Glycyrrhetinic acid-induced apoptosis in thymocytes: impact of 11beta -hydroxysteroid dehydrogenase inhibition

Hiroshi Horigome, Atsushi Horigome, Masato Homma, Toshihiko Hirano, and Kitaro Oka

Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has been proposed that glycyrrhetinic acid (GA) enhances endogenous glucocorticoid (GC) action by suppressing the metabolism of the steroid. We show here that marked involution of the thymus occurred within 24 h of a single intraperitoneal administration of GA in mice. Thymocytes from mice treated with GA exhibited DNA cleavage and mitochondrial transmembrane potential disruption, as demonstrated with agarose gel electrophoresis and flow cytometric analysis. Immunocytochemical staining revealed that CD4+CD8+ double positive cells markedly decreased after GA treatment. In contrast to GA in vivo, GA in vitro did not induce apoptosis of cultured thymocytes. These findings suggest that the apoptosis-inducing effect of GA on thymocytes is due to its indirect action. Because GA has been known to inhibit 11beta -hydroxysteroid dehydrogenase (11beta -HSD), we measured the enzyme activity in major organs and endogenous corticosterone concentration after GA treatment. The results showed a significant decrease of 11beta -HSD activity (P < 0.0001) and an increase in serum corticosterone concentration (P < 0.005). We concluded that the inhibition of hepatic 11beta -HSD activity by GA has a serious effect on GC metabolism, which results in a significant elevation of systemic GC levels. Apoptosis of thymocytes occurred as a consequence of the elevation in the level of endogenous corticosterone.

11beta -hydroxysteroid dehydrogenase inhibitor; corticosterone; glucocorticoid metabolism


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NATURAL AND SYNTHETIC glucocorticoids (GCs) are widely used to treat patients with a variety of pathological conditions. In childhood acute leukemia, the beneficial effect of GCs is known to be based on their cytocidal potency on lymphoid cells (26, 29). The cell death resulted from the GC treatment that was exhibited by the feature of apoptosis, such as cell shrinkage, nuclear condensation, and DNA fragmentation (35). GCs induce apoptosis in immature thymocytes (2, 36), bone marrow pre-B cells (21), and, under certain conditions, in peripheral mature lymphocytes both in vitro and in vivo (14, 24, 25, 37). Thus GC-induced apoptosis in specific cellular systems including immune cells might be involved in the regulation of the immune system (3, 4) and the therapeutic efficacy of GC (1).

11beta -Hydroxysteroid dehydrogenase (11beta -HSD), which promotes interconversion between biologically active 11beta -hydroxy GCs and inactive 11-oxo GCs, plays a critical role in the homeostasis of GC metabolism by regulating active GC concentrations in specific cells and tissues (31). For instance, 11beta -HSD is expressed at high levels in mineralocorticoid target tissues (32), and the enzyme protects the nonselective mineralocorticoid receptor from GC excess (33). This loss of function of 11beta -HSD causes apparent mineralocorticoid excess (8, 27), showing a hypertensive syndrome with a low level of mineralocorticoid. In this disorder, it has been proposed that an intrarenal concentration of corticosterone would be abnormally high as a consequence of its deficient metabolism by 11beta -HSD and saturation of mineralocorticoid receptors by GC (34). Hennebold et al. (10) have shown that 11beta -HSD plays a functional role in specific lymphoid organs regulating immunologic activities via regulation of GC concentrations.

Glycyrrhetinic acid (GA; Fig.1), a saponin isolated from licorice root (Glycyrrhizae radix), has been known to inhibit 11beta -HSD activity (17, 20, 22). However, action mechanisms for clinical effects of GA on peptic ulcers and chronic viral hepatitis (6) have not been elucidated yet. It was reported in early literature that GA enhances endogenous GC action by suppressing the metabolism of GCs (18), and a recent report showed that GA administration to normal mice inhibits 11beta -HSD activities in immune tissues such as the thymus, spleen, and peripheral lymph nodes in a dose-dependent manner (10). To our knowledge, however, there has been no report that demonstrates GC-induced apoptosis of immune cells via intervention of GA in GC metabolism. Our interest has been focused on a hypothesis that GA induces thymocyte apoptosis via the elevation of endogenous corticosterone concentration in mice.


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Fig. 1.   Chemical structure of glycyrrhetinic acid (GA).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice and reagents. Male C57BL/6 mice (4 wk of age) were obtained from Charles River (Tokyo, Japan). Animals were housed in a room apart from other colonies and were maintained with food and water available at liberty. GA (1.0-5.0 mg) and corticosterone (4.0 mg), both of which were purchased from Sigma (St. Louis, MO), were administered in vivo as a fine suspension in olive oil (100 µl/animal) by a single intraperitoneal injection. Control animals received vehicle (olive oil; Wako Biochemical, Osaka, Japan) alone. The mice were killed by ether anesthesia.

Thymocyte culture. The mice thymuses were minced, gently pressed to release thymocytes in ice-cold PBS, and passed through a nylon mesh (35 µm) and washed. The cells were cultured according to a modified method of another report (5). The thymocytes were briefly suspended in ice-cold RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml of penicillin, and 100 µg/ml of streptomycin. Viable cells were then counted with a trypan blue dye exclusion test and were adjusted to 1 × 106 cells/ml for culture. The thymocytes were then placed in 24-well flat-bottom plates (Iwaki Glass, Tokyo, Japan) and incubated with 1 × 10-11 to 1 × 10-4 M of GA for 6 h in a 5% CO2-95% air atmosphere at 37°C. The GA was dissolved in ethanol, and a final ethanol concentration in culture was adjusted at 1%. Ethanol was also used in the control wells.

Agarose gel electrophoresis of fragmented DNA. The extraction of thymocyte DNA and gel electrophoresis were performed by the techniques similar to previous reports (13). In brief, 1 × 106 cells were solubilized with 100 µl of lysis buffer (10 mM Tris-hydrochloride buffer, pH 8.0, containing 0.1 mM EDTA and 150 mM sodium chloride) and placed on ice for 10 min. The sample was centrifuged at 20,000 g for 20 min. The supernatant was placed in a 1.5-ml microtube and was treated with 2 µl of 10 mg/ml RNase A at 37°C for 30 min, followed by treatment with 2 µl of 50 mg/ml proteinase K at 37°C for 60 min. The sample was then extracted with an equal volume of phenol-chloroform with DNA isolating-gel (Iwaki Glass) and was precipitated by an addition of 10× vol of 3 M sodium acetate and equal volume of ethanol at -20°C. After the evaporation of the ethanol, the DNA was resuspended in a TE buffer (10 mM Tris-hydrochloride, pH 8.0, containing 1 mM EDTA). The DNA samples (10 µl/lane) were electrophoretically separated on 2.0% agarose gels containing 0.5 µg/ml ethidium bromide (Sigma) with a Mupid-2 (Advance, Tokyo, Japan). The DNA was then visualized with an ultraviolet transilluminater.

Flow cytometric analysis. Detection of apoptotic cells and phenotype analysis were performed in the following two ways by flow cytometry. 1) Cell cycle analysis: cells were fixed for more than 30 min at -20°C in 70% ethanol at a density of 2 × 106 cells/ml. After being washed with PBS, 100 µl of cell suspension were treated with 2 µl of 10 mg/ml RNase A in PBS at 37°C for 30 min. For an assessment of DNA fragmentation, the cells were then stained with 0.2 µg/ml of propidium iodide in PBS for 30 min. The percentage of apoptotic cells corresponding to the amount of fragmented DNA in the hypoploid sub-G0/G1 cell cycle peak was calculated. 2) Detection of mitochondria-specific dye and T cell subtype: 1,000,000 cells were stained with chloromethyl-X-rosamine (CMXRos; Molecular Probes, Eugene, OR) at 37°C for 30 min. CMXRos (1 mM) was prepared as a stock solution in dimethylsulfoxide and stocked at -20°C. The stock solution was then diluted in a 100-nM working solution with PBS. After being stained with CMXRos, the cells were washed in an ice-cold PBS supplemented with 2% heat-inactivated FBS and incubated for 20 min at 4°C with a phycoerythrin-conjugated anti-CD4 monoclonal antibody (0.1 ng/106 cells in 100 µl; PharMingen, San Diego, CA) and with FITC-conjugated anti-CD8 monoclonal antibody (0.1 ng/106 cells in 100 µl; PharMingen).

After staining, a total of 20,000 nongated cells were analyzed with a FACSCalibur analyzer, and obtained histograms or dot plot data were calculated by CellQuest software (Becton Dickinson, Mountain View, CA).

Determination of 11beta -HSD activity in tissue homogenate. Each 20 mg of tissues (liver and kidney), thymocytes, and thymic stromal cells taken from mice killed 24 h after administration of GA were placed in a 1.5-ml microtube poured with 400 µl of ice-cold 0.25 M sucrose. The tissues were homogenized by sonication. The concentration of protein in the homogenate was determined by Lowry's method. 11beta -HSD activities were determined with a method previously described (11). Next, enzyme assay tubes containing 840 µl of 0.1 M Tris-hydrochloride buffer including 0.01% Triton-X (pH 8.5), 50 µl of 5 mM NADP+ (for liver) or NAD+ (for kidney) (Sigma), and 200 µl of 0.3 mM cortisol as a substrate were incubated at 37°C. The incubation time for assaying both the liver and kidney homogenates was 30 min and for assaying the homogenate fraction thymocytes and thymic stromal cells was 15 h. The enzyme reaction was terminated by the addition of 100 µl of 5% sulfuric acid. The tubes were voltexed vigorously and placed on ice for HPLC analysis.

Concentrations of cortisol and its 11-oxo metabolite, cortisone, in the incubation mixtures were used to calculate the enzymatic activity of 11beta -HSD. This was determined by HPLC with a syringe-type extraction-injection device, Extrashot (Kusano Sci., Tokyo, Japan), as described previously (11). Briefly, 5 µl of incubation mixture were loaded onto Extrashot, which was then attached to the sample-loading injector of the HPLC system. Subsequently, 130 µl of dichloromethane were injected into the system through Extrashot. The mobile phase was H2O-methanol-dichloromethane-n-hexane (0.1:8.0:30.0:61.9, vol/vol) at a flow rate of 1.5 ml/min. The ratio of the levels of cortisol and its metabolite cortisone, i.e., "cortisone concentration to cortisol concentration," was determined, and the specific 11beta -HSD activity in tissue was calculated as picomoles of cortisone generated per minute per micrograms of protein.

The HPLC system used in this assay procedure consisted of a solvent delivery pump (TWINCLE, Jasco, Tokyo, Japan), a syringe-loading sample injector (Model-7125, Rheodyne, Cotati, CA), a silica-gel column (LiChrosorb Si-60, Merck, Darmstadt, Germany), an ultraviolet detector (UVIDEC-100-III, Jasco), and a single pen recorder (Pantos U-228, Nippon Denshi, Tokyo, Japan). The wavelength of the detector was set at 245 nm, and the sensitivities were 0.005-0.01 AUFS.

Quantitative analysis of serum corticosterone. Extraction of serum corticosterone with diatomaceous earth column extraction, namely rapid flow fractionation, was performed by a procedure described in a previous report (12). Briefly, the serum samples were spiked with 50 ng of prednisone as an internal standard was introduced into the rapid flow fractionation columns. The column system was treated with 7 ml of dichloromethane. The effluent was collected in a glass tube and left to evaporate to dryness. The residue was reconstituted with 15 µl of dichloromethane for injection onto the same HPLC system as described in Determination of 11beta -HSD activity in tissue homogenate. The HPLC was conducted with the mobile phase solvent of H2O- methanol-dichloromethane-n-hexane (0.1:4.0:30.0:61.9, vol/vol).

Statistical analyses. Values are expressed as means ± SD and compared by multiple comparison (Dunnett) or unpaired Student's t-test. Calculated P values of <0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GA effects on thymus weight. There was no significant difference in the body weight between mice injected with a single dose of 2.5 mg GA and the vehicle-treated control mice (Table 1). Although a slight decrease in the weight was observed in the 24 h after treatment in both of the groups, there was no significant difference between them (Table 1). The mice injected with 2.5 mg of GA showed a significant decrease in thymus weight 24 h after the administration compared with the control mice given the vehicle alone (P < 0.002; Table 1).

                              
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Table 1.   Effect of GA administration on mouse body weight and thymus weight

GA induces thymocyte apoptosis in vivo. Apoptotic thymocytes from mice treated by GA were examined with flow cytometry. An increase in the percentages of apoptotic cells was seen in a dose-dependent manner as shown in Fig. 2. After 24 h of GA treatment, the number of apoptotic cells significantly increased in the mice treated with 2.5 mg GA compared with the control mice (Fig. 3; P < 0.001). The mean percentage of apoptotic cells in the GA-treated group was 36.3 ± 11.8%, whereas that in the corresponding control was 1.7 ± 0.5%. Apoptotic cells showed no signs of increase at 6 h after administration of GA (Fig. 3). The thymocytes from the mice treated with corticosterone showed significantly higher percentages of apoptotic cells after 6 h of administration compared with the control mice (P < 0.001), whereas apoptotic cells had barely been detected after 24 h of corticosterone treatment (Fig. 3).


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Fig. 2.   Dose-dependent induction of thymocyte apoptosis by GA administration. Significant differences were observed at doses 2.0, 2.5, and 5.0 mg vs. control (dose: 0 mg). * P < 0.01 by Dunnett's test.



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Fig. 3.   Detection of apoptotic thymocytes in GA- or corticosterone-treated mice. Thymocytes were dissected from animals 6 (A) or 24 h (B) after administration of each agent. Thymocytes were then stained with propidium iodide after ethanol fixation and then analyzed with flow cytometry. Values are means ± SD (%) of propidium iodide- negative cells (n = 3). * P < 0.001 vs. control given vehicle alone.

DNA fragmentation in the thymocytes that were prepared from the mice treated with the vehicle, GA, or corticosterone was analyzed with agarose gel electrophoresis. Intraperitoneal administration of 2.5 mg GA resulted in an apparent fragmentation of thymocyte DNA compared with that of the control 24 h after the treatment (Fig. 4). However, thymocytes from mice treated with GA for 6 h did not show any signs of apoptosis (data not shown). The corticosterone-treated mice also showed significant amounts of DNA fragments after the 6 h of administration, whereas after the 24 h of administration the fragmentation had scarcely been detected (data not shown).


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Fig. 4.   Agarose gel electrophoresis of thymocyte DNA from GA- or vehicle-treated mice. Lane 1, DNA size marker; lane 2, vehicle-treated control 24 h after administration; lane 3, GA (2.5 mg)-treated mouse 24 h after administration; lane 4, vehicle-treated control 6 h after administration; lane 5, corticosterone (4 mg)-treated mouse 6 h after administration.

The mitochondria transmembrane potential also decreased significantly 24 h after the GA treatment (Fig. 5). Staining for CD4 and CD8 revealed that there was a marked reduction in the number of CD4+CD8+ double positive cells by GA treatment, whereas the number of CD4+CD8- or CD4-CD8+ single positive cells and double negative cells remained unchanged (Table 2).


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Fig. 5.   Typical histogram of thymocytes exhibiting mitochondrial parameter of apoptosis induced by GA. Thymocytes were stained with chloromethyl-X-rosamine (CMXRos) and analyzed with a flow cytometer. A: thymocytes from vehicle-treated mouse. B: thymocytes from GA-treated mouse. Data are representative of 3 experiments.


                              
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Table 2.   Effect of GA on phenotypic characterization of thymocytes

GA failed to induce thymocyte death in vitro. Thymocytes prepared from agent-untreated mice were cultured in the presence of serial concentrations of GA in vitro. After 6 h of incubation with 10-11 to 10-4 M of GA, the thymocytes did not show any signs of apoptosis as analyzed with flow cytometry (Fig. 6A). The GA did not induce apoptosis in the thymocytes even after 24 h (data not shown). Thymocyte apoptosis by corticosterone was induced dose dependently in vitro (Fig.6B).


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Fig. 6.   Effect of GA (A) or corticosterone (GC, B) on apoptotic cell ratio of murine thymocytes in vitro. Values are means ± SD of apoptotic cell population (n = 6).

Inhibition of hepatic 11beta -HSD activity and rise in serum corticosterone concentration in GA-treated mice. Finally, we examined dehydrogenation activity of 11beta -HSD in tissues and serum corticosterone concentration in mice treated with 2.5 mg of GA. Table 3 shows the inhibitory effect of GA on 11beta -HSD activity in liver, kidney, thymocytes, and thymic stromal cells. Maximal inhibition of activity by GA was obtained in all of these tissues 3 h after administration. At 24 h after the administration of GA, 11beta -HSD activities in thymocytes, thymic stromal cells, and kidney were recovered or relatively increased when compared with those of the control animals, whereas the activity in the liver was still suppressed significantly (P < 0.0001; Table 3). In the thymocytes and thymic stromal cells, 11beta -HSD activities significantly increased after the 24 h of administration (P < 0.02 compared with control). Renal 11beta -HSD activity at 24 h after the administration did not show significant differences when compared with the control mice. We also measured the serum concentration of corticosterone after the administration of GA. As shown in Table 3, the mean serum corticosterone concentration in the group treated with 2.5 mg of GA was 410 ± 143 ng/ml, which was significantly higher than that in the control group (138 ± 55 ng/ml; P < 0.005).

                              
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Table 3.   Effects of GA on circulating corticosterone levels and 11beta -HSD activity in thymocytes, thymic stromal cells, liver, and kidney


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

From the data described in RESULTS, it could be postulated that GA administration induced thymocyte apoptosis by elevating levels of endogenous corticosterone via inhibition of GC-metabolizing enzyme 11beta -HSD. These considerations were supported by the experimental findings that 1) thymocytes from GA-administered animals exhibited biochemical and morphological features of apoptosis, 2) GA administration markedly suppressed 11beta -HSD activity in the liver resulting in increased levels of circulating GC, 3) administration of GC also induced thymocyte apoptosis not only in vivo but also in vitro, 4) apoptosis induced by GC occurred faster than that induced by GA, and 5) GA had no effect on cultured thymocytes in vitro.

The induction of thymocyte apoptosis by GA was qualitatively documented by agarose gel electrophoresis of fragmented DNA extracted from thymocytes of GA-treated mice. The GA administration resulted in cleavage of cellular DNA to make a ladder structure of DNA fragments accompanied with thymus involution. To confirm the features of cell death, the thymocytes were also examined with flow cytometry. Apoptotic cells containing a relatively small amount of DNA were dramatically increased after 24 h of GA administration. Moreover, the apoptotic features of cells were accompanied by a disruption of inner mitochondrial transmembrane potential (19). We observed here that the mitochondrial transmembrane potential was disrupted in thymocytes from GA-treated mice. These results suggest that the involution of the thymus by GA occurred as a consequence of the death of the thymocytes. Phenotypic analysis of the thymocytes stained by specific antibodies revealed that CD4+CD8+ double positive cells were the main target of the GA action. It has been reported that in vivo treatment of animals with GC or anti-Fas antibody causes massive apoptosis in CD4+CD8+ thymocytes (23, 30), which coincides with our present observation of GA action. We also investigated whether GA induces thymocyte apoptosis in vitro and concluded that the apoptosis-inducing effect of GA was not detected in vitro and did not come from the direct effect of GA on the thymocytes.

In the present study, induction of apoptosis with intraperitoneal administration of corticosterone was observed 6 h after the treatment, and its effect disappeared after 24 h. These observations were consistent with those of Ishii et al. (15) who reported that the number of apoptotic cells was maximal after 8 h and disappeared after 18 h of GC administration. In contrast, we did not detect thymocyte apoptosis 6 h after administration of GA but found a marked increase in apoptotic thymocytes 24 h after GA administration. Thus, although GA induced DNA fragmentation in the thymocytes in vivo, the time required was longer than that of the corticosterone. This time lag of GA action to induce thymocyte apoptosis may also support the hypothesis that GA action involved two steps, i.e., 11beta -HSD inhibition and an elevation in circulating corticosterone. GC has been reported to induce thymocyte apoptosis (4, 5), and our data of Fig. 6B coincide with these observations. It was also reported that elevated levels of corticosterone induce apoptosis in thymocytes, and this effect of corticosterone could be abolished by the GC-receptor antagonist RU-38486 (9, 28).

Dougherty et al. (7) reported that long-term GC treatment caused a significant increase in the activity of 11beta -HSD in murine thymus. In our experiment, the activities of 11beta -HSD in thymocytes and thymic stromal cells decreased significantly and subsequently increased within 24 h after a single administration of GA. The early decrease of activity was well correlated with the subsequent elevation of the corticosterone concentrations. The surviving thymocytes at 24 h after GA treatment were thought to have escaped from apoptosis in the circumstance of the elevated corticosterone. Other evidence was that there was a relatively high amount of activity of 11beta -HSD, which inactivated the cytocidal corticosterone. A similar observation has been reported by Jellink et al. (16) in the case of long-term administration of corticosterone resulting in induction of 11beta -HSD accompanied by cell survival. Thus the elevation of the specific 11beta -HSD activity in thymus after GA administration is understandable as a result of survival of CD4+CD8- or CD4-CD8+ single positive thymocytes, which may have relatively high activity of 11beta -HSD.

11beta -HSD should play an important role in the steroid conversion in thymus. We demonstrated here that administration of GA intraperitoneally resulted in a significant increase in serum corticosterone concentrations. Serum concentration of corticosterone increased up to 410 ± 143 ng/ml after 24-h treatment by GA. We also found here that 11beta -HSD activity concomitantly decreased in a dose-dependent manner in liver by GA and that this inhibition persisted even after 24 h. On the other hand, renal 11beta -HSD activity was not inhibited by GA administration in the present study. Therefore, inhibition of hepatic 11beta -HSD activity might have a serious effect on GC metabolism, which results in significant elevation of GC systemic levels.

In conclusion, GA-induced thymocyte apoptosis might be due, at least in part, to inhibition of liver 11beta -HSD and subsequent increase in the level of serum corticosterone, which results in the initiation of apoptosis of thymocytes. Thus the data suggest that pharmacological or physiological interruption of GC metabolism influences the immune system via alteration of the thymus cell population.


    ACKNOWLEDGEMENTS

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Japan (Grant no. 09672337) and Tsumura Company.


    FOOTNOTES

We also wish to thank T. Kido, T. Keira, S. Horie, and, especially, S. Uda for experimental and technical assistance.

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. §1734 solely to indicate this fact.

Address for reprint requests and correspondence: T. Hirano, Dept. of Clinical Pharmacology, School of Pharmacy, Tokyo Univ. of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan (E-mail: hiranot{at}ps.toyaku.ac.jp).

Received 28 December 1998; accepted in final form 21 May 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 277(4):E624-E630
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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