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(Received for publication, October 28,
1994; and in revised form, December 12, 1994) From the
The regulation of sodium-dependent L-alanine transport
is described for the first time in intestinal cells. Substrate analogue
inhibition patterns and Dixon analyses indicated that uptake occurred
via transport system B, an epithelial cell variant of systems
B
The Na Animal studies have suggested that the intact small intestine can
modify amino acid
uptake(8, 9, 10, 11, 12) ,
although the cellular mechanism is unknown. Up-regulating transport
provides a means to supply developing intestinal epithelial cells with
amino acids during their rapid growth phase along the crypt to the
villous axis. Control of uptake is also means to prevent nutrient
extraction from the environment as the rate-limiting step in whole-body
interorgan amino nitrogen metabolism (2, 3, 4, 9) . Nonetheless, studies
are lacking that describe the regulation of intestinal membrane
transport systems that serve only neutral amino acids. Furthermore, the
lack of a reported well defined regulated neutral amino acid
transporter in intestinal has impeded the successful cloning of such an
epithelial membrane carrier. Re-evaluation of the SAAT1 clone,
originally thought to represent system A, has resulted in its
reassignment as an SGLT2 variant of the sodium/glucose
cotransporter(13, 14) . The purpose of this study
was to investigate the regulation of sodium-dependent L-alanine transport in Caco-2 cells. This established human
cell line is favorably recognized as a useful in vitro model
for intestinal epithelial cell studies because these cells undergo
spontaneous enterocytic differentiation in culture and mimic the in
vivo crypt to villous maturation process following
passaging(15, 16) . It has been shown that the Caco-2
transport characteristics for a variety of ions and organic nutrients
closely resemble those of the intestine or its epithelium (16, 17, 18) . Our results indicate that
Caco-2 cells regulate carrier-mediated sodium-dependent transport of L-alanine by changing the membrane capacity to transport
alanine via system B, and that this regulation involves de novo protein synthesis under the control of protein kinase C. It is
anticipated that the present report describing an in vitro model of up-relatable system B activity may aid in the successful
first cloning of an intestinal carrier polypeptide that exclusively
serves neutral amino acids.
Caco-2 cells were passaged
following treatment with 0.05% trypsin and 0.02% EDTA. Cells were
reseeded at a density of 4.5
Figure 1:
Initial time course of 50 µM and 5 mML-[
The uptake of 50 µM alanine
was measured in uptake media containing NaCl concentrations ranging
from 0 to 137 mM, with choline serving as Na
Figure 2:
Na
Figure 3:
Alanine uptake inhibition by amino acid
analogues in day 3 cells versus day 9 cells. Sodium-dependent
50 µML-[
Figure 4:
Dixon plot of the effect of MeAIB on
sodium-dependent L-[
Figure 5:
The effect of phorbol ester (TPA) dose on
50 µML-[
The phorbol ester effect was
time-dependent, as demonstrated in Fig. 6. Continual incubation
of Caco-2 cells in serum-free media containing 0.5 µM TPA
for at least 6 h was required to stimulate system B activity. Maximal
stimulation was attained by 24 h. Brief pulses (1-15 min) of 0.5
µM TPA in serum-free media, chased by sodium-dependent
alanine uptake measurements during the ensuing 24 h period, were
without affect on system B activity (data not shown).
Figure 6:
The effect of phorbol ester (TPA)
exposure time on alanine uptake. Day 3 cells were exposed to serum-free
DMEM containing or lacking 500 nM TPA for various times, then
50 µML-[
Figure 7:
The
effect of protein synthesis inhibitors and modifiers of protein kinase
C activity on phorbol ester-stimulated or control sodium-dependent
alanine uptake. Cells were exposed to serum-free DMEM containing
phorbol ester (500 nM TPA) and/or other reagents for 24 h, and
then 50 µML-[
Figure 8:
The effect of cell age and phorbol ester
(TPA) on sodium-dependent alanine uptake. Caco-2 cells of increasing
cell age post-passaging were exposed to serum-free DMEM containing or
lacking 500 nM TPA for 24 h, and then 50 µML-[
Figure 9:
Hofstee plot of the effect of cell age and
phorbol ester (TPA) on sodium-dependent alanine uptake kinetics. Cells
2 or 9 days postpassaging were exposed to DMEM lacking or containing
500 nM TPA for 24 h, and then initial uptake rates of L-[
Nonlinear regression analysis of the Na The intestinal regulation of neutral (dipolar or
zwitterionic) amino acid absorption has been predicted by whole animal
and tissue
studies(8, 9, 10, 11, 12) ,
yet the cellular mechanism responsible for transport regulation has not
been reported. The present study represent the first description of the
regulation of neutral amino acid transport by intestinal membranes. Our
results indicate that (i) the predominant sodium-dependent L-alanine uptake system present in cultured intestinal Caco-2
cells is the system B transporter predicted by Christensen (5) to exist in absorptive epithelia; (ii) expression
of system B activity is modulated as a function of Caco-2
differentiation status (e.g. cell age postpassaging); (iii) Caco-2 cells regulate carrier-mediated sodium-dependent
transport of L-alanine by changing the membrane capacity (V
System A is characteristically defined by
exclusive uptake of, or inhibition by MeAIB or AIB(1) . Our
results indicated that the absolute uptake rate of
[ System
B
On the other hand, alanine uptake activities expressed as a function
of cell age (Fig. 2, Fig. 3, and Fig. 8) were
reflected in the nearly 6-fold increase in sodium-dependent system B
transport capacity (V Differentiation-dependent changes
in transporter capacities have been reported for sodium/glucose and
H
The phorbol ester up-regulation of system B activity
involved de novo protein synthesis. Transcription and/or
translation events could be implicated because actinomycin D and
cycloheximide each blocked the increase in system B V
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3582-3587
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
and ASC. System B served >95% of the
Na
-dependent alanine uptake in both undifferentiated
(2 days postpassaging) and differentiated (>9 days postpassaging)
states of the human Caco-2 cultured intestinal cell line.
(Methylamino)isobutyric acid-inhibitable system A transport accounted
for <5% of total alanine uptake. System B activity was greater in
undifferentiated cells compared with the differentiated state, and
activity at any differentiation state was stimulated by
12-O-tetradecanoylphorbol-13-acetate (TPA). The maximal
stimulation, determined by TPA dose-response/exposure time data, was
attributable to a change in cell transport capacity (V
), with K
unaffected. The V of system B was
greater in 2-day-old cells (2.79 ± 0.21 nmol min
mg of protein; K = 164 ± 26 µM alanine),
decreasing to V = 0.51 ± 0.03 nmol min mg of protein (K= 159 ± 14
µM) in 9-day-old cells. Regardless of differentiation
status, the sodium-activation Hill coefficient was 1.06 ± 0.10,
and the alanine passive diffusion permeability coefficient was 0.53
± 0.08 µl min mg of
protein. Phorbol ester up-regulated the V of system B in 2-day-old cells to V = 6.32 ± 0.37 nmol
min mg of protein (K = 169 ± 18
µM), and in 9-day-old cells to V = 1.42 ± 0.05 nmole min mg of protein (K = 180 ± 10 µM). Phorbol ester
stimulation of transport occurred after at least 6 h of continual
exposure, and was blocked by the protein kinase C inhibitors
chelerythrine or photoactivated calphostin C. Protein synthesis
inhibitors cycloheximide and actinomycin D each blocked the phorbol
ester up-regulation of system B activity. It is concluded that Caco-2
cells regulate carrier-mediated sodium-dependent transport of L-alanine by changing the membrane capacity to transport
alanine via system B and that this regulation involves de novo protein synthesis under the control of protein kinase C.
-dependent transport of neutral amino
acids by intestinal cells is catalyzed by variety of transport systems
(for review see (1, 2, 3, 4) ). In
addition to the ``ubiquitous'' transporters strictly serving
neutral amino acids found in cells throughout the body (e.g. systems A, ASC), it is thought that the intestine possess two
systems uniquely characteristic of epithelial membranes, namely the
Imino system and system B, first reported by
us(2, 3, 4, 5, 6, 7) .
Chemicals
Dulbecco's modified
Eagle's medium (DMEM), (
)fetal bovine serum, sodium
bicarbonate, penicillin, streptomycin, nonessential amino acids,
trypsin, EDTA, Me
SO, HEPES, and Tris were of the highest
grade from Sigma. The 0.2-µm filters used to sterilize media were
from Millipore Co. Bedford, MA. L-[
H]Alanine and
[H]MeAIB were obtained from Amersham Corp.
Liquiscint scintillation fluid was from National Diagnostics, Atlanta,
GA. The protein assay reagent was from Bio-Rad. The established human
intestinal epithelial cell line Caco-2 was initially obtained as
passage 16 from American Type Culture Collection, Rockville, MD. The
cells were cultured in 6-well tissue culture dishes (Falcon type 3046),
or 100-mm tissue culture dishes (Falcon type 3003). Chelerythrine
chloride was obtained from LC Services Corporation, Woburn, MA.
Calphostin C was from Kamiya Biomedical Co., Thousand Oaks, CA. All
other reagents were obtained from Sigma. Caco-2 Cell Cultures
Caco-2 stocks (passages
19-40 stored in MeSO under liquid nitrogen), were
harvested from Falcon tissue culture dishes containing DMEM, 4.5
g/liter glucose, 0.584 g/liter glutamine, 10% fetal bovine serum, 3.7%
sodium bicarbonate, 100 IU/ml penicillin, 100 µg/ml streptomycin,
and 1% nonessential amino acids(15, 16, 19) .
Cells were grown in a humidified incubator at 37 °C in 10%
CO/90% O.
10
cells/100-mm dish
for future subculturing, or seeded in the 6-well cluster Falcon tissue
culture dishes at a density of 3.86 10
cells/35-mm
well for transport experiments. The day of seeding was designated as
day 0. The growth medium was changed daily, and cultures were inspected
daily using a phase contrast microscope. Amino Acid Uptake Measurements
Amino acid
uptake was measured in cells ranging in age from 2 days postseeding
(undifferentiated) through 16 days postseeding (differentiated).
Cultures were confluent on about day 5. Studies designed to compare
transport in cells 2-9 days post-seeding were conducted using
cells started from the same seeding parent cells. Transport activity
was measured at room temperature (23 ± 1.0 °C). Following
pretreatment of cells with various agents (described below), cells were
rinsed with ``uptake buffer'' (23 °C) composed of 137
mM NaCl (or choline chloride), 10 mM HEPES/Tris
buffer (pH 7.4), 4.7 mM KCl, 1.2 mM MgSO
,
1.2 mM KHPO, and 2.5 mM CaCl. The uptake was initiated by the addition of 1 ml
of this buffer containing also L-[H]alanine (2 µCi/ml, 1 µM to 5 mM) or [H]MeAIB (2 µCi/ml,
50 µM). Culture dishes were continuously shaken by an
orbital shaker (1 Hz) during the uptake period. Uptake was arrested by
aspirating the uptake buffer and washing 3 times with ice-cold buffer
lacking substrate. Radioactivity of isotope extracted from the cells
with 1 ml 1 N NaOH was neutralized with acetic acid, and then
assayed by liquid scintillation spectrometry. Protein in the NaOH
extract was measured using the Bio-Rad protein assay. Initial rates of
transport activity were determined during the linear uptake period (2
min), with zero time points serving as blanks. Uptake rates are
expressed as mol of alanine/min/mg of cell protein. Treatments
To treat cells with various agents
(cycloheximide, actinomycin D, chelerythrine, calphostin C, phorbol
ester), growth medium was first replaced with serum-free media (i.e. DMEM containing nonessential amino acids, penicillin,
and streptomycin, but lacking fetal bovine serum) for 2 h at 37 °C
in the humidified incubator. The cells were then exposed to each agent
at various times and concentrations described below. Pretreatment
buffers were replenished every 6 h. Caco-2 cells remained healthy
(viability >99% by dye exclusion) during at least 24 h of exposure
to serum-free media. 12-O-Tetradecanoylphorbol-13-acetate
(TPA) and phorbol 12,13-dibutyrate were prepared from MeSO
stocks, giving < 0.5% MeSO in final media exposed to
cells. This concentration of MeSO did not influence uptake. Data Analysis
All experiments were conducted
at least in triplicate (including the zero time blanks), and all
experiments were confirmed using at least two independently passaged
generations of cells. Experimental means are reported ± S.E.
Comparisons of means were made by analysis of variance with pairwise
multiple comparisons by the Newman-Keuls method at p <
0.05. Transport kinetic parameters were obtained by fitting data to the
Michaelis-Menten equation (20) by linear or nonlinear
regression analysis.
Sodium Dependence and Hill Activation
Coefficient
Uptake of L-[H]alanine was linear up to at least
10 min at concentrations of 50 µM or 5 mM in
either 137 mM NaCl or 137 mM choline media (Fig. 1). Alanine uptake measured in media containing 137 mM mannitol or 137 mM chloride or gluconate salts of
K or Li were not significantly
different from rates with choline Cl (p < 0.05; data not
shown). For all subsequent measurements, the
Na-dependent fraction of alanine total uptake was
obtained by subtracting the uptake measured in choline chloride medium
from the total uptake measured in NaCl medium during the linear uptake
period of 0-2 min.
H]alanine
uptake in the presence or absence of Na. In this
example, alanine total uptake was measured in Caco-2 cell cultures 2
days postpassaging in DMEM containing 137 mM NaCl or 137
mM choline chloride. Points are means with
S.E.
substitute. As shown in Fig. 2, alanine uptake rates
increased as a hyperbolic function of NaCl concentration in cells both
2 and 9 days postpassaging, and the sodium-dependent uptake rates were
greater in day 2 cells compared with day 9 cells. Nonlinear regression
analyses of the data fit to the Hill equation (20) gave the
same Na-activation Hill coefficient (n = 1.06 ± 0.10 at each cell age, while the V was greater in day 2 cells than in day 9
cells. For 50 µM alanine uptakes, the day 2 cell V = 525 ± 20 pmol min mg of protein, and 
K
= 10.5 mM; day 9
cell V = 149 ± 10 pmol
min mg of protein, K = 28 mM.
activation of
Na-dependent alanine uptake. Initial uptake rates of
50 µML-[H]alanine were
measured in cells 2 and 9 days postseeding. The uptake media contained
various concentrations of NaCl (with choline replacing sodium).
Sodium-dependent uptake is shown. For both cell ages, the sodium
activation apparent Hill coefficient n = 1.06 ±
0.10. Points are means with S.E.
Amino Acid Analogue Inhibition
The uptake
rates of 50 µML-[H]alanine
were measured in both day 3 and day 9 cells in media containing 137
mM NaCl or choline chloride with 5 mM unlabeled amino
acid analogues (or mannitol control). As Fig. 3indicates, the
relative pattern of inhibition was the same at both cell ages, but
absolute uptake rates were consistently greater in day 3 cells compared
with day 9 cells. Plotting the inhibited alanine uptake rates in day 9
cells as a function of inhibited rates in day 3 cells for all
inhibitors gave a straight line with a slope of 2.5.
Na-dependent [H]alanine
transport was strongly inhibited by cysteine, threonine, serine,
glutamine, and asparagine, with weaker inhibition by other neutral
(dipolar) amino acid analogues (Fig. 3). Dixon analyses of
glutamine (K
= 35 µM), glycine (K = 4.5 mM), and phenylalanine (K = 5.9 mM) inhibition of
sodium-dependent L-[H]alanine uptake
(data not shown) indicated classic competitive inhibition by these
analogues (20) . It is notable that the bicyclo amino acid BCH
partially inhibited sodium-dependent alanine uptake. MeAIB and cationic
amino acids inhibited <10% of sodium-dependent alanine uptake.
H]alanine uptake
was measured in the presence of 5 mM single amino acids.
Linear regression of the points gave the dottedline with slope of 2.5; also shown with dashedlines are the 95% confidence intervals for the regression. Inhibitor
symbols, X, MeAIB; B, BCH; U, AIB; Z, mannitol (with or without 5 mM dithiothreitol); J, cystine; C, cysteine + dithiothreitol; A, Ala; F, Phe; G, Gly; H, His; I, Ile; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln, R, Arg; K, Lys; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
Caco-2 Interactions with
MeAIB
Sodium-dependent 50 µM [H]MeAIB uptake rates measured in day 2
Caco-2 cells (0.014 ± 0.001 pmol min mg of
protein) were at least 2 orders of magnitude lower
than comparable 50 µM [H]alanine
uptake rates (e.g.Fig. 1and Fig. 2).
Furthermore, as shown in Fig. 4, Dixon analysis of the effect of
unlabeled MeAIB on [H]alanine uptake indicated
that there was negligible interaction of MeAIB with system B in Caco-2
cells.
H]alanine uptake.
Radiolabeled alanine uptake was measured at three concentrations in the
presence of increasing concentrations of unlabeled MeAIB in choline and
sodium uptake media. There was no convergence of the lines. Data
represent means ± S.E.
Phorbol Ester Stimulates System B
Activity
Caco-2 cell sodium-dependent alanine transport via
System B was stimulated by TPA and phorbol 12,13-dibutyrate. On the
other hand, dibutryl cAMP (500 µM) and forskolin (10
µM) each failed to influence alanine uptake activity (data
not shown). The dose-response of TPA is shown in Fig. 5. In
Caco-2 cells preincubated in serum-free media, the peak stimulation of
System B activity was observed at [TPA] = 0.5
µM. Similar data were obtained for phorbol
12,13-dibutyrate (data not shown).
H]alanine uptake.
Alanine initial uptake rates were measured in 137 mM Na or choline uptake media after exposing Caco-2
cells (day 2) to various doses of TPA in serum-free DMEM for a 24-h
period. Data represent means ± S.E.
H]alanine initial
uptake rates were measure in Na (
, 
), or
choline (
, 
) uptake media. Data represent means ±
S.E.
Blocked TPA Stimulation of System B
Activity
Sodium-dependent L-[H]alanine uptake rates were
significantly increased by TPA compared with control (p <
0.05), and this effect was abolished by cycloheximide (10
µM) in the incubation medium (Fig. 7), suggesting
that de novo protein synthesis was involved in up-regulating
system B activity. Actinomycin D (500 ng/ml) also blocked the phorbol
ester stimulation of System B, suggesting possible transcription
involvement. Fig. 7also demonstrates that the specific protein
kinase C inhibitors (21, 22) chelerythrine (6.6
µM) or fluorescent light activated-calphostin C (50
nM) in the serum-free incubation media containing TPA during
24 h prior to the uptake experiments and also attenuated the TPA
stimulation of the system B activity (p < 0.05). System B
transport activity in cells with unactivated calphostin C (i.e. no fluorescent light) was not significantly different from TPA
alone (p > 0.05).
H]alanine
uptake rates were measured in sodium and choline uptake media.
Sodium-dependent alanine uptake rates were significantly stimulated by
TPA, compared with control (*, p < 0.05), and this effect
was inhibited by cycloheximide (10 µM), actinomycin D (500
ng/ml), chelerythrine (6.6 µM), or fluorescent
light-activated calphostin C (50 nM). Unactivated calphostin C (i.e. no fluorescent light) did not significantly influence
the stimulatory effect of TPA (*, p > 0.05). Data represent
means ± S.E.
The Effect of Cell Age and Phorbol Ester on
Sodium-dependent Alanine Transport
Fig. 8demonstrates
that sodium-dependent alanine uptake is greatest in undifferentiated,
newly passaged Caco-2 cells, and thereafter declines as the culture
attains confluence (at about day 5-6) and undergoes
differentiation (
day 9). In addition, phorbol ester stimulated
sodium dependent alanine uptake activity at all Caco-2 cell ages from 1
through 35-days-old postpassaging, compared with control cells not
exposed to TPA (Fig. 8). Finally, the data of Fig. 8indicate that the sodium-independent uptake of alanine
remained constant regardless of cell age or TPA exposure.
H]alanine initial uptake rates
were measure in NaCl or choline chloride uptake media. Data represent
means ± S.E.
The Effect of Cell Age and Phorbol Ester on System B
Transport Kinetics
Day 2 and day 9 Caco-2 cells were
preincubated with or without 500 nM TPA in serum-free medium
for 24 h, and then alanine transport kinetics were measured in uptake
media containing 137 mM NaCl or choline chloride over the
alanine concentration range of 1 µM to 5 mM.
Eadie-Hofstee transformations (20) of the sodium-dependent
uptake components are shown for each case in Fig. 9. For all
cases of uptake, regardless of cell age or exposure to TPA, nonlinear
regression analysis of the total uptake in sodium media gave a passive
diffusion permeability coefficient p = 0.53 ±
0.08 µl min mg of protein .
H]alanine in uptake media containing
137 mM Na or choline Cl were measured in a
manner similar to that shown in Fig. 12. The Hofstee plot of the
sodium-dependent component of uptake gave single straight lines with
parallel slopes for each experimental condition. Data represent means
± S.E.
-dependent
component of uptake in day 2 control cells (i.e. not exposed
to TPA) gave a V of 2.79 ± 0.21 nmol
min mg of protein and K
= 164 ± 26 µM alanine. For day 9 cells not exposed to TPA, the V dropped to 0.51 ± 0.03 nmol
min mg of protein, and K remained relatively unaffected at 159 ±
14 µM alanine. For 2-day-old cells exposed to TPA, the V was increased to 6.32 ± 0.37 nmol
min mg of protein, while the K remained unaffected (180 ± 10
µM), relative to cells not exposed to TPA. Finally, in day
9 cells exposed to TPA, V = 1.42 ±
0.05 nmol min mg of protein, and K = 169 ± 18 µM.
) of system B activity; and (iv) that
this regulation involves de novo protein synthesis under the
control of protein kinase C. Assignment of System B
The results indicated that a
single transport system was responsible for sodium-dependent alanine
uptake in Caco-2 cells and that this system was present in both the
undifferentiated and differentiated states (cell age postpassaging).
Alanine uptake was strongly Na-dependent in both day 2
and day 9 Caco-2 cells (Fig. 4); neither K nor
Li substituted for Na in activating
alanine uptake. The sodium activation Hill coefficient of unity (Fig. 2) implicated a 1:1 Na/alanine activation
stoichiometry for secondary active transport (symport) in both day 2
and day 9 cells(23) . The analogue inhibition pattern for both
the day 2 and day 9 cells was the same whether the absolute uptake
rates or transport capacity changed as a function of cell
differentiation status (Fig. 3). Together with the kinetic
analyses giving a shift in V, but not K, with advancing cell age (see
``Results''; Fig. 9), the data indicated that a single
sodium-dependent alanine transport system was regulated in Caco-2
cells. Functional studies in a variety of cell types indicate that
sodium-dependent alanine uptake could possibly be served by several
different transport systems, although all systems do not necessarily
occur in a given cell type. In membranes of intestinal cells, the
primary candidates for this sodium-activated transporter include
systems A, B, B, or
ASC(1, 2, 3, 4) . Our data support
the notion that the sodium-dependent uptake of alanine in Caco-2 cells
was solely by system B.H]MeAIB uptake in Caco-2 cells was several
orders of magnitude less than that of [H]alanine
uptake, and that MeAIB poorly blocked alanine uptake (Fig. 3).
Furthermore, Dixon analysis (Fig. 4) indicated a lack of
competitive interaction between MeAIB and alanine uptake. These
combined observations indicated that system A provides a minimal, if
any, contribution to alanine uptake in Caco-2 cells. is developmentally regulated in blastocysts and
mediates uptake of both cationic and neutral amino acids(24) .
The neutral substrates include BCH and amino acids branching at the
and 
carbon positions(1, 24) . We
previously speculated that B may be ontogenetically
related to system NBB (neutral brush border), a system that is found
only in absorptive epithelia, and which serves neutral but not cationic
amino acids(3, 5) . Several studies have subsequently
shown that the NBB inhibition pattern is expressed uniquely in the
brush-border membranes of renal or intestinal epithelial cells of a
variety of vertebrates and
invertebrates(3, 4, 5, 6, 7, 25) .
Following discussions with Christensen, we subsequently changed our
original naming of ``system NBB'' (6) to
``system B'' (3, 4) to reflect its
relationship to B and to be consistent with the
Christensen nomenclature(5) . It is notable that the analogue
inhibition pattern of Fig. 3was minimally affected by cationic
amino acids, and the neutral analogues inhibited alanine uptake in the
pattern reminiscent of B. The apparent K of about 160-180 µML-alanine measured in Caco-2 cells (Fig. 9) was
similar to that for system B reported for apical membranes isolated
from intestinal epithelial cells(6) . System ASC is a related
pathway found in virtually all cells types, but intestinal ASC activity
is constitutively low, is not regulated, and is apparently restricted
to the basolateral membranes of epithelial
cells(2, 5, 6) . Furthermore, classic system
ASC shows less tolerance to glycine and phenylalanine than the apical
membrane system B(5, 6, 7) . In concert, the
previous and present observations assign system B as the
sodium-dependent alanine uptake pathway in Caco-2 cells. Differentiation-dependent Regulation
Our
results indicated that the simple passive diffusion component of
alanine uptake was the same (p = 0.53 ± 0.08
µl min mg of protein)
regardless of differentiation status (cell age) and that passive
diffusion contributed very little to the total uptake. For example, in
day 2 cells, passive diffusion contributes only about 5% to total
uptake at 160 µM alanine. This indicates that the
mechanism regulating alanine uptake into Caco-2 cells does not involve
modifications in the membranes' passive permeability to alanine. ) in day 2 cells compared
with day 9 cells, with no change in apparent K (Fig. 9). As described above, the
Na-activation Hill number was the same (n = 1.06 ± 0.10) for both day 2 and day 9 cells (Fig. 2), suggesting that the differentiation-dependent
modification of transport activity may not involve modifications in
sodium-activation sites of system B carrier proteins. These
observations suggest that the activity differences were likely caused
by a change in the number of copies of functional transporters in the
membrane rather than by modification of existing transporter affinities
to either alanine substrate or activator Na. The
linear relationship of the analogue inhibitor data of Fig. 3,
taken with the other kinetic indicators of differentiation-dependent
decrease in uptake capacity (Fig. 1, Fig. 2, and
10-12) are consistent with concept that sodium-dependent alanine
transport in Caco-2 cells occurs primarily via a single transport
system (system B), and that the membrane capacity for transport by this
system is greater in newly passaged undifferentiated cells compared
with day 9 differentiated cells./dipeptide transport in Caco-2
cells(17, 18) , and confirmed by us (data not shown).
However, in these cases transport activity increased with
advancing cell age, in direct contrast to the present finding of a
decrease in alanine transport with advancing cell age postpassaging.
These diametrically opposed observations rule out the possibility that
the differentiation-associated transport regulation was due to
nonspecific membrane effects, such as changes in ion electrochemical
gradients. These observations also indicated that glucose, dipeptide,
and alanine uptakes in Caco-2 cells are likely independently regulated.
The differentiation-related decrease in system B transport activity
paralleled the differentiation-related decrease in cell proliferation
rates measured by [H]thymidine incorporation
(data not shown). This may reflect the cells' anabolic
requirement for free amino acids during rapid growth that occurs in the
undifferentiated state, relative to the demand for
glucose(17) . Role of Protein Kinase C and de Novo Protein
Synthesis
The up-regulation of system B activity likely involved
protein kinase C because the tumor promoter phorbol ester TPA
stimulated sodium-dependent alanine uptake in Caco-2 cells (Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9),
and this stimulation was blocked by chelerythrine or photoactivated
calphostin C (Fig. 7). Chelerythrine specifically inhibits the
catalytic domain of protein kinase C with an IC
several
orders of magnitude less than that for protein kinase A or any other
protein kinase(21, 22) . Photoactivation of calphostin
C generates a short lived species that permanently inactivates the
phorbol ester binding portion of protein kinase C with an IC of about 50 nM(22, 26) . In a separate
set of experiments (data not shown), forskolin (10 µM) or
dibutryl cAMP (500 µM) did not affect alanine uptake
activities in our Caco-2 cells, further reinforcing the notion that
protein kinase A was likely not involved in regulating system B
transport activity. Phorbol ester stimulated system B uptake at any
given cell age (Fig. 8), even in the differentiated state (
day 9). The stimulation was due to an increase in the membrane capacity (V) to transport alanine via system B (Fig. 9), and not due to a change in transport affinity (K). The long time period required for TPA
up-regulation (Fig. 6) precludes rapid (minutes)
post-translational modifications such as phosphorylations of existing
carrier polypeptides. We are currently in the process of investigating
the possible autocrine/paracrine/endocrine factors that could trigger
the rise in Caco-2 protein kinase C activity leading to augmented
alanine uptake. that was stimulated by TPA (Fig. 7). Cycloheximide or
actinomycin D blocked the stimulation effect of TPA only after a
continual 24-h exposure period (Fig. 7); exposure to the protein
synthesis inhibitors for periods less than 6 h were ineffective in
blocking TPA stimulation (data not shown). Although it is tempting to
speculate that the change in transport capacity (V) was due simply to increased copies of the
system B carrier polypeptide, we cannot rule out the possibility of a
more complex scenario involving transcription regulators and/or
transporter regulatory subunits. Such an alternative means of
regulation could be analogous to the hypothetical model of SGLT1
carrier regulation by RS1 putative regulatory subunits (27) proposed for intestinal apical membranes. Investigating
and confirming any model of regulation awaits the cloning of system B
carrier polypeptides and any related regulatory factors. At the present
time, there have been no transporters cloned that are solely
responsible for sodium-dependent neutral amino acid
transport(28) . Although the SAAT1 clone (13) was
originally thought to be the system A transporter, further scrutiny
revealed that this clone was actually the SGLT2 variant of the
Na/glucose cotransporter(14) . It is hoped
that the present report describing a well-defined in vitro model of system B activity regulation will lead to successful
cloning of a neutral amino acid transporter of epithelial cells.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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