

they mediate release of noradrenaline, and on dopaminergic

axon terminals of the striatum, where they mediate release

of dopamine. The receptors appear to represent two subtypes

of the NMDA receptor because: (i) only the receptor

mediating NA release can be activated by quinolinate plus

glycine, NMDA plus gp120 or quinolinate plus gp120; and

(ii) the effect of NMDA plus glycine on the release of NA,

but not that on the release of DA, can be inhibited by

decreasing pH and by blocking PKC. These differential

responses may re¯ect structural differences. One possibility

is that the receptors, which both contain NR2B subunits,

display different splice variants of the NR1 subunit. In

particular, the NMDA receptor on noradrenergic neurones

might contain an NR1 subunit lacking both the N1 and the

C1 cassettes.

Quinolinic acid is present in normal postmortem human

brain (Moroni et al. 1984) at levels similar to those of rats

(Stone 1993 and references therein). The concentrations

found to be active here and in many other in vitro studies are

clearly much higher than those existing in normal human

brain tissue. However, during CNS in¯ammation and HIV-1

infection, the brain levels of quinolinate were found to be

largely increased (Achim et al. 1993; Martin et al. 1993;

Beagles et al. 1998). The extraordinarily high potency of

gp120 in enhancing selectively the effects of NMDA

receptor agonists, including quinolinate, at noradrenergic

terminals, and the rises of quinolinate levels during patho-

logical conditions, together with the absence of any speci®c

removal system for both quinolinic acid and gp120, could

play a role in the symptomatology of AIDS dementia.

Acknowledgements

This work was supported by the Italian Ministry of Health

(Contributo ISS, Programma Nazionale di Ricerca sull'AIDS,

Progetto Patologia Clinica e Terapia dell'AIDS 1998±99). The

authors thank Maura Agate for excellent assistance in preparing

the manuscript.

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