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Endocrinology Vol. 140, No. 9 3955-3961
Copyright © 1999 by The Endocrine Society

ARTICLES

Promoter-Specific Regulation of the Brain-Derived Neurotropic Factor Gene by Thyroid Hormone in the Developing Rat Cerebellum1

Noriyuki Koibuchi, Harumi Fukuda2 and William W. Chin

Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Noriyuki Koibuchi, Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Thorn 1009, Boston, Massachusetts 02115. E-mail: koibuchi{at}rascal.med.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I–IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IMPORTANCE of thyroid hormone (T3 and T4; TH) to the growth and development of many organs, including the central nervous system, is well documented (1, 2). Deficiency of TH during a critical period causes marked neuronal abnormalities, collectively known as cretinism in humans. However, the molecular mechanisms of TH action in the developing brain are not fully understood. TH exerts its major effect by binding to a TH receptor (TR), a ligand-dependent transcription factor, which then binds to target DNAs known as TH-response elements, with a retinoid X receptor. The TR/retinoid X receptor complex then recruits a number of corepressor and coactivator complexes in a ligand-dependent manner to result in activation of basal transcriptional machinery and stimulation of transcription of associated genes (3).

To study the molecular mechanisms of TH action on brain development, the rodent cerebellum may be an excellent model. In the rodent cerebellum, neuronal development is largely postnatal (4), and perinatal hypothyroidism dramatically affects the morphogenesis of this region (5, 6, 7, 8). Growth, dendritic arborization, and dendritic spine number of Purkinje cells are markedly decreased (8). Synaptic formation between the Purkinje and granule cells in the molecular layer are also dramatically reduced (5, 7). The rate of proliferation of granule cells in the external granule cell layer is diminished, and their migration into the internal granule cell layer (IGL) is retarded (6). Most of these abnormalities cannot be rescued unless TH is replaced within 2 weeks after birth (9). Based on these observations and the fact that TRs are expressed in Purkinje and granule cells during development (10, 11), these cells are considered to be critical targets of TH. However, despite the fact that many genes are known to be altered by perinatal hypothyroidism (12, 13, 14, 15, 16, 17), a TR-regulated gene(s) that plays a critical role in abnormal neurogenesis in hypothyroid animal has not yet been identified.

Brain-derived neurotropic factor (BDNF) belongs to the neurotropin family, which plays an essential role in the development of the peripheral and central nervous systems (18). Neurotropins also include nerve growth factor, neurotropin-3 (NT-3), and NT-4/5. In the developing cerebellum, BDNF is expressed in both granule and Purkinje cells (14). BDNF secreted from Purkinje and granule cells acts on granule cells to promote axonal elongation and to enhance survival (19, 20). BDNF also increases the expression of NT-3 that is stimulated by TH (21).

In the hypothyroid cerebellum, BDNF messenger RNA (mRNA) levels are suppressed, and grafting cell lines expressing BDNF into the fourth ventricle in part, but not completely, prevents hypothyroidism-induced abnormal cerebellar development (14). These results indicate that the TH effect is at least partially exerted through BDNF. Further, BDNF knockout mice exhibit delayed migration of granule cells and decreased arborization of Purkinje cell dendrites (22), which are also seen in hypothyroid animals (8). Whether TH directly regulates the expression of the BDNF gene is not known.

Given the important role of BDNF in TH-mediated cerebellar development, we decided to study the regulation of the BDNF gene by TH by first analyzing the effect of TH on its promoter. As shown in Fig. 1Go, the rat BDNF gene consists of five exons (23, 24). Exons I–IV are 5'-untranslated exons, associated with different promoters. Exon V contains the open reading frame for the BDNF protein. There are also two polyadenylate [poly(A)] addition sites. Thus, BDNF mRNAs consist of one of four untranslated exons and exon V as a result of different promoter usage and alternative splicing (23, 24). These multiple promoters are alternatively used in a tissue-specific manner (25). In the developing brain, different promoters have specific regulation patterns but are expressed coordinately in each brain region, suggesting that each promoter has both distinct and common regulatory elements (26).



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  		Figure 1. Schematic representation of the structure of the BDNF gene and its transcripts (23 24 ). The open reading frame (ORF) of BDNF protein is located in exon V. The 5'-untranslated exons are shown by hatched boxes. Two poly(A) addition sites are also shown.

 
We had initially performed a transient transfection study, using reporters containing several promoters associated with exon I or II and the luciferase complementary DNA (cDNA), in neuronal and nonneuronal cell lines. Surprisingly, the reporter activity was suppressed by TH with both promoters; note that BDNF mRNA is suppressed in the hypothyroid animal (Fukuda, H., N. Koibuchi, and W. W. Chin, unpublished observations). Thus, we hypothesized that each BDNF gene promoter might contain different regulatory elements that allow cell-specific and development stage-specific regulation of multiple BDNF transcripts (25, 26).

To understand this apparent paradox, we studied TH regulation of the different BDNF gene promoters. We used a ribonuclease (RNase) protection assay (RPA) to measure the expression of the different BDNF mRNAs during cerebellar development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatment
Timed pregnant Sprague Dawley rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were housed individually under controlled temperature and illumination. Food and water are available ad libitum. Newborn rats were rendered hypothyroid by administering 0.05% (wt/vol) propylthiouracil (PTU) in the drinking water to their mothers from the 15th day of conception until all pups were killed. Some newborn pups received a daily injection of T4 (0.02 µg/g BW) dissolved in saline from the day after birth until the day of death. Other pups received vehicle injections. We have previously shown that this T4-injected rat is comparable to the euthyroid rat regarding the changes in body weight and plasma TSH concentration (15, 27). The rats were killed with 100% CO2 on postnatal days (P) 2, 7, 15, and 30, and the cerebellum and cerebral cortex were dissected out. Cerebellum and cerebral cortex were also obtained from some of their mothers. These were frozen on dry ice and stored in liquid nitrogen until use.

Preparation of exon-specific BDNF probes
DNAs containing rat BDNF genomic fragment were provided by Drs. O. Ohara and T. Kitamura, Shionogi Research Laboratories (Osaka, Japan) (24). To prepare the exon-specific probes, the DNA fragments were amplified using PCR. A sense (5'-CTCCCTCACTTTTTCTGCGAAC-3', nucleotides 734–755) (23) primer with BamHI linker at the 5'-site, and an antisense (5'-CGGGATCCTCCCTCACTTTTTCTGGGAAC-3', nucleotides 996–959) (23) primer that contains the internal HindIII site for the exon I-specific probe were used. For the exon II-specific probe, a sense (5'-CGGAGCGTTTGGAGAGCCAGC-3', nucleotides 2045–2065) (23) primer with BamHI linker at the 5'-site and an antisense (5'-CGGCTTACACCACCCCGGTGG-3', nucleotides 2221–2201) (23) primer with KpnI linker at the 5'-end were used. For the exon III-specific probe, a sense (5'-CGTGCGAGTATTACCTCCGCC-3', nucleotides 859–879) (23) primer with BamHI linker at the 5'-site and an antisense (5'-CTGCTCTGGGGAAGACCGGTC-3', nucleotides 935–915) (23) primer with KpnI linker at the 5'-site were used. For the exon IV-specific probe, a sense (5'-CTGAGCTCTGGGTGCCCGCCG-3', nucleotides 1789–1809) (23) primer with BamHI linker at the 5'-site and an antisense (5'-CACGCTCCTGGTCCCTGCGCC-3', nucleotides 2026–2006) (23) primer with KpnI linker at the 5'-site were used. For the exon V-specific probe, a sense (5'-CCTACCCAGCTGTGCGGACCC-3', nucleotides 226–246) (28) primer with BamHI linker at the 5'-site and an antisense (5'-ACCCGGGAAGTGTACAAGTCC-3', nucleotides 415–395) (28) primer with KpnI linker at the 5'-site were used. Each probe was designed to lack any known multiple transcription start sites (23, 24). The PCR fragments were digested with the appropriate restriction endonucleases and subcloned into pBluescript II KS+.

To make a radiolabeled riboprobe, each plasmid was linearized with BamHI. Then, riboprobes were transcribed using T3 RNA polymerase (Promega Corp., Madison, WI), and [32P]UTP was incorporated to label the probe. The predicted sizes of exons I, II, III, IV, and V probes were 260, 177, 178, 238, and 190 bases, respectively. For a control, radiolabeled riboprobe for rat cyclophilin (103 bases; Ambion, Inc. Austin, TX) was also prepared.

RNA extraction and RPA
Total RNA from pooled cerebellum and cerebral cortex (7–10 brains for P2, 5–7 for P7, 2–3 for P15, 1–2 for P30, and 1 for adult) was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (29) and stored at -80 C until use.

RPA was performed using the RPA II kit (Ambion, Inc.), as reported previously (17). Twenty-microgram samples were hybridized with exon I/exon II, exon III/exon IV, or exon V, (300,000 cpm/probe·sample). The riboprobe for cyclophilin (20,000 cpm/sample) was also hybridized together with each probe. Hybridization was carried out overnight at 45 C, followed by RNase A/RNase T1 digestion. The protected fragments were separated on a 5% polyacrylamide/8 M urea gel. Then, the gel was dried and subjected to phosphorimaging (Molecular Dynamics, Inc., Sunnyvale, CA) overnight for quantitative analysis of protected bands and then to x-ray film at -80 C for 10 days. The amount of mRNA was internally standardized using cyclophilin mRNA levels, and the difference in each hybridization was standardized using the same adult RNA samples hybridized together.

Differences between treatment groups were examined by ANOVA. Post-hoc comparison was made using Duncan’s new multiple range test. The results were considered significant at P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the promoter-specific differential expression of the BDNF gene in vivo, RPA was performed with radiolabeled probes specific for each BDNF exon. Newborn pups were rendered hypothyroid by administering PTU to their mothers, and some pups received daily T4 replacement. By P15, the body weights of T4-replaced pups became significantly greater than those of their littermates, which did not receive T4 replacement. These results are consistent with previous studies using the same treatment (15, 17, 27).

Figure 2Go shows representative results of the RPA. Quantification was performed using a phosphorimager. The data were normalized using cyclophilin mRNA and adult brain mRNA (see Materials and Methods). In the cerebellum, exon I mRNA was down-regulated, whereas exon II–IV mRNAs were up-regulated during development. Exon II mRNA was most strongly expressed; on P30, the amount of exon II mRNA in T4-replaced euthyroid animals was approximately 5- and 8-fold greater than those of exons III and IV, respectively. The most striking effect of TH was seen on P15; the strength of the hybridization signal for exon II was always higher in T4-replaced animals. The expression of exon III and IV mRNAs was detected on P15 and P30 in TH-replaced animals, whereas the expression was not seen by P30 in hypothyroid animals.



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  		Figure 2. Representative autoradiograms of a RPA of exon I–V mRNAs using 20 µg total RNA from cerebellum or cerebral cortex. Hypothyroid (H) animals were obtained by administering PTU to their mother from the 15th day of conception. T4-replaced (T) animals received daily injection of T4 (0.02 µg/g BW) from the day after birth until the day of death. Mice were were killed on postnatal day (P) 2, 7, 15, or 30 to dissect out cerebellum and cerebral cortex for total RNA extraction.

 
On the other hand, in the cerebral cortex, all exon mRNAs were up-regulated during development. Exon III mRNA was most strongly expressed in the cerebral cortex; on P30, with its hybridization signal 20-, 8.5-, and 5.5-fold greater than those of exons I, II, and IV mRNA, respectively. We consistently observed a weak repression of exon I mRNA expression by TH during development. No apparent TH effect was seen in the expression of the other exon mRNAs.

In the cerebellum, exon I mRNA was down-regulated during development, and TH repressed its expression on P2 (Fig. 3Go). In the cerebrum, on the other hand, the mRNA was developmentally up-regulated, and TH tended to repress the expression of exon I mRNA, but the extent varied among the experiments and did not reach statistical significance (Fig. 3Go).



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  		Figure 3. Change in exon I mRNA content in the cerebellum and cerebral cortex of hypothyroid ({square}) and T4-replaced ({blacksquare}) animals. Shown are the mean ± SEM of arbitrary units obtained by scanning the density of each band using a PhosphorImager (Molecular Dynamics, Inc.; n = 5 determinations obtained from different animals). The amount of mRNA was internally standardized using cyclophilin mRNA levels, and the difference in each hybridization was standardized using the same adult RNA samples as standards. Note that there was no expression of exon I mRNA on P15 and P30 in the cerebellum or on P2 and P7 in the cerebrum. *, P < 0.01 vs. hypothyroid animals of the same age.

 
Exon II mRNA was up-regulated during development in both regions (Fig. 4Go). This exon is the most strongly expressed among the noncoding exons of the BDNF gene in the cerebellum. TH treatment resulted in a 3-fold increase compared with that in the hypothyroid animal on P15. A significant increase (80%) by TH replacement was also seen on P30 (Fig. 4Go). On the other hand, there was no effect of TH on exon II mRNA in the cerebrum (Fig. 4Go).



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  		Figure 4. Change in exon II mRNA content in the cerebellum and cerebral cortex of hypothyroid ({square}) and T4-replaced ({blacksquare}) animals. Shown are the mean ± SEM of arbitrary units obtained by scanning the density of each band using a PhosphorImager (Molecular Dynamics, Inc.; n = 5 determinations obtained from different animals). The amount of mRNA was internally standardized using cyclophilin mRNA levels, and the difference in each hybridization was standardized using the same adult RNA samples as standards. Note that the error bars are not seen at some points because the errors are less than 5% of the mean value. *, P < 0.01 vs. hypothyroid animals of the same age.

 
The patterns of exon III and IV mRNA expression were essentially similar (Figs. 5Go and 6Go). Both were up-regulated during development in both regions. Like exon II mRNA, the effect of TH was seen only in the cerebellum. Exon III and IV mRNAs were detected on P15 and P30 in TH-replaced animals, whereas the expression was not detected by P30 in hypothyroid animals. On P30, each exon mRNA was significantly greater in TH-replaced animals.



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  		Figure 5. Change in exon III mRNA content in the cerebellum and cerebral cortex of hypothyroid ({square}) and T4-replaced ({blacksquare}) animals. Shown are the mean ± SEM arbitrary units obtained by scanning the density of each band using a PhosphorImager (Molecular Dynamics, Inc.; n = 5 determinations obtained from different animals). The amount of mRNA was internally standardized using cyclophilin mRNA levels, and the difference in each hybridization was standardized using the same adult RNA samples as standards. Note that there was no expression of exon III mRNA on P2, P7, and P15 in the hypothyroid rat cerebellum or on P2 and P7 in the T4-replaced rat cerebellum. Also note that the error bars are not seen at some points because the errors are less than 5% of the mean value. *, P < 0.01 vs. hypothyroid animals of the same age.

 


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  		Figure 6. Change in exon IV mRNA content in the cerebellum and cerebral cortex of hypothyroid ({square}) and T4-replaced ({blacksquare}) animals. Shown are the mean ± SEM arbitrary units obtained by scanning the density of each band using a PhosphorImager (Molecular Dynamics, Inc.; n = 5 determinations obtained from different animals). The amount of mRNA was internally standardized using cyclophilin mRNA levels, and the difference in each hybridization was standardized using the same adult RNA samples as standards. Note that there was no expression of exon IV mRNA on P2, P7, and P15 in the hypothyroid rat cerebellum; on P2 and P7 in the T4-replaced rat cerebellum; or on P2 and P7 in the cerebrum. Also note that the error bars are not seen at some points because the errors are less than 5% of the mean value. *, P < 0.01 vs. hypothyroid animals of the same age.

 
Exon V mRNA contains the open reading frame encoding the BDNF protein and, therefore, is common to all BDNF transcripts. In the cerebellum, the changes largely reflect mRNA derived from exon II usage, consistent with its greater abundance in this tissue; we saw a 3-fold increase on P15 and a 1.8-fold difference on P30 by TH replacement (Fig. 7Go). On the other hand, in the cerebrum, the changes reflect exon III usage; we did not see any effect of TH during the postnatal development on exon V mRNA.



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  		Figure 7. Change in exon V mRNA content in the cerebellum and cerebral cortex of hypothyroid ({square}) and T4-replaced ({blacksquare}) animals. Shown are the mean ± SEM arbitrary units obtained by scanning the density of each band using a PhosphorImager (Molecular Dynamics, Inc.; n = 5 determinations obtained from different animals). The amount of mRNA was internally standardized using cyclophilin mRNA levels, and the difference in each hybridization was standardized using the same adult RNA samples as standards. Note that the error bars are not seen at some points because the errors are less than 5% of the mean value. *, P < 0.01 vs. hypothyroid animals of the same age.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have demonstrated the brain region-specific and developmental stage-specific expression of BDNF mRNA containing different noncoding exon transcripts. The effect of TH was mainly seen in the cerebellum; exon I mRNA was weakly down-regulated, whereas exon II–IV mRNAs were up-regulated. As each noncoding exon mRNA is associated with a different gene promoter, the results of the present study probably indicate the differential regulation of BDNF gene promoter activity during cerebellar and cerebral development.

In the developing cerebellum, the expression of many genes is known to be altered by perinatal hypothyroidism (12, 13, 14, 15, 16, 17). Interestingly, after the critical period, the expression of many genes altered by perinatal hypothyroidism return to levels seen in the euthyroid animal despite apparent morphological alterations (12, 13, 15, 17), although some of these genes are known to be regulated directly by TR (30, 31). The changes in expression of these genes do not reflect those in TRs, as the pattern of change in TR expression by perinatal hypothyroidism is different from those in other genes known to be altered in hypothyroid animals (17, 32). A key TR-regulated gene(s) that plays a major role in abnormal neurogenesis in hypothyroid animals has not yet been identified. Considering the critical role of BDNF in cerebellar development, the results of our present study indicate that the BDNF gene may be one such candidate, playing a key role in hypothyroidism-induced abnormal cerebellar development.

As exon II–IV mRNAs are regulated by TH, each promoter might contain a TH response element. However, we cannot conclude that these promoters are directly regulated by TH based solely on the present results. Our preliminary transfection study was also not helpful, because we saw only TH-dependent repression on promoters with exons I and II, probably due to the tissue and developmental stage specificity of each promoter. There are other possibilities, including 1) TH alters the stability of BDNF mRNAs; and/or 2) TH regulates the expression of other transcription factors that bind specifically to each promoter. TH is capable of modulating the stability and poly(A) tail length of TSH ß-subunit mRNA (33) in part by modulating the interaction of a RNA-binding protein to its 3'-untranslated region (34). Further, TH is capable of modulating the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element located in the 5'-untranslated region (35). The mRNA sequence to which TH-regulated RNA-binding protein binds contains a consensus region, UUA(or G)AAU(or A)GUGUUU (34). We have identified a region homologous to such a sequence within the exon II sequence (AUGCAAGUGUUU, nucleotides 2176–2187) (23). Therefore, BDNF mRNA may be also regulated at the posttranscriptional level by TH via a similar mechanism through the 5'-untranslated region of each BDNF mRNA. Other approaches need to be considered to determine the exact mechanism of TH action in BDNF gene expression. However, we believe that the present study has provided an important step toward our understanding of BDNF gene regulation by TH.

During postnatal cerebellar development, which subsets of BDNF mRNAs are expressed in which subset of cells are not clear. In the cerebellar cortex, BDNF mRNA is not detectable by in situ hybridization during the first two postnatal weeks (36), although BDNF immunoreactivity is strong in the Purkinje cells and relatively weak in the granule cells in the IGL as early as P8 (22). By P20, BDNF mRNA is strongly expressed in granule cells in the IGL and weakly in Purkinje cells (14, 36). By RPA analysis, a previous study also failed to detect BDNF mRNA in the developing cerebellum during the first 2 postnatal weeks (26) probably due to detection sensitivity, as smaller amounts of RNA (10 µg) were used. Although the amount of BDNF in the cerebellum during this postnatal period is low, BDNF probably plays an important role in the initial postnatal cerebellar development, as abnormal cerebellar development is already evident by P14 in BDNF-/- mice (22). The present study has clearly shown that the BDNF is expressed as early as P2 in the developing cerebellum, which may be critical for normal cerebellar development. However, it may be difficult to study the differential expression of each subset of BDNF mRNA in each cerebellar cell type during early postnatal cerebellar development because of their limited level of expression.

The physiological significance of multiple transcripts of BDNF is not known. Specific promoter usage in each subset of cells may confer the cell-specific and developmental stage-specific differential expression of BDNF gene. Further, it is known that the 5'-untranslated region is involved in regulating the efficiency of translation and/or stability of mRNA, which, in turn, also plays an important role in the control of gene expression (37). The small (40S) subunit of ribosome is bound initially at the 5'-end of mRNA to initiate translation (37). The binding of the 40S ribosomal subunit to this site is altered by a RNA-binding protein(s) (38) and/or by secondary structure within the 5'-noncoding region (39). Therefore, although all BDNF gene transcripts generate the same protein, different 5'-untranslated regions may mediate altered stability or translational efficiency of each mRNA, with corresponding differences in protein levels. As mentioned above, TH may be involved in regulation of the translational efficiency and/or stability of BDNF gene transcripts by altering the interaction with RNA-binding proteins.

In summary, during cerebellar development, all BDNF gene noncoding exon mRNAs (I–IV) are detected by RPA, but their expression patterns are different. Daily TH replacement induced a 3-fold increase in exon II mRNA on P15. On P30, exon II mRNA was still higher in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on both days. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were seen starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons were differentially regulated during development, the expression of each mRNA was not altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.


   Acknowledgments
 
The authors thank Drs. Osamu Ohara and Tadahisa Kitamura, Shionogi Research Laboratories (Osaka, Japan), for generously providing us plasmids containing rat BDNF genomic fragments.


   Footnotes
 
1 This work was supported in part by an American Thyroid Association research grant (to N.K.), the William Randolph Hearst Fund (to N.K.), and NIH Grant RO1-DK-54343–01 (to W.W.C.). Back

2 Present address: Department of Physiology, Dokkyo University School of Medicine, Mibu, Tochigi 321–02, Japan. Back

Received December 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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