(Received for publication, February 8, 1995; and in revised form, May 3, 1995)
From the
The hepatic transport systems mediating bile acid uptake and
excretion undergo independent, stage-specific expression during
development in the rat. In this study, the mechanisms underlying
ontogenic regulation of both the Na-dependent
basolateral bile acid transporter and canalicular bile acid
transporter/ecto-ATPase were examined. Steady state mRNA levels for the
basolateral transporter were less than 20% of adult values prior to
birth, increased to 35% on the first postnatal day, and reached adult
levels by 1 week of age. This was paralleled by transcription rates,
which were low prior to birth, reached 47% by day 1, and were maximal
by 1 week of age. Steady state mRNA levels for ecto-ATPase were 12% of
adult values prior to birth and showed a 2-fold increase by the first
day of life. Thereafter, there was a gradual increase in mRNA for this
transporter, with adult levels being reached at 4 weeks of age.
Transcription rates paralleled this increment, although adult levels
were reached earlier. Surprisingly, for both transporters, the full
complement of protein was present well before adult levels of mRNA were
reached. The basolateral protein was expressed at 82% of adult levels
on the first day of life but was of lower apparent molecular mass (39
kDa), a difference that persisted until 4 weeks of age. N-Glycanase digestion suggested that this difference could be
fully accounted for by N-linked glycosylation. The ecto-ATPase
protein was present at 33% of adult levels prior to birth, 77% by 1
day, and 84% of adult levels by 1 week of age. Unlike the basolateral
transporter, the apparent molecular weight of this protein did not
change during development. In summary, the ontogeny of bile acid
transporters on the plasma membrane of the hepatocyte is complex and
appears to be regulated at transcriptional, translational, and
post-translational levels.
The process of bile formation is immature at birth and is related, in part, to a reduced capacity of the developing liver for bile acid uptake and excretion(1) . A key finding from our previous studies employing highly purified plasma membrane vesicles has been that hepatocyte transport mechanisms for bile acids are developmentally regulated and are expressed independently on the basolateral and canalicular membranes at specific times during the perinatal period(2, 3) . The mechanisms underlying the developmental expression of these transport activities are unknown but are likely to involve transcriptional, translational, and post-translational events. The differential timing for the expression of these transporters would indicate that different modes of regulation may be required for the ontogenesis of each system.
Bile acid uptake
across the basolateral membrane occurs largely via a sodium-dependent
cotransport system, which exhibits a sodium to bile acid ratio of
greater than 1:1(4) . A cDNA encoding the rat liver sodium/bile
acid cotransporter (Ntcp) has recently been cloned by Hagenbuch and
associates (5, 6) using the Xenopuslaevis oocyte expression system. The transporter has a
molecular mass of 50 kDa and is glycosylated(7) .
Sodium-dependent bile acid uptake is not present in the rat
throughout most of gestation but is abruptly expressed on fetal day 20 (2) . It could not be determined from these studies whether the
bile acid carrier was absent or present but not functional during fetal
development. Following birth, there is a progressive rise in the V for taurocholate uptake to reach 75% of the
adult rate by 4 weeks of age(8) . These functional studies were
interpreted as being consistent with a reduced number or translocation
rate of specific taurocholate carriers during development.
In preliminary studies we have reported that mRNA transcripts for the sodium-dependent cotransporter were low during fetal life and then roughly paralleled the increase in transport activity postnatally(9) . However, in these studies, a limited range of age groups was examined. Moreover, it is uncertain how steady state mRNA levels for the transporter correlate with rates of transcription or with the amount of transport protein detected within the plasma membrane.
Excretion of bile acids at the canalicular membrane is the
rate-limiting step in hepatocellular transport of bile
acids(10) . Several distinct carrier mechanisms for bile acids
have now been identified in studies employing canalicular membrane
vesicles. The predominant low affinity, high capacity system is sodium
independent, saturable, and driven by the membrane electrical
potential(11) . This mechanism is ontogenically regulated in
that the transport activity is not detected in neonatal rat canalicular
membrane vesicles during the first week of life but is expressed at
near adult levels by day 14 of postnatal age(3) . A high
affinity, low capacity transporter for conjugated bile acids, which is
ATP dependent, has also been described (12) . In preliminary
studies, we have found that this transporter is expressed during the
first week of life at approximately 60% of the adult transport V. The molecular identities of these transport
systems have not been precisely defined, but several groups have
identified a
100-kDa bile acid transport protein using affinity
chromatographic techniques(13, 14, 15) .
Sequence analysis of the 100-kDa protein purified from canalicular
membranes by bile acid affinity chromatography in our laboratory showed
it to be identical to a rat liver ecto-ATPase/cell CAM
105(16, 17) . We have also previously demonstrated
canalicular membrane localization of this protein in developing and
adult animals in immunofluorescence studies using a polyclonal
antibody(3, 14) . Several studies have shown recently
that transfection of a full-length or truncated ecto-ATPase cDNA
resulted in de novo synthesis of immunoreactive proteins by
COS cells and their correct targeting to the plasma membrane, but only
the full-length construct conferred on the cells the capacity to pump
out taurocholate with efflux characteristics comparable to the
potential-sensitive system defined in canalicular membrane vesicles (18) .
The availability of cDNA and monospecific antibody probes for Ntcp and the ecto-ATPase/bile acid transporter now allows an in depth analysis of their development, including steady state mRNA and protein levels as well as rates of transcription. A complex pattern of regulation was found for both transport systems involving transcriptional, translational, and, for the basolateral transporter, possibly post-translational mechanisms.
The cDNA for cyclophilin was kindly provided by
William Pandak, Jr. (Medical College of Virginia), and the cDNA for
-fetoprotein was provided by Sanjay Gupta (Albert Einstein College
of Medicine, New York).
Nuclear transcription
reactions were carried out by the method of Diamond and Goodman (23) with minor modifications. Nuclei (2
10
) were incubated for 30 min at 37 °C in 20 mM Tris-Cl (pH 8.0), 150 mM KCl, 5 mM MgCl
, 3.5 mM dithiothreitol, 20% glycerol,
0.05 M EDTA, 1 mM ATP, CTP, and GTP, and 100 µCi
of [
P]UTP (3000 Ci/mmol, DuPont NEN) in a final
volume of 200 µl. Nuclei were treated sequentially with DNase I and
proteinase K and extracted with phenol:chloroform:isoamylalcohol
(25:24:1). The RNA was precipitated twice with sodium acetate and
isopropanol. The pellet was washed twice with ethanol, resuspended in
STE buffer (10 mM EDTA, 100 mM NaCl, 20 mM
Tris-Cl), and passed through a Sephadex G-50 spin column (Boehringer
Mannheim).
Nascent RNA transcripts were hybridized to cDNAs
immobilized on nylon filters (Genescreen, Dupont NEN).
-Fetoprotein was included as a positive control, as the
transcription rate of this protein has been reported during the
developmental period studied(24) . Linearized plasmid vector
was included as a negative control. Ntcp cDNA was linearized with SacI, and ecto-ATPase and
-fetoprotein with PstI. Filters were prehybridized at 45 °C for 24 h with
50% deionized formamide, 5
Denhardt's solution, 4
SSC, 50 mM PIPES, 2 mM EDTA, pH 8.0, 0.1% SDS, and
200 µg/ml salmon sperm DNA and yeast tRNA. Hybridization was
carried out for 48 h at 45 °C. The filters were then washed
sequentially as follows: 2
SSC, 0.5% SDS twice for 20 min at 45
°C; the same buffer with RNase A (20 µg/ml) and RNase T1 (700
units/ml) for 30 min at 37 °C; 2
SSC, 0.5% SDS at 65 °C
for 30 min three times; and 1
SSC, 0.5% SDS for 30 min at 65
°C. After rinsing in 2
SSC, the filters were exposed in a
PhosphorImager cassette for 3-7 days at room temperature and then
to X-OMAT film (Kodak) at -70 °C for 1 week. The bands were
quantitated by PhosphorImager using ImageQuaNT software.
To determine factors that contribute to age-related changes in bile acid transport activity across the basolateral and apical membranes of the hepatocyte, steady state mRNA levels for the basolateral sodium-dependent transporter (Ntcp) and canalicular bile acid transporter/ecto-ATPase were determined at various stages of pre- and postnatal development by Northern analysis. These experiments were intended to determine whether the minimal transport activity during early life was due to a paucity of mRNA or inefficient translation of a relatively abundant message.
The Ntcp probe hybridized to a single 1.7-kb message at each stage of development (Fig. 1A). Transcripts encoding Ntcp were not detectable by Northern analysis through most of gestation but were demonstrated at levels markedly below the adult just prior to birth. mRNA levels increased significantly between 1 and 7 days of life. Ntcp mRNA levels (Fig. 1B) were less than 20% of the adult prior to birth and increased to about 35% of the adult by the first postnatal day. By 1 week of age, adult levels were achieved and remained relatively constant thereafter.
Figure 1:
Northern blot analysis of Ntcp
expression in rat liver during development. 2 µg of mRNA, isolated
from livers of rats sacrificed at various stages of development, was
loaded onto a 1.2% agarose gel, transferred to nylon membrane, and
hybridized to a rat Ntcp P cDNA probe. The upperhalf of panelA demonstrates the
gradual increase in Ntcp mRNA levels, while cyclophilin, a
constitutively expressed message, shows little change. Lanes are labeled with the age of the animal (df, days fetal; d, postnatal day; w, weeks). The data are quantified
in panelB. Each bar in the histogram
represents the mean ± S.E. of three to five experiments using
mRNA made from separate litters.
To determine how levels of specific
mRNA correspond to relative transcription rates for the Ntcp gene,
nuclear run-on assays were performed using nuclei isolated from fetal,
neonatal, and adult liver. Transcription of the gene for
-fetoprotein, whose pattern of development has been well studied,
was used for comparison. In these experiments, all RNAs undergoing
transcription at the time of nuclear isolation are labeled and then
hybridized with the Ntcp and control cDNA probes. Nuclear run-on assays (Fig. 2, A and B) showed that transcription of
the Ntcp gene was low (5% of the adult) in the 21-day fetus. The rate
of transcription increased almost 10-fold by the first day of life and
approached adult levels by 1 week. In contrast, transcription of the
-fetoprotein gene was high in the fetus and neonate and fell to
undetectable levels in the adult as previously reported(24) .
Figure 2:
Relative transcription rates of Ntcp
during development. Nuclei isolated from livers of rats (21 days fetal,
newborn day 1, 1 week, and adult) were incubated with
[P]UTP, and NTPs. The nuclei were lysed, and RNA
isolated was hybridized to cDNAs immobilized on nylon filters. PanelA shows the increase in relative transcription
rate of Ntcp, while transcription rates for
-fetoprotein (AFP) were markedly reduced with hepatic maturity as
previously reported(24) . There is no hybridization to the
plasmid vector alone. PanelB shows quantitation of
these results. Each bar in the histogram represents the mean
± S.E. of at least three independent
experiments.
Fig. 3A shows a representative Northern blot
depicting canalicular bile acid transporter/ecto-ATPase mRNA abundance
during development. The probe recognizes a major 4.4-kb transcript
(long form). The faint band at 2.8 kb represents the short
isoform, which parallels the long form during development as previously
described by Cheung et al.(27) . Steady state mRNA
levels for the transporter were low prior to birth and, similar to the
Ntcp message, increased on the first day of life. Transcript levels (Fig. 3B) were approximately 12% of adult values prior
to birth and 24% of the adult on the first day of life. There is a
gradual increase during the suckling period to reach the levels of
adult by 4 weeks of age.
Figure 3:
Northern blot of ecto-ATPase expression in
rat liver during development. 2 µg of mRNA, isolated from livers of
rats sacrificed at various stages of development, was loaded onto a
1.2% agarose gel, transferred to nylon membrane, and hybridized to a
rat ecto-ATPase P cDNA probe. PanelA shows the gradual increase in mRNA levels, while cyclophilin, a
constitutively expressed message, shows little change. The major
transcript is the 4.4-kb-long isoform; the
2.8-kb transcript is
the short isoform, which parallels the longer one during
development(27) . Lanes are labeled with the age of
the animal (df, days fetal; d, postnatal day; w, weeks). The data are quantified in panelB. Each bar in the histogram represents the mean
± S.E. of three to five experiments using mRNA made from
separate litters.
Transcription of bile acid transporter/ecto-ATPase gene was also measured directly in several representative age groups using nuclear run-on assays (Fig. 4, A and B). A pattern similar to the transcription of the Ntcp gene was observed in that there was an increase in transcriptional activity as development proceeded. The rate of transcription was 9 and 30% of the adult rate by fetal day 21 and postnatal day 1, respectively. Adult rates of transcription were achieved by 7 days of age.
Figure 4:
Relative transcription rates of
ecto-ATPase during development. Nuclei isolated from livers of rats (21
days fetal, newborn day 1, 1 week, and adult) were incubated with
[P]UTP and NTPs. The nuclei were lysed, and RNA
isolated was hybridized to cDNAs immobilized on nylon filters. PanelA shows the gradual increase in relative
transcription rate of ecto-ATPase, while transcription rates for
-fetoprotein (AFP) were markedly reduced with hepatic
maturity as previously reported(24) . There is no hybridization
to the plasmid vector alone. PanelB shows
quantitation of these results. Each bar in the histogram
represents the mean ± S.E. of at least three independent
experiments.
It is well known that sodium-dependent
bile acid transporter activity on the basolateral membrane and
potential-dependent transport activity on the canalicular membrane are
developmentally regulated. The V for the
sodium-dependent transporter is 23, 36, and 45% of adult values at 22
days fetal, 1 day, and 2 weeks of age, respectively. Mature function is
not reached until 4 weeks postnatally(28) . In contrast, there
is an abrupt increase in the V
of
potential-sensitive transporter to adult levels between 7 and 14 days
postnatally(2, 3) . Because the patterns of mRNA
expression do not precisely mirror the observed developmental changes
in activity of these transporters, age-related changes in the quantity
or electrophoretic mobility of the basolateral and canalicular bile
acid transport proteins was examined, using specific antibody detection
of Western blots.
Fig. 5A depicts a representative
Western blot of liver homogenates prepared from rats of varying ages
probed with antibody to the basolateral transporter. Immunoreactive
protein was detectable at approximately 8% of the adult just prior to
birth and increased dramatically to 82% of the adult level by the first
postnatal day (Fig. 5B). The levels of the basolateral
protein remained constant thereafter. Similar results were obtained on
immunoblot analysis of liver basolateral membranes (Fig. 5C), indicating that the protein was not being
synthesized and sequestered intracellularly during this period of
development. However, the molecular mass of the protein detected in
developing rat liver was only 39 kDa compared to the 50-kDa
protein observed on immunoblots of adult basolateral membranes. This
difference persisted until at least 4 weeks of age.
Figure 5:
Western blot analysis of Ntcp. Liver
homogenates from rats of various age groups were separated by
SDS-polyacrylamide gel electrophoresis and, after transfer to a
nitrocellulose membrane, were incubated with a fusion protein antibody
to rat Ntcp. PanelA depicts a single band of 39
kDa during development, which increases to 50 kDa in the adult. The
signal was quantified with laser densitometry, and these results are
depicted in panelB. Each bar represents
mean ± S.E., n = 3. Samples were assayed in
duplicate. Protein levels can be seen to increase abruptly after birth
and reach adult levels by 1 week of age. PanelC shows Western blotting of basolateral membranes prepared from the
same age groups. The pattern of development is identical, suggesting
that protein is transported to the membrane and not sequestered
intracellularly.
Since transport activity does not mature fully until about 4 weeks of age, a change in the electrophoretic mobility of the transport protein may be of functional importance. Several sites for N-linked glycosylation are present on examination of the Ntcp amino acid sequence(6) . Fig. 6shows the results of an experiment to further determine whether differences in glycosylation are responsible for age-related differences in the relative molecular mass of the protein. Deglycosylation of the transport protein in both the adult and 4-week-old animal was carried out enzymatically using N-glycanase. On deglycosylation, both proteins migrated at the same rate, suggesting that N-glycosylation is responsible for the difference in the apparent molecular weight of the proteins between developing and mature animals.
Figure 6:
N-Deglycosylation of Ntcp
proteins from 4-week-old and adult rats. Basolateral membrane
preparations from adult and 4-week-old rats were N-deglycosylated using N-glycanase. Untreated
controls were run in parallel. The firstlane represents untreated adult membranes. Upon deglycosylation (secondlane), the protein is reduced to 33 kDa.
Deglycosylation of basolateral membranes from 4-week-old rats (lane3) also results in a protein of
33 kDa when compared
to untreated membranes (lane4).
The results of Western blot analysis
of the canalicular bile acid transporter ecto-ATPase protein are shown
in Fig. 7A. A protein of 100 kDa was detected, and
there was no apparent change in the electrophoretic mobility of protein
during prenatal and postnatal development. The level of the protein, as
measured by densitometry (Fig. 7B), was about 30% of
the adult level in the 21-day-old fetus and increased approximately
2-fold by postnatal day 1. The canalicular protein reached adult levels
by 7 days of age and remained unchanged thereafter. The level of
protein correlated reasonably well with transport activity since we
have previously shown that potential-dependent transport activity
matures between 1 and 2 weeks of postnatal life (3) and that
the V
for ATP-dependent transport at 1 week is
66% of the adult. (
)
Figure 7: Western blot of ecto-ATPase during development. Liver homogenates from rats of various age groups were separated by SDS-polyacrylamide gel electrophoresis and, after transfer to a nitrocellulose membrane, were probed with a polyclonal antibody to rat ecto-ATPase. PanelA depicts a single band of 100 kDa, and there is no change in molecular mass during development. The signal was quantified with laser densitometry, and these results are shown in panelB. Each bar represents mean ± S.E. (n = 3). Protein levels increase abruptly after birth and reach adult levels by 1 week of age.
The recent cloning of the cDNA for the sodium taurocholate
cotransporting polypeptide (Ntcp) and the availability of specific
antibodies directed against the transporter have now allowed an in
depth analysis of the mechanisms that underly the developmental
regulation of the predominant hepatocyte uptake mechanism for bile
acids. The pattern of regulation has proven to be considerably more
complex than might have been predicted from the gradual increase in
transport activity during development. Consistent with functional
studies, transcripts encoding Ntcp were not detectable through most of
gestation but were present at levels markedly below the adult just
prior to birth. mRNA levels for the transporter increased to adult
levels by 7 days of age even though transport activity, as estimated by V, is only about 25% of the adult level at that
time. Relative transcription rates for the Ntcp gene, as estimated by
nuclear run-on assays, corresponded reasonably well to the amount of
mRNA present at each developmental stage and also approached adult
levels by 1 week. Thus, these data indicate that gene transcription
rate is an important determinant in the maturation of the Ntcp
transport system.
Quantitation of Ntcp protein in basolateral
membranes by Western blotting demonstrated an even more complex pattern
of regulation. Immunoreactive Ntcp protein was found to be unexpectedly
near adult levels shortly after birth. However, the molecular mass of
the protein detected in basolateral membranes from developing rat liver
was significantly less than that seen in mature liver (39 versus 50 kDa). This difference persisted until at least 4
weeks of age. Further experiments showed that incomplete glycosylation
was responsible for the age-related change in the molecular mass of the
protein. In light of the demonstration of adult levels of Ntcp mRNA and
protein concentrations in neonatal liver but deficient transport
activity until after weaning, an immaturity of post-translational
processing may explain the lower functional capacity of Ntcp during
development. A similar discrepancy in the molecular mass has also been
observed recently with the brush border membrane
Na
/bile acid cotransporter of neonatal compared with
adult rat ileum(29) . This recently cloned polypeptide is
similar in structure and function to Ntcp.
Developmental changes in N-linked oligosaccharides of glycoproteins in rat liver have been characterized (30) and occur because enzymes involved in glycosylation of proteins, such as galactosyl transferase and sialyl transferase, themselves undergo ontogenic regulation(31, 32) . Further, functionality of the carbohydrate moiety has been suggested for the sodium and chloride-coupled glycine transporter from pig brain stem(33) . Thus, incomplete glycosylation during development may represent a further level of regulation of the Ntcp in that its transport function may be altered, possibly by interference with its normal conformation within the basolateral membrane or its capacity to bind transported substrates.
The pattern of ontogenic regulation for the canalicular bile acid transporter/ecto-ATPase was also complex. Low rates of transcription and mRNA levels were present prior to birth. Although the adult rate of transcription for the gene was present by 7 days of age, a more gradual increase in the amount of bile acid transporter/ecto-ATPase mRNA occurred during the suckling period to achieve adult levels by 4 weeks of age. These data suggest that transcripts encoding the bile acid transporter/ecto-ATPase may be less stable during development.
Western blot analysis of canalicular membranes showed bile acid transporter/ecto-ATPase protein levels of about 30% of the adult in the 21-day-old fetus, which increased about 2-fold by the first postnatal day. Adult levels of the transporter were achieved by 7 days of age and remained constant. There was no apparent change in molecular weight during pre- and postnatal development. Although it is unclear whether the bile acid transporter/ecto-ATPase is involved in the potential-dependent and/or ATP-dependent components of bile acid excretion, the adult levels of protein detected by 7 days correlated reasonably well with the maturation of both transport systems by 7-14 days postnatally. Factors other than glycosylation may be important in contributing to the developmental maturation of this transport system. For example, it has been demonstrated in transfection and site-directed mutagenesis studies that phosphorylation sites on the cytoplasmic tail of the bile acid transporter/ecto-ATPase are essential for transport activity(34) . Developmental change in the ability to phosphorylate this protein may be a critical determinant in achieving full transport capacity after the protein is expressed in the canalicular membrane.
These studies point to the complexity of events during development leading to full transport capacity for bile acids across the basolateral and canalicular membrane of the hepatocyte. The different timing for the perinatal expression of carrier proteins and transport activities on these domains indicates that distinct arrays of transcription factors and post-transcriptional mechanisms may be required for the ontogenesis of each transport system. Transcriptional activation during the perinatal period plays a central role in regulating the mRNA abundance for both transporters; however, specific factors inducing expression of membrane transporters for bile acids have not yet been identified.