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Introduction
Animal and imaging data support a novel model of schizophrenia that putative brain dopamine (DA) function imbalance,
that is, the subcortical D2 receptor
(D2R) hyperfunction and prefrontal cortical (PFC)
D1 receptor (D1R) dysfunction, may be
sequential to NMDA receptor (NMDAR) hypofunction in the
PFC[1,2]. NMDAR are ligandgated ion channels and are
composed of multiple subunits (NR1, NR2A-D, and
NR3A-C)[3]. A remarkable property of NMDAR is its high permeability to
the calcium ion (Ca2+). Several studies have shown that neuro-leptics may affect influence NMDAR directly or indirectly.
Haloperidol (Hal), a typical neuroleptic, is a potent antagonist of
D2R. The cyclic AMP (cAMP) pathway is enhanced by Hal
via its D2R blockage and it can phosphorylate the NR1 subunit of the NMDAR, which is necessary for neuroleptic-mediated
gene expression and may contribute to the therapeutic
bene-fits as well as side effects of neuroleptics
treatment[4]. Clozapine (Cloz), an atypical neuroleptic, is effective in
treating positive and negative syndromes of schizophrenia and
refractory schizophrenia. In contrast to Hal, Cloz treatment
induces relatively few extrapyramidal side
effects[5]. A number of neurotransmitters and receptors, including Glu, DA,
could contribute to Cloz's atypical profile and clinical
efficacy[6]. Cloz, but not Hal, can facilitate Glu
receptor-mediated neurotransmission in the
PFC[7].
A direct interaction of neuroleptics with NMDAR has
been demonstrated. Hal has been shown to inhibit NMDAR
function in a subunit-selective manner with
NR2B-containing receptors displaying more sensitivity to the drug than
non-NR2B-containing receptors[8]. Cloz, on the other hand,
inhibits both NR2A and NR2B-containing
NMDAR[9]. It is noted that these effects of Hal and Cloz would be
counteractive to their therapeutic effects since blockade of NMDAR
exacerbates symptoms in
schizophrenia[10].
(-)-Stepholidine (SPD) is a tetrahydroprotoberberine
alkaloid isolated from the Chinese herb
Stephania. Our
previous studies have shown that SPD is an agonist at
D1R, but acts as antagonist at
D2R[2]. SPD exhibits a profile similar
to that of atypical
neuroleptics[2,11_13]. More recently, it has
been shown that SPD possesses agonist action on
D1R in the PFC pyramidal glutamic (Glu) neuron, by which it
modulates firing activity of nucleus accumbens
neurons[14]; it mimics the effect of Cloz, preferentially increasing c-fos
expression in the PFC via DA and non-DA
systems[15]. Moreover, it has been found that SPD (0.1_10 µmol/L) can potently
enhance synaptically-evoked NMDA EPSC (excitatory postsynaptic currents) in PFC neurons recorded in brain
slices[16]; SPD (50 µmol/L) also increased the amplitude
under the similar brain slices
experiments[17]. However, little is known about the effect of SPD on NMDA receptors by
direct or indirect mechanisms at the molecular level.
This study is intended to elucidate whether SPD has a
similar pharmacological property to NMDAR as Cloz or
whether it is different from Cloz and Hal, using
Ca2+ imaging techniques for studying NMDAR function. The calcium
imaging technique has an advantage in studying NMDAR
function without change of intracellular conditions, which is
different from patch clamp recording. The latter might cause
alterations in the concentrations of intracellular constituents,
such as protein kinase or phosphatase (important
constituents in modulating NMDAR activity). The direct effects of
SPD were compared with Hal and Cloz on cloned NMDAR
transiently expressed in HEK293 cells.
Materials and methods
Materials Dulbecco's modified Eagle's medium (DMEM,
Gibco, Grand Island, NY, USA), newborn calf serum (Si Ji
Qing, Hangzhou, China), penicillin and streptomycin (Watson
Biotechnologies, Shanghai, China), Fura-2 acetoxymethyl
ester (Fura-2 AM, Molecular Probes, Eugene, OR, USA),
pluronic-127 (Sigma, St Louis, MO, USA), l-Glu acid sodium
and glycine (Gly, Sino-American Biotechnology, Beijing,
China), and Hal and Cloz (RBI, Natick, MA, USA) were used
in the experiments. (-)-Stepholidine was obtained from
Shanghai Institute of Materia Medica (Chinese Academy of
Sciences, Shanghai, China). (-)-Stepholidine and Hal were
dissolved in DMSO at a final concentration of 100 mmol/L,
and Cloz in 0.1 mol/L HCl at a final concentration of 100
mmol/L. All stock solutions were stored under -20
oC and diluted with extracellular solution immediately before use.
Cell culture and transfection The HEK293 cells, a
generous gift from Dr Gang PEI (Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai, China), were
maintained in DMEM supplemented with 10% newborn calf
serum, 100 U/mL penicillin, and 100 U/mL streptomycin. The
cells were plated at 2.5×104 onto acid-washed,
poly-L-lysine coated glass coverslips (20 mm×20 mm). Transfections were
performed when cell confluence reached about 80%. Rat
NR1a, NR2A, or NR2B (generous gifts from Dr John WOODWARD, Medical University of South Carolina,
Charleston, SC, USA), and enhanced green fluorescent
protein (EGFP) plasmids were delivered at a ratio of 1:1:1 by the
calcium-phosphated precipitation method with the
modification of an additional 1 mmol/L ketamine to prevent
excitotoxity during transfections[18]. After transfection and
incubation for 24 h, the cells were used for the experiments.
EGFP was used to identify the transfected cells.
Calcium imaging[19] The transfected cells were
incubated with Fura-2 AM (4 μmol/L with 0.025% pluronic-127)
for 45 min at 37 °C in extracellular solution (135 mmol/L NaCl,
5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 10mmol/L glucose, and 5
mmol/L HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid); pH (6.8). After washing 3 times with
Fura-free extracellular solution (pH 7.2), the dishes were
mounted on the stage of an Olympus BX51WI upright
microscope (Tokyo, Japan) and perfused continuously with
modified Ringer's solution at a flow rate of 1.2_1.5 mL/min.
l-Glu/Gly were applied via a computer-controlled Y-tube
(outer j=100 µm) situated 80 µm above the cells. The
cells were exposed to the alternating 340 nm and 380 nm light from
a xenon lamp via DG4 (Sutter Instrument Company, Novato,
CA, USA) every 2 s during and immediately after Glu/Gly
application, and the images were acquired via a charge
coupled device (CCD) camera (CoolSNAP HQ, Roper
Scien-tific, Duluth, GA, USA). To minimize UV exposure, images
were taken every 15_60 s between drug applications. Ratio
images were generated using MetaFlour software from
Universal Imaging (West Chester, PA, USA). Intracellular
calcium concentrations
([Ca2+]i) were determined by the ratio of
the intensity of the wavelengths at 340 and 380 nm.
NMDA-dependent increases in
[Ca2+]i were calculated by
subtracting the average baseline value from the peak value obtained
during Glu/Gly application. SPD, Hal, and Cloz were applied
throughout the bath.
Data analysis Data were analyzed using Origin 6.0
(OriginLab Corporation, Northampton, MA, USA) and
expressed as mean±SEM. The statistical significance of a drug's
effect was determined by comparing measures before and
after drug injection using ANOVA followed by a
post-hoc Tukey test[20].
Results
Calcium imaging study of NMDA receptor function
To establish stable Ca2+ response induced
by l-Glu/Gly, and to protect cells from potential damage due to UV exposure,
[Ca2+]i induced by Glu/Gly was recorded at 2 s intervals and basal
[Ca2+]i were recorded at 15_60 s intervals. Pseudocolour
ratio images at wavelengths of 340 and 380 nm were taken
(Figure 1A). Cells were locally perfused with Glu/Gly (100/10
µmol/L) for 3 s. An increase of
[Ca2+]i was detected in cells
expressing NR1a/NR2A or NR1a/NR2B receptors. The peak
of [Ca2+]i was 3_7 s after Glu/Gly treatment and then returned
to basal levels following washout for 300 s.
The local perfusion of Glu/Gly for 3 s could induce the
reproducible increases in
[Ca2+]i performed every 400 s
duration (Figure 1B). Bath applications of
(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)
(1 µmol/L), a specific NMDAR antagonist, significantly
blocked Glu/Gly-induced Ca2+ influx (80.3%±1.4% of
inhibition, P<0.01, n=15; Figure 1C).
Effects of SPD, Hal, and Cloz on the NR2B subunit of
NMDA receptors In the cells expressing the NR1a/NR2B
receptor, both Hal and Cloz significantly inhibited the
increase in [Ca2+]i induced by Glu/Gly. The inhibition induced
by the 2 drugs could be observed at concentrations as low
as 0.1_1 µmol/L (Figure 2). In contrast, SPD
elicited only limited inhibition at a concentration of 100 µmol/L
(18.1%±
2.4% inhibition, P<0.01, n=26; Figures 2, 3). SPD produced
no significant effect at less than 100 µmol/L (0.1
µmol/L, P=
0.97, n=12; 1 µmol/L, P=0.52,
n=7; and 10 µmol/L, P=0.15,
n=10).
Although both Hal and Cloz inhibited the function of the
NR1a/NR2B receptor, their effects were not entirely identified.
Hal was much more potent than Cloz. Hal (0.1 µmol/L)
inhibited Glu/Gly-induced Ca2+ influx
(P<0.01, n=21), while Cloz produced no inhibition at the same concentration. However,
when the concentration reached 10 µmol/L, Cloz inhibited
the Glu/Gly-induced Ca2+ influx (Figure 3). The speed for Hal
and Cloz operation also differed. Hal produced its peak
effect at about 5 min after onset, while the maximal inhibition
induced by Cloz required a 30 min perfusion. However, the
effects of both Hal and Cloz were much less potent than that
of MK-801 (Figure 2C).
Effects of SPD, Hal, and Cloz on the NR2A subunit of
NMDA receptors Cloz, not SPD (100 µmol/L) or Hal (50
µmol/L), significantly inhibited the function of the
NR1a/NR2A receptor (Figure 4A, 4B). Cloz produced an average
of 31.2%±4.2% (P<0.01, n=9) and 59.2%±5.8%
(P<0.01, n=9) inhibition at 10 µmol/L and 100 µmol/L, respectively. A
similar result was observed with the NR1a/NR2B
receptors. Cloz also showed a slow onset in its effect on the NR1a/NR2A
receptors (Figure 4A, lower section), which took 30 min to
reach the maximum effect.
Discussion
The results of the present study found that SPD did not
interact with the NR1a/NR2A or NR1a/NR2B receptors at
low concentrations, unless 100 µmol/L SPD elicited certain
inhibitory effects on NR1a/NR2B receptor-mediated
Ca2+ influx. It appeared that SPD did not interact directly with
NMDAR. In contrast with SPD, Hal and Cloz produced
significant inhibitory effect on NMDAR-mediated
Ca2+ influx. However, Hal selectively blocked the NR1a/NR2B receptors,
whereas Cloz inhibited both NR1a/NR2B- and
NR1a/NR2A-mediated responses.
In a previous study[8], Hal inhibited the function of
NMDAR in a subunit-selective manner, with the
NR2B-containing receptor more sensitive to the drug. However, Cloz
inhibited Ca2+ influx mediated by both the NR2A and NR2B
subunits of NMDAR[9]. Our results of Hal and Cloz were
similar to the above mentioned both reports. Therefore, the
different effects of the 3 drugs on NMDAR function may
imply that SPD, Hal, and Cloz have different mechanisms in
its antipsychotic use. In other words, Hal and Cloz have
direct interaction with NMDAR, while SPD has an indirect
one[21].
Our results showed that the significant inhibitory effect
on Ca2+ influx by Hal was observed at 0.1 µmol/L, while Cloz
was at 1 µmol/L. However, the clinic plasma concentra-
tion of Hal and Cloz could reach 8_35 ng/mL (0.021_0.093
µmol/L)[22] and 350 ng/mL (1.07
µmol/L)[23,24], respectively, at which the 2 drugs may display their inhibitory effects on
NMDAR-mediated Ca2+ influx. So what is the meaning of
this inhibitory effect? There are two aspects with a
contradiction. The inhibitory effect induced by Hal or Cloz is
implicated to be beneficial for the negative symptoms of
schizophrenia[25,26]. Alternatively, the inhibition of Hal and
Cloz on NMDAR would limit their clinical therapeutic
effect[27,28]. It may be suggested that the inhibition induced by Hal
or the Cloz D2R blockade is the result of the inhibitory effect
on the PFC NMDAR; subsequently, the inhibition would
deteriorate the therapeutic effect, based on the DA function
imbalance hypothesis[1,2].
As to the indirect interaction between SPD and NMDAR,
we obtained definite evidence requiring postsynaptic
density-95 (PSD-95)[21]. In an in
vitro study, when PSD-95 was co-expressed with
D1R and NMDAR in HEK293 cells, the activation of
D1R could enhance NMDAR-mediated
Ca2+
influx[21]. SPD hence could not display its enhancing effect
on NMDAR under such experimental conditions without
PSD-95. In the present study, SPD also could not affect
NMDAR directly in this in vitro expression system for lack
of D1R and other components.
On the other hand, in the experiment of brain PFC slices,
including endogenic D1R and PSD-95, the patch clamp study
recently demonstrated that SPD could increase the
amplitude[17] or
frequency[16] of sEPSC recording from the NMDAR
of PFC pyramidal cells, respectively. Furthermore, the
D1R agonist of SPD is mediated via intracellular protein kinase A
and protein kinase C signaling
pathways[16]. These results also strongly support that the
D1R agonist of SPD enhances the NMDAR effect. Nevertheless, the enhancement of SPD
on the NMDAR is obviously different from the inhibition
elicited by Hal or Cloz on the NMDAR. According to these
findings, 3 possible pathways have being proposed to
explain how SPD can enhance NMDAR-mediated
neurotransmission in the PFC. First, SPD possesses a unique
D1R agonist effect[2], and the activation of
D1R has been shown to enhance NMDAR-mediated responses in PFC
neurons[16,29]. Second, SPD may act through the
D1R to either enhance synaptic Glu release or to increase the response of the cell to
NMDAR activation[16,17], as Cloz has been shown to increase
spike-dependent presynaptic Glu
release[30]. Third, SPD could result in a part effect through non-DA
systems[15]. However, which class of non-DA receptors is responsible
for the enhancing effect of SPD on NMDAR is still unknown.
Our results indicate that unlike Hal and Cloz, SPD did not
directly affect the function of NMDAR at a low concentration.
It is presumed that the modulatory effect of SPD on NMDAR
function is an indirect effect via the
D1R[16,21].
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