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Am J Physiol Regul Integr Comp Physiol 287: R21-R26, 2004; doi:10.1152/ajpregu.00310.2003
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Oxidative Stress

Fullerene derivatives protect against oxidative stress in RAW 264.7 cells and ischemia-reperfused lungs

Ya-Wen Chen,1 Kuo Chu Hwang,2 Cheng-Chieh Yen,3 and Yih-Loong Lai1

1Department of Physiology, National Taiwan University College of Medicine, Taipei 100; 2Department of Chemistry, National Tsing Hua University, Hsinchu 300; and 3Department of Occupational Safety and Health, College of Health Care and Management, Chung Shan Medical University, Taichung 402, Taiwan

Submitted 4 June 2003 ; accepted in final form 22 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fullerene derivatives have often been used as effective scavengers for reactive oxygen species (ROS). This study was designed to test whether polyhydroxylated fullerene derivatives [C60(OH)7±2] protect against oxidative stress in cultured RAW 264.7 cells and ischemia-reperfused (IR) lungs. In RAW 264.7 cells, sodium nitroprusside (SNP, 1 mM) and H2O2 (400 µM) caused a marked (90%) decrease in cell viability, and this decrease was dose dependently reversed by pretreatment with C60(OH)7±2 (10–50 µM). The increase in ROS production induced by SNP and H2O2 was significantly suppressed by C60(OH)7±2. Also, the decrease in mitochondrial membrane potential induced by SNP and H2O2 was significantly reversed by C60(OH)7±2. However, high concentration of C60(OH)7±2 (1 and 1.5 mM) lead to cell death (apoptosis or necrosis). In the isolated rat lung, the increases in pulmonary artery pressure and capillary filtration pressure induced by SNP during IR were reversed significantly by C60(OH)7±2 (10 mg/kg). These results indicate that polyhydroxylated fullerene derivatives C60(OH)7±2 at low concentrations protect against oxidative stress in RAW 264.7 cells and IR lungs.

antioxidants; nitric oxide; reactive oxygen species

LUNG TRANSPLANTATION has become an accepted and effective treatment for a variety of end-stage pulmonary diseases (10). Despite optimal lung preservation techniques, the lung remains extremely vulnerable to ischemia-reperfusion (IR) injury (6). IR injury occurs in the context of lung transplantation when blood supply is reintroduced to the ischemic graft at the completion of the implantation procedure (26). The alveolar oxygen represents a cofactor for the generation of reactive oxygen species (ROS), and continuous oxygen supply might thus enhance IR injury (21). IR lung injury plays a significant role in clinical situations, including lung transplantation as well as pulmonary thromboendarterectomy, reexpansion of collapsed lungs, and fibrinolysis after lung embolism (21). The pathophysiology of IR injury in the lung involves alveolar-capillary barrier leakage, neutrophil migration, interstitial and alveolar edema, tissue inflammation, and cell injury and death, alteration of nitric oxide synthase (NOS) activity, ROS production, lipid peroxidation, and proinflammation cytokine release (tumor necrosis factor-{alpha}, interferon-{gamma}, and interleukin-1) (26).

Recently, many studies pointed out that IR injury could be attenuated by antioxidant therapy. In 1985, a novel allotrope was reported in which 60 carbon atoms (C60) were arranged as a truncated icosahedron, with 60 vertices and 32 faces, 12 of which were pentagonal and 20 hexagonal (2, 12). Fullerene is a condensed aromatic ring with extended {pi} systems (11). Because the inventors commemorated the builder of American Rice University, Buckminister Fuller, C60 was termed Buckministerfullerene and is now commonly abbreviated to fullerenes (12). Various fullerene-based compounds have been prepared and diverse uses were sought for them. Some were incorporated into photovoltaic cells and nanotubes. Others were tested for biological activity, including antiviral, antioxidant, and chemotactic activities, and a neuroprotective agent in a mouse model of amyotrophic lateral sclerosis (2). According to these effects of fullerenes, we investigated whether fullerene derivatives [C60(OH)7±2] protect against oxidative stress in RAW 264.7 cells and IR lungs. We used sodium nitroprusside (SNP) and H2O2 to elevate NO and oxidants, respectively, in the culture medium of RAW 264.7 cells. In addition, experimental procedure for IR was carried out to examine whether SNP enhances injury in isolated rat lungs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Synthesis of C60(OH)7±2 derivatives. Polyhydroxylated C60 was synthesized according to the literature procedure (3). In brief, C60 in a benzene solution was nitrated by addition of NO2 gas, which was generated by mixing sodium nitrite with concentrated nitric acid. After removal of the solvent, the polynitro C60 derivatives solid was then hydrolyzed by sodium hydroxide aqueous solution. Desorption chemical ionization (DCI) mass spectroscopy shows the presence of up to nine hydroxyl groups. Element analysis shows that the polyhydroxylated C60 contains an average of 7 ± 2 hydroxyl groups.

Cell culture. The murine macrophage RAW 264.7 cell line was cultured in RPMI 1640 (2 g/l sodium bicarbonate) plus 10% fetal bovine serum. The cells were cultured in a 10-cm2 dish at 37°C flushing with a gas mixture of 5% CO2-95% air for 24 h.

Cytotoxicity. Cells were washed with fresh media from dishes and cultured in 24-well plates (2x105 cells/well) and then were mixed with SNP (Sigma, St. Louis, MO), hydrogen peroxide (H2O2, Hayashi Pure Chemical), or fullerene derivatives [C60(OH)7±2]. After 24 h, the medium was removed and replaced with fresh medium containing 3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT, Sigma), 30 µl (2 mg/ml), according to the method of Hansen et al. (9) with some modifications. After incubation for 4 h, the medium was removed and added to 1 ml DMSO [(CH3)2SO, Sigma] dissolving blue formazan crystal and then the well-mixed mixture (150 µl) was transferred to 96-well plates. The fluorescence was detected by using an ELISA reader at an absorption band of 570 nm.

Analysis of mitochondrial transmembrane potential, cell death, and the generation of ROS. Changes in mitochondrial transmembrane potential and cell viability were monitored according to the double staining method of Shenker et al. (22) and Chen et al. (2) using the flow cytometry. ROS were monitored according to the method of LeBel et al. (17) using the flow cytometry. Briefly, the RAW 264.7 cell line was exposed to [C60(OH)7±2] for 24 h and then to SNP (1 mM) for 24 h or hydrogen peroxide (H2O2) for 2 h. Fluorescence was measured after 3,3'-dihexyloxacarbocyanine (DiOC6, Sigma) (100 nM) and propidium iodide (PI, Sigma) (50 µg/ml) double staining the cells for 30 min at 37°C. The detector DiOC6 was with positive charge. If mitochondria were subjected to damages, the ability to pump hydrogen ions outside of the mitochondrial membrane would be suppressed, and then the cell fluorescence intensity would be reduced. On the other hand, PI could move across the damaged cell membrane, bind to DNA, and produce fluorescence. To assess ROS generation by flow cytometry, cells were treated with 80 µM 2',7'-dicholorofluorescein diacetate (DCF-DA, Sigma) for 30 min at 37°C. The lipophilic DCF-DA could be transported across the cell membrane to the cytosol and enzymatically converted to hydrophilic 2,7-dichlorofluorescein (DCFH) by cytosolic esterase. Peroxide could oxidize DCFH and release fluorescence.

In an additional study, cells (4x105/ml) were cultured in complete medium and added to 12-well plates (1 ml/well) containing various concentrations (10–1,000 µM) of C60(OH)9±2 for 24 h. Then culture was changed with a fresh medium containing SNP (1 mM) for 24 h. To detect apoptosis using the Annexin V/PI (BioVision Research Products, Mountain View, CA) method (27), the cells were washed twice with PBS and stained with Annexin V and PI for 20 min at room temperature. The cells were washed again twice with PBS and their apoptosis levels were determined by measuring the fluorescence via flow cytometric analysis. Five thousand cells were analyzed per sample.

Isolated perfused lungs. This was carried out mainly according to our previous method (15). Thirty-two male Wistar rats weighing 250 ± 10 g were divided evenly into four groups: control; SNP; C60(OH)7±2; and C60(OH)7±2+SNP. Each animal in the C60(OH)7±2- treated group was intraperitoneally injected with C60(OH)7±2 (10 mg/kg) for 3 days just before the study, whereas each animal in the control group was intraperitoneally injected with saline. SNP was given 20 min before the initiation of ischemia (see below). On the day of the study, animals were anesthetized with intraperitoneal injection of pentobarbital sodium (30 mg/kg) and their tracheae were intubated through tracheostomy with 14-gauge intravenous catheters. The intubated tracheal tube was then connected to a volume-controlled ventilator, and the animal was ventilated at a rate of 60 breaths/min, a tidal volume of 10 ml/kg, and a positive end-expiratory pressure of 2 cmH2O with a gas mixture of 75% O2-25% room air. Then, a median laparosternotomy was performed and 300 U of heparin was injected into the inferior vena cava. The main pulmonary artery was cannulated through a right ventriculotomy, and the left atrium was cannulated via a left ventriculotomy. The cannulated catheters, flared at the tip, were sutured in place. The heart, lungs, and mediastinal structures were removed en bloc and suspended from a strain gauge force-displacement transducer (Grass FT-03) inside a humidified chamber to monitor weight changes (15). Mean pulmonary arterial (Pa) and left atrial pressures (Pv) were continuously measured by pressure transducers (P23 ID Gould-Statham). The lungs were perfused with a mixture of Krebs-Henseleit (K-H) buffer (Sigma). The buffer contained (in g/l): 2.0 D-glucose, 0.141 anhydrous MgPO4, 0.16 KH2PO4, 0.35 KCl, 6.9 NaCl, 0.373 CaCl2 2H2O, and 2.1 NaHCO3. The experimental protocol included baseline, ischemia, and reperfusion periods. The initiation of the ischemia period was begun with stopping the pulmonary perfusion, and that of reperfusion was carried out with starting perfusion of the lungs again. SNP (500 µM) was given 20 min before the ischemia period.

Measurement of pulmonary capillary pressure. Pulmonary capillary pressure (Pc) was estimated by using the venous occlusion technique (4). Venous occlusion was performed by clamping the outflow tubing. During the occlusion period, Pa and Pv rapidly equilibrated, and Pc was taken as the equilibrium pressure 3 s after the occlusion.

Measurement of pulmonary capillary filtration coefficient. After the lungs reach an isogravimetric state, the venous reservoir was rapidly elevated to increase Pv by 10 cmH2O. The increase in lung weight was recorded over time ({Delta}wt/{Delta}t). The initial 3-min period of weight gain represents vascular distension and recruitment and is not a reflection of capillary permeability. The {Delta}wt/{Delta}t between 4 and 10 min represents increased transvascular fluid flux secondary to increased capillary permeability. This later {Delta}wt/{Delta}t was analyzed by using linear regression of the log10-weight change per min (5). The initial rate of weight gain was calculated by extrapolation of {Delta}wt/{Delta}t to time zero.

Wet-to-dry weight ratios. At the completion of the experiment, all lungs were dissected free of nonpulmonary tissue and weighed, and then dried to a constant weight at 60°C. Wet-to-dry (W/D) was obtained by dividing the wet weight by the final dried weight.

Statistical analysis. Values are given as means ± SE. One-way analysis of variance was used to establish differences among groups or subgroups. If significant difference existed among groups or subgroups, Newman-Keuls test was used to differentiate differences between any two groups or subgroups. To analyze the difference between before and after a treatment on the same sample, the paired Student’s t-test was employed. Difference was considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of C60(OH)7±2 on SNP- and H2O2-induced damage in RAW 264.7 cells. Exposure to SNP, which was added to increase NO for 24 h caused a concentration-dependent decrease in cell viability (Fig. 1). After exposure to C60(OH)7±2 (5–1,500 µM), SNP (1 mM)-induced RAW 264.7 cell damage was reversed (Fig. 2). Similarly, exposure to H2O2 for 24 h caused a concentration-dependent decrease in cell viability (Fig. 3), which was also reversed by preexposure to C60(OH)7±2 (not shown).



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  		Fig. 1. Cytotoxicity of RAW 264.7 cells exposed to various doses of sodium nitroprusside (SNP) for 24 h. *Significant difference (P < 0.05) compared with the vehicle control.

 


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  		Fig. 2. Cytotoxicity of RAW 264.7 cells exposed first to various doses of C60(OH)7±2 (doses are as indicated) and then to a fixed dose of SNP (1 mM) for 24 h. *Significant difference (P < 0.05) compared with the vehicle control.

 


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  		Fig. 3. Cytotoxicity of RAW 264.7 cells exposed to various doses of hydrogen peroxide (H2O2) for 24 h. *Significant difference (P < 0.05) compared with the vehicle control.

 
Effects of C60(OH)7±2 on ROS elimination. In the absence of SNP, RAW 264.7 cells were exposed first to C60(OH)7±2 for 24 h. The fullerenes dose dependently decreased ROS level (Fig. 4). In addition, in cells pretreated with C60(OH)7±2 for 24 h and then exposed to H2O2 for 2 h, H2O2-induced ROS production was decreased by C60(OH)7±2 in a dose-dependent manner (Fig. 4). The action of SNP was similar to that of H2O2 (not shown). At high concentration of C60(OH)7±2 (1,000–1,500 µM), C60(OH)7±2 suppressed 0.1 mM SNP-induced increase in ROS level.



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  		Fig. 4. Attenuation of H2O2 (exposure for 2 h)-induced reactive oxygen species production by the fullerene derivatives C60(OH)7±2 (preexposure for 24 h). *Significant difference (P < 0.05) compared with all the same C60(OH)7±2 concentration groups with different treatments. #Significant difference (P < 0.05) compared with the vehicle control group in the same treatment. DCF-DA, 2',7'-dicholorofluorescein diacetate.

 
Effects of C60(OH)7±2 on membrane potential of RAW 264.7 cells. Higher doses, but not lower doses, of the fullerenes significantly decreased membrane potential (Fig. 5). When 0.1 mM SNP was added, lower doses (5, 10, and 100 µM) of C60(OH)7±2 increased DiOC6 fluorescence, but higher doses of C60(OH)7±2 decreased mitochondrial permeability. After 1 mM SNP was added, 10 µM of the C60(OH)7±2 increased membrane potential, but higher doses of the C60(OH)7±2 decreased mitochondrial permeability transition state (Fig. 5).



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  		Fig. 5. Effects of C60(OH)7±2 on the mitochondrial membrane potential damage induced by SNP. *Significant difference (P < 0.05) compared with all the same C60(OH)7±2 concentration groups with different treatments. #Significant difference (P < 0.05) compared with the vehicle control group in the same treatment. DiOC, 3,3'-dihexyloxacarbocyanine.

 
Effects of C60(OH)7±2 on cell death. After cells were pretreated with C60(OH)7±2 for 24 h, we found that lower doses (5 µM) of the fullerenes decreased cell death, but higher doses (1,000 µM and 1,500 µM) of the fullerene increased cell death (Fig. 6). To determine whether C60(OH)7±2 protects against cell death induced by SNP, we also used flow cytometry to detect DNA fragments. RAW 264.7 cells were exposed first to the fullerenes for 24 h and then exposed to SNP for 4 h. With 5 and 10 µM of C60(OH)7±2, the cell death decreased, respectively, 15.6 and 57.4%, when exposed to 0.1 mM SNP. Also, the cell damage induced by higher doses of the fullerenes (1,000 and 1,500 µM) was reversed 86.61 and 18.77%, respectively, by SNP (0.1 mM) (Fig. 6). On the other hand, 1 mM SNP-induced cell damage was reversed by low doses (5 and 10 µM) of the fullerenes (Fig. 6). After 24 h, the cell viability did not increase with longer exposure to C60(OH)7±2.



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  		Fig. 6. Effects of C60(OH)7±2 on cell death induced by SNP. *Significant difference (P < 0.05) compared with all the same C60(OH)7±2 concentration groups with different treatments. #Significant difference (P < 0.05) compared with the vehicle control group in the same treatment.

 
After fullerene treatment for 24 h, cells apoptosis decreased significantly with low doses (10–100 µM) of C60(OH)9±2 (Fig. 7). After 24 h pretreatment with C60(OH)9±2, 10 and 1,000 µM of C60(OH)9±2 significantly protected 1 mM SNP-induced apoptosis (Fig. 7).



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  		Fig. 7. Apoptosis of RAW 264.7 cells exposed first to various doses of C60(OH)7±2 (doses are as indicated) and then to a fixed dose of SNP (1 mM) for 24 h. *Significant difference (P < 0.01) compared with the vehicle control.

 
Effects of C60(OH)7±2 on Pa. During the baseline state, the administration of SNP increased Pa (Fig. 8). After different treatments, the trend of Pa was SNP >vehicle >C60(OH)7±2. During 1–20 min of the IR period, both IR- and SNP-induced elevation in Pa was attenuated by C60(OH)7±2 (Fig. 8).



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  		Fig. 8. Pulmonary artery pressure (Pa) in isolated-reperfused lungs. C60(OH)7±2, the fullerene derivatives. *Significant difference (P < 0.05) compared with all the same time points with different treatments. #Significant difference (P < 0.05) compared with the vehicle control.

 
During the baseline state, Pc of the SNP group increased significantly compared with the vehicle and the C60(OH)7±2 groups (Fig. 9). The elevated Pc caused by IR and SNP was attenuated by the addition of C60(OH)7±2 during 1–15 and 5 min of the IR period, respectively, indicating that the fullerene derivatives reduce IR- and SNP-induced increases in Pc.



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  		Fig. 9. Capillary filtration pressure (Pc) in isolated-reperfused lungs. C60(OH)7±2, the fullerene derivatives. *Significant difference (P < 0.05) compared with all the same time points with different treatment groups. #Significant difference (P < 0.05) compared with the SNP group.

 
Pulmonary capillary filtration coefficient and W/D ratio. There were no significant differences in baseline capillary filtration coefficient (Kfc) values among groups (Fig. 10). However, Kfc increased significantly after IR in all groups. The W/D ratio increased significantly to 11.24 ± 0.51 in the SNP group compared with that of the vehicle group (8.10 ± 0.81).



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  		Fig. 10. Fluid filtration coefficient (Kfc) in isolated-reperfused lungs. C60(OH)7±2, the fullerene derivatives. *Significant difference (P < 0.05) compared with the respective before ischemia-reperfusion (IR) period.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
High doses of C60(OH)7±2 (50–1,500 µM) increased cell viability and attenuated SNP-induced ROS production and mitochondrial membrane potential damage. Whereas low doses of C60(OH)7±2 decreased SNP-induced apoptosis, effects of C60(OH)7±2 on H2O2-induced alterations in cell viability and ROS production were similar to those of SNP-induced changes. Also, C60(OH)7±2 attenuated SNP-induced increases in Pa and Pc. Several features of these results will be discussed below.

Three types of oxidative stress were used in this study. SNP and H2O2 were used for the in vitro cell culture, whereas IR and SNP were employed in isolated lungs. It is well known that H2O2 (7) and IR process (19) are important generators for ROS. SNP is a precursor of NO. Reacted with superoxide, a high concentration of NO is easy to form monooxynitrite or peroxynitrite. Peroxynitrite is a potent form of ROS, with severe cellular damaging ability (18).

In this study, we explored possible cellular mechanisms by using RAW 264.7 cells. Our results indicate that SNP and H2O2 (Fig. 4) produced an increase in ROS, which, in turn, caused cell damage. The cell damage was indicated by decreases in cell viability (Figs. 1 and 3) and mitochondrial membrane potential (Fig. 5), as well as increases in the binding of PI with DNA fragment (Fig. 6) and apoptosis (Fig. 7). Also, the process of IR causes increases in ROS (16, 19) in the isolated-perfused lung. Increased ROS are expected to induce the production and release of vasoconstrictors. Consequently, these vasoactive mediators, such as thromboxane, endothelin, and leukotrienes, could then cause vasoconstriction at both the artery (Fig. 8) and capillary (Fig. 9).

We (14) and others (13, 20, 25) found that fullerene derivatives are potent antioxidants and/or free radical scavengers. A C60 molecule contains 30 CC double bonds (12). Two bridged type substitution groups will use up one CC double bond. For C60 and its derivatives, the chemical reactivity is due to its double bonds. C60 is known to be able to react with up to 34 methyl radicals (20). The antioxidant effect of C60 is related to the number of reactive sites and the distant location to the reactive sites. The higher the number of reactive sites and the closer the distance will turn out to be the more effective antioxidant effects of fullerene derivatives. We confirmed in this study that C60(OH)7±2 is an effective antioxidant by demonstrating that C60(OH)7±2 attenuated ROS levels during the baseline or after treatment with either SNP, H2O2, or IR. In an attempt to investigate the relationship between the production of ROS- and IR-induced lung injury, we thus tested here if C60(OH)7±2 indeed could attenuate SNP-, H2O2-, or IR-induced alterations in RAW 264.7 cells and the in situ lungs.

Our results indicated that low concentrations of the fullerene inhibited SNP- and H2O2-induced cell damage and apoptosis but augmented mitochondrial membrane potential stabilization. Also, this low concentrations of C60(OH)7±2 inhibited SNP- and IR-induced lung injury and elevations in both Pa and Pc. On the other hand, although high concentrations of C60(OH)7±2 increased cell viability and decreased ROS production, it inhibited mitochondrial membrane potential. There was a discrepancy in cell death between the MTT and PI methods detecting cell death at high concentration of C60(OH)7±2. Using MTT, high concentration of C60(OH)7±2 caused an increase in cell viability (Fig. 2), whereas the same high concentration of the fullerene induced an increase in cell death detected by PI (Fig. 6). It is possible that high concentration of the fullerene induces increases in both the permeability of MPT pore (8, 23) and DNA fragment. The former increase led to an increase in the permeability of MTT and thus the cell viability. The latter increase shows the increase in cell death by the PI method. Therefore, the actual result caused by high concentration of the fullerene should be an increase in DNA fragment and cell death. In this study, we could not differentiate whether the cell death is caused by apoptosis or necrosis. However, according to the characteristics defined by Fischer et al. (7), C60(OH)7±2-induced cell death should be mainly apoptosis. We also demonstrated that high concentration of the fullerene caused an increase in apoptosis (Fig. 7). Therefore, low concentrations of C60(OH)7±2 provide mainly beneficial effects in tested cells and isolated lung, whereas high concentrations of the fullerene provide mixed effects. Thus low concentrations of the fullerene derivatives should be used to suppress ROS.

Finally, the efficacy of C60(OH)7±2 is mentioned briefly here. Compared with the frequently used antioxidant N-acetyl-cysteine, our results suggest that C60(OH)7±2 is more efficient. For example, inhibition of cerebral ischemia induced mixed lineage kinase-3 activation; an in vivo pretreatment (intraperitoneal injection) with 100 mg/kg (0.613 mM/kg) of N-acetyl-cysteine was required (24). In our study, the effective in vivo pretreatment dose of C60(OH)7±2 was 10 mg/kg (0.01145 mM/kg). In the in vitro study, the effective concentration (to prevent ERK activation) of N-acetyl-cysteine was 3 mM (1) compared with our 50–100 µM (low doses) of C60(OH)7±2 in this study (Figs. 1–3).

In summary, we found that low concentrations of C60(OH)7±2 prevented SNP- and H2O2-induced cell damage, a decrease in mitochondrial membrane potential, and increases in Pa and Pc. However, high concentrations of C60(OH)7±2 inhibited mitochondrial membrane potential. Our results suggest that low concentrations of the antioxidant fullerene derivative C60(OH)7±2 have beneficial effects and attenuate IR-induced lung injury. Due to its antioxidant characteristics, the fullerene derivatives might be used to prevent ROS-induced pulmonary diseases such as sepsis-related lung injury, respiratory distress syndrome, fibrosis, emphysema, and asthma. More studies are needed to demonstrate its potential benefits in humans, however.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This investigation was supported by the National Science Council (NSC89–2320-B002–078).


   ACKNOWLEDGMENTS
 
We thank C.-F. Huang for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-L. Lai, Dept. of physiology, College of Medicine, National Taiwan Univ., No. 1, Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan (E-mail: tiger{at}ha.mc.ntu.edu.tw).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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