©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differentiation- and Protein Kinase C-dependent Regulation of Alanine Transport via System B (*)

(Received for publication, October 28, 1994; and in revised form, December 12, 1994)

Ming Pan (§) Bruce R. Stevens (¶)

From the Department of Physiology, College of Medicine, University of Florida, Gainesville, Florida 32610

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(max)), with K unaffected. The V(max) 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(max) = 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(max) of system B in 2-day-old cells to V(max) = 6.32 ± 0.37 nmol min mg of protein (K = 169 ± 18 µM), and in 9-day-old cells to V(max) = 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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

Chemicals

Dulbecco's modified Eagle's medium (DMEM), (^1)fetal bovine serum, sodium bicarbonate, penicillin, streptomycin, nonessential amino acids, trypsin, EDTA, Me(2)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-[^3H]Alanine and [^3H]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 Me(2)SO 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(2)/90% O(2).

Caco-2 cells were passaged following treatment with 0.05% trypsin and 0.02% EDTA. Cells were reseeded at a density of 4.5 times 10^6 cells/100-mm dish for future subculturing, or seeded in the 6-well cluster Falcon tissue culture dishes at a density of 3.86 times 10^5 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(4), 1.2 mM KH(2)PO(4), and 2.5 mM CaCl(2). The uptake was initiated by the addition of 1 ml of this buffer containing also L-[^3H]alanine (2 µCi/ml, 1 µM to 5 mM) or [^3H]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 Me(2)SO stocks, giving < 0.5% Me(2)SO in final media exposed to cells. This concentration of Me(2)SO 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.


RESULTS

Sodium Dependence and Hill Activation Coefficient

Uptake of L-[^3H]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.


Figure 1: Initial time course of 50 µM and 5 mML-[^3H]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(max) was greater in day 2 cells than in day 9 cells. For 50 µM alanine uptakes, the day 2 cell V(max) = 525 ± 20 pmol min mg of protein, and K(0.5) = 10.5 mM; day 9 cell V(max) = 149 ± 10 pmol min mg of protein, K(0.5) = 28 mM.


Figure 2: Na activation of Na-dependent alanine uptake. Initial uptake rates of 50 µML-[^3H]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-[^3H]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 [^3H]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(i) = 35 µM), glycine (K(i) = 4.5 mM), and phenylalanine (K(i) = 5.9 mM) inhibition of sodium-dependent L-[^3H]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.


Figure 3: Alanine uptake inhibition by amino acid analogues in day 3 cells versus day 9 cells. Sodium-dependent 50 µML-[^3H]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 [^3H]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 [^3H]alanine uptake rates (e.g.Fig. 1and Fig. 2). Furthermore, as shown in Fig. 4, Dixon analysis of the effect of unlabeled MeAIB on [^3H]alanine uptake indicated that there was negligible interaction of MeAIB with system B in Caco-2 cells.


Figure 4: Dixon plot of the effect of MeAIB on sodium-dependent L-[^3H]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).


Figure 5: The effect of phorbol ester (TPA) dose on 50 µML-[^3H]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-[^3H]alanine initial uptake rates were measure in Na (circle, bullet), or choline (box, ) uptake media. Data represent means ± S.E.



Blocked TPA Stimulation of System B Activity

Sodium-dependent L-[^3H]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).


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-[^3H]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 (geq 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.


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-[^3H]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 .


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-[^3H]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(max) of 2.79 ± 0.21 nmol min mg of protein and K(m) = 164 ± 26 µM alanine. For day 9 cells not exposed to TPA, the V(max) dropped to 0.51 ± 0.03 nmol min mg of protein, and K(m) remained relatively unaffected at 159 ± 14 µM alanine. For 2-day-old cells exposed to TPA, the V(max) was increased to 6.32 ± 0.37 nmol min mg of protein, while the K(m) remained unaffected (180 ± 10 µM), relative to cells not exposed to TPA. Finally, in day 9 cells exposed to TPA, V(max) = 1.42 ± 0.05 nmol min mg of protein, and K(m) = 169 ± 18 µM.


DISCUSSION

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(max)) 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(max), but not K(m), 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.

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 [^3H]MeAIB uptake in Caco-2 cells was several orders of magnitude less than that of [^3H]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 alpha and beta 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(m) 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.

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(max)) in day 2 cells compared with day 9 cells, with no change in apparent K(m) (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 [^3H]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 (geq day 9). The stimulation was due to an increase in the membrane capacity (V(max)) to transport alanine via system B (Fig. 9), and not due to a change in transport affinity (K(m)). 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.

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(max) 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(max)) 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.

To whom correspondence should be addressed: Dept. of Physiology, College of Medicine, University of Florida, P. O. Box 100274, Gainesville, FL 32610-0274. Tel.: 904-392-4480; Fax: 904-846-0270.

(^1)
The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; MeAIB, (methylamino)isobutyric acid; TPA, 12-O-tetradecanoylphorbol-13-acetate; BCH, 2-aminobicyclo[2.2.1]heptane-2carboxylic acid.


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