©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Ontogenic Regulation of Basolateral and Canalicular Bile Acid Transport Proteins in Rat Liver (*)

(Received for publication, February 8, 1995; and in revised form, May 3, 1995)

Winita Hardikar (§) Meenakshisundaram Ananthanarayanan Frederick J. Suchy (¶)

From the Section of Gastroenterology/Hepatology, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(max) 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(max). 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.


EXPERIMENTAL PROCEDURES

Materials

The sources of materials used were as follows. Reagents of analytical grade were purchased from Sigma, J. T. Baker, Inc. (Phillipsburg, NJ), and American Bioanalytical (Natick, MA). Restriction enzymes were from New England Biolabs (Beverly, MA).

The cDNA for cyclophilin was kindly provided by William Pandak, Jr. (Medical College of Virginia), and the cDNA for alpha-fetoprotein was provided by Sanjay Gupta (Albert Einstein College of Medicine, New York).

Animals

Male (200-250 g) and timed-pregnant female Sprague-Dawley rats were obtained from Camm Laboratories (Wayne, NJ). Rats were housed at constant temperature (20 °C) with alternating 12-h light-dark cycles and free access to standard rat chow and water. Livers from adult males were harvested after portal perfusion with ice-cold normal saline. Dams were anesthetized with 6% pentobarbital, and fetal livers (days 18, 20, and 21) were removed. Neonatal (day 1) and suckling (week 1, 2, 3, and 4) rats were killed by decapitation, prior to hepatectomy. Liver tissue was either used fresh or snap frozen in liquid nitrogen and stored at -70 °C prior to use.

Northern Blotting

Total RNA was made by the acid guanidium thiocyanate-phenol chloroform extraction method(19) . mRNA was purified using biotinylated-oligo(dT) and streptavidin-labeled magnetic beads (Poly(A)Tract, Promega). Probes were labeled with P using a random-primed labeling method (Life Technologies, Inc.), and unincorporated nucleotides were removed by Sephadex G-50 chromatography. RNA blotting to nylon membrane (Genescreen, DuPont NEN) and hybridization with P cDNA probes were performed as previously described(20) . The blots were washed sequentially as follows: 2 SSC at room temperature, 2 SSC, 0.5% SDS at 60 °C, 0.1 SSC at room temperature, and exposed to a PhosphorImager cassette at room temperature and then X-OMAT film (Eastman Kodak Co.) at -70 °C for 1-5 days. Cyclophilin, a constitutively expressed message, was used to correct for loading(21) . The Ntcp probe was a 0.9-kb (^1)EcoRI fragment (nucleotides +261 to +1187) of the cDNA (6) . The ecto-ATPase probe was a 1.3-kb XbaI-PstI fragment (nucleotides -243 to +1093) of the cDNA(16) . The cyclophilin probe was a full-length (743 base pairs) cDNA in pBI vector. Bands were quantitated by PhosphorImager using ImageQuaNT (Molecular Dynamics) software according to instructions from the manufacturer. Membranes were also exposed to AR film (Kodak) for 1-3 days at -70 °C. The results given at each age are the mean of three to five separate samples (each sample being made from a separate litter) and are corrected for loading using cyclophilin.

Nuclear Transcription Assays

Nuclei were made from fresh livers using a modification of the method described by Sasaki et al.(22) Livers were weighed, suspended in 10 volumes of homogenization buffer (0.25 M sucrose, 10 mM HEPES, pH 8.0, 10 mM MgCl(2), 2 mM dithiothreitol, 0.1% Triton X-100), and homogenized using a Potter-Elvehjem homogenizer at 700 rpm for seven strokes. The homogenate was then filtered through two layers of gauze and centrifuged at 700 g for 5 min. The pellet was resuspended in 2.5 ml of homogenization buffer and layered on top of a sucrose cushion (3.1 M sucrose, 1 mM MgCl(2), 1 mM HEPES, pH 6.8), After centrifugation at 50,000 g for 80 min at 4 °C, the nuclear pellet was resuspended in storage buffer (20 mM Tris, pH 8.0, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 2 mM dithiothreitol) and counted using a methylene blue stain. Aliquots were stored at -70 °C.

Nuclear transcription reactions were carried out by the method of Diamond and Goodman (23) with minor modifications. Nuclei (2 10^7) were incubated for 30 min at 37 °C in 20 mM Tris-Cl (pH 8.0), 150 mM KCl, 5 mM MgCl(2), 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). alpha-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 alpha-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.

Western Blotting

Western analysis was performed on liver homogenates and purified basolateral membrane preparations (25) . Only liver homogenates were used for the ecto-ATPase studies because of the large number of animals that would be required to prepare canalicular membranes from fetal and neonatal animals. Protein concentrations were determined according to Bradford (26) with bovine serum albumin as a standard. Antibody to the basolateral bile acid transporter was made by immunizing New Zealand White rabbits with a fusion protein comprising the C terminus (amino acids 309-357) of the transporter and schistosomal glutathione transferase(7) . Specificity of this antibody was demonstrated on Western blots of basolateral membranes, using pre-immune sera, as well as by preabsorption of the sera with the fusion protein. Further, the ability of this antibody to detect Ntcp protein on the basolateral membrane of hepatocytes was demonstrated by immunofluorescence(7) . Antibody to the ecto-ATPase was made to a 100-kDa protein isolated from a bile acid affinity column and further purified by SDS-polyacrylamide gel electrophoresis(14) . The secondary antibody in each case was a horseradish peroxidase-conjugated goat anti-rabbit IgG (ICN). An enzyme chemiluminescent method was used for detection (ECL, Amersham Corp.) and quantitated by laser densitometry using an LKB Ultroscan XL densitometer (Pharmacia Biotech Inc.).

Deglycosylation

Deglycosylation of Ntcp from 4-week-old and adult animals was carried out using highly purified basolateral membrane preparations (25) from these age groups with N-glycanase (Genzyme, Cambridge, MA), according to the instructions from the manufacturer. Briefly, basolateral membrane preparations were diluted in 0.5 M Tris-Cl, pH 8.0, with 0.5% SDS and 50 mM 2-mercaptoethanol. After denaturing, 7.5% Nonidet P-40 and 0.3 units per 20 µg of protein of N-glycanase were added and incubated overnight at 37 °C. After stopping the reaction, the proteins were analyzed on an SDS-polyacrylamide gel in parallel with an undigested control, which was similarly incubated.


RESULTS

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 alpha-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 alpha-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 alpha-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 alpha-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(max) 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(max) 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(max) for ATP-dependent transport at 1 week is 66% of the adult. (^2)


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.




DISCUSSION

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(max), 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.


FOOTNOTES

*
This study was supported in part by U. S. Public Health Grant HD20632 (to F. J. S.) and Liver Center Grant DK34989. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Fulbright post-doctoral fellowship and the Ackman Fellowship (University of Melbourne).

To whom correspondence should be addressed: Section of Gastroenterology/Hepatology, Dept. of Pediatrics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4649; Fax: 203-785-7194.

(^1)
The abbreviations used are: kb, kilobase(s); PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
M. Ananthanarayanan and F. J. Suchy, unpublished observations.


REFERENCES

  1. Suchy, F. J., Bucuvalas, J. C., and Novak, D. A. (1987) Semin. Liver. Dis. 7,77-84 [Medline] [Order article via Infotrieve]
  2. Suchy, F. J., Bucuvalas, J. C., Goodrich, A. L., Moyer, M. S., and Blitzer, B. L. (1986) Am. J. Physiol. 251,G665-G673
  3. Novak, D. A., Sippel, C. J., Ananthanarayanan, M., and Suchy, F. J. (1991) Am. J. Physiol. 260,G743-G751
  4. Yamazaki, M., Sugiyama, Y., Suzuki, H., Iga, T., and Hanano, M. (1992) J. Hepatol. 14,54-63 [Medline] [Order article via Infotrieve]
  5. Hagenbuch, B., Lubbert, H., Stieger, B., and Meier, P. J. (1990) J. Biol. Chem. 265,5357-5360 [Abstract/Free Full Text]
  6. Hagenbuch, B., Stieger, B., Foguet, M., Lubbert, H., and Meier, P. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,10629-10633 [Abstract]
  7. Ananthanaryanan, M., Ng, O. C., Boyer, J. L., and Suchy, F. J. (1994) Am. J. Physiol. 267,G637-G643
  8. Suchy, F. J., Courchene, S. M., and Blitzer, B. L. (1985) Am. J. Physiol. 248,G648-G654
  9. Boyer, J. L., Hagenbuch, B., Ananthanarayanan, M., Suchy, F. J., Stieger, B., and Meier, P. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,435-438 [Abstract]
  10. Boyer, J. L. (1971) Am. J. Physiol. 221,1156-1163 [Medline] [Order article via Infotrieve]
  11. Meier, P. J., Meir-Abt, A. S., and Boyer, J. L. (1987) Biochem. J. 242,465-469 [Medline] [Order article via Infotrieve]
  12. Adachi, Y., Kobayashi, H., Kurumi, Y., Shouji, M., Kitano, M., and Yamamoto, T. (1991) Hepatology 14,655-659 [Medline] [Order article via Infotrieve]
  13. Reutz, S., Fricker, G., Hugentobler, G., Winterhalter, K., Kurz, G., and Meier, P. J. (1987) J. Biol. Chem. 262,11324-11330 [Abstract/Free Full Text]
  14. Sippel, C. J., Ananthanarayanan, M., and Suchy, F. J. (1990) Am. J. Physiol. 258,G728-G737
  15. Muller, M., Ishikawa, T., Berger, U., Klunemann, C., Lucka, L., Schreyer, A., Kannicht, C., Reutter, W., Kurz, G., and Keppler, D. (1991) J. Biol. Chem. 266,18920-18926 [Abstract/Free Full Text]
  16. Lin, S. H., and Guidotti, G. (1989) J. Biol. Chem. 264,14408-14414 [Abstract/Free Full Text]
  17. Lin, S. H. (1989) J. Biol. Chem. 266,14403-14407
  18. Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter, D. H. (1993) J. Biol. Chem. 268,2083-2091 [Abstract/Free Full Text]
  19. Chromczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159 [CrossRef][Medline] [Order article via Infotrieve]
  20. Sambrook, K. J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  21. Pandak, W. M., Vlahcevic, Z. R., Chiang, J. Y. L., Heuman, D. M., and Hylemon, P. B. (1992) J. Lipid. Res. 33,659-668 [Abstract]
  22. Sasaki, K., Cripe, T. P., Koch, S. R., Andreone, T. L., Petersen, D. D., Beale, E. G., and Granner, D. K. (1984) J. Biol. Chem. 259,15242-15251 [Abstract/Free Full Text]
  23. Diamond, D. L., and Goodman, H. M. (1985) J. Mol. Biol. 181,41-62 [Medline] [Order article via Infotrieve]
  24. Shafritz, D. A. (1988) Semin. Liv. Dis. 8,285-292 [Medline] [Order article via Infotrieve]
  25. Blitzer, B. L., and Donovan, C. B. (1984) J. Biol. Chem. 259,9295-9301 [Abstract/Free Full Text]
  26. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  27. Cheung, P. H., Thompson, N. L., Earley, K., Culic, O., Hixson, D., and Lin, S-H. (1993) J. Biol. Chem. 268,6139-6146 [Abstract/Free Full Text]
  28. Suchy, F. J., Ballistreri, W. F., Heubi, J. E., Searcy, J. E., and Levin, R. S. (1981) Gastroenterology 80,1037-1041 [Medline] [Order article via Infotrieve]
  29. Shneider, B. L., Dawson, P. E., Chrisitie, D. M., Hardikar, W., Wong, M. M., and Suchy, F. J. (1995) J. Clin. Invest. 95,745-754 [Medline] [Order article via Infotrieve]
  30. Kato, S., Oda-Tamai, S., and Akamatsu, N. (1988) Biochem. J. 253,59-66 [Medline] [Order article via Infotrieve]
  31. Oda-tamai, S., Kato, S., and Akamatsu, N. (1989) Biochem. J. 261,371-375 [Medline] [Order article via Infotrieve]
  32. Oda-tamai, S., Kato, S., and Akamatsu, N. (1991) Biochem. J. 280,179-185 [Medline] [Order article via Infotrieve]
  33. Nunez, E., and Aragon, C. (1994) J. Biol. Chem. 269,16920-16924 [Abstract/Free Full Text]
  34. Sippel, C. J., Fallon, R. J., and Perlmutter, D. H. (1994) J. Biol. Chem. 269,19539-19545 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.