Am J Physiol Gastrointest Liver Physiol 297: G781–G791, 2009.
First published July 30, 2009; doi:10.1152/ajpgi.90605.2008.
Luminal L-glutamate enhances duodenal mucosal defense mechanisms via
multiple glutamate receptors in rats
Yasutada Akiba,1,2,3 Chikako Watanabe,2,3 Misa Mizumori,2,3 and Jonathan D. Kaunitz1,2,3
1
Greater Los Angeles Veterans Affairs Healthcare System; 2Department of Medicine, School of Medicine, University
of California; 3Brentwood Biomedical Research Institute, Los Angeles, California
Submitted 20 October 2008; accepted in final form 23 July 2009
intracellular alkalinization; mucus secretion; bicarbonate secretion;
monosodium glutamate; taste receptor
THE DUODENAL MUCOSA HAS multilayered, multistep defense
mechanisms to counter mucosal injury attributable to constant
exposure to luminal concentrated acid and high PCO2 attributable to gastric acid and secreted HCO⫺
3 (24). These mechanisms coordinately regulate premucosal, mucosal, and submucosal components, including mucus and HCO⫺
3 secretion, intracellular pH (pHi) regulation and cellular buffering, and
submucosal neuronal activation and blood flow response. Because duodenal luminal pH rapidly changes between 2 and 7
as a result of the constant mixture of secreted HCO⫺
3 with
jets of antrally propelled gastric acid, the duodenal mucosa
Address for reprint requests and other correspondence: Y. Akiba, Bldg. 114,
Suite 217, West Los Angeles VA Medical Ctr., 11301 Wilshire Blvd., Los
Angeles, CA 90073 (e-mail: [email protected]).
http://www.ajpgi.org
must rapidly adjust its defense mechanisms according to
luminal pH (23).
We have examined the upper gastrointestinal (GI) defense
factors in response to luminal acid, using fluorescent microscopy and Doppler flowmetry where epithelial pHi, mucosal
blood flow, and mucus gel thickness (MGT), corresponding to
mucus secretion rate, are simultaneously measured in the
gastroduodenum of living rats (6, 7, 9, 25, 34, 43). In duodenum, luminal acid is sensed by the “capsaicin pathway,”
consisting of acid-induced intracellular acidification, H⫹ secretion via the basolateral Na⫹/H⫹ exchanger-1, activation of
capsaicin-sensitive afferent nerves, the release of vasoactive
mediators, and an increase of mucosal blood flow and mucus
secretion, followed by delayed cyclooxygenase (COX)/prostaglandin (PG)-dependent mucus and HCO⫺
3 secretion (24).
These data demonstrate that the duodenal mucosa “tastes”
luminal acidity using epithelial ion transporters and neuronal
acid sensors. Furthermore, luminal CO2 is also sensed by the
capsaicin pathway, facilitated by epithelial carbonic anhydrases (5, 31), suggesting that luminal H⫹ and CO2 provide
equivalent acid loads that signal protective effector mechanisms.
Although the five basic tastes (sweet, salty, sour, bitter, and
umami) have been recognized for centuries, taste receptor
families have only recently been cloned from the lingual taste
buds (26). Of interest, these receptors are also present in the GI
tract. In addition to salty taste detected by epithelial Na⫹
channels and sour taste by H⫹-gated ion channels, a heterodimer of the taste receptor, type 1, member 2 (T1R2) and 3
(T1R3) is a sweet receptor, expressed in the small intestinal
mucosa (19, 28, 29). Bitter taste receptors consisting of the
taste receptor, type 2 (T2R) family are expressed in the GI tract
(47). This suggests that the intestinal mucosa, in addition to the
tongue, also senses the luminal content to presumably detect
nutrients and expel unfavorable substances to maintain physiological processes such as digestion, absorption, secretion, and
motility.
The receptor for L-glutamate (L-Glu), the primary nutrient
conferring umami taste, is a heterodimer of T1R1 and T1R3
(32, 48) or alternately the metabotropic L-Glu receptor
(mGluR) 1 or 4 (15, 44). These receptors belong to G proteincoupled receptor superfamily. These receptors and their cognate G proteins, ␣-gustducin, are localized in the epithelial
cells in the GI tract (28, 29, 38, 40), suggesting that the mucosa
directly taste luminal content to release mediators or conduct
the luminal information to internal milieu. Indeed, luminal
L-Glu stimulates gastric vagal afferents with release of nitric
oxide and 5-hydroxytryptamine (5-HT) with the involvement
of 5-HT3 receptor (46), suggesting a nutrient-sensing pathway
for L-Glu in the upper GI tract related to known protective
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Society
G781
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Akiba Y, Watanabe C, Mizumori M, Kaunitz JD. Luminal
enhances duodenal mucosal defense mechanisms via
multiple glutamate receptors in rats. Am J Physiol Gastrointest Liver
Physiol 297: G781–G791, 2009. First published July 30, 2009;
doi:10.1152/ajpgi.90605.2008.—Presence of taste receptor families in
the gastrointestinal mucosa suggests a physiological basis for local
and early detection of a meal. We hypothesized that luminal Lglutamate, which is the primary nutrient conferring fundamental
umami or proteinaceous taste, influences mucosal defense mechanisms in rat duodenum. We perfused the duodenal mucosa of anesthetized rats with L-glutamate (0.1–10 mM). Intracellular pH (pHi) of
the epithelial cells, blood flow, and mucus gel thickness (MGT) were
simultaneously and continuously measured in vivo. Some rats were
pretreated with indomethacin or capsaicin. Duodenal bicarbonate
secretion (DBS) was measured with flow-through pH and CO2 electrodes. We tested the effects of agonists or antagonists for metabotropic glutamate receptor (mGluR) 1 or 4 or calcium-sensing receptor
(CaSR) on defense factors. Luminal L-glutamate dose dependently
increased pHi and MGT but had no effect on blood flow in the
duodenum. L-glutamate (10 mM)-induced cellular alkalinization and
mucus secretion were inhibited by pretreatment with indomethacin or
capsaicin. L-glutamate effects on pHi and MGT were mimicked by
mGluR4 agonists and inhibited by an mGluR4 antagonist. CaSR
agonists acidified cells with increased MGT and DBS, unlike Lglutamate. Perfusion of L-glutamate with inosinate (inosine 5⬘-monophosphate, 0.1 mM) enhanced DBS only in combination, suggesting
synergistic activation of the L-glutamate receptor, typical of taste
receptor type 1. L-leucine or L-aspartate had similar effects on DBS
without any effect on pHi and MGT. Preperfusion of L-glutamate
prevented acid-induced cellular injury, suggesting that L-glutamate
protects the mucosa by enhancing mucosal defenses. Luminal Lglutamate may activate multiple receptors and afferent nerves and
locally enhance mucosal defenses to prevent subsequent injury attributable to acid exposure in the duodenum.
L-glutamate
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L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
Table 1. PCR primers used
Product
Size, bp
Sequence
5⬘-TTGGTCTACTGCTGGGCTTT-3⬘
5⬘-AGCGTGGTCAGTGTTGTCAG-3⬘
414
NM053305
5⬘-AACAACACGGTCCCTGTCTC-3⬘
5⬘-GGCAGAAGCATGAGAAGACC-3⬘
439
XM001074791
5⬘-GAGGCCACTCTCAACCAGAG-3⬘
5⬘-CCAGCTGAAATTCTGCAACA-3⬘
477
NM130818
5⬘-GCTCCTGGATTTCCTCATCA-3⬘
5⬘-CGTACCATTCCGCTTTTGTT-3⬘
216
NM017011
5⬘-GATATTGTCCGAGCCCTCAA-3⬘
5⬘-CTCCAACACCCTCCTGATGT-3⬘
255
NM022666
5⬘-TCTTGTGGAGTGGGTTCTCC-3⬘
5⬘-GGCCTTGACGATAGGTGTGT-3⬘
353
NM016996
5⬘-GTGGGCCGCCCTAGGCACCA-3⬘
5⬘-TGGCCTTAGGGTTCAGAGGG-3⬘
241
NM031144
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T1R1
sense
antisense
T1R2
sense
antisense
T1R3
sense
antisense
mGluR1
sense
antisense
mGluR4
sense
antisense
CaSR
sense
antisense
␤-actin
sense
antisense
Accession
Number
epithelial cells. After BCECF loading, fluorescent microspheres diluted to 0.05% wt/vol with prewarmed Krebs buffer were placed over
the mucosa to delineate the luminal surface of the gel layer. Blood
flow was measured via serosally placed right angle probe with laser
Doppler flowmetry (Transonic, Ithaca, NY).
BCECF fluorescence was visualized with 495- and 450-nm excitation and 515-nm emission (green filter set; Chroma, Brattleboro,
VT), whereas fluorescent microspheres were visualized with 575-nm
excitation and 600-nm emission (red filter set) using a ⫻10 objective
mounted on a Zeiss MPS microscope and recorded with a cooled
charge-coupled device video camera (Hamamatsu Orca-EN;
Hamamatsu USA, Bridgewater, NJ). Images were captured, digitized,
recorded, and analyzed using Apple G4 microcomputer and image
analysis software (OpenLab; Improvision, Lexington, MA). pHi was
calculated from fluorescence intensity ratio and in vitro calibration
T1R, taste receptor type 1; mGluR, metabotropic glutamate receptor; CaSR,
calcium-sensing receptor.
efferent neural circuitry. Thus we hypothesized that luminal
L-Glu affects mucosal defense mechanisms via L-Glu receptors
in the upper GI tract, similar to the acid-sensing pathways.
Here we show that luminal L-Glu enhances duodenal mucosal defenses mediated via multiple L-Glu receptors and activation of capsaicin and COX/PG pathways and that L-Glu protects the duodenal mucosa from acid-induced injury.
MATERIALS AND METHODS
Chemicals and animals. 2⬘,7⬘-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acid, its acetoxy methyl ester (BCECF-AM),
and propidium iodide (PI) were obtained from Molecular Probes
(Eugene, OR). Two-micron pink fluorescent microspheres were obtained from Bangs Laboratories (Fishers, IN). VU0155041 (cis-2[[(3,5-dichlorophenyl)amino]carbonyl]cyclohexane carboxylic acid)
sodium salt was obtained from Tocris Bioscience (Ellisville, MO).
Monosodium L-Glu, D-glutamate (D-Glu), L-aspartate (L-Asp), L-alanine (L-Ala), L-leucine (L-Leu), inosine 5⬘-monophosphate (IMP),
spermine, L-2-amino-4-phosphonobutyric acid (L-AP4), (S)-2-amino2-methyl-4-phosphonobutyric acid (MAP4), (S)-3,5-dihydroxyphenylglycine (S-DHPG), 1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid
(AIDA), HEPES and other chemicals were obtained from Sigma Chemical (St. Louis, MO). Krebs solution contained (in mM) 136 NaCl, 2.6
KCl, 1.8 CaCl2, and 10 HEPES at pH 7.0. Osmolarity was adjusted to
isotonic by reducing NaCl concentration. pH of Krebs solution after
dissolving the compound was adjusted to pH 7.0. All solutions were
prewarmed at 37°C in a water bath, and temperature was maintained
by a heating pad. All studies were performed with approval of the
Veterans Affairs Institutional Animal Care and Use Committee (VA
IACUC). Male Sprague-Dawley rats weighing 200 –250 g (Harlan,
San Diego, CA) were fasted overnight but had free access to water.
Measurements of pHi, blood flow, and MGT in duodenum. Simultaneous measurements of pHi of duodenal epithelial cells using
fluorescent ratio technique, duodenal blood flow with laser Doppler
flowmetry, and MGT with fluorescent microspheres were performed
as described elsewhere (7, 9, 30). Under isoflurane anesthesia (2%),
the exposed, chambered duodenal mucosa was incubated with 10 ␮M
BCECF-AM in pH 7.0 Krebs buffer for 15 min to load the duodenal
AJP-Gastrointest Liver Physiol • VOL
Fig. 1. Effect of luminal L-glutamate (L-Glu) on mucosal defense factors in rat
duodenum. Intracellular pH (pHi) of epithelial cells, mucus gel thickness, and
blood flow were simultaneously measured in vivo using fluorescence microscopy and flowmetry. Luminal perfusion of L-Glu (0.1–10 mM) dose dependently increased pHi (A) and mucus gel thickness (B) but had no effect on
duodenal blood flow (C). Each data point represents mean ⫾ SE (n ⫽ 6 rats).
*P ⬍ 0.05 vs. pH 7.0 Krebs group.
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L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
was ligated just proximal to its insertion into the duodenal wall and
cannulated with a PE-10 tube to drain the juice. After stabilization
with continuous perfusion of pH 7.0 saline for ⬃30 min, the time was
set as t ⫽ 0. The duodenal loop was perfused with pH 7.0 saline from
t ⫽ 0 min until t ⫽ 10 min (basal period). The perfusate was then
changed to pH 7.0 Krebs buffer from t ⫽ 10 min until t ⫽ 35 min
(challenge period), with or without chemicals. At t ⫽ 10 min, the
system was gently flushed so as to rapidly change the composition of
the perfusate.
Experimental protocol. To examine the effect of luminal L-Glu on
mucosal defense mechanisms, L-Glu (0.1–10 mM) was luminally
perfused over the duodenal mucosa while pHi, blood flow, and MGT
were measured. D-Glu (10 mM) was used as a negative control. In
duodenal HCO⫺
3 secretion experiments, L-Glu (10 mM) was perfused
through a duodenal loop during the challenge period.
Some animals were pretreated with high dose of capsaicin (125
mg/kg sc) 10 –14 days before the experiments to selectively denervate
the afferent nerves or were pretreated with indomethacin (5 mg/kg ip)
1 h before the experiments to inhibit COX activity as previously
described (6). The duodenal mucosa of capsaicin- or indomethacintreated rats was superfused with L-Glu (10 mM) as described above.
To evaluate the involvement of T1Rs as an amino acid receptor in
L-Glu effects, the effects of L-Asp, L-Ala, or L-Leu (each 10 mM) on
pHi/blood flow/MGT or HCO⫺
3 secretion were examined. Because the
luminal concentration of L-Ala (1.9 mM) or L-Leu (3 mM) in the
human jejunum 3 h after a protein-rich meal is similar to that of L-Glu
(2.6 mM) (1), we chose L-Ala and L-Leu in addition to another
excitatory amino acid L-Asp to compare their effects with L-Glu.
Because L-Glu-stimulated lingual umami perception is synergistically
enhanced by the addition of IMP (41), and because the T1R1/T1R3
heterodimer is synergistically activated with amino acid ⫹ IMP (32),
the additional effect of IMP on mucosal defense factors (pHi/blood
Fig. 2. Effect of luminal amino acids on pHi and mucus gel thickness in rat duodenum. D-glutamate (D-Glu, 10 mM), L-aspartate (L-Asp, 10 mM), L-alanine
(L-Ala, 10 mM), or L-leucine (L-Leu, 10 mM) had no effect on pHi (A and C) and mucus gel thickness (B and D). Each data point represents mean ⫾ SE (n ⫽
6 rats).
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curve as previously described (9). MGT was determined by measuring
traveling distance between focal planes of the green fluorescent cell
surface and the red fluorescent microspheres using a digital z-axis
measuring device (Quick-Check; Metronics, Bedford, NH) (7).
After BCECF was loaded, fluorescent microspheres were applied
on the gel surface, and blood flow was stabilized with pH 7.0 Krebs
buffer perfusion for ⬃30 min, the time was set as t ⫽ 0. The proximal
duodenal mucosa was isolated by the chamber and topically perfused,
preventing the contamination of pancreatic secretion. The chambered
duodenal mucosa was perfused with pH 7.0 Krebs from t ⫽ 0 min
until t ⫽ 10 min (basal period), followed by with pH 7.0 Krebs with
or without chemicals from t ⫽ 10 min until t ⫽ 20 min (challenge
period), then with pH 7.0 Krebs from t ⫽ 20 min until t ⫽ 35 min
(recovery period). All measurements were conducted every 5 min.
Measurement of duodenal HCO3⫺ secretion. Duodenal loops were
prepared and perfused as previously described (10, 31). In brief, under
isoflurane anesthesia (2%), the proximal duodenal loop (perfused
length 2 cm) was perfused with pH 7.0 normal saline by using a
peristaltic pump (Fisher Scientific, Pittsburgh, PA) at 1 ml/min. The
perfusate was bubbled with 100% O2 and stirred and warmed at 37°C
with a heating stirrer (Barnstead International, Dubuque, IA). The pH
of the perfusate was kept constant at pH 7.0 with a pH stat (models
PHM290 and ABU901; Radiometer Analytical, Lyon, France). Furthermore, to eliminate the buffer action of agonists or antagonists,
which would over- or underestimate the titration volume using pH
stat, two sets of flow through pH and CO2 electrodes (Lazar Research
Laboratories, Los Angeles, CA) were connected in the perfusion loop
where pH and [CO2] were simultaneously and continuously measured.
Because the input (perfusate) [CO2] is ⬃0, the effluent [CO2] and pH
were used to calculate the total CO2 output equivalent to the secreted
HCO⫺
3 as previously described (10, 31). To prevent contamination of
the perfusate from bile or pancreatic juice, the pancreaticobiliary duct
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EFFECTS ON MUCOSAL DEFENSES
Fig. 3. Effect of afferent denervation or cyclooxygenase inhibition on L-Gluinduced intracellular alkalinization and mucus secretion in rat duodenum. Rats
were pretreated with capsaicin (Cap-t, 125 mg/kg sc) or indomethacin (Indo-t
5 mg/kg, sc). A: pHi. Cap-t and Indo-t inhibited L-Glu-induced intracellular
alkalinization, while L-Glu acidified the cells in Cap-t rats. Each data point
represents mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs group, †P ⬍
0.05 vs. L-Glu group. B: mucus gel thickness. Cap-t and Indo-t reduced
L-Glu-induced mucus secretion. Each data point represents mean ⫾ SE (n ⫽
6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs group, †P ⬍ 0.05 vs. L-Glu group.
flow/MGT or HCO⫺
3 secretion) was examined. IMP (0.1 mM) alone
or IMP with L-Glu, L-Asp, L-Ala, or L-Leu (each 10 mM) was
superfused or perfused during the challenge period.
To test the role of mGluRs in L-Glu effects, we examined the
effects of a selective group I mGluR (mGluR1 and 5) agonist
S-DHPG (0.1 mM) or a selective group III mGluR (mGluR4, 6, 7,
and 8) agonist L-AP4 (0.1 mM) on mucosal defense factors. The
effects of coperfusion of a selective group I mGluR antagonist
AIDA (0.1 mM) or a selective group III mGluR antagonist MAP4
(0.1 mM) with L-Glu (10 mM) were also examined. L-AP4 and
MAP4 were predicted to be selective for mGluR4. Furthermore, a
highly selective mGluR4 agonist was tested. VU0155041 (10 ␮M),
which is a potent allosteric modulator of mGluR4, but also acts as
a highly selective allosteric agonist of mGluR4 (35), was superfused.
To investigate the role of calcium-sensing receptor (CaSR) in L-Glu
effects, the effects of perfusion of high Ca2⫹ Krebs solution (4 mM)
or a CaSR agonist spermine (1 mM) (16) on mucosal defense factors
were examined.
Measurement of duodenal epithelial cell injury. Epithelial cellular
injury was assessed by in vivo in situ PI staining as previously
reported (4). The mucosa was superfused with a PI-containing Krebs
solution (1 ␮M) to stain injured cell nuclei. After BCECF images
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Fig. 4. Expression of L-Glu receptors in rats. A: RT-PCR analysis for taste
receptor 1 subtypes (T1R1, 2, 3), metabotropic glutamate receptor subtypes
(mGluR1, 4), and calcium-sensing receptor (CaSR) in the mucosa (m) and
muscle layer (s) of the esophagus (Ö), fundic stomach (F), gastric antrum (A),
proximal duodenum (B), dorsal root ganglia (DR) and nodose ganglia (NG) in
rats. 1 of 3 experiments is represented. B: real-time PCR for T1Rs, mGluRs,
and CaSR in the villi, crypt, and muscle layer of the proximal duodenum.
Relative expression of each receptor for ␤-actin was calculated from threshold
cycle (Ct) values. Each data point presents mean ⫾ SE (n ⫽ 3 rats).
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were captured, PI fluorescence was visualized by illuminating the
mucosa at 535 nm with emission detected at 590 nm. PI-positive dots
corresponding to injured cell nuclei were counted in each image
(microscopic field) observed with ⫻10 objective lens. The mean of
the number obtained from three microscopic fields was defined as the
number for the given time period. We have correlated PI-positive cell
number in vivo with a cellular injury scale on the basis of the
examination of hematoxylin-eosin conventionally stained sections (4).
After BCECF loading followed by the stabilization with pH 7.0 Krebs
superfusion, the mucosa was superfused with pH 7.0 Krebs for 10 min
with or without L-Glu (10 mM) during the basal period (t ⫽ 0 –10
min), followed by superfusion of pH 1.8 acid solution for 5 min
(challenge period, t ⫽ 10 –15 min), then with pH 7.0 Krebs for 15 min
(recovery period, t ⫽ 15–30 min). PI images were captured every 5
min.
Expression of L-Glu receptors in rat tissues. To investigate the
expression of L-Glu receptors in the rat tissues, RT-PCR analysis was
performed for T1R isoforms, mGluR isoforms, and CaSR. Three rats
were euthanized by the terminal exsanguinations under sodium pentobarbital anesthesia (50 mg/kg ip). The lower esophagus, fundus and
antrum of stomach, proximal duodenum, Th1-Th8 dorsal root ganglia
(DR), and nodose ganglia (NG) were removed and used for RT-PCR
L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
RESULTS
Effect of luminal L-Glu on duodenal mucosal defense factors. Duodenal perfusion with pH 7.0 Krebs buffer solution
alone showed the stable pHi, MGT, and blood flow (Fig. 1,
A–C). Luminal perfusion with L-Glu (0.1–10 mM) dose dependently increased pHi (Fig. 1A). Luminal L-Glu also increased
MGT, followed by the gradual decrease of gel thickness after
L-Glu removal (Fig. 1B), consistent with the increased mucus
secretion (7). In contrast, luminal L-Glu had no effect on blood
flow (Fig. 1C). These results suggest that luminal L-Glu enhances duodenal defense mechanisms.
Luminal perfusion of D-Glu or L-Asp (10 mM) had no effect
on pHi or MGT in the duodenum (Fig. 2, A and B). Furthermore, L-Ala or L-Leu (10 mM) also had no effect on pHi or
MGT (Fig. 2, C and D). None had any effect on blood flow.
These results suggest that L-Glu is a potent effector on pHi and
MGT.
Effect of capsaicin or indomethacin pretreatment on L-Gluinduced effects in rat duodenum. Because the duodenal mucosal defense mechanisms involve the activation of capsaicin-sensitive afferent nerves and/or COX (24), we next
⫺
Fig. 5. Effect of amino acid and inosine monophosphate (IMP) on duodenal HCO⫺
3 secretion in rats. Duodenal HCO3 secretion is expressed as total CO2 output.
⫺
Perfusion of L-Glu (10 mM) or IMP (0.1 mM) had a little effect on HCO⫺
3 secretion, while L-Glu coperfused with IMP synergistically augmented HCO3 secretion
(A). Similar synergism was observed with perfusion of L-Asp (10 mM) (B) or L-Leu (10 mM) (D), whereas L-Ala (10 mM) increased HCO⫺
secretion
without
3
the synergy with IMP (C). Each data point represents mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs group, †P ⬍ 0.05 vs. the corresponding amino acid
group.
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as previously described (5). Furthermore, to sublocalize the presence
of the target mRNA in the esophagus, stomach, and duodenum, these
tissues were separated from the muscle layers and the mucosa by
sharp dissection under a Zeiss stereomicroscope in ice-cold RNA
stabilizing solution (RNA-Later; Qiagen, Valencia, CA) followed by
RNA extract. The PCR primers used in the present study were listed
in Table 1, and ␤-actin was used as an internal control. An aliquot of
the RT reaction product served as a template in 40 cycles with 30 s at
95°C, 30 s at 58 – 64°C, and 30 s at 72°C. Furthermore, after
dissection of villi, crypt, and muscle layer of the duodenum under a
stereomicroscope, real-time PCR was performed with SYBR green
fluorescence using a thermal cycler (MyiQ single-color real-time PCR
detection system; Bio-Rad, Hercules, CA). Threshold cycle (Ct) value
of each target mRNA was compared with Ct value of ␤-actin using
⌬Ct method for reference gene. The expression level of each receptor
was presented as fold induction per 103 copies of ␤-actin. Each primer
set showed 89 –98% PCR efficiency in brain or tongue tissue as
positive controls, except for T1R2 (⬃70%), presumably attributable
to lower expression.
Statistics. All data are expressed as means ⫾ SE. Data were
derived from six rats in each group. Comparisons between groups
were made by one-way ANOVA followed by Fischer’s leastsignificant difference test. P values of 0.05 were taken as significant.
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Fig. 6. Effect of L-Glu and IMP on pHi and mucus gel thickness in rats.
A: coperfusion of IMP (0.1 mM) with L-Glu (10 mM) inhibited L-Glu-induced
intracellular alkalinization. Each data point represents mean ⫾ SE (n ⫽ 6 rats).
*P ⬍ 0.05 vs. pH 7.0 Krebs group, †P ⬍ 0.05 vs. L-Glu group. B: IMP had no
enhancing effect on L-Glu-induced mucus secretion. Each data point represents
mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs group, †P ⬍ 0.05 vs.
L-Glu group.
mM) partially inhibited L-Glu-induced alkalinization but had
no effect on L-Glu-induced mucus secretion (Fig. 7, D and E).
These results suggest that L-Glu-induced pHi and MGT increases are mediated mainly via mGluR4 and that mGluR1/5 is
partially involved in only L-Glu-induced cellular alkalinization.
To further confirm the role of mGluR4 in L-Glu effects, we
tested the highly selective compounds for mGluR4. Cells were
alkalinized by a highly selective mGluR4 agonist VU0155041
(10 ␮M) (Fig. 7A). VU0155041 also stimulated mucus secretion (Fig. 7B). Another highly selective mGluR4/8 agonist
(1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (0.1
mM) also mimicked L-Glu effects on pHi and MGT (data not
shown). These results further suggest that L-Glu increases pHi
and MGT most likely via mGluR4.
We also tested the effects of the mGluR1/5 agonist S-DHPG
(0.1 mM) or the mGluR4 agonist L-AP4 (0.1 mM) on duodenal
⫺
HCO⫺
3 secretion. S-DHPG or L-AP4 had no effect on HCO3
secretion (Fig. 7C), suggesting that, at least, mGluR1 and 4
were not involved in the regulation of duodenal HCO⫺
3 secretion.
Effect of CaSR agonists on duodenal mucosal defense factors. We next examined the effect of CaSR agonists on pHi,
blood flow, and MGT. Perfusion of high Ca2⫹ Krebs solution
(4 mM) or spermine (1 mM) decreased pHi (Fig. 8A) but
increased MGT (Fig. 8B) and blood flow (data not shown),
mimicking the effect of luminal acid (2) but unlike L-Glu.
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examined the effect of L-Glu on the duodenal mucosal
defenses in capsaicin- or indomethacin-pretreated rats. Either treatment had no effect on pHi and MGT during the
basal period (Fig. 3, A and B). Pretreatment with indomethacin inhibited L-Glu-induced cellular alkalinization (Fig.
3A) and reduced L-Glu-induced mucus secretion (Fig. 3B).
Pretreatment with capsaicin actually decreased pHi during
L-Glu perfusion (Fig. 3A) and reduced L-Glu-induced mucus
secretion (Fig. 3B). Blood flow was unchanged in any
groups (data not shown). These results suggest that cellular
alkalinization and mucus secretion induced by luminal LGlu are dependent on capsaicin-sensitive afferent nerves
and COX activity in rat duodenum.
Expression of L-Glu receptors in the rat tissues. RT-PCR
revealed high T1R1 and T1R3 expression in esophageal, gastric, and duodenal mucosa and in sensory neurons, with less
T1R2 expressed (Fig. 4A). mGluR1 and 4 and CaSR were also
expressed in the fundic and duodenal mucosa. Furthermore,
real-time PCR revealed that T1R1 and 3, mGluR1 and 4, and
CaSR were expressed in the duodenal villi (Fig. 4B).
Effect of luminal L-Glu on duodenal HCO⫺
3 secretion. Because HCO⫺
3 secretion is one of the most important, wellstudied defense factors against luminal acid in duodenum,
we hypothesized that luminal L-Glu stimulates duodenal
HCO⫺
3 secretion. Luminal perfusion of L-Glu (10 mM) significantly but slightly increased HCO⫺
3 secretion (Fig. 5A), which
was robustly enhanced by the addition of IMP (0.1 mM) to
⫺
L-Glu. IMP alone had a little effect on HCO3 secretion. These
results are consistent with the synergistic effect of L-Glu and
IMP, well-described for L-Glu sensing by the taste buds (33).
Furthermore, L-Asp or L-Leu also modestly increased HCO⫺
3
secretion with robust enhancement by the addition of IMP (Fig.
5, B and D), similar to L-Glu. These results suggest that
⫺
L-Glu-associated HCO3 secretion is likely mediated via T1R1/
T1R3. In contrast, L-Ala alone increased HCO⫺
3 secretion
without further significant enhancement by IMP (Fig. 5C).
Lack of synergy of L-Ala with IMP may be due to a different
affinity for T1R1/T1R3 (32), submaximal effect of L-Ala alone,
or a nonspecific effect.
The synergy between L-Glu and IMP prompted us to examine the effect of L-Glu and IMP on pHi and MGT. The addition
of IMP inhibited L-Glu-induced cellular alkalinization
(Fig. 6A), suggesting that L-Glu-induced pHi modulation is
closely linked with HCO⫺
3 secretion. The addition of IMP to
L-Glu did not augment the peak value of MGT (Fig. 6B).
Nevertheless, the addition of IMP with L-Asp, L-Leu, or L-Ala
had no effect on pHi, MGT, or blood flow (data not shown),
further suggesting that the activation of T1R1/T1R3 does not
directly affect pHi or MGT.
Effect of mGluR agonists or antagonists on duodenal mucosal defense factors. Next, we examined the effect of mGluR
agonists on pHi and MGT or the effect of mGluR antagonists
on L-Glu-induced cellular alkalinization and mucus secretion in
the duodenum. Perfusion of the mGluR4 agonist L-AP4 (0.1
mM) increased both pHi and MGT (Fig. 7, A and B), mimicking the effects of L-Glu. In contrast, the mGluR1/5 agonist
S-DHPG (0.1 mM) temporally increased pHi but had no effect
on MGT (Fig. 7, A and B). Coperfusion of the mGluR4
antagonist MAP4 (0.1 mM) with L-Glu abolished L-Glu-induced cellular alkalinization and inhibited L-Glu-induced mucus secretion, whereas the mGluR1/5 antagonist AIDA (0.1
L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
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Furthermore, perfusion of high Ca2⫹ or spermine increased
duodenal HCO⫺
3 secretion (Fig. 8C).
Effect of L-Glu on acid-induced epithelial injury in duodenum. Because luminal L-Glu enhanced mucosal defense mechanisms, we examined the protective effect of L-Glu on acidinduced injury. Acute epithelial injury was invoked in a microscopic model by perfusion of a superphysiological pH 1.8
acid solution for 5 min with injury measured in vivo in situ PI
staining (4). Exposure to a pH 1.8 solution progressively
increased the number of PI-positive nuclei within the mucosa
(Fig. 9). Preperfusion of L-Glu (10 mM) reduced the increase
AJP-Gastrointest Liver Physiol • VOL
of PI-positive nuclei, suggesting that luminal L-Glu protects the
mucosa from acid-induced injury in the duodenum.
DISCUSSION
We demonstrated that luminal L-Glu increased pHi and
MGT, but not blood flow, in the duodenal mucosa. L-Gluinduced cellular alkalinization and mucus secretion were mediated by capsaicin- and indomethacin-sensitive pathways in
the duodenum, corresponding to the activation of capsaicinsensitive afferent nerves and COX activity, respectively. Lu297 • OCTOBER 2009 •
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Fig. 7. Effect of mGluR agonists or antagonists on duodenal mucosal defense factors. A and B: effects of (S)-3,5-dihydroxyphenylglycine (S-DHPG) (0.1 mM),
L-2-amino-4-phosphonobutyric acid (L-AP4) (0.1 mM), or VU0155041 (10 ␮M) on pHi of epithelial cells (A) and mucus gel thickness (B) were examined in
rat duodenum. Each data point represents mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs group. C: duodenal loop was perfused, and HCO⫺
3 secretion
was measured with pH and CO2 electrodes. HCO⫺
3 secretion is expressed as total CO2 output. Each data point represents mean ⫾ SE (n ⫽ 6 rats). D and E: effects
of 1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid (AIDA) (0.1 mM), or (S)-2-amino-2-methyl-4-phosphonobutyric acid (MAP4) (0.1 mM) on L-Glu (10
mM)-induced pHi (D) and mucus gel thickness (E) increases were examined. Each data point represents mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs
group, †P ⬍ 0.05 vs. L-Glu group.
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L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
Fig. 8. Effect of CaSR agonists on duodenal mucosal defense factors. A and
B: effects of perfusion of high Ca2⫹ solution (4 mM) or spermine (Sp) (1 mM)
on pHi of epithelial cells (A) and mucus gel thickness (B) were examined in rat
duodenum. pH 7.0 Krebs solution containing 1.8 mM Ca2⫹ was perfused as
control. Each data point represents mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH
7.0 Krebs group. C: effect of Ca2⫹ (4 mM) or spermine (1 mM) on duodenal
HCO⫺
3 secretion measured via duodenal loop perfusion was examined. Each
data point represents mean ⫾ SE (n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs
group.
minal L-Glu enhanced mucosal defense mechanisms via multiple L-Glu receptors (Fig. 10). L-Glu-induced pHi and MGT
increases were mimicked by mGluR4 agonists and inhibited by
an mGluR4 antagonist. mGluR1/5 was partially involved in the
L-Glu effect on pHi. In contrast, the synergy between L-Glu and
IMP for augmentation of duodenal HCO⫺
3 secretion implicates
the involvement of the T1R1/T1R3 heterodimer. This is the
first study to demonstrate the involvement of L-Glu sensing by
the upper GI mucosa in the enhancement of mucosal defense
AJP-Gastrointest Liver Physiol • VOL
Fig. 9. Effect of L-Glu on acid-induced epithelial injury in rat duodenum.
Epithelial injury was assessed in vivo in situ by propidium iodide (PI) staining.
Pretreatment with luminal L-Glu (10 mM) reduced pH 1.8 acid-induced
increase of PI-positive cell number. Each data point represents mean ⫾ SE
(n ⫽ 6 rats). *P ⬍ 0.05 vs. pH 7.0 Krebs group, †P ⬍ 0.05 vs. L-Glu group.
297 • OCTOBER 2009 •
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mechanisms and in the protection of the mucosa from acidinduced injury.
Cellular alkalinization is an important defense factor against
luminal acid. Cellular alkalinization attributable to epithelial
⫺
cell basolateral HCO⫺
3 uptake before HCO3 secretion protects
the epithelial cells from acid-induced injury, as demonstrated
using models of cystic fibrosis transmembrane conductance
regulator (CFTR) dysfunction (4, 8, 22). We demonstrated that
L-Glu-induced cellular alkalinization contributes to the protection of epithelial cells from acid-induced injury. Mucus secretion is another mucosal defense factor, which is thought to
create a premucosal buffering zone, damping direct acid exposure to the epithelial cells (20). Our study also implicates
L-Glu-induced mucus secretion in the mucosal protection.
Mucosal defense mechanisms in the upper GI tract are
mediated by capsaicin-sensitive afferent nerves and COX (24).
Capsaicin and PGE2 stimulates HCO⫺
3 secretion with the
augmented epithelial cellular buffering and stimulates mucus
secretion in the duodenum (3, 42), consistent with our result
that L-Glu-induced increase of pHi and MGT was inhibited by
pretreatment with capsaicin or indomethacin in the duodenum.
These two pathways also increase blood flow in the duodenum
(6, 27), inconsistent with the observed lack of effect of L-Glu
on blood flow. Because intracellular acidification, not alkalinization, is correlated with blood flow increase (9), L-Gluinduced cellular alkalinization may fail to increase blood flow,
despite the involvement of capsaicin and COX pathways.
Possible candidates for the L-Glu receptor are taste receptor
heterodimer (T1R1 and 3) (32, 48) or mGluR (1 or 4) (15, 44),
as reported in the taste buds for umami perception, or possibly
the CaSR (17). The T1R1/3 detects amino acids including
L-Glu, L-Asp, L-Ala, and L-Leu with differing affinity synergistically enhanced by IMP (32). The mGluR recognizes L-Glu
selectively, yet synergism with IMP has not been confirmed.
CaSR nonselectively binds L-type amino acids including L-Glu
and L-Asp but is not synergistic with IMP (17). The relatively
specific effects of L-Glu on pHi and mucus secretion in the
duodenum suggest that mGluRs are most likely L-Glu receptors. Our results suggest that mGluR4 mainly mediates L-Gluinduced pHi and MGT increases, whereas mGluR1 may have
L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
G789
partial role in pHi regulation. In contrast, the synergistic effect
of L-Glu, L-Asp, or L-Leu with IMP on duodenal HCO⫺
3
secretion suggests that T1R1/3 is the corresponding receptor.
Nevertheless, L-Asp, L-Leu, or L-Ala alone had no effect on pHi
and MGT, unlike L-Glu, suggesting that T1R1/3 is only involved in the regulation of HCO⫺
3 secretion. Furthermore, lack
of effect of L-Asp, L-Leu, or L-Ala with IMP on pHi and MGT
suggests that T1R1/3 activation is indirectly involved in pHi
and MGT regulation. In contrast, CaSR agonists acidified cells
and increased MGT, blood flow, and HCO⫺
3 secretion, like
luminal acid, but unlike L-Glu. It is still possible that the CaSR
is involved in L-Glu-induced mucus and HCO⫺
3 secretion. The
selective inhibitors for T1R1/T1R3 and CaSR will clarify their
contribution to L-Glu-induced augmentation in mucosal defense factors.
Inhibition of cellular alkalinization by L-Glu with IMP is still
unclear. Because our results suggest that the modulation of pHi
and HCO⫺
3 secretion by luminal L-Glu is differently mediated
via mGluRs and T1Rs, respectively, the close link between pHi
and HCO⫺
3 secretion may explain this phenomenon. A rapid
increase in the rate of HCO⫺
3 secretion may reduce cellular
HCO⫺
3 concentration, resulting the inhibition of cellular alkalinization. L-Glu and mGluR agonists had discrepant effects on
pHi and HCO⫺
3 secretion. Although both agonists alkalinized
cells, L-Glu increased HCO⫺
3 secretion, whereas mGluR agonists did not. Cellular alkalinization may not have been of
sufficient magnitude to support measurable HCO⫺
3 secretion,
which may also need the participation of other secretory
components such as CFTR (4). The mechanism by which
CaSR stimulation acidified cells accompanied by increased
mucus and HCO⫺
3 secretion is also unclear. Acid superfusion
of the mucosa acidifies epithelial cells, presumably via CO2
entry while increasing mucus secretion and blood flow (7).
Because CaSR activation produces comparable effects, we can
speculate that the observed cellular acidification is attributable
to augmented HCO⫺
3 secretion with reduction of cellular
HCO⫺
3 concentration or to independent effects of CaSR activation. Activation of CaSR acidifies in vitro microperfused
mouse medullary thick ascending limb cells, which express
CaSR at the basolateral membrane (11), consistent with our
AJP-Gastrointest Liver Physiol • VOL
finding. This suggests that intracellular acidification by CaSR
activation triggers mucus and HCO⫺
3 secretion, similar to the
effect of luminal acid.
L-Glu receptors have not been fully localized in the gut.
Because intestinal endocrine cells as well as enterocytes are
functionally implicated in luminal chemosensing (28, 29, 37,
38), L-Glu receptors are likely to be expressed in the intestinal
endocrine cells or enterocytes. Nevertheless, the localization of
T1Rs in the GI tract is controversial. T1R1 and T1R3 are
located on the apical membrane of rat jejunal enterocytes (28),
whereas T1R3 is only expressed in the endocrine cells in
human and mouse duodenum (29). Although each expression
was detected by PCR in this study, lack of specific antibodies
for T1Rs limits to clearly localize T1Rs in the GI tract. In
contrast, CaSR has been localized mainly on the basal surface
of enterocytes, gastric surface cells, and parietal cells as well as
in the myenteric plexus (14, 18, 39). Furthermore, all groups of
mGluRs are expressed in DR and NG (12, 13, 36), suggesting
the mGluR expression on the afferent nerves in the GI tract,
consistent with our findings that L-Glu effects on pHi and MGT
are mediated via mGluRs and capsaicin-sensitive afferent
nerves. Detailed localization of these receptors in the GI tract
may further clarify the mechanisms underlying L-Glu-induced
effects on mucosal defense factors.
Luminal L-Glu signaling is of uncertain functional significance. Because L-Glu is the most predominant amino acid in
proteins (21), a sharp rise of luminal L-Glu concentration can
signal protein ingestion, readying the gut for gastric and
pancreatic secretion of peptidases (45). Although the luminal
concentration of some other amino acids reached the similar
level of L-Glu after a protein-rich meal in human jejunum (1),
L-Asp, L-Ala, or L-Leu had no effect on pHi and MGT. The
specificity for luminal L-Glu among the 20 amino acid in
the stimulation of rat gastric vagal afferents accompanied by
the release of 5-HT and nitric oxide (46) also support the
observed protective effects of L-Glu on pHi and MGT. In
contrast, these amino acids including L-Glu likely share the
regulation of HCO⫺
3 secretion, suggesting cooperative mucosal
protection during protein digestion. Because luminal L-Glu
enhanced mucosal defenses and reduced acid-induced epithe297 • OCTOBER 2009 •
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Fig. 10. Summary diagram: effects of luminal L-Glu on
mucosal defense factors in rat duodenum. 1) L-Glu
increases pHi and mucus gel thickness (MGT) mainly
via mGluR4 without changes of blood flow (BF), mimicked by L-AP4 and VU0155041, inhibited by MAP4,
capsaicin pretreatment (Cap-t), or indomethacin (Indo-t).
L-Glu-induced effects may involve mGluR1, partially
mimicked by S-DHPG and reduced by AIDA. *Note
that L-Glu does not likely increase duodenal bicarbonate
secretion (DBS) via mGluRs. 2) L-Glu increases DBS,
enhanced by IMP, probably via the T1R1/3 heterodimer,
mimicked by L-Asp, L-Leu, or L-Ala. L-Glu-induced
changes in pHi and MGT are unlikely to be mediated by
T1R1/3. **Note that IMP inhibits L-Glu-induced pHi
increase and sustained L-Glu-induced MGT increase.
3) CaSR activation by high Ca2⫹ solution or spermine
decreases pHi and increases MGT, BF, and DBS, not
mirroring L-Glu-induced effects. Blue arrows, stimulation; red lines, inhibition; ⫹, positive allosteric effect by
IMP; dashed line, possible involvement; parenthesis,
partial involvement.
G790
L-GLUTAMATE
EFFECTS ON MUCOSAL DEFENSES
lial injury in the duodenum, luminal L-Glu signaling including
L-Glu-specific and -nonspecific amino acid sensing may precondition or prime the mucosa for subsequent acid exposure
and presumably protein digestion.
In conclusion, luminal L-Glu enhanced the mucosal defense
mechanisms in the duodenum, via activation of most likely
mGluR4 and T1R1/3, capsaicin-sensitive afferent nerves and
COX pathway, protecting the mucosa from injury attributable
to luminal acid. The umami taste receptors may be involved in
the mucosal physiological responses for luminal amino acids,
especially L-Glu.
ACKNOWLEDGMENTS
We thank Jenifer Kugler for assistance with manuscript preparation.
This work was supported by a research grant from Ajinomoto, Japan (Y.
Akiba), Department of Veterans Affairs Merit Review Award, NIH-NIDDK
R01 DK54221 (J. Kaunitz), and the animal core of NIH-NIDDK P30 DK0413
(J. E. Rozengurt).
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EFFECTS ON MUCOSAL DEFENSES