G-00096-2005-R3

L-type amino acids stimulate gastric acid secretion by activation of the

calcium-sensing receptor in parietal cells

Stephanie M. Busque 1, 4, Jane E. Kerstetter 4, John P. Geibel 1, 2*, Karl Insogna3*

Department of Surgery1, and Department of Cellular and Molecular Physiology2, and

Medicine3, Yale University, School of Medicine, and the University of Connecticut,

School of Allied Health4. *These two authors contributed equally to this work

Keywords: stomach, pH, H,K-ATPase, rat, mouse, gastric glands.

Address correspondence to:

John Geibel, M.D., D.Sc.Dept. of SurgeryBML 265310 Cedar StreetNew Haven, CT 06520john.geibel@yale.edu

FINAL ACCEPTED VERSION

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (June 16, 2005). doi:10.1152/ajpgi.00096.2005

Copyright © 2005 by the American Physiological Society.

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ABSTRACT

Parietal cells are the primary acid secretory cells of the stomach. We have previously

shown that activation of the CaSR by divalent (Ca2+) or trivalent (Gd3+) ions stimulates

acid production in the absence of secretagogues by increasing H+,K+-ATPase activity.

When over-expressed in HEK-293 cells, the CaSR can be allosterically activated by L-

amino acids in the presence of physiological concentrations of extracellular Ca2+ (Ca2+o;

1.5-2.5 mM). To determine if the endogenously expressed parietal cell CaSR is

allosterically activated by L-amino acids, we examined the effect of the amino acids L-

phe, L-trp and L-leu on acid secretion. In ex vivo whole stomach preparations, exposure

to L-phe resulted in gastric luminal pH significantly lower than controls. Studies using

D-phe (inactive isomer), failed to elicit a response on gastric pH. H+,K+-ATPase activity

was monitored by measuring the intracellular pH (pHi) of individual parietal cells in

isolated rat gastric glands and calculating the rate of H+ extrusion. We demonstrated that

increasing Ca2+o in the absence of secretagogues caused a dose dependent increase in H+

extrusion. These effects were amplified by addition of amino acids at various Ca2+o

concentrations. Blocking the H2 receptor with cimetidine or inhibiting system L amino

acid transport with BCH did not affect the rate of H+ extrusion in the presence of L-phe.

These data support the conclusion that amino acids, in conjunction with a physiological

Ca2+o concentration, can induce acid secretion independent of hormonal stimulation via

allosteric activation of the stomach CaSR.

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Introduction

Regulation of acid secretion is complex, requiring many receptor-ligand

interactions. In the classical pathway of acid secretion, gastrin release is induced from

antral G-cells causing a subsequent efflux of histamine from enterochromaffin-like cells

(ECL cells). Histamine then binds to its cognate receptor (H2R) on the basolateral

membrane of the parietal cell. The resultant cascade of events include a rise in cytosolic

Ca2+ (Ca2+i) and cAMP levels, which trigger both chloride and H+ efflux from the apical

cell surface via a chloride conductance channel and the H+,K+-ATPase (13; 14; 22; 25;

29). Upon activation by secretagogues (gastrin, histamine, acetylcholine) the H+,K+-

ATPase, located on tubular vesicular elements within the cytosol, traffics to the apical

cell surface where the vesicles subsequently fuse with canaliculi along the apical

membrane resulting in HCL secretion into the lumen of the gland (11; 13; 22; 25; 29).

The role of nutrients in regulating parietal cell acid production is less well

understood. Previous work has suggested that intragastric or intraduodenal

administration of L- but not D-amino acids can modulate gastric acid secretion in the

absence of classical hormonal intermediaries such as gastrin and histamine in humans and

animals (15-17; 23). Intravenous infusion of amino acids stimulated acid secretion before

elevated serum gastrin levels were detected, this effect was also observed in human

subjects who had undergone a vagotomy (15; 16; 19). Serum amino acid levels in these

subjects were comparable to those observed post-prandially after a steak meal, suggesting

that physiologic amino acid concentrations affect acid production in vivo (19).

The calcium-sensing receptor (CaSR) is a member of family C of G-protein

coupled receptors, and is expressed on many tissues throughout the body, with high

G-00096-2005-R3

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expression in gastric parietal cells (3-5; 12; 20). Recent studies have confirmed that

CaSR is also expressed in gastrin-secreting G cells, and plays an active role in gastrin

release (2; 4; 5). The CaSR functions as a “biosensor” for divalent and trivalent ions,

such that small changes in the concentrations of these ions can alter receptor activity. In

addition to its biosensor function, it has also been shown that certain antibiotics, as well

as polyamines can activate the receptor. Conigrave et al. demonstrated that CaSR activity

can be regulated by aromatic L-amino acids at sub-stimulatory concentrations of Ca2+o, as

evidenced by a rise in Ca2+i concentration, suggesting allosteric modification (7; 9). In

HEK-293 cells over-expressing the CaSR, L-phenylalanine, L-tryptophan or L-histidine

in the presence of physiological Ca2+o concentrations (1.5-2.5 mM), caused a significant

rise in Ca2+i, indicative of receptor activation. The D-isoforms, as well as branched chain

amino acids such as L-leu, had little or no effect (7; 9).

While the above data establish a mechanism by which amino acids could

theoretically modulate parietal cell acid secretion, the ability of amino acids to stimulate

gastric acid secretion in parietal cells has not been experimentally determined. Therefore,

we sought to determine i the endogenously expressed CaSR could be allosterically

activated by physiological levels of amino acids. In these studies we chose to use

phenylalanine and tryptophan (two of the more potent amino acids in regards to CaSR

activation), in the presence of various Ca2+o concentrations, to determine the effects on

parietal cell acid secretion. We demonstrate by measuring gastric pH ex vivo in whole

stomach preparations and acid secretory rates in parietal cells in the presence of L-phe or

L-trp and physiological levels of Ca2+o, that allosteric activation of the CaSR by amino

G-00096-2005-R3

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acids lead to enhanced H+,K+-ATPase activity independent of the hormonal status of the

tissue.

METHODS

Ex vivo whole stomach pH measurements

Rats

Male Sprague-Dawley rats weighing approximately 200-300 g were housed in

climate and humidity-controlled, light-cycled rooms and fed standard chow with free

access to water. Prior to experimentation, animals were fasted for 18-24 hours to reduce

basal acid secretion. Animals were sacrificed with an overdose of ether or isoflurane, and

an abdominal incision was made. The stomach was ligated at the esophageal and

duodenal junctures and excised. Using a syringe, 1 ml of non-buffered, isotonic saline

(140 mM) was infused into the lumen of the stomach. This volume of saline did not

distend the stomach, thus avoiding potential stimulation of acid secretion by stretch. The

preparations were then placed in either oxygenated HEPES-buffered Ringer’s solution

(HEPES), or this same solution containing 50 mM L- or D-phe; pH 7.4, and maintained

at 37˚C. When amino acids were added to the solutions, we adjusted the mannitol

concentration and reduced the glucose concentration to 5 mM to maintain a final

osmolarity of 290 mOsms (see Table 1). All solutions were checked on a freezing point

osmometer (Precision Systems, Inc.; Natick, MA, USA). Decreasing the glucose

concentration by half did not adversely affect tissue viability or acid secretion. After a 1

hour incubation period, the stomach contents were aspirated and the pH was recorded. It

G-00096-2005-R3

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was necessary to add an additional 1 ml of non-buffered saline to the stomach aspirate

after incubation to ensure sufficient volume to accurately measure pH.

In vitro pH measurements in parietal cells of freshly isolated gastric glands

Male Sprague-Dawley rats were sacrificed as described above, stomachs were

excised and immediately rinsed with ice-cold HEPES to remove any residual food

particles and kept on ice until use. Small segments of stomach mucosa were prepared

and later transferred to the stage of a dissecting microscope where individual gastric

glands were hand-dissected using a previously described technique (28). Individual

glands were subsequently placed on cover slips coated with Cell-Tak (cell adhesive, BD

Biosciences; Bedford, MA) prior to beginning the experiments. Isolated gastric glands

were loaded with 10 µM BCECF (2’,7-bis(2-carbocyethyl)-5-(and 6)-carboxyfluorescein-

acetomethyl ester; Molecular Probes) a pH sensitive dye (28). Following a 15 minute

dye-loading period, gland preparations were placed in a perfusion chamber maintained at

37˚C on the stage of an inverted microscope (Olympus IX50) attached to a digital

imaging system (Universal Imaging Corp; Downingtown, PA), and perfused with HEPES

to remove any non de-esterified dye. The BCECF was excited at 490 ± 10 nm and 440 ±

10 nm and the emitted fluorescence light measured at 535 ± 10 nm using an intensified

charge-coupled device camera (28). Data were collected every 15 seconds, and the ratio

490/440 nm was initially recorded as arbitrary pH units, which were then converted to

absolute pH using a high K+/nigericin calibration technique (26; 28).

Proton extrusion by individual parietal cells was monitored by observing recovery

of pHi after acid loading the cells with a Na+ free HEPES solution containing 20 mM

G-00096-2005-R3

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NH4Cl. Parietal cells were subsequently superfused with Na+ free HEPES, which

abolished all Na+/H+ Exchanger (NHE) activity, trapping H+ within the cytosol and

initiating an immediate drop in pHi. Under these conditions, the only potential H+

extrusion pathway is via H+,K+-ATPase activation. Intracellular pH recovery rates were

measured in Na+ free HEPES solutions containing: a) 30 mM L-phe + 0.5, 1, 2, or 5 mM

Ca2+o; b) 0.5, 1, 2, or 5 mM Ca2+

o only; c) 30 mM L-trp, L-leu or D-phe + 2 mM Ca2+o; d)

3 mM L-phe and 2 mM Ca2+o +/- 10 mM BCH (2-amino-2-norbornane-carboxylic acid;

Sigma); or e) 30 mM L-phe + 2 mM Ca2+o +/- 100 µM cimetidine or omeprazole. As

noted, with the addition of amino acid to these solutions, the glucose and mannitol

concentrations used in Table 1 were reduced to maintain a constant osmolarity.

Intracellular pH recovery rates were calculated from the same initial starting pH to

eliminate potential variations in the individual intracellular buffering power of cells under

the different experimental conditions. The recovery rates are expressed as the ∆pH

units/min, and were calculated over the pH range of 6.8-7.2.

Statistical Analysis

An unpaired, Student’s t-test was used to test for differences in pH of ex vivo

whole stomach preparations, and to test for significant differences in recovery rates for

the in vitro parietal cell pHi experiments.

G-00096-2005-R3

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RESULTS

L-phenylalanine directly stimulates gastric acid production ex vivo

To determine if L-phe could elicit changes in gastric acid secretion in the whole

organ, we examined luminal pH in freshly isolated rat stomachs after incubation in

HEPES, or this same solution in the presence of 50 mM L- or D-phe. As illustrated in

Figure 1, in the presence of 50 mM L-phe, mean luminal pH was significantly lower than

in stomach preparations incubated in HEPES alone (2.69 ± 0.22 vs. 4.02 ± 0.37, n=9 for

each; p = 0.003). In the presence of 50 mM D-phe, the inactive isomer, mean luminal pH

was 3.49 ± 0.32 (n = 6), which was not significantly different from the control group (p =

0.13).

In vitro intracellular pH measurements

To explore the cellular mechanism by which L-phe stimulates acid secretion, we

measured pHi in parietal cells of hand-dissected rat gastric glands. As shown in Figure 2

panel A, in the absence of acid secretagogues but in the presence of 2 mM Ca2+o, there is

little recovery of pHi after parietal cells were exposed to an acid load in a Na+ free,

NH4Cl-containing solution, resulting in a fall in pHi to approximately 6.8. As illustrated

in Figure 2 panel B, addition of 30 mM L-phe in the presence of 2 mM Ca2+o

significantly increased the rate of pHi recovery, indicative of H+,K+-ATPase activity and

H+ extrusion from the cell. When Ca2+o is reduced to 0.5 mM as illustrated in Figure 2

panel C, the robust pHi recovery induced by 30 mM L-phe is completely abolished.

Perfusion with 30 mM D-phe, the inactive isomer, in the presence of 2 mM Ca2+o did not

elicit pHi recovery (Figure 2 panel D). These data demonstrate the need for a permissive

G-00096-2005-R3

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level of Ca2+o for amino acids to exert their effect in vitro, and that this effect is stereo-

specific (7; 9).

To more fully explore the interaction between amino acids and Ca2+o in parietal

cells, studies were conducted in isolated glands exposed to 0.5, 1, 2, or 5 mM Ca2+o in

the presence or absence of 30 mM L-phe (Figure 3). In the presence of physiological

Ca2+o concentrations (1 or 2 mM) and L-phe, the rates of H+ extrusion were comparable

(0.074 ± 0.007 and 0.083 ± 0.005 ∆pH units/min, respectively, n= 32 and 51 cells).

Removal of L-phe and perfusion with 1 or 2 mM Ca2+o resulted in a reduction in the H+

extrusion rate from 0.074 ± 0.007 to 0.051 ± 0.007 ∆pH units/min and 0.083 ± 0.005 to

0.041 ± 0.006 ∆pH units/min, respectively. Reducing Ca2+o concentration to 0.5 mM

completely inhibited H+ extrusion (-0.020 ± 0.008 ∆pH units/min, n=16 cells). Addition

of L-phe had only a minimal effect on ∆pHi (0.005 ± 0.004 ∆pH units/min, n=29 cells).

Exposing glands to 5 mM Ca2+o alone significantly raised ∆pHi, (0.074 ± 0.01 ∆pH

units/min, n=17 cells). This rate was further increased in the presence of L-phe (0.159 ±

0.02 ∆pH units/min, n=32 cells). The ability of amino acids to augment acid secretion at

submaximal Ca2+o concentrations is highly suggestive of allosteric activation.

To confirm and extend our findings with L-phe, we tested additional amino acids

that are known to either strongly activate or elicit weak modulatory effects on the CaSR

(7-9). As shown in Figure 4, panel A, exposing parietal cells to 30 mM L-trp, an amino

acid known to activate the CaSR, resulted in rates of H+ extrusion very similar to those

observed with 30 mM L-phe (0.082 ± 0.013 vs. 0.083 ± 0.005 ∆pH units/min). Perfusion

with a weak activator, L-leu, or the inactive isomer, D-phe, resulted in pHi recovery rates

significantly lower than that observed with L-phe (0.024 ± 0.006 and 0.015 ± 0.003 vs.

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0.083 ± 0.005 ∆pH units/min, respectively, p<0.0001*). These results also suggest

allosteric activation of the CaSR by amino acids, since the profile for allosteric activation

was identical in rank order potency to that shown by Conigrave et al. in HEK-293 cells

over-expressing the CaSR (7-9).

To confirm that L-phe was acting via the CaSR and not via the system L amino

acid transporter, we used BCH (2-amino-2-norbornane-carboxylic acid), a known

inhibitor of the L amino acid transport system (6; 18; 24; 27). For these studies, we used

3 mM L-phe and 2 mM Ca2+o in the presence or absence of 10 mM BCH, a concentration

that competitively blocks the system L transport system. The mean H+ extrusion rate in

the presence of BCH was not different from the rate in the absence of BCH (0.059 ±

0.004 vs. 0.062 ± 0.006, n= 33 and 30 cells, respectively, (see Figure 4, panel B).

Figure 5 summarizes mean H+ extrusion rates in the presence of 30 mM L-phe,

and either cimetidine or omeprazole. In the presence of 100 µM cimetidine, the H2R

antagonist, the H+ extrusion rates induced by 30 mM L-phe was comparable to that seen

with L-phe alone, (0.078 ± 0.007 vs. 0.083 ± 0.005 ∆pH units/min, n=30 and 51

respectively). In a separate series of experiments, cells were perfused with 100 µM

omeprazole in the presence of 30 mM L-phe and 2 mM Ca2+o. The rate of H+ extrusion

was significantly reduced from 0.083 ± 0.005 to 0.03 ± 0.006 ∆pH units/min

(p<0.0001*), confirming the role of the H+,K+-ATPase in this H+ extrusion pathway.

These data clearly demonstrate that L-phe-induced H+ extrusion from parietal cells

occurs via H+,K+-ATPase activation, which is histamine-independent and omeprazole-

sensitive.

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Discussion

Experimental work in animals and humans has established that amino acids can

stimulate gastric acid secretion (15-17; 19; 23). However, the molecular mechanism(s)

by which amino acids elicit this effect have not yet been fully elucidated. The CaSR is

expressed in parietal cells of gastric glands and can be activated by divalent and trivalent

cations to induce acid secretion in isolated rat gastric glands (3-5; 12; 20). The CaSR is

also expressed in gastric G cells, where activation of the receptor results in gastrin

release, ultimately stimulating gastric acid production (2; 4; 5).

The endogenous ligands for the CaSR in the stomach are not known. Our data

strongly suggest that exposure to amino acids at physiological Ca2+o concentrations

enhance the rate of H+ extrusion in a manner consistent with allosteric activation of the

CaSR. Previous work in HEK-293 cells stably over-expressing the CaSR demonstrated

that L-amino acids augmented the rise in Ca2+i observed in the presence of 1.5-2.5 mM

Ca2+o, supporting the notion that amino acids are allosteric activators of this receptor (7;

9). Our findings in parietal cells endogenously expressing the CaSR are entirely

consistent with the same conclusion, namely that CaSR-dependent acid secretion occurs

via allosteric activation by L-amino acids.

Our group has recently identified a functional system L heteromeric amino acid

transporter comprised of the LAT2-4F2hc dimer in mouse parietal cells (Geibel,

unpublished observation). We therefore felt it was important to exclude the possibility

that amino acids were exerting their effect on H+ extrusion independent of CaSR

G-00096-2005-R3

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activation, namely via system L amino acid transport. However, we did not observe an

inhibitory effect of BCH on L-phe induced acid extrusion, excluding a contribution of

this transporter to the enhanced H+ extrusion observed in the presence of amino acids.

Our data strongly support the conclusion that L-amino acids, specifically L-phe

and L-trp, can regulate gastric acid secretion. We chose these particular amino acids

since they have been shown to be potent allosteric activators of the CaSR in HEK-293

cells transfected with CaSR (7; 9). In our initial studies, we measured luminal pH in

freshly isolated whole stomach preparations bathed in HEPES or this same solution with

50 mM L- or D-phe. A concentration of 50 mM L-phe was used to ensure that a

sufficient amount of amino acids would diffuse across the stomach serosa to reach the

basolateral aspect of the parietal cells, emulating the postprandial rise in serum amino

acids (1; 10). We observed a significant decrease in pH of the gastric aspirate from

preparations bathed in L-phe as compared to those in buffer alone. Incubation with 50

mM D-phe, however, did not induce acid secretion.

To explore the cellular mechanism for the effect of amino acids on acid secretion

observed in the above experiments, we used freshly isolated gastric gland preparations.

Although as noted, the CaSR is expressed in antral G cells of the gastric gland, we tested

our hypothesis in parietal cells, the primary acid secretory cells of the stomach (2; 4; 5).

In our experimental system, CaSR-mediated activation of the H+,K+-ATPase and thus H+

extrusion is observed as a rise in pHi (net proton extrusion from cell to lumen of the

gland). In the absence of acid secretagogues, administration of 30 mM L-phe or 30 mM

L-trp in the presence of 2 mM Ca2+o in the gastric gland perfusate produced a significant

increase in pHi and the rate of H+ extrusion from acid-loaded parietal cells (Figure 3c;

G-00096-2005-R3

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black bar, and Figure 4). Reducing the Ca2+o concentration to 1 mM in the continued

presence of L-phe had no effect on H+ extrusion (Figure 3b vs. c; black bars). Further

decreasing Ca2+o to 0.5 mM, reduced H+ extrusion to a rate significantly lower than that

observed with either 1 or 2 mM Ca2+o alone (Figure 3a, black bar vs. b and c open bars).

The rate of H+ extrusion with 0.5 mM Ca2+o plus 30 mM L-phe was significantly lower

than the rate observed with 1 or 2 mM Ca2+o plus 30 mM L-phe (a vs. b and c, black

bars). These data demonstrate a dependence of amino acid-induced acid secretion on

Ca2+o. The finding that use of an inactive isomer, D-phe, was devoid of any activity

supports the specificity of this amino acid effect.

The molecular details of how amino acids interact with the CaSR are not known. Recent

evidence indicates that the CaSR’s “Venus Fly Trap” (VFT) domain is required for L-

amino acid sensing by the CaSR, although it has not yet been directly determined that

amino acids bind there (21). Nonetheless, the fact that we observed additivity of Ca2+o

and amino acids even at high concentrations of Ca2+o is consistent with the idea of

separate binding sites for calcium and amino acids (7; 9; 21). The possibility of two

separate binding sites in no way precludes an allosteric interaction of amino acids and

Ca2+o in activating this receptor. The enhanced rates of acid secretion at sub-maximal

Ca2+o (Figure 3), the parallel potencies to those observed by Conigrave (Figure 4, panel

A) and the absolute requirement for a permissive concentration of Ca2+o (Figure 3) are all

consistent with this idea.

To exclude any hormonal contribution to the effects observed with L-phe,

all in vitro experiments were performed in the absence of known acid secretagogues such

as histamine and/or gastrin. Consistent with the conclusion that L-phe is acting via a

G-00096-2005-R3

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hormone independent pathway, inhibiting the H2R with cimetidine did not affect the rate

of H+ extrusion induced by 30 mM L-phe and 2 mM Ca2+o (see Figure 5). The fact that

omeprazole blocks the effect of amino acids on H+ extrusion shows that the amino acid

effect on the CaSR works via the H+,K+-ATPase (see Figure 5).

Existence of a novel acid secretory pathway initiated by physiologic levels of

amino acids and Ca2+o illustrates the complexities of amino acid stimulated acid secretion

by parietal cells. It is apparent that no single pathway is solely responsible for acid

secretion by the stomach. Inhibiting CaSR activity specifically or studying animals with

conditional deletion of the receptor in the parietal cells may help to elucidate the

importance of this pathway in acid secretion. The in vivo role of amino acids in

modulating acid secretion may combine indirect effects of CaSR-mediated G cell gastrin

release, and direct effects on parietal cell CaSR acid secretion. This could ultimately

explain why some individuals who consumer high protein diets experience symptoms of

acid reflux, and more importantly why some patients experience breakthrough acid

secretion despite acid suppression therapy with H2R antagonists. In light of these

findings, clarifying the role of nutrient sensing by the CaSR may help to identify new

treatments for acid reflux disease through dietary manipulation of CaSR activity.

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Acknowledgements

This study was supported by grants from NIH DK50230, 17433, 60069 to John Geibel,

the USDA 2005-00806 to Karl Insogna, and in part by the Yale Core Center for

Musculoskeletal Disorders (P30 AR46032).

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Table 1. Composition of solutions (mM) Final osmolarity of all solutions was 290 mOsms. As detailed in the Methods, all solutions were adjusted with mannitol to maintain a constant osmolarity*. Glucoseconcentration was reduced to 5 mM when L-phe, L-trp, L-leu or D-phe were added to a solution.

G-00096-2005-R3

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Figure legends

Figure 1: L-phe stereo-selectively stimulates gastric acid secretion in freshly isolated rat whole stomach preparations.

Ex vivo rat whole-stomach preparations were incubated in HEPES-buffered Ringer’s

(control; n=9, open bar); HEPES-buffered Ringer’s + 50 mM L-phe (n= 9, black bar); or

HEPES-buffered Ringer’s + 50 mM D-phe (n=6, shaded bar). Stomach preparations

incubated with L-phe had a significantly lower pH than those in HEPES-buffered

Ringer’s alone (p=0.003*). Incubation with D-phe did not affect pH in comparison to the

control (p=0.13).

Figure 2: Effect of L-phe and extracellular Ca2+on pHi in rat parietal cells

Recovery of pHi was calculated in a Na+-free HEPES solution +/- amino acid after

intracellular acidification with a Na+-free NH4Cl-containing solution for all experimental

protocols. A. Perfusion of gastric glands with a Na+-free NH4Cl-containing solution

causes intracellular acidification. There was reduced recovery of pHi when the acidified

glands were subsequently perfused with Na+ free HEPES containing only 2 mM Ca2+o

(control). B. Perfusion with Na+-free HEPES containing 30 mM L-phe + 2 mM Ca2+o

(arrow indicates addition of amino acid), results in a substantial rise in pHi, indicative of

H+,K+-ATPase activation and H+ extrusion from the cell. C. Reducing Ca2+o to 0.5 mM

in the presence of 30 mM L-phe (arrow) does not induce pHi recovery. D. Perfusion with

the inactive isomer, 30 mM D-phe, and 2 mM Ca2+o (arrow) does not result in a

significant increase in the rate of H+ extrusion from the cell.

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Figure 3: Ca2+o dose dependent effects on L-phe stimulated H+ extrusion.

Mean pHi recovery rates were calculated in Na+ free HEPES solutions containing 0.5, 1,

2 or 5 mM Ca2+o in the presence of absence of 30 mM L-phe. At 1 and 2 mM Ca2+

o, H+

extrusion rates were comparable to each other (b. and c.; open bars; n = 16 and 25 cells,

respectively). Adding 30 mM L-phe significantly increased pHi recovery rates under

these conditions (b. and c.; black bars; n= 32 and 51 cells, respectively). Reducing Ca2+o

to 0.5 mM results in a decrease in pHi, and adding L-phe does not significantly increase

the H+ extrusion rate at this low Ca2+o concentration (a.; open and black bars, n= 16 and

29 cells, respectively). Perfusing with 5 mM Ca2+o results in H+ extrusion rates

comparable to that seen with L-phe + 2 mM Ca2+o (d.; open bar vs. c., black bar); while

adding L-phe in the presence of 5 mM Ca2+o resulted in a two-fold increase in H+

extrusion in comparison to 5 mM Ca2+o alone (d.; open vs. black bars, n=17 and 32 cells,

respectively).

Figure 4: CaSR-mediated H+ extrusion is amino acid and isomer-specific.

A. Mean pHi recovery rates were calculated in Na+ free HEPES solutions containing 2

mM Ca2+o and the indicated amino acids all at 30 mM. Perfusion with either of the

aromatic amino acids L-phe or L-trp resulted in comparable H+ extrusion rates. Perfusion

with the weak activators, D-phe or L-leu, gave pHi recovery rates that were substantially

lower than L-phe or L-trp, (n=17 or 58 cells, respectively). B. Mean pHi recovery rates

in the presence of 2 mM Ca2+o and 3 mM L-phe. Blocking system L amino acid transport

with 10 mM BCH, a competitive inhibitor of the system L transporter, did not effect H+

G-00096-2005-R3

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extrusion rates induced by 2 mM Ca2+o and 3 mM L-phe (n=30 and 33 cells,

respectively).

Figure 5: Amino acid stimulation of parietal cell acid secretion is histamine

independent and H+,K+-ATPase dependent.

Mean pHi recovery rates were calculated in Na+ free HEPES solutions containing 2 mM

Ca2+o and 30 mM L-phe. In the presence of 100 µM cimetidine, the mean pHi recovery

rate was comparable to that seen with L-phe alone (n=30 cells). Blocking H+,K+-ATPase

activity with 100µM omeprazole effectively inhibited H+ extrusion (p<0.0001*).

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Table 1.

7.07.47.47.4pH (37˚C)

32.232.232.232.2HEPES

-101010Glucose

5***Mannitol

1121CaCl2

1.21.21.21.2MgSO4

105555KCl

-20--NH4Cl

32.8132.8132.8-NMDG

---115NaCl

High K+

CalibrationNa+ free HEPES +NH4Cl

Na+ free HEPES

HEPES

G-00096-2005-R3

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Figure 1.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

1

*

pH

control L-phe D-phe

G-00096-2005-R3

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Figure 2.

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

8

0 200 400 600 800

Time (s)

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

8

0 200 400 600 800

Time (s)

6.4

6.6

6.8

7

7.2

7.4

7.6

0 200 400 600 800Time (s)

6.4

6.6

6.8

7

7.2

7.4

7.6

0 200 400 600 800 1000

Time (s)

A. B.

C. D.

G-00096-2005-R3

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Figure 3.

-0.1

0.0

0.1

0.2

∆pH

uni

ts/m

in Ca2+only

30mM L-phe

0.5mM Ca2+ 1mM Ca2+ 2mM Ca2+ 5mM Ca2+

a. b. c. d.

G-00096-2005-R3

Figure 4.

0.100.

A

24

L-phe

D-phe

L-trp

L-leu

0.000

0.025

0.050

0.075

**

∆pH

uni

ts/m

in

G-00096-2005-R3

Figure 4.

B.

25

3mM L

-phe

BCH + 3m

M L-p

he0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

∆pH

uni

ts/m

in

G-00096-2005-R3

26

Figure5.

L-p

he

cimet

idin

e

omep

razo

le0.000.010.020.030.040.050.060.070.080.09

∆pH

uni

ts/m

in

*

G-00096-2005-R3

27

References

1. Adibi SA and Mercer DW. Protein digestion in human intestine as reflected

in luminal, mucosal, and plasma amino acid concentrations after meals. J

Clin Invest 52: 1586-1594, 1973.

2. Buchan AM, Squires PE, Ring M and Meloche RM. Mechanism of action of

the calcium-sensing receptor in human antral gastrin cells. Gastroenterology

120: 1128-1139, 2001.

3. Caroppo R, Gerbino A, Debellis L, Kifor O, Soybel DI, Brown EM, Hofer

AM and Curci S. Asymmetrical, agonist-induced fluctuations in local

extracellular [Ca(2+)] in intact polarized epithelia. EMBO J 20: 6316-6326,

2001.

4. Chang W and Shoback D. Extracellular Ca2+-sensing receptors--an

overview. Cell Calcium 35: 183-196, 2004.

5. Cheng I, Qureshi I, Chattopadhyay N, Qureshi A, Butters RR, Hall AE,

Cima RR, Rogers KV, Hebert SC, Geibel JP, Brown EM and Soybel DI.

Expression of an extracellular calcium-sensing receptor in rat stomach.

Gastroenterology 116: 118-126, 1999.

G-00096-2005-R3

28

6. Chillaron J, Roca R, Valencia A, Zorzano A and Palacin M. Heteromeric

amino acid transporters: biochemistry, genetics, and physiology. Am J

Physiol Renal Physiol 281: F995-1018, 2001.

7. Conigrave AD, Franks AH, Brown EM and Quinn SJ. L-amino acid sensing

by the calcium-sensing receptor: a general mechanism for coupling protein

and calcium metabolism? Eur J Clin Nutr 56: 1072-1080, 2002.

8. Conigrave AD, Mun HC, Delbridge L, Quinn SJ, Wilkinson M and Brown

EM. L-amino acids regulate parathyroid hormone secretion. J Biol Chem

279: 38151-38159, 2004.

9. Conigrave AD, Quinn SJ and Brown EM. L-amino acid sensing by the

extracellular Ca2+-sensing receptor. Proc Natl Acad Sci U S A 97: 4814-4819,

2000.

10. Feldman EJ and Grossman MI. Liver extract and its free amino acids

equally stimulate gastric acid secretion. Am J Physiol 239: G493-G496, 1980.

11. Forte JG. Recent concepts of the mechanism of hydrochloric acid secretion. J

Indian Med Prof 12: 5637-5647, 1966.

G-00096-2005-R3

29

12. Geibel JP, Wagner CA, Caroppo R, Qureshi I, Gloeckner J, Manuelidis L,

Kirchhoff P and Radebold K. The stomach divalent ion-sensing receptor scar

is a modulator of gastric acid secretion. J Biol Chem 276: 39549-39552, 2001.

13. Hersey SJ and Sachs G. Gastric acid secretion. Physiol Rev 75: 155-189, 1995.

14. Hirschowitz BI, Keeling D, Lewin M, Okabe S, Parsons M, Sewing K,

Wallmark B and Sachs G. Pharmacological aspects of acid secretion. Dig Dis

Sci 40: 3S-23S, 1995.

15. Isenberg JI and Maxwell V. Intravenous infusion of amino acids stimulates

gastric acid secretion in man. N Engl J Med 298: 27-29, 1978.

16. Konturek SJ, Tasler J, Cieszkowski M and Jaworek J. Comparison of

intravenous amino acids in the stimulation of gastric secretion.

Gastroenterology 75: 817-824, 1978.

17. Konturek SJ, Tasler J, Obtulowicz W and Cieszkowski M. Comparison of

amino acids bathing the oxyntic gland area in the stimulation of gastric

secretion. Gastroenterology 70: 66-69, 1976.

18. Mastroberardino L, Spindler B, Pfeiffer R, Skelly PJ, Loffing J, Shoemaker

CB and Verrey F. Amino-acid transport by heterodimers of 4F2hc/CD98 and

members of a permease family. Nature 395: 288-291, 1998.

G-00096-2005-R3

30

19. McArthur KE, Isenberg JI, Hogan DL and Dreier SJ. Intravenous infusion

of L-isomers of phenylalanine and tryptophan stimulate gastric acid

secretion at physiologic plasma concentrations in normal subjects and after

parietal cell vagotomy. J Clin Invest 71: 1254-1262, 1983.

20. Mitsuma T, Rhue N, Kayama M, Mori Y, Adachi K, Yokoi Y, Ping J,

Nogimori T and Hirooka Y. Distribution of calcium sensing receptor in rats:

an immunohistochemical study. Endocr Regul 33: 55-59, 1999.

21. Mun HC, Franks AH, Culverston EL, Krapcho K, Nemeth EF and

Conigrave AD. The Venus Fly Trap Domain of the Extracellular Ca2+-

sensing Receptor Is Required for L-Amino Acid Sensing. J Biol Chem 279:

51739-51744, 2004.

22. Prinz C, Kajimura M, Scott D, Helander H, Shin J, Besancon M, Bamberg

K, Hersey S and Sachs G. Acid secretion and the H,K ATPase of stomach.

Yale J Biol Med 65: 577-596, 1992.

23. Richardson CT, Walsh JH, Hicks MI and Fordtran JS. Studies on the

mechanisms of food-stimulated gastric acid secretion in normal human

subjects. J Clin Invest 58: 623-631, 1976.

G-00096-2005-R3

31

24. Rossier G, Meier C, Bauch C, Summa V, Sordat B, Verrey F and Kuhn LC.

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of

kidney and intestine. J Biol Chem 274: 34948-34954, 1999.

25. Sachs G, Prinz C, Loo D, Bamberg K, Besancon M and Shin JM. Gastric

acid secretion: activation and inhibition. Yale J Biol Med 67: 81-95, 1994.

26. Singh SK, Binder HJ, Geibel JP and Boron WF. An apical permeability

barrier to NH3/NH4+ in isolated, perfused colonic crypts. Proc Natl Acad Sci

U S A 92: 11573-11577, 1995.

27. Wagner CA, Lang F and Broer S. Function and structure of heterodimeric

amino acid transporters. Am J Physiol Cell Physiol 281: C1077-C1093, 2001.

28. Waisbren SJ, Geibel J, Boron WF and Modlin IM. Luminal perfusion of

isolated gastric glands. Am J Physiol 266: C1013-C1027, 1994.

29. Yao X and Forte JG. Cell biology of acid secretion by the parietal cell. Annu

Rev Physiol 65: 103-131, 2003.

G-00096-2005-R3

32