Short Communication

The Influence of Oxygen-Glucose
Deprivation on Nitric Oxide and Intracellular Ca²⁺ in Cultured
Hippocampal Neurons

ZHANG
Mu, NING Gang-Min, HONG Di-Hui, YANG Yong, KUTOR John, ZHENG Xiao-Xiang*

( Department
of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China )

Abstract        Nitric
oxide (NO) was speculated to play an important role in the pathophysiology of
cerebral ischemia. In this study, the effect of oxygen-glucose deprivation
(OGD) on the cellular production of NO was investigated in cultured hippocampal
neurons. Intracellular Ca²⁺ was also detected as its closely
relationship with NO. The generation of NO and changes in intracellular Ca²⁺
were evaluated using confocal laser scanning microscopy with diaminofluorescein
diacetate (DAF-2 DA), an NO probe, and Fluo-3, a Ca²⁺ probe
respectively. Extracellular glutamate level was also measured by HPLC with
fluorescence detection. Results showed that OGD induced an increase in NO
production and intracellular Ca²⁺ concentration ([Ca²⁺]_i),
the rise of DAF-2 and Fluo-3 fluorescence intensity was about 160% and 270%
respectively; an increase of about 100% in glutamate level was observed after
20 min of OGD. NMDA inhibitor MK-801 significantly reduced the OGD-induced
elevation of [Ca²⁺]_i and NO, DAF-2 and Fluo-3
fluorescence intensity uptake was inhibited by 69% and 74% respectively. The
increase in NO production was also attenuated by extracellular Ca²⁺
elimination and calmodulin (CaM) antagonist trifluoperazine dose-dependently.
These results indicated that NO production increased during oxygen-glucose
deprivation, and was greatly modulated by glutamate release, intracellular Ca²⁺
change and Ca²⁺-CaM pathway.

Key words     nitric oxide;
calcium; glutamate; calmodulin; hippocampal neuron; oxygen-glucose deprivation

Nitric oxide
(NO) was produced endogenously by the conversion of L-arginine to citrulline by
NO synthase (NOS)[1]. It was known that at the early stage of cerebral
ischemia, endothelial NOS (eNOS) and neuronal NOS (nNOS) were extremely active
and produce large amounts of NO[2]. There were increasing evidences that
nNOS-derived NO was an important mediator in ischemic brain injury[3,4].
Selective nNOS inhibitor 7-nitroindazole, which did not influence eNOS activity
but effectively diminish nNOS activity, was neuroprotective in models of focal
ischemia[5]. Additionally, neuronal cultures from nNOS null transgenic mice
were markedly resistant to combined oxygen-glucose deprivation compared with wild-type
cultures[6]. It was also proposed that excessive NO production ordinarily might
result from ischemic depolarization, glutamate transmitter release, N-methyl-D-aspartate
(NMDA) ionotropic receptor stimulation, elevation of [Ca²⁺]_i
and downstream nNOS activation[7－9]. nNOS is a kind of Ca²⁺/CaM-dependent enzyme and the
increase in intracellular calcium in ischemia neurons leads to a persistent
activation of nNOS, then resulting in continuous NO production.

In present
study, the extracellular glutamate (Glu) level induced by oxygen-glucose
deprivation (OGD) and further the effect of NMDA inhibitor MK-801 on NO
production in cultured hippocampal neurons were first investigated. Secondly,
the impacts of intracellular Ca²⁺ and CaM activity on the NO generation
during early stage of ischemia were further studied using confocal laser
scanning microscopy (CLSM) to supply a close understanding on the intracellular
processes and the relationship between the NO and Ca²⁺/CaM system.

1    Materials
and Methods

1.1   Animals
and drugs

Sucking
Sprague-Dawley rats (1－3 day-old) were from Zhejiang Center of Laboratory Animals.
Fluo-3/AM was purchased from Molecular Probe. 4, 5-diaminofluorescein diacetate
(DAF-2 DA) was from Calbiochem. TFP and MK-801 was from Sigma. Fetal calf serum
was from Hangzhou Sijiqing Biological Engineering Materials Co. All other
chemicals used were of analytical purity.

1.2   Measurement
of intracellular Ca2+ and NO in cultured hippocampal neurons

Primary
hippocampal cell cultures from 1－3 day-old sucking Sprague-Dawley rats were prepared as described
previously[10]. Briefly, hippocampi were rapidly dissected, dissociated by
trypsin digestion and gentle trituration, and plated onto pre-coated (poly-D-lysine,
10 mg/L) glass coverslips in DMEM containing 10% fetal calf serum. The culture
media was refreshed every 3 d. On the third day in culture, the media was
supplemented with 5-fluoro-2′-deoxyuridine to block glial proliferation. Cells
of 8－10 d in
culture were used for experiment.

NO generation in
neurons was monitored by labeling with 4,5-diaminofluorescein diacetate (DAF-2
DA) that was de-esterified intracellularly to DAF-2. NO provided the third
nitrogen to form a triazo ring from the two amino groups of the nonfluorescent
DAF-2 and converted it to diaminotriazolofluorescein (DAF-2T) that could be
monitored at 490 nm excitation and 530 nm emission[11]. The intracellular Ca²⁺
were labeled by Fluo-3. Cells were incubated with 10 μmol/L DAF-2 DA or 5
μmol/L Fluo-3/AM at 37 °C for 30 min. The fluorescence was measured using a
confocal laser scanning microscopy (Zeiss LSM510, Germany). Both Fluo-3 and
DAF-2 were excited at 488 nm laser and emissions between 515－560 nm were obtained. Images of 512×512 pixels were acquired with a 20× objective. Typically one image was
acquired every 5 min before OGD and every 1 min during OGD.

During
oxygen-glucose deprivation, cellular edema might induce swelling of the
neurons, in this case the neurons of interest would be out of focus. To
eliminate this influence, the maximum pinhole available (1000 μm) was used to
enlarge the optical section, thus keeping the neuron in focus through out the
entire experiment, though it might reduce the resolution to some degree[12].
Because the integrated fluorescent signal was related to the morphology of the
neuron, to test whether the cell swelling induced by OGD affect fluorescence
measurements, the DAF-2 fluoresence change in hyposmotic fluid was also
detected. Hyposmotic treatment was done by dilution of the artificial cerebrospinal
fluid (ACSF)：(126 mmol/L
NaCl, 3.75 mmol/L KCl, 2 mmol/L CaCl₂, 1 mmol/L MgCl₂, 26
mmol/L NaHCO₃, 1.25 mmol/L KH₂PO₄, 10 mmol/L
glucose, pH 7.4) with distilled water (1:1).

Combined OGD was performed as described[13]
by complete exchange of media with deoxygenated, glucose-free ACSF, which was
gassed with 95% N₂/5% CO₂. The cells were designed into 5
groups: (1) Control group ( normal ACSF); (2) OGD group (deoxygenated,
glucose-free ACSF); (3) OGD+Ca²⁺-free group (Ca²⁺ removed
from ACSF and 0.5 mmol/L EGTA was added); (4) OGD+MK-801 group; (5) OGD+TFP
group. MK-801 and TFP were added 10 min before OGD and was maintained during
OGD.

1.3   Determination
of Glu by HPLC

Samples obtained
at the 20th min after the stimulation of OGD were filtered with millipore
filters (M_r: 10 000, Microcon). Glu levels were measured by
OPA－β-mercaptoethanol
precolumn derivatization, reversed-phase gradient elution and fluorescence
detection. Because samples should be first derivatized to their fluorescent
isoindoles, 20 μL dialysate samples and 10 μL OPA (Sigma) derivating fluid were
allowed to react for 1 min at room temperature. The HPLC employed buffer A, 0.1
mol/L KH₂PO₄ buffer : methanol=65:35; and buffer B, 0.1
mol/L KH2PO4 buffer : methanol=10:90. The two-buffer HPLC system
(Shimadzu-10AVP, Japan) was coupled to a fluorescent detector (RF-10AXL,
Shimadzu, Japan). Separation was achieved on a C18 column (Hypersil, BDS, 5 μm). 20 μL of the reaction mixture was
injected onto the column and separated with a gradient elution. The flow rate
was 1 mL/min; Ex/Em=357 nm / 455 nm. The areas under the peaks of the Glu were
used for calculating the concentrations.

1.4   Statistical
analysis

Experiments were
repeated on 5－8 different
coverslips. Data are presented as x±s. Statistical significance was
evaluated with Student’s t-test. Significance was accepted when P<0.05.

2    Results

An example of
the DAF-2 fluorescence change of a single dissociated neuron in hyposmotic
medium was shown in Fig.1(A), in which the fluorescence intensity had no
significant change in hyposmotic medium compared with that in control medium
(Fig.1).

Fig.2 exhibited
progressive increase in DAF-2 and Fluo-3 fluorescence intensity in the OGD
group, indicating that ischemia leaded to increased [Ca²⁺]_i
and NO generation in the neuron compared with the control group (P<0.01). The increases of DAF-2 and Fluo-3 fluorescence intensity were (162±26)% and (272±59)%, respectively. In Ca²⁺-free group, when Ca²⁺
was removed from the media, the rise in [Ca²⁺]_i and NO
were significantly lower than in OGD group. In the control group, the DAF-2
fluorescence even showed a slight decrease in intensity, which was probably due
to photobleaching or leakage of the dye.

To study the avenues responsible
for NO production during ischemia, the Glu release and theeffect of
NMDA-subtype Glu receptor antagonist MK-801 were tested. After 20 min OGD, the
extracelluar Glu concentration was (3.53±1.12) μmol/L, which was about 2-fold
to control group (Fig.3). As shown in Fig.4, application of 10 μmol/L MK-801
markedly blocked the elevation of [Ca²⁺]_i and NO (P<0.01).

Fig.1       DAF-2
fluoresence change in hyposmotic ACSF during 20 min

(A) Hyposmotic ACSF. (B) Normal ACSF as a control. (C) Statistical comparision
of these two groups. There was no significant difference between two groups
(P>0.05). (n=17－20). Control, normal ACSF; Hypo, hyposmotic ACSF. Scale bar: 10 μm.

Fig.2       Effect of
OGD on NO and [Ca²⁺]_i fluorescence in hippocampal neurons

(A) DAF-2 fluorescence change on OGD. (B) Fluo-3 fluorescence change on
OGD. (C) Statistical change of NO. (D) Statistical change of [Ca2+]i. The
increase in DAF-2 and Fluo-3 fluorescence in OGD group indicated increase in NO
and intracellular Ca2+ with OGD. Each data point represents the x±s. ( n=20－22 ). Results were
analyzed by two-tailed Student’s 
t-test. *P<0.05, **P<0.01 vs. control group. ^#P<0.05, ^##P<0.01 vs. OGD group. Scale bar: 10 μm.

Fig.3       Effects of
OGD on Glu release

After 20 min OGD, the
extracellular Glu concentration was significantly enhanced compared with that
of control group. Values were represented as x±s (n = 8). **P<0.01 vs. Control.

Fig.4       MK-801 (10
μmol/L) blocked the elevation of [Ca2+]i and NO induced by OGD.

The increases of DAF-2 and
Fluo-3 fluorescence intensity were significantly lower than OGD group. Values
were represented as x±s (n=20). **P<0.01.

When the neuron was treated by TFP,
a CaM inhibitor, the NO production induced by OGD was dose-dependently inhibited.
The increase of DAF-2 fluorescence intensity induced by OGD was (162±26)%. When
the neurons were treated by TFP with the concentrations of 10 μmol/L, 50 μmol/L
and 100 μmol/L, the increases of fluorescence intensity were (154±31)%,
(121±22)% and (116±19)%, respectively (Fig.5).

Fig.5       Effects of
TFP on OGD-induced NO production

TFP dose-dependently
inhibited the OGD-induced NO production. Values were represened as x±s (n=20－22). **P<0.01 vs. OGD.

3    Discussion

Nitric oxide is
a gaseous free radical and has been shown to be a physiological
neurotransmitter or neuromodulator. Because of its instability and low
concentration in biological systems, it had been difficult to perform direct NO
detection with satisfactory sensitivity. For the measurement of cellular NO
production, direct measurement of released NO by electrodes or measurement of
its metabolite-nitrite by Griess method has been used[14]. DAF-2 was developed
recently as a NO-sensitive fluorescent dye and had been widely used for
real-time bioimaging of NO with fine temporal and spatial resolution[15].
Culture systems were well suited for investigating altered ion regulation,
because, in contrast to in vivo situation, the extracellular conditions could
be manipulated and controlled rigorously. In this study, the neurons exhibited
progressive increase in DAF-2 fluorescence during OGD deprivation, indicating
an increase in NO generation, compared with the control group (Fig.2). It was
demonstrated by this experiment that imaging technique using DAF and CLSM was
convenient and efficient in detecting NO activity in the neuron during
ischemia.

There were some
evidences that NO production increases during ischemia. Olesen et al.[16]
reported that 7 min after ligaturing the carotids (two-vessel occlusion [2-VO]
procedure) an increase in NO was observed in hippocampus. Zhang et al.[17]
reported an increase of NO at 2－6 min after middle cerebral artery (MCA) occlusion. The results in
this experiment were consistent with these reports. As shown in Fig.2(C), there
was a sharp rise of NO in the first 10 min of OGD, followed by a gradual
increase. NO generation was modulated by the administration of NO substrate,
such as oxygen and L-arginine. After more substrate was being used, the NO
generation became slower. This might be the reason for the gradual rise of NO
in group 2.

As shown in
Fig.2(D), the OGD induced Ca²⁺ increase in hippocampal neurons was
partially prevented by Ca²⁺-free medium. Correspondingly, Ca²⁺-free
medium markedly inhibited the OGD-induced increase in NO [Fig.2(C), P<0.01], suggesting that the inhibition of Ca²⁺ might cause the decrement of
NO synthesis. Experimental evidence had supported a major role of increased
intracellular Ca²⁺ in the induction of neuronal damage during cerebral
ischemia. However, the source of Ca²⁺ rise had not been fully
elucidated. There were reasonable evidences that most of [Ca^2+]_i
increase was derived from the extracellular space and partial due to liberation
of Ca²⁺ from intracellular stores[13,18,19]. In this experiments, it
was shown that when the extracellular calcium was removed, the increase in
intercellular Ca²⁺ during OGD was significantly lower, and the
relative increases in Fluo-3 intensity was inhibited by 82% (from 274% to
132%). So, it could be concluded that most elevated Ca²⁺ during OGD
was derived from the extracellular space. On other hand, considerable
elevations of Fluo-3 and DAF-2 fluorescence intensity were also induced by OGD
in Ca²⁺-free solution (Fig.2), the fluorescence intensity of Fluo-3
increased to 132% and DAF-2 to 119%, which indicated that ischemic increase in
Ca²⁺ was due to both influx and intracellular release and the
increase in DAF-2 fluorescence in Ca²⁺-free solution was due to Ca²⁺
release from intracellular store sites. All these results showed that, during
OGD, a major route of Ca²⁺ entry into the cytosol was the plasma
membrane Ca²⁺ channels and Ca²⁺ release from internal
stores also contributed significantly to the intracellular concentration of
free Ca²⁺.

Glu is the most
abundant excitatory neurotransmitter in the human CNS. Experiments showed that
Glu concentration significantly increased during ischemia, which resulted from
both excessive presynaptic release and failure of reuptake[7]. Excess Glu leads
to prolonged activation of its receptor and then induces overload of
intracellular Ca²⁺[20]. It was known that Ca²⁺ entry and
increased cytosolic Ca²⁺ were very largely mediated by NMDA
receptors[18] and experiments implicated NO as a mediator of glutaminergic neurotoxicity
acting through NMDA receptors[9, 21]. In this study, the effects of MK-801 in
OGD were investigated. MK-80l was a non-competitive NMDA receptor antagonist.
It can reduce Ca²⁺ influx by inhibiting the opening of ion channel
in NMDA receptor complex. After application of 10 μmol/L MK-801, the
OGD-induced increases of [Ca²⁺]_i and NO were markedly
blocked. These results suggested that excessive stimulation of NMDA receptors
allowed influx of Ca2+ into neurons and thereby stimulated NOS producing abnormally
to result in an increased level of NO.

TFP is often
used as a CaM inhibitor and is a useful pharmacological tool for determining
whether calmodulin is involved in a cell response. The effects of TFP at the
concentration of 10, 50 and 100 μmol/L were evaluated. The results showed that
TFP dose-dependently inhibited the increase in DAF-2 fluorescence (Fig.5),
indicating a role of calmodulin binding for the activation of NOS in OGD. CaM
is a Ca²⁺-binding protein and is highly abundant in the mammal central
nervous system. It modulates the action of numerous Ca²⁺ dependent
enzymes, including NOS. nNOS is composed of an oxygenase domain that binds
heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that
binds FAD, FMN, and NADPH. CaM binding lied between the two domains, which
activated nNOS catalytic functions and triggers electron transfer between
flavin and heme groups[22]. The results suggested that TFP could reduce NO
production through inhibiting CaM activity. It was also reported that TFP
reduced the lesion in Wistar rats by 87%, 24 h after 2-h temporary focal
ischemia[23]. However, the mechanism of TFP’s protective effect was not very
clear. In this experiment, the protective effect of TFP was probably partial
due to the fact that it could decrease NO production in ischemia and eliminated
the lesion induced by NO.

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Received: January 24, 2003   Accepted: March 18, 2003

This work is supported by the grants from
the National Nature Science Foundation of China (No. 30170275), Science and
Technology Department of Zhejiang Province (No. 011106239), and the Key
Laboratory for Biomedical Engineering of Ministry of Education of China

*Corresponding author: Tel,
86-571-87951091; Fax, 86-571-87951676; e-mail, [email protected]