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Pflugers Arch – Eur J Physiol (2006) 451: 738–748
DOI 10.1007/s00424-005-1507-2

EPITHELIAL TRANSPORT

Philipp Kirchhoff Æ Mital H. Dave Æ Christine Remy

Ortrud Kosiek Æ Stephanie M. Busque
Matthias Dufner Æ John P. Geibel Æ Francois Verrey
Carsten A. Wagner

An amino acid transporter involved in gastric acid secretion

Received: 5 May 2005 / Accepted: 4 August 2005 / Published online: 25 November 2005

Springer-Verlag 2005

Abstract Gastric acid secretion is regulated by a variety substrate and a competitive inhibitor of system L amino
of stimuli, in particular histamine and acetyl choline. In acid transport, abolished the stimulation of acid
addition, dietary factors such as the acute intake of a secretion by glutamine or cysteine suggesting that this
protein-rich diet and the subsequent increase in serum stimulation required the uptake of amino acids by system
amino acids can stimulate gastric acid secretion only L. In the absence of histamine glutamine also stimulated
through partially characterized pathways. Recently, we H+-extrusion, whereas glutamate did not. Also, phen-
described in mouse stomach parietal cells the expression ylalanine was effective in stimulating H+/K+-ATPase
of the system L heteromeric amino acid transporter activity. Glutamine did not increase intracellular Ca2+
comprised of the LAT2-4F2hc dimer. Here we address levels indicating that it did not act via the recently
the potential role of the system L amino acid transporter described amino acid modulated Ca2+-sensing receptor.
in gastric acid secretion by parietal cells in freshly iso- These data suggest a novel role for heterodimeric amino
lated rat gastric glands. RT-PCR, western blotting and acid transporters and may elucidate a pathway by which
immunohistochemistry confirmed the expression of 4F2- protein-rich diets stimulate gastric acid secretion.
LAT2 amino acid transporters in rat parietal cells. In
addition, mRNA was detected for the B0AT1, ASCT2,
and ATB(0+) amino acid transporters. Intracellular pH
measurements in parietal cells showed histamine-in- Introduction
duced and omeprazole-sensitive H+-extrusion which
was enhanced by about 50% in the presence of gluta- Gastric acid secretion is a tightly regulated process of
mine or cysteine (1 mM), two substrates of system H+-extrusion by the gastric H+/K+-ATPase localized
L amino acid transporters. BCH, a non-metabolizable in parietal cells. Classically, two stimulatory pathways
have been defined: (1) a neuronal via the vagus nerve
through release of acetyl choline and (2) a biphasic
P. Kirchhoff and M.H. Dave contributed equally to this study and endocrine pathway [21, 26, 32]. The endocrine pathway
therefore share first authorship depends on the release of gastrin from antral G cells
P. Kirchhoff Æ M. H. Dave Æ C. Remy Æ M. Dufner
leading to the stimulation of histamine-containing
F. Verrey Æ C. A. Wagner (&) enterochromaffin-like (ECL) cells [21, 26, 32]. ECL cells
Institute of Physiology and Center for Integrative Human then release histamine, which in turn initiates the direct
Physiology (CIHP), University of Zurich, Winterthurerstr. 190, insertion and activation of H+/K+-ATPases into the
8057 Zurich, Switzerland apical membrane of parietal cells and subsequent acid
E-mail: Wagnerca@[Link]
Tel.: +41-1-6355032 secretion mainly via a cAMP and PKA-dependent
Fax: +41-1-6356814 pathway. The exposure to histamine, however, also
causes a simultaneous rise in intracellular Ca2+ in
P. Kirchhoff parietal cells [5]. This elevation in intracellular Ca2+ has
Department of Visceral Surgery, University of Zurich,
Zurich, Switzerland been associated with increased acid secretion. In addi-
tion to these classical pathways, several other pathways
P. Kirchhoff Æ O. Kosiek Æ S. M. Busque Æ J. P. Geibel are able to stimulate or enhance gastric acid secretion.
Departments of Cellular and Molecular Physiology, Activation of the divalent cation receptor, CaSR or
Yale University, New Haven, USA
SCAR, has recently been described to stimulate gastric
J. P. Geibel acid secretion independent of other pathways [9, 10]. A
Departments of Surgery, Yale University, New Haven, USA protein-rich meal, and the subsequent rise in blood
 739

amino acid levels, is another stimulus known to increase handled according to the humane practices of animal
gastric acid secretion [11, 13, 15, 19]. This stimulatory care established by the Yale Animal Care and Use
effect does not increase serum gastrin levels and is fully Committee and the Swiss Kantonales Veterinäramt,
operative even after vagotomy [13, 15]. These results Zürich. Prior to experiments, animals were fasted for
imply that amino acids may have direct effects on pari- 12 h with free access to water.
etal cells and may modulate gastric acid secretion
through hitherto unrecognized mechanisms.
Recently, we have described that in mouse stomach, RNA extraction and RT-PCR
several amino acid transporter subunits are expressed on
mRNA and protein level [8]. All these subunits belong to Male Wistar rats were killed, and stomach, kidney, and
the novel family of heteromeric amino acid transporters. brain were collected. Stomach mucosa was scraped off
The structural and functional hallmarks of these trans- on ice and all the samples were rapidly frozen. Total
porters are that they consist of two subunits, a light chain mRNA was extracted from 30 mg of tissue using the
and a heavy chain, and that these transporters mostly RNA Aqueous 4PCR kit (Ambion) according to the
obey an antiport mechanism leading to the exchange of manufacturer’s instruction. For RNA extraction, tissue
intracellular against extracellular amino acids [6, 28–30]. was thawed in RNALater solution (Ambion), trans-
Thus, the activity of these transporters does not lead to the ferred to lysis buffer, and homogenized on ice with an
accumulation of amino acids but rather to a widening of Elvehjem potter. RNA was bound on columns and
the range of transported amino acids and equilibration of treated with DNAse for 15 min at 30C temperature to
intra- and extracellular amino acids. The main subunits reduce genomic DNA contamination. The quantity and
identified in mouse stomach were the heavy chain 4F2hc purity of the total eluted RNA were assessed by spec-
(also named CD98, SLC3A2) and the associated heavy trometry and on agarose gels. Each RNA sample was
chain LAT2 (SLC7A8) which together form one isoform diluted to 200 ng/ll and 1 ll used as a template for
of the system L transport activity. The activity of system L reverse transcription using the Taqman reverse tran-
has been characterized as the Na+-independent transport scription kit (Applied Biosystems, USA).
of large neutral amino acids in exchange for other intra- For reverse transcription, 200 ng RNA template was
cellular neutral amino acids [17]. Pharmacologically, diluted in a 20-ll reaction mix that contained (final
system L amino acid transport activity can be competi- concentrations): RT buffer (1x), MgCl2 (5.5 mM), ran-
tively inhibited by BCH [6, 14, 20, 30] which itself is a dom hexamers (2.5 lM), RNAse inhibitor (0.4 U/ll),
transport substrate but cannot be metabolized. The the multiscribe reverse transcriptase enzyme (1.25 U/ll),
localization of 4F2hc and LAT2 in mouse stomach was deoxyNTP mix (500 lM each) and RNAse-free water.
restricted to the acid secretory parietal cells in agreement PCR primers were designed using the primer express
with earlier reports describing system L-like amino acid program to yield amplicons between 50 and 450 bps
transport activity in these cells [24]. (Table 1). Taqman universal PCR Mastermix (2· con-
In order to address the role of system L amino acid centrated) was used (Applied Biosystems 4304437) con-
transport in parietal cells, we examined the influence of taining AmpliTaq Gold DNA Polymerase, dNTPs with
amino acids transported by the system L on the main dUTP and optimized buffer components. One microlitre
function of parietal cells, the extrusion of H+ by the cDNA (200 ng), 1.4 ll primers (each 25 lM), and 10 ll
H+/K+-ATPase. Similar to mouse stomach, system L mastermix were mixed with water to a total reaction
amino acid transporter subunits were identified in rat volume of 20 ll. PCR products were mixed with 10·
stomach parietal cells. Addition of glutamine or cyste- Bromophenol blue dye and loaded on a 4% ethidium
ine, substrates of system L, stimulated H+-extrusion by bromide/agarose gel and bands were visualized under
the gastric H+/K-ATPase in freshly isolated gastric UV light.
glands as confirmed by omeprazole sensitivity. The
stimulation was prevented by BCH, a known competi-
tive inhibitor of this transport system. These results Membrane preparation and western blot analysis
suggest that system L amino acid transporter may be
involved in modulating gastric acid secretion after a After removing the kidneys, brain, and stomach from
protein-rich meal and may represent part of a sensing Wistar rats, gastric mucosa was scraped off and then
mechanism for amino acid concentrations in blood. suspended in an ice-cold K-HEPES buffer (200 mM
mannitol, 80 mM K-HEPES, 41 mM KOH, pH 7.5)
with pepstatin, leupeptin, K-EDTA, and phenylmeth-
Material and methods ylsulfonyl fluoride (PMSF) added as protease inhibitors.
To obtain crude membrane fractions, the kidneys, brain,
Animals and gastric mucosa were homogenized on ice and cen-
trifuged at 1,000 g for 10 min; the supernatant was
Wistar Rats 250–300 g (Charles River) were housed in collected and centrifuged at 100,000 g for 60 min at 4C.
climate- and humidity-controlled, light-cycled rooms, The pellets containing the plasma membranes were
fed standard chow with free access to water, and resuspended in a homogenization buffer and stored at
740

80C until use. Fifty microgram of membrane proteins

ENSRNOG00000026501 205 bp
Accession number/expected size
was solubilized in a Laemmli sample loading buffer and

Table 1 Primers used to amplify the 4F2hc, LAT1, LAT2, BAT1, ASCT2, and ATB(0+) gene products from total RNA isolated from rat stomach mucosa, kidney, and brain
separated on an 8% SDS polyacrylamide gel. The pro-
teins were transferred to PVDF membranes, with
unspecific protein binding blocked by PBS containing

XM_233305 161 bp
AB024400 111 bp
AJ132846 649 bp
5% non-fat dry milk. The membranes were incubated

AB015432 73 bp
AB015433 68 bp
with either a polyclonal rabbit anti-LAT1 (1:5,000)
(friendly gift of N. Thompson, Brown University, RI,
USA [3], rabbit anti-LAT2 antibody (1:5,000) [8, 20] or
goat anti-4F2hc (Santa Cruz Biotech, 200 lg/ml, 1:1,000
[8]) for 2 h at room temperature. The membranes were
then washed three times, blocked for 1 h and again
incubated for 1 h at room temperature with the sec-
ondary goat anti-rabbit or donkey anti-goat antibodies
5¢-CGGGTAAAGAGGAAGTAGATG (1085–1106)
5¢-AAAGGATGTCAGGAGTCCTGC (1190–1210)

5¢-TGGATCATGGCTAACACG CT (1657–1676)

1:5,000 linked to alkaline phosphatase (Promega). The

5¢-CAGGGTACTCCAGCCACAAT (1360–1379)
5¢-GGACAACTTCTGGTGCAGGTT (754–774)

5¢-GAACCCTCATGCGTTTCATT (1568–1587)
protein signal was detected with the CDP Star chemi-
lumescensce system (Roche Diagnostics).

Immunohistochemistry

Male Wistar rats (200–250 g) were anesthetized with

ketamine/xyalzine i.p. and perfused through the left
ventricle with PBS followed by paraformaldehyde–ly-
sine–periodate (PLP) fixative [16]. The stomachs were
removed, cleaned from food residues, and fixed over-
Reverse primer

night at 4C by immersion in PLP. Stomachs were then

washed three times with PBS and the sections were cut at
a thickness of 5 lm after cryoprotection with 2.3 M in
PBS for at least 12 h. Immunostaining was carried out
as described previously [12]. Sections were incubated
with 1% SDS for 5 min, washed three times with PBS
and incubated with PBS containing 1% bovine serum
5¢-ATCCGGAAGCACTAGCTCAA (1426–1445)
5¢-GTATTTGAATGCCACTGGCA (1141–1160)

5¢-TCCACATTTGGTGGAGTCAA (1576–1595)

5¢-CCCAACATCTTCTGGCAAGT (1174–1193)

albumin for 15 min prior to the primary antibody. The

5¢-CCTGCTCGGCTTCATCCAGAT (702–722)

5¢-GCCTGATCGGAGGTGCAGCC (457–476)

primary antibodies (mouse monoclonal anti-human b

gastric H+/K+-ATPase (Affinity Bioreagents, CA,
USA) and rabbit anti-LAT1 (affinity purified, friendly
gift of N. Thompson, Brown University, RI, USA [3]),
rabbit anti-mouse LAT2 [20] and goat anti-4F2hc (Santa
Cruz Biotech, 200 lg/ml [8]) were diluted 1:50, 1:200 or
1:400, respectively, in PBS and applied overnight at 4C.
Sections were then washed twice for 5 min with high
NaCl PBS (PBS + 2.7% NaCl), once with PBS, and
incubated with the secondary antibodies (donkey–anti-
Forward primer

rabbit Alexa 546, donkey–anti-goat Alexa 488 or don-

key–anti-mouse Alexa 488, Molecular Probes, Oregon)
at a dilution of 1:1,000 and 1:200, respectively, for 1 h at
room temperature. Sections were then washed twice with
high NaCl PBS and once with PBS before mounting
with VectaMount (Vector laboratories, Burlingame,
CA). The specimens were viewed with a Nikon E-800
microscope or a Leica SP1 UV CLSM confocal micro-
ATB(0+) (SLC6A14)

scope.
B0AT1 (SLC6A19)
ASCT2 (SLC1A5)
4F2hc (SLC3A2)
LAT1 (SLC7A5)
LAT2 (SLC7A8)

Isolation of gastric glands

The stomach was opened longitudinally after removal

Gen

and the corpus and antrum were isolated and sliced into
 741

Table 2 Composition of
solutions used for intracellular Standard HEPES Na+-free HEPES Na+-free HEPES + NH4Cl High K+ calibration
pH measurements in single
parietal cells in freshly isolated NaCl 125 – – –
rat gastric glands NMDG – 125 105 32.8
NH4Cl – – 20 –
KCl 3 3 3 105
MgSO4 1.2 1.2 1.2 1.2
All concentrations are given CaCl2 1 1 1 1
in mM. NMDG is N-methyl-D- KH2PO4 2 2 2 –
glucamine, all solutions were Glucose 5 5 5 –
titrated to pH 7.4 at 37C using HEPES 32.2 32.2 32.2 32.2
either NaOH or KOH. NMDG pH 7.4 7.4 7.4 6.5, 7.0
was titrated with HCl

0.5 cm2 sections, and washed with cold Ringer solution All the experiments were performed in the nominal
to remove residual food particles. The tissues were absence of bicarbonate. The initial solution was a
transferred to the stage of a dissecting microscope. HEPES-buffered Ringer solution (125 mM NaCl, 3 mM
Individual glands were isolated using a hand-dissection KCl, 1 mM CaCl2, 1.2 mM MgSO4, 2 mM KH2PO4,
technique as described previously [12]. Following isola- 32.2 mM HEPES, pH 7.4). Cells were acidified by using
tion, individual isolated glands were either allowed to the NH4Cl (20 mM) prepulse technique and washed into
adhere to cover slips that had been pre-coated with Cell- a Na+-free solution (Na+ was replaced by equimolar
Tak (Collaborative Research, Bedford, MA) and were concentrations of N-methyl-D-glucamine) as described
transferred to the stage of an inverted microscope. previously [12]. The composition of all solutions used is
given in Table 2.
All chemicals were obtained from Sigma Chemicals;
Digital imaging for intracellular pH and Ca2+ Omeprazole was a kind gift of Dr. K. Andersson
measurements AstraZeneca, Mölndal, Sweden.

Coverslips with isolated gastric glands were transferred

to a thermostatically controlled perfusion chamber Results
(3 ml/min flow rate), maintained at 37C on an
inverted microscope (Zeiss Axiovert 200), and equipped Expression of system L amino acid transporters
with a video imaging system (Visitron, Munich, in rat stomach
Germany) for the duration of the experiment. Isolated
gastric glands were incubated in a HEPES-buffered RT-PCR was used to test for the presence of mRNA
Ringer solution containing either 10 lM of the encoding subunits of system L heteromeric amino acid
pH-sensitive dye BCECF-AM (2¢,7¢)-bis-(2-carboxyeth- transporters in rat stomach mucosa. Total RNA was
yl)-5-(and-6)-carboxy-fluorescin, aceto-methyl ester or extracted from rat stomach mucosa, kidney, and brain.
10 mM of the Ca2+-sensitive dye Fluo-4 (Molecular The kidney and brain were included as positive con-
Probes, Eugene, OR) for 10 min as described previously trols as these organs are known to express several
[12] . Following loading, the chamber was flushed with a subunits of system L heteromeric amino acid trans-
HEPES buffered Ringer solution to remove all non-de- porters [8, 30]. RT-PCR products of the expected size
esterfied dye. BCECF and Fluo-4 were successively were found for all three known subunits, the 4F2hc
excited at 440±10 nm and 490±10 nm for BCECF and heavy chain and the LAT1 and LAT2 light chains in
at 488±10 nm, and the resultant fluorescent signal was all three organs were tested (Fig. 1a). In addition, we
monitored at 535±10 nm and 520±10 nm, respec- tested for the presence of other amino acid trans-
tively, using an intensified CCD camera. Individual porters capable of transporting amino acids with
regions of interest were outlined and simultaneously similar substrates as system L such as ASCT2
monitored during the course of the study. A minimum (SLC1A5), B0AT1 (SLC6A19), and ATB(0+)
of seven cells or regions was selected per gland. All data (SLC6A14) [1]. mRNA for all three transporters was
including the individual images for all wavelengths were detected in rat stomach. The presence of mRNAs of
recorded to the hard disk which allowed us to return to 4F2hc, LAT1, LAT2, and ATB(0,+) in gastric mu-
the individual images after the experiment for further cosa has been described similarly in mouse or human
analysis. stomach tissue [8, 23].
Intensity ratio data of 490/440 were converted to pH Western blotting with rat brain, kidney, and stomach
values by using the high K+/nigericin calibration tech- mucosa confirmed the expression of 4F2hc and LAT2
nique [25]. Data are expressed as DpH/min, percentages protein in rat stomach mucosa yielding again bands of
were calculated using Gauss’ law of error propagation, the expected size (Fig. 1b). These results demonstrated
and tested for significance using the Student’s t test. the expression of the subunits 4F2hc and LAT2 subunits
742

forming the system L heteromeric amino acid trans- 0.023±0.001 units pH/min (Fig. 3a, c, Table 3, n=33
porter in rat stomach mucosa. cells, five glands, four animals). Histamine was present
in all solutions during the experiments.
Addition of 1 mM glutamine or 1 mM cysteine to all
Localization of system L amino acid transporters solutions (including the 10 min preincubation with
in rat parietal cells histamine) resulted in an increase of the alkalinization
rate by about 100–200% (0.039±0.003 U pH/min for
Immunohistochemistry confirmed the presence of 4F2hc glutamine and 0.068±0.006 U pH/min for cysteine,
and LAT2 in rat stomach, whereas no specific signal for respectively).
LAT1 could be detected (Fig. 2). The localization of Gastric glands were stimulated with histamine and
4F2hc and LAT2 was restricted to a subset of cells and incubated with omeprazole, a specific inhibitor of the
to the basolateral side of these cells. Double-labelling for H+-extruding H+/K+-ATPase, to confirm that gluta-
LAT2 and the gastric H+/K+-ATPase b subunit mine increased the rate of H+-extrusion via this pump.
showed clearly that LAT2 is expressed together with the Omeprazole reduced the rate of pHi recovery to
H+-extruding ATPase in parietal cells as described 0.002±0.002 U pH/min demonstrating that glutamine
previously in mouse stomach [8]. stimulated H+/K+-ATPase activity (Fig. 4, n=37 cells,
six glands, four animals).
Thus, these results suggested that substrates of the
Acid extrusion is increased by system L amino acid system L amino acid transporter can modulate hista-
transporter substrates mine-stimulated gastric H+/K+-ATPase activity and
enhance proton excretion.
Freshly isolated rat gastric glands were used for intra-
cellular pH measurements to assess the activity of the
gastric H+/K+-ATPase as the rate of intracellular Glutamine or phenylalanine alone stimulate
alkalinization (DpH/min) in the absence of bicarbonate H+/K+-ATPase activity
and Na+ after an acid load (20 mM NH4Cl prepulse)
as described previously [31]. Glands were already Stimulation of H+/K+-ATPase activity was also
preincubated for 10 min with histamine (100 lM) prior observed in the presence of glutamine (1 mM) even
to the measurements to stimulate H+/K+-ATPase without prior activation of H+-extrusion by histamine
activity which resulted in an alkalinization rate of (Fig. 5). Control glands without exposure to histamine

Fig. 1 Expression of system L amino acid transporter subunits in could be detected in stomach whereas LAT1 was only observed in
rat stomach. a mRNA was isolated from rat kidney (K), stomach rat brain. The weak bands detected in kidney and stomach are
mucosa (S), and brain (B), and RT-PCR was performed for the probably not LAT1 and unspecific. c PCR products for the BAT1,
three subunits 4F2hc, LAT1, and LAT2. Only single PCR products ATB(0+), and ASCT2 transporters in kidney, stomach, and brain.
were found for each gene in all three organs. b Western blotting of mRNA for all three transporters was detected in stomach. BAT1
crude membranes (50 lg protein/lane) prepared from rat kidney and ASCT2 were also present in brain and kidney, whereas
(K), stomach mucosa (S), and brain (B). Only 4F2hc and LAT2 ATB(0+) was not
 743

Fig. 2 Localization of system L

subunits 4F2 and LAT2 in rat
gastric parietal cells. a–c
Localization of 4F2hc (green)
(a) and LAT2 (red) (b) in rat
stomach gastric glands. (c)
overlay of 4F2hc and LAT2.
Original magnification 200·. d,
e High magnification (400· (d),
600· (e)) of single parietal cells
showing the colocalization of
LAT2 (red) and the b subunit of
the H+/K+-ATPase (green) in
the same cells. LAT2 localizes
to the basolateral membrane,
whereas the H+/K+-ATPase
subunit is seen in intracellular
vesicles and tubulovesicular
structures as previously
described. Stomachs were taken
from fasted animals

or glutamine showed an intracellular alkalinization rate ATPase activity (Fig. 5, Table 3). It has been previously
of 0.007±0.001 U pH/min, whereas an addition of suggested that amino acids stimulate gastric acid secre-
glutamine increased this rate to 0.052±0.005 U pH/min. tion by releasing histamine from neighbouring ECL cells
Also L-phenylalanine, another substrate of system and thus activate parietal cells. To test for glutamine-
L, increased the intracellular alkalinization rate to induced histamine-mediated stimulation of H+/K+-
0.066±0.003 U pH/min. (Fig. 5, Table 3). In contrast, ATPase activity, gastric glands were preincubated for
addition of 1 mM L-glutamate which is not transported 10 min with the H2-receptor antagonist cimetidine
by system L to all solutions had no effect on H+/K+- (100 lM) and all the experiments were performed in the
744

Fig. 3 Stimulation of intracellular alkalinization by amino acids in b Original tracing of intracellular pH measurement of a parietal cell
gastric parietal cells. a Original tracing of intracellular pH stimulated with 1 mM glutamine. c Summary bar of the rate of
measurement in a single parietal cell in rat isolated gastric gland. intracellular alkalinization of histamine stimulated rat gastric
Glands were stimulated with 100 lM histamine and cells acidified parietal cells in the absence and presence of 1 mM glutamine or
with NH4Cl in the absence of Na+. The rate of intracellular Na+- cysteine. Data are presented as mean ± SEM
independent alkalinization (dashed line) was calculated (DpH/min).

continuous presence of cimetidine. Addition of gluta- activity and that they may act from an intracellular site
mine (1 mM) stimulated H+-extrusion to a similar either as intact amino acids or after being metabolized.
extent in the absence and presence of cimetidine BCH does not surrogate for glutamine or cysteine at this
(0.044±0.004 U pH/min.) (Fig. 6, Table 3) demon- intracellular site.
strating that the stimulatory effect of glutamine does not
require H2 receptors or release of histamine from ECL
cells. Intracellular Ca2+ measurements in parietal cells

We have recently shown that the activation of the

Stimulation of acid extrusion by glutamine and cysteine divalent cation receptor, CaSR, can also stimulate H+/
requires system L amino acid transport activity K+-ATPase activity even in the absence of any other
stimulus such as histamine [9]. The affinity of the CaSR
The stimulatory effect of glutamine and cysteine on H+/ for divalent cations and its subsequent activation can be
K+-ATPase activity was further examined in the pres- modulated by L-amino acids binding to the receptor and
ence of BCH, a specific inhibitor of system L amino acid shifting the activation curve to the left, i.e., activation
transport activity. BCH while being transported itself occurs already at lower concentrations of divalent ca-
cannot be metabolized [6, 14, 18, 20, 30] and thus acts as tions [2, 7]. Activation of the Ca2+-sensing receptor in
a competitive inhibitor. BCH (10 mM) was used in turn increases intracellular Ca2+, a stimulus known to
excess over glutamine or cysteine (both 1 mM) and be involved in the activation and stimulation of the
completely prevented the stimulatory effect of both pump [4, 5]. In order to examine if glutamine stimulated
amino acids on pHi-recovery (H+-extrusion)(Fig. 7, the activity of the gastric H+/K+-ATPase by increasing
Table 3). These results thus, indicate that both the intracellular Ca2+, intracellular Ca2+ was measured
amino acids need to be transported by a system L-like using the Ca2+-sensitive dye Fluo-4. Glands were not
amino acid transporter to stimulate H+/K+-ATPase stimulated with histamine. Superfusion of isolated gas-
 745

Table 3 Summary of
intracellular pH measurements pHi recovery DpH/min n
in single parietal cells in freshly
isolated rat gastric glands Control 0.007±0.001 93 cells
10 glands
7 animals
L-Glutamine 0.052±0.005 95 cells
8 glands
3 animals
L-Phenylalanine 0.066±0.003 66 cells
5 glands
3 animals
L-Glutamate 0.003±0.001 108 cells
8 glands
3 animals
L-Glutamine + cimetidine 0.044±0.004 101 cells
6 glands
3 animals
Histamine 0.023±0.001 33 cells
5 glands
4 animals
Histamine + L-Glutamine 0.039±0.003 82 cells
10 glands
7 animals
Histamine +L-Cysteine 0.068±0.006 76 cells
8 glands
4 animals
Histamine +L-GlutamineOmeprazole 0.002±0.002 37 cells
6 glands
4 animals
Histamine +L-GlutamineBCH 0.015±0.002 45 cells
4 glands
3 animals
Histamine +L-CysteineBCH 0.012±0.002 42 cells
5 glands
All data are given as 4 animals
mean ± SEM

tric glands with 1 mM glutamine did not alter the acids on acid-secretory parietal cells by a mechanism
intensity of the Fluo-4 signal. However, when 500 lM recognizing the stereo-specificity of amino acids.
Gd3+, a potent activator of the Ca2+-sensing receptor in The recent identification of system L amino acid
gastric parietal cells [4, 9], was applied a rapid increase in transporter in parietal cells provided a novel pathway.
intensity of the Fluo-4 signal was observed, indicative of System L amino acid is stereo-selective accepting only
an increase in intracellular Ca2+ [4, 9] (Fig. 8). Thus, L-isomers and transports most neutral amino acids that
glutamine at this low concentration does not stimulate have been found to stimulate gastric acid secretion upon
H+/K+-ATPase activity by acting on the Ca2+-sensing intravenous infusion, namely, phenylalanine, cysteine,
receptor. glutamine, and leucine. Our results demonstrate that
system L amino acid transport could indeed be involved
in amino acid stimulated gastric acid secretion: (1) sys-
Discussion tem L amino acid transporter subunits 4F2hc and LAT2
are specifically expressed in parietal cells in mouse and
Gastric acid secretion is stimulated through a variety of rat stomach, (2) glutamine, phenylalanine, and cysteine
factors including neurotransmitters, hormones, meta- stimulated gastric H+/K+-ATPase activity, (3) the
bolic factors, and several nutrients including amino stimulatory effect is specific for system L substrates and
acids [32]. The mechanism by which nutrients stimulate does not occur with glutamate, (4) stimulation is abol-
and regulate gastric acid secretion is not fully under- ished by BCH, an inhibitor of system L amino acid
stood. A protein-rich diet or the intravenous infusion of transport, (5) the effect did not require H2-receptors
a solution containing either single amino acids or a mix ruling out the involvement of ECL cells, and (6) amino
of different amino acids has been shown to represent a acids did not act via the recently described Ca2+-sensing
potent stimulus for gastric acid secretion [11, 13, 15, 19]. receptor as evident from intracellular Ca2+-measure-
This stimulatory effect is observed only with L-amino ments. However, it should also be noted that phenylal-
acids but not with their D-stereomers [13, 15]. In anine, glutamine, and cysteine are substrates of BAT1
addition, gastric acid secretion occurs in the absence of and ATB(0+) and glutamine and cysteine of ASCT2.
gastrin release and after vagotomy [13, 15]. Taken Only ATB(0+) is sensitive to BCH whereas BAT1 and
together, these results suggest a direct effect of amino ASCT2 not. Thus, the data on amino acid selectivity and
746

Fig. 5 Glutamine or phenylalanine alone stimulates H+/K+-

ATPase activity. The intracellular Na+-independent alkalinization
rate was measured in rat gastric glands left either untreated
(control), incubated with 1 mM glutamine, 1 mM phenylalanine or
1 mM glutamate. Incubation with 1 mM glutamine or phenylala-
nine strongly stimulated the Na+-independent alkalinization rate
whereas glutamate had no effect, mean ± SEM

as a metabolic fuel for ATP synthesis and subsequently

contribute to H+/K+-ATPase activity. Glucose, lactate,
and some amino acids have previously been shown to
support aminopyridine accumulation in parietal cells
possibly as substrates for ATP synthesis [22].
Thus, the system L amino acid transporter may be
part of a sensing mechanism for which it represents the
amino acid uptake system across the plasma membrane,
whereas the intracellular mechanism that senses the
amino acids remains to be identified. The fact that a
Fig. 4 Amino acids stimulate omeprazole-sensitive H+/K+-AT- mammalian plasma membrane transporter forms part of
Pase activity. a Original tracing of an intracellular pH measurement a metabolic-sensing mechanism has been extensively
of a histamine-stimulated rat parietal cell in the presence of 1 mM studied in the insulin secreting b-cells of the pancreas
glutamine and the specific H+/K+-ATPase inhibitor omeprazole
(100 lM). No intracellular Na+-independent alkalinization could
be observed. b Bar graph summarizing intracellular pH-recovery
rates (DpH/min) of histamine stimulated rat parietal cells in the
presence of 1 mM glutamine or 1 mM glutamine/100 lM omep-
razole. The data for histamine and histamine plus glutamine from
Fig. 3 are shown again for better comparison, mean ± SEM

inhibition by BCH fit best to system L but do not

completely rule out the involvement of the three above-
mentioned amino acid transporters.
The mechanism by which amino acids transported by
system L stimulate acid secretion remains unclear. It
appears, however, that the sensor mechanism must be
localized intracellularly as it requires the uptake of
amino acids into parietal cells which can be blocked by
BCH. The sensing mechanism might involve the
metabolism of the transported amino acids as BCH is Fig. 6 H2-receptors are not involved in the effect of glutamine on
transported by system L but cannot be metabolized. parietal cell acid secretion. Rat gastric glands were left untreated
Interestingly, BCH even decreased the acid secretion (control) or incubated with 1 mM glutamine in the absence or
below control. This could be due to the exchange func- presence of the H2-receptor antagonist cimetidine (100 lM).
Cimetidine did not affect the stimulation by glutamine demon-
tion of LAT2-4F2hc that mediates the efflux of amino strating that H2-receptors are not mediating the effect. The data of
acids stimulated by the uptake of BCH. The amino acids control and glutamine are shown again for better comparison,
tested here could at least in the case of glutamine also act mean ± SEM
 747

where glucose is taken up via the GLUT2 glucose

transporter and then metabolized to generate an ATP-
based signal initiating insulin secretion [27]. The mech-
anism described here in gastric parietal cells may be of a
similar nature and the system L mediated uptake of
amino acids into parietal cells may provide the initial
step mediating the increased gastric acid secretion after
the ingestion of a protein-rich meal.
In summary, an isoform of the system L amino acid
transporter is expressed in acid-secreting gastric parietal
cells and its substrates stimulate H+/K+-ATPase
activity in isolated gastric glands. The stimulatory effect
requires transport of system L-specific amino acids and
Fig. 7 Inhibition of system L amino acid transport activity with does not depend on H2 or CaSR receptors. This defines
BCH prevents stimulatory effect of amino acids on H+/K+- a novel pathway mediating the stimulatory effect of
ATPase activity. Incubation of rat gastric glands with BCH
(10 mM), a competitive inhibitor of system L amino acid transport amino acids on gastric acid secretion.
activity, abolished the stimulatory effect of glutamine (1 mM) and
cysteine (1 mM) on the rate of intracellular pH alkalinization. The Acknowledgements This study was supported by grants from the
data for histamine, histamine plus glutamine and histamine plus Theodor and Ida Herzog-Egli foundation to F.V. and C.A.W., by
cysteine are shown again for better comparison, mean ± SEM the Hartmann Müller foundation to P.K. and C.A.W., by the
EUGINDAT project of the sixth Framework of the EU to F.V.
and C.A.W., and by the NIH to J.P.G. (DK-50230, DK-14669,
DK-17433, DK-60069). We thank N. Thompson, Brown Univer-
sity, RI, USA for providing us with the anti-LAT1 antibody.

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