(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 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.
The Na-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) .
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 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.
Figure 1:
Initial time course of 50 µM and 5 mML-[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.
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 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.
Figure 2:
Na 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.
Figure 3:
Alanine uptake inhibition by amino acid
analogues in day 3 cells versus day 9 cells. Sodium-dependent
50 µML-[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.
Figure 4:
Dixon plot of the effect of MeAIB on
sodium-dependent L-[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.
Figure 5:
The effect of phorbol ester (TPA) dose on
50 µML-[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.
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-[H]alanine initial
uptake rates were measure in Na
(
,
), or
choline (
,
) uptake media. Data represent means ±
S.E.
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-[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.
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-[H]alanine initial uptake rates
were measure in NaCl or choline chloride uptake media. Data represent
means ± S.E.
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-[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.
Nonlinear regression analysis of the Na-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.
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) of system B activity; and (iv) that
this regulation involves de novo protein synthesis under the
control of protein kinase C.
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
[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.
System
B 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.
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) 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.
Differentiation-dependent changes
in transporter capacities have been reported for sodium/glucose and
H/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) .
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 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.