LECTURE
Luminal acid elicits a protective duodenal mucosal response
Jonathan D. Kaunitz1 and Yasutada Akiba1,2
1Greater Los Angeles Veterans Affairs Healthcare System; CURE: Digestive Diseases Research Center; andDepartment of Medicine, School of Medicine, University of California Los Angeles, Los Angeles, CA, USA(Received for publication on November 22, 2001)

Abstract. Measuring duodenal epithelial intracellular pH (pHi ), blood ﬂow and mucus gel thickness(MGT), we studied duodenal defense mechanisms in vivo so as to more fully understand the protective mucosal response to luminal acid. Exposure of the mucosa to physiologic acid solutions promptly
lowered pHi , followed by recovery after acid was removed, indicating that acid at physiologic concentrations readily diffuses into, but does not damage duodenal epithelial cells. Cellular acid then exits thecell via an amiloride-inhibitable process, presumably sodium-proton exchange (NHE1). MGT andblood ﬂow increase promptly during acid perfusion; both decrease after acid challenge and are inhibited by vanilloid receptor antagonists or by sensory afferent denervation. Bicarbonate secretion does
not increase during acid perfusion but increases following acid challenge. Inhibition of cellular alkali
uptake by anion transport inhibitors lowers pHi , and increases mucosal injury, whereas inhibition of
apical alkali secretion alkalinizes pHi and diminishes injury. These observations support the following
hypothesis: luminal acid diffuses into the epithelial cells, lowering pHi . Acidic pHi increases the activityof a basolateral NHE, acidifying the submucosal space and increasing cellular alkali loading. The acidic
submucosal space activates capsaicin receptors on afferent nerves, increasing MGT and blood ﬂow.
With continued acid exposure, a new steady state with thickened mucus gel, increased blood ﬂow, anda higher cellular buffering power protects the mucosa from acid injury. After acid challenge, mucus
secretion, blood ﬂow and pHi return to normal, while bicarbonate secretion increases. Through these
integrated mechanisms, the epithelial cells are protected from damage due to repeated pulses of concentrated gastric acid. (Keio J Med 51 (1): 29–35, March 2002)
Key words: bicarbonate secretion, cellular buffering power, intracellular pH, mucosal blood ﬂow,
mucus secretion

In this review, we will systematically describe the current knowledge concerning duodenal defense mechanisms, ending with a scheme that integrates these mechanisms into a coherent protective response to luminal
acid. Moreover, we will discuss how these mechanisms
might be altered in the disease cystic ﬁbrosis, in which
the duodenum appears to be highly resistant to luminal
acid.3–5

Introduction
The location of the duodenal epithelium just distal to
the gastric antrum and proximal to the pancreaticobiliary ducts uniquely exposes it to a highly variable pHenvironment due to peristaltically conveyed pulses ofconcentrated gastric acid combining with secreted
bicarbonate. Since the duodenum does not have the inherent acid protective structural properties of its ‘neighbors to the North’, namely the stomach and esophagus,
whose intercellular junctions severely curtail transepithelial ionic permeation,1,2 the duodenum, being leaky,
has evolved alternate means for defense against acid
(Fig. 1).

Duodenal Luminal pH
Antral peristalsis and pyloric opening exposes theproximal duodenum to cyclical variations of luminal pH.
These cycles are more pronounced post-prandially, andPresented at the 1168th Meeting of the Keio Medical Society in Tokyo, September 4, 2000.
Reprint requests to: Dr. Jonathan D. Kaunitz, Greater Los Angeles Veterans Affairs Healthcare System; CURE: Digestive Diseases ResearchCenter; and Department of Medicine, School of Medicine, University of California Los Angeles, Bldg. 114, Room 217, West Los Angeles VA
Medical Center, 11301 Wilshire Blvd., Los Angeles, CA 90073 USA, e-mail: jake@ucla.edu
2 Present afﬁliation: Department of Internal Medicine, School of Medicine, Keio University
29

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Kaunitz JD and Akiba Y: Duodenal defense mechanisms

Fig. 1 Duodenal defense mechanisms. All of these mechanisms are believed to defend the mucosa against luminal acid, although bicarbonate
secretion is the most accepted.

vary luminal pH between two and seven on a scale ofminutes.6–8 These rapid shifts of pH presumably arise
from alternate exposure to gastric acid and secreted bicarbonate of epithelial and pancreatic origin. In contrast, gastric luminal pH is sustained on a minute-tominute scale, although it does of course vary over a 24
hr period. Rapid shifts of duodenal pH are likely tocreate intense stress on the epithelial cells to maintain
constant intracellular pH (pHi ) in order to maintainfunction and prevent irreversible necrosis due to intracellular acidiﬁcation. Thus, a potent defensive system
must be in place to prevent cellular acidiﬁcation duringmucosal acid challenge.
Defense Mechanisms
The most studied duodenal defense mechanism is epithelial bicarbonate secretion. Other potential defense
mechanisms include the mucus gel and mucosal blood
ﬂow. Reparative processes such as restitution from injury are beyond the scope of this review and will not be
further addressed.

restores duodenal bicarbonate secretory capacity.15,16
The mechanism by which bicarbonate is secreted fromthe epithelial cell is controversial. Formerly, it wasthought that CO2 diffusing into the cell was converted
to bicarbonate and protons by cellular carbonic hydrase.
Bicarbonate was then secreted across the apical membrane by anion exchange. More recent studies, including molecular immunolocalization and studies of pHi ,do not fully support this mechanism.17–19 What appears
to also be present is that bicarbonate is transported
from the blood across the basolateral membrane by avariant of the sodium-bicarbonate transporter, or NBC,in response to decreased pHi resulting from exposure
to luminal acid. Since inhibiting or eliminating the apical membrane chloride channel cystic ﬁbrosis transmembrane regulator (CFTR) greatly attenuates bicarbonate secretion,20,21 the CFTR has been implicated in
the mechanism of bicarbonate secretion, although it isunknown whether it serves directly as a bicarbonate
channel, or indirectly to preserve transmembrane electrical or ion gradients.
Role of alkali loading

Bicarbonate secretion
Bicarbonate secretion is a logical duodenal defense
mechanism for the following reasons: 1) Duodenal bicarbonate secretion/cm 2 epithelium is much greater thangastric bicarbonate secretion,2,9–11 2) pH electrodestudies suggest that epithelial bicarbonate secretion
creates a layer of neutral pH next to the mucosa,12–14
3) Helicobacter pylori infection complicated by duodenal ulcers is associated with diminished bicarbonate secretion, and eradication of helicobacter infection

We have re-examined the role of bicarbonate secretion in overall duodenal defense from acid, and, in doing so, have formulated a novel hypothesis with regard
to the role of bicarbonate transport. To test these possibilities, we developed a technique for the measurement of pHi , blood ﬂow, and mucus gel thickness in the
duodenum of anesthetized rats.22 With this system, we
could perfuse solutions of varying pH through a chamber placed over the exposed duodenal mucosa, thereby
simulating changes in luminal pH. With this system, weKeio J Med 2002; 51 (1): 29–35

exposed the mucosa to a brief pulse of acid, which
promptly decreased pHi . This fall of pHi , even with mildly
acidic perfusates, suggested that acid could readily penetrate the overlying mucus gel and the mucosa, therefore
calling into question the role of pre-epithelial bicarbonate neutralization in duodenal mucosal defense. With removal of the acid challenge, pHi was elevated to supernormal values, which indicated that cellular buffering
power has increased, not decreased, during acid challenge. Furthermore, a second acid challenge acidiﬁed pHi
less than the ﬁrst, further conﬁrming that acid exposure
was associated with cellular alkali loading and increased
cellular buffering power. This somewhat surprising ﬁnding was conﬁrmed by comparison with prior studies conducted in a variety of systems, in which acid pulses were
followed by pHi overshoot, indicative of cellular alkali
loading in cells containing a plasma membrane alkaliloading mechanism such as sodium-bicarbonate cotransport.23
Further studies indicated that this alkali loading wasinhibited by the stilbene anion transport inhibitor DIDS
(4,4 0 diisothiocyanostilbene-2,2 0 -disulfonic acid). When
exposed to two short acid pulses, pHi decreased lessduring the second challenge; again strongly suggestivethat cellular buffering power was increased during acid
exposure. Again, DIDS inhibited this adaptive effect.24
Our studies were thus consistent with alkali loading
being induced by luminal acid exposure by a DIDSinhibitable mechanism. This ﬁnding was expected insofar as primary isolated duodenal epithelial cells recovered from acid exposure by a mechanism consistent
with the activity of an NBC,25 and that bicarbonatesecreting pancreatic duct cells have a basolateral membrane NBC.26 Recently, our laboratory, in collaboration with Dr. Ira Kurtz, have conﬁrmed the presence ofthe pancreatic-type isoform of NBC1 (pNBC1) in thebasolateral membrane of rodent proximal duodenal
epithelial cells using immunohistochemistry.27,28
Alkali loading during acid challenge, which increases
cellular buffering power and attenuates the fall of pHi ,is an attractive means of defending the epithelium fromacid challenge. To address how bicarbonate secretionis related to this observation, we performed parallel
experiments in which bicarbonate secretion was measured in a perfused duodenal loop exposed to the same
pH perfusion sequence as the measurements of pHi .
Bicarbonate secretion was measured by the conventional acid back-titration technique, but also by measurement of total dissolved CO2 content of the efﬂuent
collected from the perfusion with a CO2 electrode. We
found that titratable alkalinity increased substantiallyduring acid perfusion. Surprisingly, total CO2 content
decreased somewhat at the same time.24 To account for
the large discrepancy between the back-titration experiments and the measurement of efﬂuent CO2 content,

31

we performed control experiments in which we measured total CO2 content in the perfusates and efﬂuent of
perfused duodena. We found that there was a ﬁnite lossof CO2 during duodenal perfusion, in agreement withprior studies29 but inadequate to explain the discrepancy.
To help interpret the data, we postulated threemeans by which acid can disappear from the lumen:
back-diffusion of acid, back-diffusion of CO2 , and neutralization by secreted bicarbonate. If all of the aciddisappearance measured by back-titration occurred bybicarbonate neutralization, the efﬂuent CO2 content
should increase to the same extent as did titratable
acidity, which was not the case. If the perfusate bicarbonate was converted into CO2 , which then backdiffused into the epithelium, we would still predict alarge increase of efﬂuent CO2 content. Hence, the bestmeans of explaining the discrepancy between efﬂuent
CO2 content and acid disappearance is by postulatingthat most of the acid loss during perfusion with pH 2
solution is due to acid back-diffusion. In that case, bicarbonate secretion must have been unchanged or perhaps decreased during acid challenge. The implicationsof these data, combined with our measurements of pHi ,support our hypothesis that increased cellular buffering, and not bicarbonate secretion, was the primary
duodenal defense mechanism from acid. Acid was notneutralized at the duodenal surface, since cellular pHi
clearly decreased during acid challenge, and since acidback-diffusion was the major means of acid loss when
perfused over the mucosa. Furthermore, since bicarbonate secretion was unchanged during acid perfusion,and only increased after acid removal, secreted bicarbonate is unlikely to be protective, since its increased
secretion is present only when it is not needed i.e. whenluminal acid is no longer present. Supporting the concept that bicarbonate secretion is not enhanced during
luminal acid stress are the observations that lowered
pHi deceases cellular bicarbonate concentration, inhibiting bicarbonate exit, and that CFTR permeability to
the related anion chloride is diminished at acidic pHi
due to lack of CFTR phosphorylation.30
We further tested this hypothesis by measuring
epithelial injury under conditions in which alkali loading is either inhibited or enhanced, in order to conﬁrm
that bicarbonate loading, and not secretion is the primary defensive mechanism. To accomplish this, DIDS,
and the anion channel inhibitor NPPB (5-nitro-2-(3phenylpropylamino) benzoic acid), added to the perfusate, respectively either decreased or increased pHi .
Changes of pHi also correlated with mucosal injury
susceptibility. The most striking ﬁnding was that NPPB
inhibited bicarbonate secretion but increased pHi anddecreased injury susceptibility. Thus, NPPB ‘uncoupled’bicarbonate secretion from mucosal protection, a novel

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Fig. 2 Sequential response of duodenal epithelial cells to luminal
acid. In the left panel, steady-state pHi ¼ @7:1 when no acid is present. In the succeeding panels to the right, luminal acid rapidly acidiﬁes
the epithelial cells. Low pHi decreases CFTR conductance andintracellular [HCO3  ], suppressing HCO3  secretion. Low pHi also
increases the activity of the basolateral sodium-bicarbonate cotransporter (pNBC1), which in turn increases cellular bicarbonate concentration. When luminal pH returns to neutrality, acid diffuses out of thecell. The excess intracellular alkali raises pHi over baseline (overshoot), which activates CFTR and DRA, which then increases bicarbonate secretion. In the disease cystic ﬁbrosis, a dysfunctional CFTR
and DRA limit apical HCO3  exit, which raises pHi .

ﬁnding that casts further doubt on the primacy ofbicarbonate secretion on mucosal protection.27 In this
proposed mechanism, shown in Fig. 2, bicarbonate
secretion occurs to remove excess alkali from the cell,when excess intracellular bicarbonate is no longer
needed after acid challenge.
Blood ﬂow
Mucosal blood ﬂow is an accepted component ofupper gastrointestinal barrier function. In the stomach,for example, interventions that attenuate the hyperemic
response to acid perfusion increase mucosal injury.31–33
The data derived from studies of the duodenum are lessconclusive, however. One potential confounder is that
stimuli of bicarbonate secretion and blood ﬂow are
co-regulated, making it difﬁcult to determine the relative importance of blood ﬂow in terms of overall barrier
function.
With our technique, we were able to measure blood
ﬂow, pHi , and mucus gel thickness simultaneously. This
technique enabled us to formulate novel conclusions
about the relative contribution of blood ﬂow to overallbarrier function. Mucosal blood ﬂow, as measured bylaser-Doppler ﬂowmetry, increased in response to acidKaunitz JD and Akiba Y: Duodenal defense mechanisms

perfusion.22,34 This response differs from the gastricmucosa, which must be either injured or pre-treatedwith gastrin or other compound in order to induce thisacid response.35,36 Our studies revealed some novel
observations about the nature of duodenal blood ﬂow
and its regulation. For example, inhibition of sodiumproton exchange (NHE) with the potent amiloride
analog dimethylamiloride inhibited the hyperemic acidresponse. Interestingly, acidiﬁcation of the cytoplasmby alternate means such as with ammonium pre-pulse
or valinomycin increased blood ﬂow, also inhibitable bydimethylamiloride.22 These studies suggested that acidmust pass through the epithelial cell and exit via by
NHE prior to eliciting a hyperemic response. In further
studies, we examined the sensing mechanisms underlying the hyperemic response. Capsazepine, an antagonist
to the recently cloned vanilloid receptor, abolished thehyperemic response to acid, conﬁrming the involvementof vanilloid receptors in the acid response. Further
studies also conﬁrmed that the hyperemic response wasmediated by a well-known pathway that includes afferent sensory nerves, nitric oxide release the neuropeptide calcitonin gene-related peptide (CGRP), but
was not inhibited by indomethacin, a non-selective inhibitor of cyclooxygenase. These studies provided data
supporting our proposed mechanism of duodenal acidinduced hyperemia, including acid diffusion into theepithelial cell, basolateral extrusion via NHE, activation of vanilloid receptors on afferent nerves, CGRP release, with activation of endothelial nitric oxide synthesis, with production of vasodilatory nitric oxide. A
scheme of proposed regulatory mechanisms for bloodﬂow is shown in Fig. 3.
Mucus secretion
The role of mucus in duodenal mucosal defense is
the subject of only a few studies. The most acceptedhypothesis is that mucus stabilizes the pre-epithelial pH
gradient, with neutral pH measured near the mucosa,
preventing acid from entering the epithelial cells.12,37–39
Mucus secretion is also co-regulated by the same neurohormonal and phamacologic stimuli that increase otherdefense mechanisms such as bicarbonate secretion andblood ﬂow, making it a likely candidate for being a secondary defense mechanism.
With our technique, we could optically and noninvasively measure mucus gel thickness in our anesthetized preparation.40 We found, for example, that
mucus gel thickness rapidly increases in response toperfused acid, but equally rapidly decreases in thickness
when the acid challenge is removed. Measurement of
efﬂuent mucus glycoprotein content was consistent withincreased sloughing of mucus into the perfusate whenmucus was rapidly secreted, indicating that there is a

Keio J Med 2002; 51 (1): 29–35

33

that the capsaicin pathway, involving acid-sensing vanilloid receptors, afferent nerves, CGRP, and nitric oxide,
regulates mucus gel secretion and that non-selective
COX inhibition with indomethacin abolishes the mucus
secretory response to all secretagogues, suggesting afundamental role of prostaglandins in duodenal mucus
secretion.42
Clinical Correlate

Fig. 3 The capsaicin pathway. Mucosal responses to luminal acid are
mediated by this pathway, which includes an acid sensor, which is avanilloid receptor, afferent nerves, and an effectors mechanism dependent on the secretion of calcitonin gene-related peptide (CGRP)
and nitric oxide synthase (NOS). Prostaglandin synthesis is also
involved in the regulation of mucus gel thickness. Italics denote
inhibitors of each component.

dynamic relation between mucus secretion and erosion,as has been previously hypothesized.41 When the secretion slowed, the rapid sloughing remained, thinning
the gel until a new steady state occurred. A scheme
depicting our concept of how mucus gel thickness isregulated is shown in Fig. 4. Further studies showed

We have formulated the ‘‘CF paradox’’27 in which
we pose the question: why are duodenal ulcers notincreased in patients with CF? Patients with CF, forexample, have high normal acid secretion,43 and hencemust take gastric antisecretory medications in order todiminish esophageal acid reﬂux and to prevent acidmediated inactivation of pancreatic enzymes.44,45 Furthermore, pancreatic and duodenal bicarbonate secretion are presumably impaired by the disease,21 and theduodenal pH is lower than normal.46 Combined withthe frequent prevalence of chronic lung disease, thesepatients should be a high risk for peptic ulceration.
Clinical experience, and the literature, however, do notsupport an increased incidence of peptic ulceration is
this population, but rather, it appears that the prevalence of peptic ulceration may actually be diminished.5
Our hypothesis that elevated cellular buffering power ispresent when the CFTR is dysfunctional is supported
by independent observations: 1) resting pHi is elevatedin cells derived from individuals with a dysfunctional
CFTR47 and 2) the apical anion exchanger DRA, theonly other known apical exit pathway for bicarbonate,
is downregulated when the CFTR is dysfunctional,48,49

Fig. 4 Dynamic regulation of mucus gel thickness in response to luminal acid. In the left panel, alkaline mucus secretion and the rate ofsloughing into the lumen are balanced. Luminal acid creates a sudden exocytotic burst of mucus secretion from goblet cells and Brunner’s
glands, which thickens the gel. The newly secreted mucus sloughs into the lumen at a higher rate, resulting in a new steady-state gel thickness.
Removal of luminal acid decreases mucus secretion, decreasing gel thickness, and also initiates synthesis of new mucus granules.

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Kaunitz JD and Akiba Y: Duodenal defense mechanisms

which would support the concept that bicarbonate is‘trapped’ in the cell in CF, raising pHi and increasing
cellular buffering power.
We propose that the impairment of duodenal bicarbonate secretion in the disease may explain why
this population may be protected from peptic ulceration. Recall that the CFTR and apical DRA are
needed for duodenal bicarbonate secretion. If the cells
can alkali load normally via a basolateral NBC1 and byin-diffusion CO2 converted to HCO3  by carbonic
anhydrase, but cannot secrete bicarbonate across theapical membrane due to defective CFTR and DRA
functioning, cellular buffering may be abnormally highdue to the dysfunction of the two major apical bicarbonate exit pathways. This increased cellular buffering
power may then protect the epithelial cells from undue
acidiﬁcation due to low luminal pH.
Summary and Conclusions
Study of the duodenal response to acid has yielded
fresh insights into how mucosal surfaces protect themselves from a hostile environment. Cellular alkali loading in response to acid is plausible and ﬁts well with theexisting body of data correlating ulcer disease with bicarbonate secretion, in that bicarbonate secretion is theend result of alkali loading, although it may not, in andof itself, be protective against acid. The alkali-loading
hypothesis also ﬁts well with the clinical observation of
the sparing of CF patients from ulcer disease. Although
the signal for alkali loading may be decreased pHi ,enhanced blood ﬂow and mucus secretion in responseto acid challenge rely on well-described pathways thatare present in many tissues,50–52 and serve as a useful paradigm for epithelial responses to environmental
stimuli.
In the future, we plan to study the proposed two-stepmechanism (separate basolateral uptake and apical exit
processes) by which duodenal bicarbonate secretion
may occur. We also would like to rigorously test thealkali-loading hypothesis in CF knockout and othermouse models, and also to further study the duodenalacid response. Other planned studies include the role ofthe high CO2 duodenal luminal environment in mucosal
acidiﬁcation. Through these studies, we hope to understand how environmentally exposed epithelia deal withpotentially damaging conditions, with the hope thatfurther understanding and insight might be gained
about diseases that affect the gastrointestinal tract andother epithelia.
Acknowledgements: We would like to thank Drs. Eli Engel, Paul
Guth, for their helpful advice, and Drs. Kotaro Kaneko, Yasu Nishizaki, and Shin Tanaka for developing the experimental system used inthese studies and for their invaluable intellectual contributions. Supported by Department of Veterans Affairs Merit Review Funding andNational Institutes of Health, Digestive Diseases and Kidney InstituteRO1 DK 54221.

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