Am J Physiol Gastrointest Liver Physiol
280: G475–G481, 2001.
Ontogenic and longitudinal activity of Na⫹-nucleoside
transporters in the human intestine
LEOCK Y. NGO*, SHIVAKUMAR D. PATIL*, AND JASHVANT D. UNADKAT
Department of Pharmaceutics, University of Washington, Seattle, Washington 98195
Received 27 June 2000; accepted in final form 21 September 2000
Na⫹-dependent nucleoside transporters; fetal intestine;
adult intestine; antiviral nucleoside drugs; longitudinal distribution
for cell survival because they
play a pivotal role in a variety of key cellular processes.
Because de novo synthesis of nucleosides is an energyintensive 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 [nitrobenzylmercaptoNUCLEOSIDES ARE ESSENTIAL
* L. Y. Ngo and S. D. Patil contributed equally to this research.
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@u.
washington.edu).
http://www.ajpgi.org
purine 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 ␮M (IC50 ⬃ 2.8 ␮M) (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 costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
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solely to indicate this fact.
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society
G475
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Ngo, Leock Y., Shivakumar D. Patil, and Jashvant D.
Unadkat. Ontogenic and longitudinal activity of Na⫹-nucleoside transporters in the human intestine. Am J Physiol
Gastrointest Liver Physiol 280: G475–G481, 2001.—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 purinespecific (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.
G476
ONTOGENY OF HUMAN INTESTINAL NA⫹-NUCLEOSIDE TRANSPORTERS
MATERIALS AND METHODS
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 twentyone 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 ␮M). 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 ␮M) 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 ␮M)
or [3H]thymidine (1 ␮M) as prototypical purine and pyrimidine nucleoside substrates, respectively, by the intestinal
BBMV in the presence and absence of inhibitors, including
inosine (100 ␮M), cytidine (100 ␮M), NBMPR (1 ␮M), and
DPA (10 ␮M) 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,Ndimethylformamide (DMF, final concentration ⱕ 0.25% vol/
vol), appropriate controls (solvent only) were conducted for
every experiment. DMF (ⱕ0.25% vol/vol) did not significantly
affect the uptake rate of permeants (P ⬎ 0.05). Unless otherwise indicated, 10-␮l aliquots of BBMV were incubated at
room temperature with 40 ␮l of incubation medium (pH 7.4)
for 10 s for adult BBMV and 20 s for fetal BBMV. Reactions
were terminated by the addition of 3 ml ice-cold STOP solution (containing, in mM: 225 NaCl, 50 HEPES-Tris buffer,
0.1 MgSO4, and 1 phloridzin), followed by three washes each
with 3 ml ice-cold STOP solution.
Data 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)
v* ⫽
V max 䡠 T
⫹ kD 䡠 T
K m ⫹ S cold ⫹ T
where v* is the initial rate of tracer uptake, T and Scold are
the tracer and unlabeled substrate concentrations, respectively, Km is the Michaelis-Menten constant, Vmax is the
maximum velocity, and kD is the rate constant of passive
diffusion.
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
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
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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. AntiAIDS 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.
ONTOGENY OF HUMAN INTESTINAL NA⫹-NUCLEOSIDE TRANSPORTERS
G477
Time Course of Uridine Uptake by Fetal BBMV
Fig. 1. Time course of [3H]uridine (2 ␮M) uptake in the brush-border
membrane vesicles (BBMV) isolated from the small intestines of 11- to
12-wk-old human fetuses (82 ⫾ 1 days; n ⫽ 9). The vesicles were
resuspended in 50 mM HEPES-Tris buffer (pH 7.4) containing 0.1 mM
MgSO4, 225 mM KCl, and valinomycin (3 ␮M). The final concentrations
in the incubation media were 50 mM HEPES-Tris buffer (pH 7.4)
containing 0.1 mM MgSO4, 2 ␮M [3H]uridine, and either 150 mM NaCl
with 75 mM KCl (■) or 225 mM KCl (F). An aliquot (10 ␮l) of BBMV was
incubated with 20 ␮l of incubation medium at room temperature at
various time intervals. Points shown are means of duplicate data
obtained from 1 batch of BBMV. Inset: uptake up to 40 s.
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.
Identity of Nucleoside Transporters Along the Human
Adult and Fetal Small Intestine
The profiles of inhibition of the uptake of [3H]thymidine (2 ␮M) or [3H]guanosine (2 ␮M) 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 ␮M) by fetal BBMV was completely
inhibited in the presence of inosine (100 ␮M) but was
not significantly inhibited by cytidine (100 ␮M). Conversely, the net Na⫹-dependent uptake of [3H]thymidine (2 ␮M) was significantly inhibited by cytidine (100
␮M; ⱖ95% inhibition) but not significantly inhibited by
inosine (100 ␮M). These data suggest that two distinct
Na⫹-dependent N1 and N2 transporters are present on
Fig. 2. Representative inhibition profiles of uptake of [3H]guanosine (2 ␮M; A) and [3H]thymidine (2 ␮M; B) by BBMV
in the presence (solid bars) and absence (open bars) of a Na⫹ gradient. BBMV were isolated from 18-wk-old fetal small
intestines (122 ⫾ 5 days old; pooled from 5 intestines). The vesicles were resuspended in 50 mM HEPES-Tris buffer (pH
7.4) containing 0.1 mM MgSO4, 225 mM KCl, and valinomycin (3 ␮M). The final concentrations in the incubation media
were 50 mM HEPES-Tris buffer (pH 7.4) containing 0.1 mM MgSO4, 2 ␮M 3H substrate, and either 150 mM NaCl with
75 mM KCl (solid bars) or 225 mM KCl (open bars) in the absence (tracer) or presence of various inhibitor concentrations as
indicated. An aliquot (10 ␮l) of BBMV was incubated with 40 ␮l of incubation medium at room temperature for 20 s. Valuese
are means ⫾ SD of triplicate determinations. Values shown above for nitrobenzylmercaptopurine riboside (NBMPR) and
dipyridamole (DPA) inhibition were after correction for the effect of solvents. *P ⬍ 0.05 vs. tracer alone.
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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 ␮M) 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.
G478
ONTOGENY OF HUMAN INTESTINAL NA⫹-NUCLEOSIDE TRANSPORTERS
Fig. 3. Representative profiles of inhibition of uptake of [3H]guanosine (1 ␮M; left) and [3H]thymidine (1 ␮M; right) by BBMV in the presence (solid
bars) and absence (open bars) of a Na⫹ gradient.
BBMV were isolated from the duodenum (A) and
distal ileum (B) of an adult small intestine. Resuspending buffers and incubation media were identical to those described in the legend to Fig. 2 except
that 1 ␮M of 3H-labeled substrates was used and
initial uptake rate was measured at 10 s. *P ⬍ 0.05
vs. tracer alone.
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.
Kinetics of Nucleoside Transport
To characterize the nucleoside transporters found in
the fetal intestine at different gestational ages, uptake
of a [3H]nucleoside (1 ␮M) 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
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the brush-border membrane of the fetal small intestine. The lack of partial inhibition of Na⫹-dependent
[3H]guanosine uptake by cytidine (100 ␮M) ruled out
the possibility of the existence of N3 and N4. In addition, NBMPR (1 ␮M) and DPA (10 ␮M) did not have an
inhibitory effect on the uptake of [3H]guanosine and
[3H]thymidine in both Na⫹-containing and Na⫹-free
media. These data suggest that Na⫹-dependent csg
and Na⫹-independent es and ei transporters are not
present on the brush-border membrane of fetal enterocytes.
In the human adult small intestine, the profiles of
inhibition of uptake of [3H]thymidine (1 ␮M) or
[3H]guanosine (1 ␮M) 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 (ⱖ90% inhibition) in the presence of
inosine (100 ␮M), and that of [3H]thymidine was significantly inhibited (ⱖ72% inhibition) by cytidine (100
␮M). Neither NBMPR (1 ␮M) nor DPA (10 ␮M) inhibited Na⫹-dependent (NBMPR only) or Na⫹-independent uptake of [3H]guanosine or [3H]thymidine by
ONTOGENY OF HUMAN INTESTINAL NA⫹-NUCLEOSIDE TRANSPORTERS
Table 1. Summary of kinetic parameters of human
intestinal N1 and N2 transporters
Fetal
Guanosine
Km
Vmax
KNa
Hill coefficient
Thymidine
Km
Vmax
KNa
Hill coefficient
11–12 wk
14–15 wk
Adult
10.7, 14.1
9.8, 2.8
14.5, 6.09
5.7, 5.7
9.38
1.08
12.0 ⫾ 1.34
63.9 ⫾ 10.9
13.0 ⫾ 2.5
1.20 ⫾ 0.12
2.06, 3.22
5.8, 6.4
5.56, 3.20
3.8, 3.8
49.6, 40.5
0.98, 0.94
2.74 ⫾ 0.54
16.1 ⫾ 3.64
26.7 ⫾ 3.5
0.95 ⫾ 0.08
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.
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 proximaldistal 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.
DISCUSSION
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
Fig. 4. [3H]guanosine (A) and [3H]thymidine (B) uptake by BBMV isolated
from the proximal and distal parts of
16- to 18-wk-old human fetal small intestines (116 ⫾ 5 days old; pooled from
6 intestines). The BBMV were resuspended in 50 mM HEPES-Tris buffer
(pH 7.4) containing 0.1 mM MgSO4,
225 mM KCl, and valinomycin (3 ␮M).
The final concentrations in the incubation media were 50 mM HEPES-Tris
buffer (pH 7.4) containing 0.1 mM
MgSO4, 1 ␮M 3H substrate, and either
150 mM NaCl with 75 mM KCl or 225
mM KCl. An aliquot (10 ␮l) of BBMV
was incubated with 40 ␮l of incubation
medium at room temperature for 20 s.
Values are means ⫾ SD of triplicate
determinations of net Na⫹-dependent
uptake.
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Values for fetal parameters are best fit means from least square
nonlinear regression analyses of the data obtained from 2 different
batches of pooled vesicles. Values for adult parameters are means ⫾
SD of 3–4 individuals (17). Km, Michaelis-Menten constant (␮M);
Vmax, maximal transport activity for nucleoside uptake (pmol/mg
protein/10 s); KNa, Na⫹ concentration required to achieve half of the
maximal uptake velocity in a Na⫹-activation study (mM).
G479
G480
ONTOGENY OF HUMAN INTESTINAL NA⫹-NUCLEOSIDE TRANSPORTERS
Fig. 5. Regional uptake of [3H]guanosine
(A) and [3H]thymidine (B) by BBMV isolated from various sections along the
length of the human adult small intestine
(n ⫽ 5). Resuspending buffers and incubation media were identical to those described in the legend to Fig. 4 except that
initial uptake rate was measured at 10 s.
Values are means ⫾ SD of triplicate determinations of net Na⫹-dependent uptake. Data for N1 (A) and N2 (B) nucleoside transport activity in any one
individual are represented by the same
symbol.
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
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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–
ONTOGENY OF HUMAN INTESTINAL NA⫹-NUCLEOSIDE TRANSPORTERS
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.
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.
REFERENCES
1. Auricchio S. Brush border enzymes. In: Pediatric Gastroenterology and Hepatology (3rd ed.), edited by Gracey M and Burke V.
Boston: Blackwell Scientific, 1993, p. 176–199.
2. Behrens CM, Enns CA, and Sussman HH. Characterization
of human foetal intestinal alkaline phosphatase. Comparison
with the isoenzymes from the adult intestine and human tumour
cell lines. Biochem J 211: 553–558, 1983.
3. Chandrasena G, Giltay R, Patil SD, Bakken A, and Unadkat J. Functional expression of human intestinal Na⫹-dependent and Na⫹-independent nucleoside transporters in Xenopus
laevis oocytes. Biochem Pharmacol 53: 1909–1918, 1997.
4. Chenu C and Berteloot A. Allosterism and Na(⫹)-D-glucose
cotransport kinetics in rabbit jejunal vesicles: compatibility with
mixed positive and negative cooperativities in a homodimeric or
tetrameric structure and experimental evidence for only one
transport protein involved. J Membr Biol 132: 95–113, 1993.
5. Flanagan SA and Meckling-Gill KA. Characterization of a
novel Na⫹-dependent, guanosine-specific, nitrobenzylthioinosine-sensitive transporter in acute promyelocytic leukemia cells.
J Biol Chem 272: 18026–18032, 1997.
6. Grand RJ, Watkins JB, and Torti FM. Development of the
human gastrointestinal tract. A review. Gastroenterology 70:
790–810, 1976.
7. Griffith DA and Jarvis SM. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1286:
153–181, 1996.
8. Harig JM, Barry JA, Rajendran VM, Soergel KH, and
Ramaswany K. D-Glucose and L-leucine transport by human
intestinal brush-border membrane vesicles. Am J Physiol Gastrointest Liver Physiol 256: G618–G623, 1989.
9. Klein RM. Small intestinal cell proliferation during development. In: Human Gastrointestinal Development, edited by E.
Lebenthal. New York: Raven, 1989, p. 367–392.
10. Koldovsky O, Heringova A, Jirsova V, Jirasek JE, and
Uher J. Transport of glucose against a concentration gradient in
everted sacs of jejunum and ileum of human fetuses. Gastroenterology 48: 185–187, 1965.
11. Lev R, Siegel HI, and Bartman J. Histochemical studies of
developing human fetal small intestine. Histochemie 29: 103–
119, 1972.
12. Malo C. Multiple pathways for amino acid transport in brush
border membrane vesicles isolated from the human fetal small
intestine. Gastroenterology 100: 1644–1652, 1991.
13. Malo C and Berteloot A. Proximo-distal gradient of Na⫹dependent D-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine. FEBS Lett
220: 201–205, 1987.
14. Moxey PC and Trier JS. Development of villus absorptive cells
in the human fetal small intestine: a morphological and morphometric study. Anat Rec 195: 463–482, 1979.
15. Moxey PC and Trier JS. Specialized cell types in the human
fetal small intestine. Anat Rec 191: 269–285, 1978.
16. Odinecs A, Nosbisch C, Keller RD, Baughman WL, and
Unadkat JD. In vivo maternal-fetal pharmacokinetics of stavudine (2⬘,3⬘-didehydro-3⬘-deoxythymidine) in pigtailed macaques
(Macaca nemestrina). Antimicrob Agents Chemother 40: 196–
202, 1996.
17. Patil S and Unadkat JD. Sodium-dependent nucleoside transport in the human intestinal brush-border membrane. Am J
Physiol Gastrointest Liver Physiol 272: G1314–G1320, 1997.
18. Patil SD, Ngo LY, Glue P, and Unadkat JD. Intestinal
absorption of ribavirin is preferentially mediated by the Na⫹nucleoside purine (N1) transporter. Pharm Res 15: 952–954,
1998.
19. Pereira CM, Nosbisch C, Winter HR, Baughman WL, and
Unadkat JD. Transplacental pharmacokinetics of dideoxyinosine in pigtailed macaques. Antimicrob Agents Chemother 38:
781–786, 1994.
20. Said HM, Redha R, and Nylander W. Biotin transport in the
human intestine: site of maximum transport and effect of pH.
Gastroenterology 95: 1312–1317, 1988.
21. Segel IH. Enzyme Kinetics. Behaviour and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems. New York:
Wiley-Interscience, 1975.
22. Tuntland T, Nosbisch C, Baughman WL, Massarella J, and
Unadkat JD. Mechanism and rate of placental transfer of
zalcitabine (2⬘,3⬘-dideoxycytidine) in Macaca nemestrina. Am J
Obstet Gynecol 174: 856–863, 1996.
23. Tuntland T, Odinecs A, Nosbisch C, and Unadkat JD. In
vivo maternal-fetal-amniotic fluid pharmacokinetics of zidovudine in the pigtailed macaque: comparison of steady-state and
single-dose regimens. J Pharmacol Exp Ther 285: 54–62, 1998.
[Corrigenda. J Pharmacol Exp Ther 286: September 1998, p.
1495.]
24. Ward JL and Tse CM. Nucleoside transport in human colonic
epithelial cell lines: evidence for two Na⫹-independent transport
systems in T84 and Caco-2 cells. Biochim Biophys Acta 1419:
15–22, 1999.
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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 120day-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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