Department of Pharmaceutics, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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The objectives of our study were to identify the types of nucleoside transporters present in the human fetal small intestine and to characterize their developmental activity, longitudinal distribution, and transport kinetics compared with those present in the adult intestine. Nucleoside uptake by intestinal brush-border membrane vesicles was measured by an inhibitor-stop rapid filtration technique. Only the purine-specific (N1; hCNT2) and the pyrimidine-specific (N2; hCNT1) Na+-dependent nucleoside transporters were found to be present on the brush-border membranes of the enterocytes along the entire length of the fetal and adult small intestines. The activity of these transporters was higher in the proximal than in the distal small intestine. Both the N1 and N2 transporters found in the fetal intestine shared similar kinetic properties (Michaelis-Menten constant and Na+-nucleoside stoichiometry) to those in the adult intestine. During the period of rapid morphogenesis (11-15 wk gestation), no temporal differences were apparent in the activity of the N1 and N2 transporters in the fetal small intestine. These findings have implications for the absorption of drugs from the amniotic fluid by the fetus after maternal drug administration of nucleoside drugs such as the antivirals zidovudine and didanosine.
Na+-dependent nucleoside transporters; fetal intestine; adult intestine; antiviral nucleoside drugs; longitudinal distribution
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INTRODUCTION |
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NUCLEOSIDES ARE ESSENTIAL for cell survival because they play a pivotal role in a variety of key cellular processes. Because de novo synthesis of nucleosides is an energy-intensive process, cells fulfill their nucleoside requirement by salvage from both inside and outside the cell. To do so, they use one or more of a family of membrane nucleoside transporters. To date, five Na+-dependent concentrative (N1, N2, N3, N4, and csg) transporters and two Na+-independent equilibrative (es and ei) transporters have been identified (7). These transporters exhibit differential requirements for a Na+ gradient, substrate specificity, and sensitivity to S-(p-nitrobenzyl)-6-thioinosine [nitrobenzylmercaptopurine riboside (NBMPR)]. Briefly, N1 (hCNT2) and N2 (hCNT1) selectively transport purine and pyrimidine nucleosides, respectively, whereas N3 has broad substrate selectivity, accepting both purine and pyrimidine nucleosides as permeants. N4 has similar substrate specificity to N2 except that it also transports guanosine. Uridine and adenosine are permeants of N1-N4 transporters. The csg transporter is unique in that it is the only Na+-dependent transporter that is highly sensitive to inhibition by low concentrations of NBMPR and dipyridamole (DPA) (5). The permeant selectivity of this transporter, found in isolated human leukemic cells, has not been clearly established. In contrast, both of the equilibrative nucleoside transporters, es (hENT1) and ei (hENT2), exhibit a broad substrate specificity, accepting both purine and pyrimidine nucleosides as permeants. However, es is sensitive to inhibition at low concentrations of NBMPR (IC50 ~ 0.4 nM), whereas ei is unaffected by NBMPR up to 1 µM (IC50 ~ 2.8 µM) (24).
Enterocytes have a limited capacity for de novo nucleoside synthesis. Therefore, they rely heavily on nucleoside transporters to salvage nucleosides from their environment to meet their metabolic demands. We have previously established, using Xenopus oocytes microinjected with mRNA isolated from the human adult intestine, that the human adult intestine expresses two Na+-nucleoside transporters, N1 and N2, and two Na+-independent transporters, es and ei (3). Although both N1 and N2 Na+-nucleoside transporters are found on the apical surface of adult jejunal enterocytes (17), the Na+-independent transporters, es and ei, are not present there. In the same study, we reported that the Na+-nucleoside transporters have greater activity in the jejunum than in the ileum.
In this study, we have determined whether nucleoside transporters are present in the human fetal intestine, the types of transporters found there, and whether their activity is developmentally regulated. In addition, we have conducted a detailed study to determine the longitudinal distribution of the nucleoside transporters along the entire length of the human adult and fetal intestine. Such information is of fundamental biological and pharmacological interest. With the rise in the incidence of viral diseases (e.g., AIDS) in pregnant women, there has been an increase in the use of antiviral nucleoside drugs in this population. Anti-AIDS nucleoside drugs such as zidovudine, didanosine, dideoxycytidine, and stavudine readily cross the placenta and accumulate in the amniotic fluid (16, 19, 22, 23). Thus the amniotic fluid serves as a reservoir from which drug can be absorbed from the fetal intestine. Understanding the types and activity of nucleoside transporters found in the intestine during the course of fetal development might shed some light on the extent of exposure of the fetus to nucleoside drugs after maternal administration. In addition, detailed characterization of the types and activity of nucleoside transporters found in the human adult intestine will also provide critical data for optimization of controlled-release formulations of nucleoside drugs.
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MATERIALS AND METHODS |
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Chemicals
[5-3H]uridine (23.6 Ci/mmol), [8-3H]guanosine (14.7 Ci/mmol), and [methyl-3H]thymidine (20 Ci/mmol) were purchased from Moravek Biochemicals, (Brea, CA). Nucleoside analogs valinomycin and phloridzin were obtained from Sigma Chemical (St. Louis, MO). All other chemicals were of the highest analytical grade available.Procurement of Human Intestines
This study was approved by the institutional review board of the University of Washington.Fetal intestines.
Human fetal small intestines of various gestational ages (9-18 wk
old) were collected from normal abortuses of both sexes, immediately
snap frozen in liquid nitrogen, and then stored at 70°C until use.
Five to twenty-one fetal intestines of similar gestational age (within
an age range of 14 days) were pooled for each transport experiment.
Except for longitudinal activity studies, in which fetal intestines
(from duodenum to cecum) were divided into two equal segments, proximal
and distal, brush-border membrane vesicles (BBMV) were isolated from
whole fetal intestines.
Adult intestines.
Human intestines (from the ligament of Treitz to the cecum) were
obtained from breathing adult organ donors (victims of vehicular or
cerebrovascular accidents in otherwise good health) of both sexes.
After the duodenum (the first foot) was separated, the small intestines
were divided into two equal segments, with the proximal half
representing the jejunum and the distal half the ileum. The jejunal and
ileal segments were subdivided into 1-foot segments, each of which was
rinsed with ice-cold saline solution to remove luminal debris. The last
2-3 feet of the intestine was defined as the distal ileum, and the
last foot was defined as the terminal ileum. The tissues were stored at
70°C until use. For longitudinal activity studies, BBMV were
prepared separately from the duodenum (1st foot), proximal jejunum (3rd
foot), midpoint of the small intestine (usually 7th-9th foot), and
distal ileum (last 2-3 feet) of each subject (total of 5 subjects)
on the same day.
Preparation of BBMV
Fetal and adult intestines were thawed on ice, and BBMV were purified by the Mg2+ precipitation method as described previously (17). Membrane potential was clamped using the K+ ionophore valinomycin (3 µM). The purity of these preparations was routinely determined by assaying enzyme marker activities for the apical (alkaline phosphatase) and basolateral (ouabain-sensitive K+-phosphatase) membranes in both the starting homogenates and the vesicle suspensions.Transport Studies
Uptake studies were performed under zero-trans conditions by the rapid filtration technique as described previously (17). The exact composition of the resuspension buffers and the incubation media are given in the legends to the figures. To determine whether Na+-dependent nucleoside transporters were present in the fetal BBMV, the uptake of [3H]uridine (2 µM) by fetal BBMV was measured in the presence and absence of a Na+ gradient (150 mM, out > in) as a function of time. In the latter case, an equivalent amount of KCl was used instead of NaCl. To distinguish the types of nucleoside transporters present along the adult and fetal intestine, we measured the uptake of [3H]guanosine (1 µM) or [3H]thymidine (1 µM) as prototypical purine and pyrimidine nucleoside substrates, respectively, by the intestinal BBMV in the presence and absence of inhibitors, including inosine (100 µM), cytidine (100 µM), NBMPR (1 µM), and DPA (10 µM) in Na+-containing or Na+-free media. Where applicable, the net Na+-dependent uptake of the labeled nucleosides was calculated as the difference in uptake in the presence and absence of Na+. Because NBMPR and DPA, inhibitors of es and ei, respectively, were dissolved in N,N-dimethylformamide (DMF, final concentrationData Analysis
Kinetic parameters of transport were estimated by applying least-square nonlinear regression analysis (WinNONLIN) to tracer displacement curves according to the following equation (4)
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Statistical Analysis
The data were analyzed using one-way ANOVA with an alpha set at 0.05. When a significant F-ratio was detected, a Student's t-test with Bonferroni correction (for multiple comparisons) was performed to detect which treatments were significantly different from the corresponding control (tracer only). ![]() |
RESULTS |
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Purity of Human Intestinal Brush-Border Membranes
Protein content was 6.0-14.4 mg protein/ml for adult BBMV and 4.4-16.9 mg protein/ml for fetal BBMV. The activity of brush-border marker enzyme alkaline phosphatase was enriched 12- to 34-fold and 12- to 19-fold for adult and fetal BBMV, respectively.Time Course of Uridine Uptake by Fetal BBMV
Na+-dependent transport of nucleosides by fetal BBMV was evident as early as 11-12 wk (82 ± 1 days; n = 9) of gestation. In the presence of a Na+ gradient (150 mM, out > in), [3H]uridine (2 µM) was transiently taken up by fetal BBMV against its own concentration gradient and peaked at 120 s (Fig. 1). When Na+ was replaced by K+, the overshoot phenomenon was abolished. The close agreement between the uptake rates at equilibrium (~30 min) in the presence and absence of a Na+ gradient suggested that there was no loss in the integrity of the BBMV. Because nucleoside uptake was found to be linear up to at least 20 s, and this time period allowed a greater differentiation from passive diffusion when compared with uptake at 10 s, this time was used in all experiments conducted with fetal intestinal BBMV.
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Identity of Nucleoside Transporters Along the Human Adult and Fetal Small Intestine
The profiles of inhibition of the uptake of [3H]thymidine (2 µM) or [3H]guanosine (2 µM) by the fetal BBMV were similar at both the 12th (81 ± 2 days; n = 21) and 18th (122 ± 5 days; n = 5) week of gestation (the findings for the latter age group are depicted in Fig. 2). Briefly, the net Na+-dependent uptake of [3H]guanosine (2 µM) by fetal BBMV was completely inhibited in the presence of inosine (100 µM) but was not significantly inhibited by cytidine (100 µM). Conversely, the net Na+-dependent uptake of [3H]thymidine (2 µM) was significantly inhibited by cytidine (100 µM;
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In the human adult small intestine, the profiles of inhibition of
uptake of [3H]thymidine (1 µM) or
[3H]guanosine (1 µM) by BBMV were similar to those
observed with the fetal intestinal BBMV. For the sake of brevity, data
obtained from the duodenum (1st foot) or the distal ileum (last
2-3 feet) of a human adult small intestine are illustrated in Fig.
3. Net Na+-dependent uptake
of [3H]guanosine was almost completely inhibited (90%
inhibition) in the presence of inosine (100 µM), and that of
[3H]thymidine was significantly inhibited (
72%
inhibition) by cytidine (100 µM). Neither NBMPR (1 µM) nor DPA (10 µM) inhibited Na+-dependent (NBMPR only) or
Na+-independent uptake of [3H]guanosine or
[3H]thymidine by adult BBMV. These profiles were
qualitatively replicated when BBMV isolated from the proximal jejunum
(3rd foot) and the midpoint (7th foot) of the intestines of the same
individual were examined or when uptake into BBMV isolated from these
four regions was examined in two additional individuals. Collectively,
N1 and N2 transporters are found on the brush-border membrane along the entire length of the adult small intestine. However, none of the other
nucleoside transporters, namely N3, N4, csg, es,
and ei is present there.
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Kinetics of Nucleoside Transport
To characterize the nucleoside transporters found in the fetal intestine at different gestational ages, uptake of a [3H]nucleoside (1 µM) into BBMV (2 separate batches per gestational age) isolated from 11- to 12- (76 ± 3 days; n = 11 and 15) and 14- to 15- (100 ± 3 days; n = 9 and 10) wk-old fetuses was measured in the presence of increasing concentrations of the corresponding unlabeled nucleoside and a constant Na+ gradient (150 mM, out > in). The uptake of both [3H]guanosine and [3H]thymidine obeyed the Michaelis-Menten relationship, suggesting that transport kinetics of N1 and N2 are saturable. In addition, the close agreement of the Km values obtained using the fetal and the adult BBMV (Table 1) suggested that the fetal N1 and N2 transporters had similar affinities for their permeants compared with the adult N1 and N2 transporters, respectively. The lower Vmax values may have been due to a lower yield of BBMV from the fetal tissue per milligram of protein or may reflect a lower maximal activity of these transporters in fetal intestines.
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The stoichiometric relationship of Na+ to nucleoside uptake by BBMV obtained from 14- to 15-wk-old fetal intestines was estimated using the Na+-activation method. Increasing the concentration of NaCl in the extravesicular medium (isosmolarity maintained with choline chloride) produced a hyperbolic stimulation in the uptake rate of [3H]guanosine and [3H]thymidine. To estimate the Hill coefficient, the Hill equation (21) was fitted to the data. The Na+-nucleoside stoichiometry was found to be 1:1 (Table 1), which suggests transport of a single nucleoside molecule with a single Na+ ion.
Longitudinal Distribution of Nucleoside Transporters Along the Human Adult and Fetal Small Intestines
The proximal-distal distribution of the N1 and the N2 Na+-nucleoside transporters along the fetal small intestines (16-18 wk old or 116 ± 5 days; n = 6) demonstrated a modest gradient; transporter activity was higher in the proximal half compared with the distal half (Fig. 4). In the adult intestine, the proximal-distal distribution of the N1 and the N2 Na+-nucleoside transporters, as illustrated by [3H]guanosine and [3H]thymidine uptake, respectively, demonstrated considerable intersubject variability in magnitude and pattern (Fig. 5). In general, the N1 transporter activity increased (by 1.7-fold in most cases) from the duodenum (1st foot) to the proximal jejunum (3rd foot) and then declined toward the distal ileum. In some individuals the proximal-distal distribution of the N2 transporter activity paralleled that of the N1 transporter, whereas in other individuals the N2 transporter activity increased along the length of the intestine and reached a peak in the distal ileum.
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DISCUSSION |
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This is the first study to examine the ontogenic profile of nucleoside transporters in the human fetal small intestine and to compare the type, functional characteristics, and regional activity of these transporters to those found in the adult small intestine. We have used 11- to 18-gestational-wk-old fetal intestines because this represents a period of the most rapid morphogenic development. Previous studies have shown that during the first few weeks of gestation, the fetal intestinal epithelium consists of proliferative, undifferentiated cells 2-3 cell layers thick (9, 15). There is no evidence of villi in the fetus at ~7 wk old (6). Villus formation begins at 8-10 wk of gestation and progresses from the duodenum toward the colon. Similarly, cellular differentiation of the intestinal epithelium to form various cell types including enterocytes begins as early as 8-10 wk of gestation and progresses in a proximal-to-distal direction. At 20 wk of gestation, the colonic villi start to disappear and the structure of enterocytes in the fetal jejunum resembles those found during adulthood (14). We initially attempted to isolate BBMV from fetal small intestine of younger age (8 wk old) by the Mg2+ precipitation method. However, because of extremely low yield of BBMV and the limited supply of 8- to 10-wk-old intestinal tissues, functional assay for nucleoside uptake could not be performed. Therefore, data obtained from an older gestational age (11-18 wk old) only are reported. For ethical reasons, fresh fetal intestinal tissue of gestational age greater than 18-20 wk cannot be obtained.
Na+-dependent uptake of nucleosides across the brush-border membrane of the fetal small intestine was clearly evident at an early stage of morphogenesis (11-12 wk of gestation; Fig. 1). Furthermore, we identified the existence of two functionally distinct subtypes of nucleoside transporters that behaved identically to the purine-specific (N1) and pyrimidine-specific (N2) Na+-dependent nucleoside transporters found in the adult intestine (Figs. 2 and 3). In addition, similar to the adult small intestine, functional activities of other Na+-dependent nucleoside transporters (N3, N4, and csg) and equilibrative transporters (es and ei) were not detected on the brush-border membrane of early differentiating (12 wk old) and maturing (18 wk old) fetal intestines. These findings suggest that the nucleoside transporters and other apical membrane proteins such as glucose transporters (10, 13), amino acid transporters (12), alkaline phosphatases (11), and disaccharidases (6) are expressed during early stages of intestinal morphogenesis. However, unlike alkaline phosphatase (2) and sucrase-isomaltase (1), for which complex differences exist between the fetal and the adult isoforms, there are no fetal-specific isoforms of the Na+-dependent transporters. The fetal N1 and N2 transporters exhibited identical substrate selectivity and high affinity for their respective prototypical substrates compared with their respective adult counterparts (Table 1). The Km values obtained from both early differentiating (11-12 wk old) and maturing (14-15 wk old) fetal intestines did not differ from those obtained from the adult intestines (Table 1). In addition, the close-to-unity values of the Hill coefficient suggest that, like the adult N1 and N2 transporters, the N1 and N2 transporters present in the fetal small intestine have a Na+:nucleoside stoichiometry of 1:1. In addition, these nucleoside transporters had a similar affinity for Na+ to their adult counterparts (Table 1). Hence, the N1 and N2 transporters present in the fetal small intestines share the same kinetic characteristics as the respective transporters found in adult intestines. Moreover, the lack of difference between the kinetics of nucleoside transport across BBMV isolated from early differentiating (11-12 wk old) and maturing (14-15 wk old) fetal intestines suggests that there is no age-specific regulation of the types and characteristics of the Na+-nucleoside transporters in the human fetal intestine during this period.
The distribution of the N1 and N2 transporters in the fetal intestine showed a modest proximal-distal gradient; transport activities in the proximal region were slightly higher than those in the distal small intestine. Limited availability of tissue precluded us from examining this gradient in detail. A similar proximal-distal gradient was also demonstrated for the adult N1 and N2 transporters. The indicated transporter activity pattern was found at subsaturating and saturating concentrations (when Vmax is observed) in both the adult and the fetal intestine (data not shown). Since Vmax is independent of the affinity of the transporter for its substrate but is dependent on the density of the transporters, we speculate that it is the density of the transporters that changes along the length of the intestine. The availability of specific antibodies to these transporters will help confirm this hypothesis. Such a proximal-distal gradient in transporter activity in the human intestine has been observed by others for glucose (8) and biotin (20). Similar to the N1 and N2 Na+-nucleoside transporters, glucose transport activity was found to be highest in the distal jejunum and lowest in the distal ileum (8). This gradient in glucose transport activity was a result of a decreased density of transporter expression as measured by Vmax. In contrast, transport activity of biotin was found to decrease in the order duodenum > jejunum > ileum. Thus the activity of distinct transporters in the adult human intestine demonstrates different patterns of distribution.
This is the first report demonstrating that the N1 and N2 Na+-nucleoside transporters are present in the human fetal intestinal brush-border membrane. Moreover, the types of transporters present in 100- to 120-day-old fetal intestine were uniform, with a modest gradation in activity along the length of the intestine. Similarly, the human adult intestine demonstrated only a modest proximal-distal gradient in activity, with the highest activity residing in the jejunum. These results should be useful in developing site-directed delivery of nucleoside drugs, which are absorbed in the human intestine via transport by the nucleoside transporters [e.g., ribavirin (18)]. In addition, the presence of nucleoside transporters in the fetal intestine may have implications for the absorption of drugs from the amniotic fluid after maternal administration of nucleoside drugs.
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ACKNOWLEDGEMENTS |
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We thank Glenda Schneider for her excellent technical assistance in conducting the uptake studies. Fetal tissue was supplied by the Birth Defect Laboratory of the University of Washington.
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FOOTNOTES |
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* L. Y. Ngo and S. D. Patil contributed equally to this research.
This research was supported in part by grants from the National Institute of Health (HD-27438 and GM-54447).
Present address for L. Y. Ngo: Schering-Plough Research Institute, 2015 Galloping Hill Rd., K-15-1-1450, Kenilworth, NJ 07033.
Present address for S. D. Patil: Knoll Pharmaceuticals, 3000 Continental Dr. North, Mt. Olive, NJ 07828.
Address for reprint requests and other correspondence: J. D. Unadkat, Dept. of Pharmaceutics, Box 357610, H272 Health Sciences Bldg., Univ. of Washington, Seattle, WA, 98195 (E-mail: jash{at}u.washington.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 June 2000; accepted in final form 21 September 2000.
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