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ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(6): 561-566 CN 31-1300/Q

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]