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(Hypertension. 1999;33:1179-1184.)
© 1999 American Heart Association, Inc.

Scientific Contributions

11ß-Hydroxysteroid Dehydrogenase in Cultured Human Vascular Cells

Possible Role in the Development of Hypertension

Haruhiko Hatakeyama; Satoru Inaba; Isamu Miyamori

From the Third Department of Internal Medicine, Fukui Medical University (Japan).

Correspondence to Haruhiko Hatakeyama, MD, Third Department of Internal Medicine, Fukui Medical University, 23-1 Matsuoka-cho, Fukui 910-1141, Japan.

	Abstract

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Abstract—11ß-Hydroxysteroid dehydrogenases (11ß-HSD) interconvert cortisol, the physiological glucocorticoid, and its inactive metabolite cortisone in humans. The diminished dehydrogenase activity (cortisol to cortisone) has been demonstrated in patients with essential hypertension and in resistance vessels of genetically hypertensive rats. 11ß-Hydroxysteroid dehydrogenase type 2 (11ß-HSD2) catalyzes only 11ß-dehydrogenation. However, a functional relationship between diminished vascular 11ß-HSD2 activity and elevated blood pressure has been unclear. In this study we showed the expression and enzyme activity of 11ß-HSD2 and 11ß-HSD type 1 (which is mainly oxoreductase, converting cortisone to cortisol) in human vascular smooth muscle cells. Glucocorticoids and mineralocorticoids increase vascular tone by upregulating the receptors of pressor hormones such as angiotensin II. We found that physiological concentrations of cortisol-induced increase in angiotensin II binding were significantly enhanced by the inhibition of 11ß-HSD2 activity with an antisense DNA complementary to 11ß-HSD2 mRNA, and the enhancement was partially but significantly abolished by a selective aldosterone receptor antagonist. This may indicate that impaired 11ß-HSD2 activity in vascular wall results in increased vascular tone by the contribution of cortisol, which acts as a mineralocorticoid. In congenital 11ß-HSD deficiency and after administration of 11ß-HSD inhibitors, suppression of 11ß-HSD2 activity in the kidney has been believed to cause renal mineralocorticoid excess, resulting in sodium retention and hypertension. In the present study we provide evidence for a mechanism that could link impaired vascular 11ß-HSD2 activity, increased vascular tone, and elevated blood pressure without invoking renal sodium retention.

Key Words: 11ß-hydroxysteroid dehydrogenase • receptors, angiotensin II • cortisol • vascular tone • hypertension, essential

	Introduction

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Vascular wall is a target tissue for glucocorticoids and mineralocorticoids. These steroids are essential for the maintenance of vascular tone.¹ They can potentiate the vasoconstrictor action of a number of pressor hormones, including -adrenergic agonists and angiotensin II (Ang II).² This potentiation is postulated to be mediated by the upregulation of receptors for these pressor hormones in vascular smooth muscle cells.³ We have previously demonstrated that vascular cells per se are steroidogenic,^{4 5 6 7 8} and the locally produced aldosterone might participate in Ang II–induced vascular hypertrophy in an autocrine/intracrine manner through type 1 mineralocorticoid receptor (MR).^{4 8}

The MR has an equal affinity for cortisol and aldosterone, despite the fact that the circulating cortisol levels are much higher than those of aldosterone.⁹ It has been proposed that the abundance of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) in the kidney, which metabolizes cortisol into cortisone with very low affinity for the MR, explains how the kidney can be a mineralocorticoid target tissue.^{10 11} A defect of 11ß-HSD activity would thus allow the MR to be occupied mostly by cortisol. In humans, two 11ß-HSD isozymes have been described and cloned. The first enzyme (11ß-HSD1) catalyzes both 11ß-dehydrogenation and the reverse oxoreduction and is a low-affinity NADPH enzyme.¹² The enzyme has been detected in a wide range of rat and human tissues including liver, lung, and testis. A second isozyme (11ß-HSD2) is present in the kidney and placenta. It is a high-affinity NAD-dependent enzyme and catalyzes only 11ß-dehydrogenation.¹³ It has been believed that in congenital 11ß-HSD deficiency (apparent mineralocorticoid excess syndrome) and after administration of 11ß-HSD inhibitors (licorice and carbenoxolone), the renal MR can be occupied mostly by cortisol, causing sodium retention and hypertension.^{14 15} Recently, it has been postulated that 11ß-HSD1 does not play a significant role in conferring ligand specificity on the MR.¹² Indeed, several mutations in the 11ß-HSD2 gene have been identified in patients with this syndrome, but none in the 11ß-HSD1 gene.¹⁶

Since local glucocorticoids (mineralocorticoids as well) within vascular wall could directly affect vascular tone, the local metabolism of glucocorticoids mediated by 11ß-HSD may be important in controlling blood pressure. Soro et al¹⁷ reported that the ratio of cortisol to cortisone metabolites in the urine was significantly higher in patients with essential hypertension. We demonstrated elevated levels of 11ß-HSD inhibitory substances in the urine of patients with low-renin essential hypertension.¹⁸ Furthermore, we reported decreased dehydrogenase activity of 11ß-HSD1 in resistance vessels of genetically hypertensive rats.^{19 20} However, a functional relationship between diminished vascular 11ß-HSD2 activity and elevated blood pressure has been unclear.

The purpose of this study was to clarify the physiological and pathophysiological significance of 11ß-HSD2 activity in human resistance vessels. We investigated the activity and gene expression of the enzyme in human coronary artery smooth muscle cells (HCASMC). Furthermore, to assess its potential role as a modulator of vascular tone, a functional relationship between the vascular 11ß-HSD2 activity and the effect of physiological concentrations of cortisol on Ang II receptor regulation was also tested by manipulating 11ß-HSD2 gene expression with an antisense DNA.

	Methods

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Materials
HCASMC (Clonetics Corp) were cultured according to the supplier's instruction. ¹Sar, [¹²⁵I]⁴Tyr, ⁸Ile-Ang II was purchased from NEN-DuPont. [1,2,6,7-³H]cortisol was from Amersham International plc. Cortisol, cortisone, spironolactone, and RU38486 were from Sigma. Ang II was from Peptide Institute. DuP 753 and PD123319 were from Yamanouchi Pharmaceutical Co.

Cell Culture
HCASMC were cultured to confluence in Dulbecco's modified Eagle's medium with 5% FCS plus 4 µg/mL of gentamicin under 5% CO₂/95% air at 37°C. Cells at 5 to 8 passages were used for the experiments; >95% of the cells were identified as smooth muscle cells by their typical "hill-and-valley" morphology and by immunofluorescence with the use of a monoclonal antibody against human -smooth muscle actin.

Detection of 11ß-HSD mRNA
Oligonucleotide primers for reverse transcription–polymerase chain reaction (RT-PCR) were synthesized with an Applied Biosystems model 392 DNA synthesizer and purified with an oligonucleotide purification column. The sequences of sense and antisense primers were 5'-CTCGAGTCGGATGGCTTTTTATG-3' and 5'-ACTTGCTTGCAGAATAGG-3' for detecting 11ß-HSD1 mRNA.^{12 13} The sequences of sense and antisense primers were 5'-ACCGTATTGGAGTTGAACAGC-3' and 5'-TCACTGACTCTGTCTTGAAGC-3' for detecting 11ß-HSD2 mRNA.^{13 21} RT-PCR experiments were also conducted to amplify the ubiquitously expressed 1 subunit of human Na,K-ATPase using the sense (5'-ATATGGAACAGACTTGAGCCG-3') and antisense (5'-GGCAATTCTTCCCATCACAGT-3') primers.²² RT-PCR was performed as described previously.⁴ A 10-µL aliquot of each RT-PCR reaction mixture was electrophoresed on a 2% agarose gel. The gel was stained with ethidium bromide and photographed.

Assay of 11ß-HSD Activity
The apparent K_m values for the dehydrogenase reaction and the reductase reaction in vascular smooth muscle cells are 100 and 300 nmol/L, respectively.²³ 11ß-HSD activities were measured by a radiometric conversion assay, as previously described.²⁴ In brief, confluent HCASMC were incubated in a hydrocortisone-free and serum-free medium containing 100 nmol/L [1,2,6,7-³H]cortisol or [1,2,6,7-³H]cortisone for 8 hours, after which steroids were extracted with chloroform and were resolved by thin-layer chromatography. Radioactivities corresponding to cortisol and cortisone were determined. Dehydrogenase and reductase activities were calculated as counts per minute for cortisone/(counts per minute for cortisol+counts per minute for cortisone)x100 and as counts per minute for cortisol/(counts per minute for cortisol+counts per minute for cortisone)x100, respectively. [1,2,6,7-³H]cortisone was prepared from [1,2,6,7-³H]cortisol as described previously.²⁵ Briefly, the labeled cortisol (10 µCi) was incubated in 1 mL of 50% aqueous acetic acid containing 1% chromium trioxide at room temperature for 20 minutes. The residue from the dichloromethane extract of the reaction products was chromatographed by thin-layer chromatography with the use of chloroform-methanol (9:1) as solvent and nonradioactive cortisol and cortisone as reference markers. The cortisone-containing resin was scraped off and eluted with ethyl acetate.

Ang II Binding
Confluent HCASMC were washed 3 times with saline and incubated with ¹Sar, [¹²⁵I]⁴Tyr, ⁸Ile-Ang II for 60 minutes at room temperature. Assay buffer consisted of 50 mmol/L Tris (pH 7.4), 100 mmol/L NaCl, 5 mmol/L MgCl₂, 0.25% BSA, and 0.5 mg/mL bacitracin. At the end of incubation, the cells were washed with saline 4 times, solubilized in 1% sodium dodecyl sulfate, and counted with a -counter. Saturation binding assays were performed with increasing concentrations of [¹²⁵I]Ang II (50 to 700 pmol/L) in the presence (nonspecific binding) or absence (total binding) of 1 µmol/L unlabeled Ang II and processed as explained above. Competition binding assays were performed with 200 pmol/L of [¹²⁵I]Ang II in the presence of increasing concentrations of unlabeled Ang II, nonpeptide Ang II type 1 receptor antagonist DuP 753, and type 2 receptor antagonist PD123319.

Antisense Oligonucleotides
A 24-mer phosphorothioate antisense oligonucleotide (AS) complement of the 5' region of human 11ß-HSD2 mRNA²⁶ containing the initiator AUG codon and, as a control, a nonsense oligonucleotide (NS) containing the same base composition but in a random, scrambled order were synthesized with an Applied Biosystems model 392 DNA synthesizer. Sequences of AS and NS were 5'-CGACGGCCAAGGCCAGCGCTCCAT-3' and 5'-TCACGCACGCGCCAACCGCGGAGT-3', respectively.

Statistical Analysis
Data are expressed as mean±SEM. The significance of differences was assessed by 1-way ANOVA and multiple comparison test. Values of P<0.05 were accepted as statistically significant.

	Results

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11ß-HSD Expression in HCASMC
We examined the expression of 11ß-HSD1 and 11ß-HSD2 genes in HCASMC. With the use of the RT-PCR method, amplified products corresponding to transcripts of both genes were detected (Figure 1). Cloning and sequence analysis of the PCR products demonstrated that both bands had the known sequences of the human 11ß-HSD1 and 11ß-HSD2 mRNA, respectively (data not shown). Although the amount of mRNA of 11ß-HSD2 seemed lower than that of 11ß-HSD1, more exact quantification of the comparative levels of 11ß-HSD1 and 11ß-HSD2 was not attempted because of possible differences in amplification efficiency with the various sets of primers.

View larger version (18K): [in this window] [in a new window]   Figure 1. 11ß-HSD expression in HCASMC. One hundred nanograms of poly (A)+ RNAs from the cells were amplified by RT-PCR as described in Methods. cDNA quantity was monitored by amplifying 1 subunit of human Na,K-ATPase mRNA (data not shown).

11ß-HSD Activity in HCASMC
To assess 11ß-HSD activities, labeled cortisol or its metabolite cortisone was added to each medium. Incubation with cortisol resulted in moderate (18±2%) conversion to cortisone after 8 hours, whereas incubation with cortisone resulted in more conversion (42±5%) to cortisol (Figure 2). Thus, HCASMC were capable of performing the dehydrogenase as well as the reverse oxoreductase phase of the reaction, and under physiological conditions these cells favored the latter phase.

View larger version (21K): [in this window] [in a new window]   Figure 2. 11ß-HSD activity in HCASMC. Confluent HCASMC in 6-well plates were incubated with 100 nmol/L [3H]cortisol or [3H]cortisone for 8 hours. Dehydrogenase and oxoreductase activities were calculated as described in Methods. Open columns represent data from experimental blanks. Data were analyzed by 1-way ANOVA and multiple comparison test (n=6).

Ang II Receptor in HCASMC
Before we investigated the cortisol effect on Ang II binding, HCASMC were tested for their ability to bind to ¹Sar, [¹²⁵I]⁴Tyr, ⁸Ile-Ang II. A saturation binding study demonstrated the specific binding of [¹²⁵I]Ang II at the concentration of 50 to 700 pmol/L in HCASMC. Scatchard analysis of the binding data revealed a single class of high-affinity (0.31±0.06 nmol/L) and low-capacity (10.8±0.5 fmol/mg protein, 468±21 sites per cell) binding sites (Figure 3A). A competition binding study showed that radioligand binding was potentially inhibited by unlabeled Ang II and the Ang II type 1 receptor selective antagonist DuP 753; however, PD123319, an Ang II type 2 receptor selective antagonist, had no effect on the binding of [¹²⁵I]Ang II at doses as high as 1 µmol/L (Figure 3B). This indicated that HCASMC exhibited high-affinity Ang II type 1 receptor, the predominant Ang II receptor subtype in vascular smooth muscle cells.²⁷

View larger version (20K): [in this window] [in a new window]   Figure 3. Binding of 1Sar, [125I]4Tyr, 8Ile-Ang II to control HCASMC. A, Saturation binding of the [125I]Ang II to control HCASMC. Confluent cells in 6-well plates were incubated with increasing concentrations (50 to 700 pmol/L) of [125I]Ang II in the absence (total binding) or presence (nonspecific binding) of 1 µmol/L unlabeled Ang II. Specific binding () was obtained by subtracting nonspecific binding () from total binding (). Inset shows a Scatchard analysis of the saturation binding. B, Inhibition of the [125I]Ang II binding to control HCASMC. Confluent cells in 6-well plates were incubated with 200 pmol/L [125I]Ang II in the presence of increasing concentrations of unlabeled Ang II (), DuP 753 (), and PD123319 (). Each point shows the mean of triplicate assays. Similar results were obtained from 2 different experiments.

Effect of Cortisol on Ang II Binding
Next we examined the effect of cortisol on Ang II binding in these cells. Incubation of HCASMC for 24 hours with cortisol resulted in concentration-dependent increases in Ang II binding, with a maximal increase (98±10%) at 1 µmol/L cortisol (Figure 4A). Lower concentrations of cortisol (1 to 10 pmol/L) had no effect on Ang II binding (data not shown). The competition binding data and the Scatchard analysis of the binding data from control cells and cells treated for 24 hours with cortisol demonstrated that Ang II receptors upregulated by cortisol (as well as basal Ang II receptors) were of the type 1 receptor, and the affinity was not significantly changed (data not shown). Upregulation of Ang II binding was completely inhibited by RU38486, a specific antagonist for glucocorticoid receptors (GR), but not by spironolactone, a selective antagonist for MR, indicating that the regulation was mediated through GR (Figure 4B). RU38486 or spironolactone alone did not alter Ang II binding (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 4. Effect of cortisol on Ang II binding in HCASMC. A, Confluent cells were incubated with increasing concentrations of cortisol in a serum-free medium for 24 hours, and then Ang II binding was performed. B, Confluent cells were incubated in the serum-free medium with 1 µmol/L cortisol in the presence of increasing concentrations of RU38486 or spironolactone for 24 hours, and then Ang II binding was performed. The values represent mean±SEM (n=6). *P<0.01 compared with control. **P<0.05, ***P<0.01 compared with cells cultured with 1 µmol/L cortisol alone. Incubation of ethanol (0.1%), the vehicle for cortisol, RU38486, and spironolactone did not alter the [125I]Ang II bindings.

Effect of 11ß-HSD2 Antisense Oligonucleotides
We next tested whether vascular 11ß-HSD2 activity could be functionally related to the cortisol effect on Ang II binding. For this, a 24-mer phosphorothioate AS and, as a control, an NS containing the same base composition but in a random, scrambled order were administered to the culture medium in which HCASMC were grown. No visible signs of toxicity were observed. As shown in Figure 5, incubation of HCASMC for 24 hours with the AS induced dose-dependent decreases in the dehydrogenase activity, with a maximal decrease (78±6%) at 10 µmol/L AS, but the oxoreductase activity was unaffected. The NS altered neither activity. Since the oxoreductase activity was unaffected, the present results indicate that the inhibitory effect of AS is due to a specific decrease of 11ß-HSD2 activity. The possibility of the effect on 11ß-HSD1 activity would be very unlikely.

View larger version (45K): [in this window] [in a new window]   Figure 5. Effect of AS on 11ß-HSD activities in HCASMC. After confluent HCASMC in 6-well plates had been incubated in the presence of the indicated concentrations of AS or NS in a 5% FCS medium for 24 hours, dehydrogenase (open columns) and oxoreductase (crosshatched columns) activities were determined as described in Methods. The values represent mean±SEM (n=6). *P<0.01 compared with cells cultured without oligonucleotides.

Effect of 11ß-HSD2 Activity on Ang II binding
After confluent HCASMC had been incubated with the AS for 24 hours, the cells were further incubated with 0.5 µmol/L cortisol for 24 hours. The AS induced dose-dependent increases in Ang II binding, with a maximal increase (48±5%) at 10 µmol/L AS (Figure 6A). The NS did not alter the Ang II bindings. We then investigated whether the upregulation was mediated through GR or MR. HCASMC were exposed to RU38486 or spironolactone and then assayed for Ang II binding under the presence of 0.5 µmol/L cortisol and 10 µmol/L AS (Figure 6B). The upregulated Ang II binding in the cells was significantly inhibited by the presence of spironolactone or RU38486. The effects of both spironolactone and RU38486 were dose dependent, with a maximal inhibition (24±3%) at 1 µmol/L spironolactone (72±6% inhibition with 1 µmol/L RU38486). Concomitant administration of both spironolactone and RU38486 completely inhibited the upregulation of Ang II binding. Mineralocorticoids are known to increase the Ang II receptor number through its action on MR.²⁸ Our results indicate that diminished vascular 11ß-HSD2 activity enhances the effect of cortisol, and the enhancement is mediated through both GR activation by cortisol as a glucocorticoid and MR activation by cortisol as a mineralocorticoid, suggesting that 11ß-HSD2 plays a significant role in conferring the ligand specificity on MR in HCASMC.

View larger version (32K): [in this window] [in a new window]   Figure 6. Effect of AS on Ang II binding in HCASMC. A, After confluent HCASMC had been incubated with the indicated concentrations of AS (black columns) or NS (crosshatched columns) in a 5% FCS medium for 24 hours, the cells were further incubated in a serum-free medium with 0.5 µmol/L cortisol for 24 hours, and then Ang II binding was performed. *P<0.05, **P<0.01 compared with control. B, After confluent HCASMC had been incubated with 10 µmol/L AS, the cells were further incubated in the serum-free medium with 0.5 µmol/L cortisol in the presence of the indicated concentrations of RU38486 (crosshatched columns) and/or spironolactone (black columns) for 24 hours, and then Ang II binding was performed. P<0.05, P<0.01 compared with cells cultured with 10 µmol/L AS and 0.5 µmol/L cortisol in the absence of the receptor antagonists. The values represent mean±SEM (n=6).

	Discussion

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We demonstrated for the first time the gene expression and the bidirectional activity of 11ß-HSD in vascular smooth muscle cells cultivated from a human resistance vessel. Previous studies indicated bidirectional activity, favoring the oxoreductase reaction 4-fold over the dehydrogenase reaction, in rat aortic vascular smooth muscle cells²³ and greater dehydrogenase reaction in rat resistance vessels than aorta.²⁹ Since dehydrogenase activity has been demonstrated to play a significant role in conferring the mineralocorticoid specificity on MR, the greater dehydrogenase activity in the present study may be related to the presence of much higher levels of MR in resistance vessels. Comparative levels of MR in various vessels are to be examined for further investigation. Glucocorticoids (and mineralocorticoids as well) increase vascular tone by potentiating the vasoconstrictor action of Ang II and -adrenergic catecholamines. We observed that HCASMC contain significant quantities of 11ß-HSD1 mRNA and enzyme activity. It has been suggested that the 11ß-HSD1 may function as a predominant 11ß-reductase, regenerating active glucocorticoids from circulating inactive 11-keto forms and modulating glucocorticoid access to GR.³⁰ An increase in available glucocorticoids could make vascular cells more responsive to circulatory vasoconstricting hormones. Thus, local glucocorticoid metabolism mediated by 11ß-HSD1 within vascular wall may be important in the control of vascular tone and may be relevant to the pathogenesis of hypertension. Furthermore, we demonstrated that diminished vascular 11ß-HSD2 activity enhances the effect of cortisol, partly by the activation of MR. However, because we administered high concentrations of cortisol into HCASMC, saturation or direct inhibition of this enzyme by excess cortisol might lead to the existence of unmetabolized cortisol acting as a mineralocorticoid.

We also demonstrated the first information on Ang II binding sites in HCASMC. This receptor is of the type 1 Ang II receptor subtype. Ang II is the active component of the renin-angiotensin system and has been demonstrated to play important roles in the hypertrophic response of the vessel wall during hypertension, as well as in the hyperplastic response that accounts for restenosis after balloon angioplasty³¹ and for accelerated coronary atherosclerosis in transplanted hearts.³² The existence of 2 distinct subtypes of Ang II receptor has been confirmed, and type 1 has now been demonstrated to be the major mediator of Ang II effects on the circulation system.²⁷ Type 1 Ang II receptor number is increased in resistance vessels from animals made hypertensive with glucocorticoids and mineralocorticoids.^{33 34}

To clarify the potential and pathophysiological role of 11ß-HSD2, we manipulated 11ß-HSD2 gene expression with an AS. Licorice and carbenoxolone have been shown to be potent inhibitors of 11ß-HSD.³⁵ However, we did not attempt to administer these agents into HCASMC because of the nonspecific inhibition of both dehydrogenase and oxoreductase reactions. The dehydrogenase activity of HCASMC was decreased by nearly 80%, but oxoreductase activity was unaffected. Although we did not perform similar experiments using 11ß-HSD1 AS, Brem et al³⁶ reported that only the oxoreductase activity was affected with 11ß-HSD1 AS in rat aortic endothelial cells, and inhibition of the oxoreductase activity decreased contractile responses of rat aortic rings to vasoconstricting hormones.³⁷ However, Stewart et al³⁸ reported decreased hepatic 11ß-HSD1 activity and gene expression in the hypertensive rat. The pathological roles played by 11ß-HSD1 in the development of hypertension need to be clarified. Nevertheless, these observations raise the possibility that the dehydrogenation is mediated in vivo by 11ß-HSD2 alone.

Mineralocorticoid target tissues such as the kidney contain 11ß-HSD activity. In congenital or acquired 11ß-HSD–deficient states, suppression of 11ß-HSD2 activity in the kidney has been believed to cause renal mineralocorticoid excess, resulting in sodium retention and hypertension. However, after administration of 11ß-HSD inhibitors, there is a discrepancy between sodium retention (which occurs in the first few days) and elevated blood pressure (which occurs only after chronic administration).³⁹ Therefore, the rise in blood pressure may be independent of renal mineralocorticoid excess. In administration of glucocorticoids to rats, increased vascular responses to pressor hormones precede the rise in blood pressure, which is not associated with renal MR.^{40 41} The present study has shown that 11ß-HSD could modulate the access of glucocorticoids to vascular receptors and influence vascular tone. We propose that vascular 11ß-HSD2 activity could influence blood pressure by this mechanism without invoking renal sodium retention.

	Acknowledgments

 
This work has been supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture and by a research grant from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan.

Received September 17, 1998; first decision October 16, 1998; accepted January 11, 1999.

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