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Introduction
Clinically, the chronic use of opioid drugs leads to the development of analgesia tolerance and dependence, but the
cellular mechanisms underlying these processes are
unknown[1,2]. Chronic co-administration of
N-methyl-D-aspartate (NMDA) receptor antagonists and morphine attenuates the development of analgesia
tolerance[3,4], showing that glutamatergic
transmission is involved in opioid tolerance and dependence. Following NMDA receptor activation,
nitric oxide (NO) synthase is stimulated to generate
NO[5]. NO synthase inhibitors have similar attenuating effects on
analgesia tolerance, supporting a role for glutamatergic transmission in opioid tolerance and
dependence[6].
Previously, we have shown that, after combined pre- and postnatal morphine treatment of female rats, the NMDA receptor
density in the hippocampal and cortical regions is significantly reduced in the 2-week-old rat brain compared to that in pups
from control rats[7]. In electrophysiological studies of hippocampal slices, we also found that the mean opening time of the
NMDA receptor is longer in 2-week-old rats after the same morphine treatment
protocol[8]. These results suggest that the
functional properties of NMDA receptors during the early life of the offspring are altered by morphine or that the increased
opening time of the postsynaptic NMDA receptors may be a result of increased glutamate secretion.
An increase in the cytosolic Ca2+ concentration
([Ca2+]i) controls a diverse range of cell functions, including
neuro-transmission, secretion, and contraction. Activation of NMDA receptors results in
Ca2+ influx and a
[Ca2+]i increase. In the present study, using the same pre- and postnatal morphine exposure protocol as before, we compared
Ca2+ homeostasis following NMDA receptor activation in cultured hippocampal neurons of neonatal rats from control and morphine-addicted
female rats.
Materials and methods
Animal treatment Adult (12-week-old) female Sprague-Dawley rats were used. All procedures employing experimental
rats were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of the National
Defense Medical Center and Triservice General Hospital, Taiwan, China. Saline or morphine was injected subcutaneously
twice a day (09:00 and 17:00 ). The starting dose of morphine was 2 mg per kg bodyweight and the dose was progressively
increased by 1 mg per kg at 7 d intervals. The rats were mated 1 week after the start of morphine administra-tion, which was
continued throughout pregnancy and for the first 2 weeks after birth; the dose of morphine was 5 mg per kg bodyweight at
birth and 6 mg per kg bodyweight for the next 14 d as described in previous
studies[8,9]. For the preparation of hippocampal
neuron cultures, pups were decapitated on the day of birth, while synaptosomes were prepared from 2-week-old pups.
Culture of hippocampal neurons Primary cultures of hippocampal neurons were prepared from neonatal rats as described
in a previous study[10]. The hippocampi were dissected from the brain and incubated for 20 min at 37
oC with trypsin (0.3 g/L), DNAase I (500 U/mL), and 10% horse serum. After mechanical dissociation, the resulting single cell suspension was
centrifuged and the cell pellet suspended at an appropriate density and plated on
poly-D-lysine (30 µg/mL)-coated 24-mm glass coverslips
(1×105 cells/coverslip). The cells were grown in a humidified incubator with 5%
CO2/
95% air at 37 oC in growth medium consisting of DMEM supplemented with 10% fetal bovine serum, 10% Ham’s F12 nutrient
mixture, 50 U/mL of penicillin, and 50 µg/mL of streptomycin (Life Technologies, Grand Island, NY, USA). Normally, 20
mmol/L KCl and 10 µmol/L cytosine arabinoside were added to cultures 24 h after plating to minimize the proliferation of
non-neuronal cells. In the case of cells prepared from morphine-exposed pups, 10 µmol/L morphine was added to the cultures
every day until use. Experiments were performed on cells at 12_14 d after plating.
Preparation of synaptosomes A crude synaptosomal fraction was prepared from the hippocampi of 2-week-old rat pups
from control and morphine-treated females as described in a previous study with some
modifications[11]. All procedures were
at 4 ºC. The hippocampi were homogenized (20 strokes, loose-fitting Dounce type homogenizer) in 9 volumes of 0.32 mol/L
sucrose, 5 mmol/L HEPES, pH 7.4, and the homogenate centrifuged at
3000×g for 2 min, then the supernatant was centrifuged
at 14 600×g for 12 min and the resulting pellet saved. The white loosely-packed layer of the pellet, the synaptosome-enriched
fraction, was carefully removed with a spatula and resuspended in 0.32 mol/L sucrose, 5 mmol/L HEPES, pH 7.4, at a protein
concentration of approximately 10 g/L and stored on ice until use. Glutamate release from the synaptosomal preparation was
measured within 6 h after preparation.
Glutamate release Glutamate release from the synaptosomal preparation was measured as described in a previous
study[12]. Immediately before measurement of glutamate release, an aliquot of synaptosomes (approximately 1 mg of protein) was
washed twice in 3 volumes of HB buffer consisting of NaCl 140 mol/L, KCl 5 mol/L, 2 mol/L
CaCl2, 20 mol/L HEPES, 5 mol/L
NaHCO3, 1 mol/L MgCl2, 1.2 mol/L
Na2HPO4, and 10 mol/L glucose, pH 7.4, by centrifugation for 10 s at
10 000×g in a bench microcentrifuge, and the pellets resuspended in 3 mL of HB buffer containing 1 mmol/L
NADP+ and 50 U/mL of glutamate dehydrogenase. The fluorescence of the NADPH generated by enzymatic catalysis in response to
stimulation was measured in a stirred cuvette using a spectrofluorimeter (Spex Industries, Edison, NJ, USA) with excitation and
emission wavelengths of 340 nm and 460 nm, respectively. The traces were calibrated by addition of 2 nmol of glutamate at
the end of each assay and expressed as nmol/mg of synaptosomal protein.
Measurement of the
[Ca2+]i The change in the
[Ca2+]i was measured using the fluorescent
Ca2+ indicator, Fura-2, as described in a previous
study[13]. Cells grown on glass coverslips were loaded with Fura-2 by incubation for 20 min at 37
oC with 5 µmol/L fura-2 AM (Molecular Probes, Eugene, OR, USA) in loading buffer consisting of 150 mmol/L NaCl, 5 mmol/L
KCl, 5 mmol/L glucose, 2.2 mmol/L CaCl2, 1 mmol/L
MgCl2, and 10 mmol/L HEPES, pH 7.4, as described in a
previous study[14]. The coverslips were then mounted in a modified Cunningham chamber attached to the stage of a Nikon
Diaphot (Shinnagawa-ku, Tokyo, Japan) inverted microscope equipped with a Nikon ×40 Fluor objective, and the
fluorescence of the cells monitored using a dual-excitation spectrofluorimeter with a photomultiplier-based detection system (Spex
Industries). Using a pinhole diaphragm placed in the image plane in front of the photomultiplier, one cell was selected per
coverslip and excited alternately with 340 and 380 nm light, and the emitted fluorescent light collected via the objective
through a 510 nm long wave-pass filter. The
[Ca2+]i was expressed as the ratio of the fluorescence using excitation at 340 nm
and 380 nm. Changes in the
[Ca2+]i in response to KCl, ATP, leu-enkephalin, or
[D-Ala2, N-Me-Phe4,
Gly5-ol]-enkephalin (DAMGO) were measured in loading buffer, whereas changes in the
[Ca2+]i in response to known or suspected NMDA
receptor agonists were measured in loading buffer lacking
Mg2 + to avoid Mg2+-dependent block of the NMDA
receptor[15] and containing 10 µmol/L
glycine, an NMDA receptor
coagonist[16]. Experiments were repeated at least six times using different
batches of cells; the results of one representative experiment are shown in the Figures 1, 2, and 3. In some experiments, the
means±SD values for the 340/380 fluorescence ratio, calculated for
n experiments, are also shown.
Results
The neuronal character of the cultured hippocampal neurons used was judged by the extent of the NMDA- or
KCl-induced [Ca2+]i increase. In hippocampal neurons prepared from control rats, the addition of 100 µmol/L NMDA or 50
mmol/L KCl resulted in an increase in the
[Ca2+]i from a basal ratio level of 1.2±0.1
(n=37) to 1.9 ± 0.2 (n=6) or 2.5±0.3
(n= 9), respectively, (Figure 1A, traces a and b), showing activation of ionotropic NMDA receptors and voltage-sensitive
Ca2+ channels. The
[Ca2+]i remained at this level as long as NMDA or KCl was present, and reproducible
[Ca2+]i increases were evoked by repetitive stimulation. In contrast, the amplitude of the
[Ca2+]i increase progressively decreased if the cells were
repetitively stimulated by the G protein-coupled receptor agonists, ATP, DAMGO, and leu-enkephalin (Figure 1A, traces
c_e), showing desensitization. We next determined whether chronic pre/postnatal morphine exposure modified the effect of
glutamate on the [Ca2+]i. As shown in Figure 1B, glutamate caused a dose-dependent
[Ca2+]i increase in cells of pups from
both control and morphine-treated rats. The
EC50 values for glutamate were indistinguishable, being 1.0 µmol/L for control
cells and 0.9 µmol/L for the morphine-exposure cells. The same was true for the response to NMDA, the
EC50 values being 9.0 µmol/L and 8.2 µmol/L, respectively (Figure 1C). We then examined the effect of NO on the NMDA-induced
[Ca2+]i increase in control and morphine-exposed neurons. At NMDA concentrations lower than 30 µmol/L, S-Nitrosoglutathione
(GSNO) (300 µmol/L), an NO donor, potentiated the action of NMDA to the same extent in both groups (Figure 1C), while the
other NO donors, 3-morpholinosydnonimine (SIN1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP),
had no effect (Figure 1D) suggesting that it was the glutathione, and not the NO, liberated during GSNO degradation that
activated the NMDA receptor.
GSNO alone was as effective as NMDA in inducing a
[Ca2+]i increase, the amplitudes of the increase induced by GSNO or
MMDA being indistinguishable (Figures 1C, 1D and 2). In addition, the effect of GSNO was inhibited by addition of
Mg2+ or the NMDA receptor antagonist, AP-5, to the bathing solution (Figure 2A, traces a and b, and 2B, trace b), suggesting that the
NMDA receptor was activated by GSNO. Glutathione had a similar effect to GSNO in terms of effectiveness and blockade of
the effect by Mg2+ and AP-5 (Figure 2B, trace c). As shown in Figure 2C, the effect of both GSNO and glutathione on the
increase of [Ca2+]i was concentration-dependent, the
EC50 values being 56 and 414 µmol/L, respectively, in both the control
and morphine-addicted groups.
These results suggest that glutathione activates the NMDA receptor and NO then modulates its activity. To clarify the
role of NO and whether the sulfhydryl groups of the receptor were required for
Ca2+ flux, we next examined the effect of N-
ethylmaleimide on the NMDA-induced
[Ca2+]i increase in the absence or presence of GSNO and found that it had no effect
(Figure 3A), showing that sulfhydryl groups were not involved.
As glutathione is a tripeptide of glutamate, cysteine, and glycine, it was possible that glutamate, generated by glutathione
degradation, activated the NMDA receptor. To exclude this possibility, we measured the glutamate-, glutathione-, and
GSNO-induced [Ca2+]i increases in the presence of 20 U/mL of glutamate pyruvate transaminase and 10
mmol/L pyruvate to scavenge glutamate and found that the effect of glutamate was markedly inhibited, while those of
glutathione and GSNO were unaffected (Figure 3B). We also examined the effect of other glutathione derivatives on the
[Ca2+]i. When we examined whether the unmodified sulfhydryl group of glutathione was required for the
[Ca2+]i increase, we found
that S-methylglutathione, S-ethylglutathione,
S-propylglutathione, S-butylglutathione, or oxidized
glutathione failed to induce a
[Ca2+]i increase (Figure 3B, traces a and b), showing that this group was required. The
glutathione-derived dipeptides, Glu-Cys and Cys-Gly, were ineffective (Figure 3B trace d), as were the antioxidants, dithiothreitol
and mercaptoethanol (Figure 3B, trace c).
We finally examined whether glutathione acted as an NMDA receptor agonist in inducing glutamate secretion from
synaptosomes. As shown in Figure 4A, the fluorescence of NADPH, generated by glutamate dehydrogenase in the presence
of glutamate, showed a marked increase when the synaptosomes were permeabilized by digitonin, as all the glutamate in the
nerve terminals leaked out. A high KCl solution (50 mmol/L), which evoked an increase in the
[Ca2+]i via voltage-sensitive
Ca2+ channels (Figure 1A, trace b), induced glutamate secretion, as shown by the increased
NADPH fluorescence. Glutathione also stimulated glutamate secretion, although to a slightly lower extent than 50 mmol/L
KCl. The effect of glutathione was dose-dependent, with an
EC50 in the control group of 580 µmol/L (Figure 4B), consistent
with the value for inducing the
[Ca2+]i increase (Figure 2C). The potency of glutathione was very similar in the
morphine-treated group (EC50 609 µmol/L).
Discussion
To characterize the effect of NO on the interrelationship between chronic morphine exposure and NMDA receptor
signaling, we compared its effect on the NMDA-induced
[Ca2+]i increase in the control and chronic morphine-treated groups.
As shown in Figure 1, although GSNO potentiated the action of NMDA on the
[Ca2+]i at an NMDA concentration lower than
30 µmol/L, this potentiation was not altered by chronic morphine treatment. Other NO donors tested did not have such effect
(Figure 1). These results show that NO does not modulate glutamatergic transmission in hippocampal neurons of neonatal
rats from control or morphine-addicted female rats and that the potentiating effect of GSNO is attributable to its glutathione
moiety, rather than NO. Our previous study showed that the duration of synaptic NMDA receptor-mediated currents in the
hippocampus of the 2-week-old offspring of morphine-treated rats is extended, presumably resulting in increased
Ca2+ entry through NMDA receptor
channels[8]. However, the present study, using the same pre/postnatal exposure protocol, showed
that the [Ca2+]i increase evoked by activation of NMDA receptor channels was not significantly different in control and
morphine-treated rats (Figure 1) and that pre/postnatal morphine treatment has no effect on glutamate release from the
hippocampal nerve terminals of the offspring (Figure 4). This difference may be because of the fact that hippocampal slices
prepared from 2-week-old offspring were used in the previous study, while, cultured hippocampal neurons prepared from
neonatal rats were used in the present study.
Glutathione, acting as a neuromodulator, displaces
iono-tropic glutamate receptor ligands from their binding sites
and regulates calcium influx through the NMDA
receptor[17_19]. Our results showed that glutathione also acted as an NMDA
receptor agonist. Not only did it cause a dose-dependent
[Ca2+]i increase and the response was blocked by
Mg2+ and AP-5 (Figure 2), but it also evoked glutamate release from nerve terminals (Figure 4). Of all the glutathione derivatives and
antioxidants tested, glutathione was the only species to activate the NMDA receptor (Figure 3). S-nitrosylation of NMDA
receptor thiol groups by NO results in channel
inactivation[20]. Furthermore, in a ligand binding study on pig cerebral cortical
synaptic membranes, GSNO was shown to act as an NMDA receptor
ligand[21]. If the effect of GSNO on the
[Ca2+]i were attributable to its degradation product, glutathione, GSNO would be expected to be less effective than glutathione, because
the NO released by GSNO breakdown would inactivate the NMDA channel once activated by glutathione and, in addition,
GSNO would not be completely degraded to glutathione. However, this was not the case, as GSNO was actually more potent
than glutathione, the EC50 values for inducing a
[Ca2+]i increase being 56
µmol/L and 414 µmol/L for GSNO and glutathione,
respectively (Figure 2). Pretreatment of the cells with N-ethylmaleimide did not inhibit the potentiation effect of GSNO on the
NMDA-induced [Ca2+]i increase, ruling out the possibility that the effect was because of nitrosylation caused by GSNO.
Thus, GSNO itself also acts as an NMDA receptor agonist. The effect of GSNO was inhibited by the addition of
Mg2+ or an NMDA receptor antagonist (Figure 2). In addition, repetitive stimulation with either GSNO or glutathione induced
[Ca2+]i increases (Figure 2), a typical character of NMDA receptor
activation[14], and both retained their ability to increase the
[Ca2+]i in the presence of a glutamate scavenger (Figure 3) suggesting that the molecules themselves, rather than the degradation
product, glutamate, are the active species. Taken together, our data show that GSNO and glutathione are endogenous
NMDA receptor agonists.
In conclusion, in an attempt to characterize the interrelationship between chronic pre- and post-natal morphine exposure
and the glutamatergic neurotransmission, we measured NMDA receptor agonist-induced
[Ca2+]i increase and glutamate
secretion in hippocampal neurons of the offspring of morphine-addicted female rats. Our results indicate that combined pre-
and postnatal morphine exposure does not modulate NMDA receptor signaling in the cultured hippocampal neurons. However,
we also found that GSNO and glutathione act as an endogenous NMDA receptor agonist.
Acknowledgments
We thank Dr Thomas BARKAS for helpful discussion.
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