Am J Physiol Gastrointest Liver Physiol 285: G804–G812, 2003.
First published July 3, 2003; 10.1152/ajpgi.00150.2003.
Critical role for NHE1 in intracellular pH regulation
in pancreatic acinar cells
David A. Brown,1 James E. Melvin,2 and David I. Yule1
1
Department of Pharmacology and Physiology and 2Center for Oral Biology, School of Medicine
and Dentistry, University of Rochester Medical Center, Rochester, New York 14642
Submitted 1 April 2003; accepted in final form 26 June 2003
physiological role is to
synthesize, package in granules, and secrete zymogens
in response to stimulation (57). In addition, pancreatic
acinar cells also secrete a NaCl-rich primary fluid (21,
41). Although this fluid secretion is minor compared
with the copious secretion by the ductal system of the
pancreas (2, 21), the acinar fluid secretion plays an
important role in hydrating the secreted contents of
the granules and effectively “flushing” the zymogens
from the secretory end piece into the ductal system.
The currently held model for acinar fluid secretion has
been most extensively developed in salivary acinar
cells (7, 8, 29, 53); however, a majority of the processes
involved are common to other acinar cells such as the
exocrine cells of the pancreas (42). In the model, Ca2⫹mobilizing agonists play a key role in initiating fluid
secretion by activating Ca2⫹-sensitive Cl⫺ channels
located in the luminal membrane (3, 4, 29, 40). Subsequently, to maintain the electrical gradient for Cl⫺
movement, Ca2⫹-activated K⫹ channels in the basal
and lateral membranes are also activated (41, 42). To
effectively sustain secretion, intracellular Cl⫺ levels
must be replenished, and this is accomplished by a
basolateral Na⫹-K⫹-2Cl⫺ cotransporter and by a basolateral HCO3⫺/Cl⫺ exchanger (23, 24, 30). A consequence of the exit of HCO3⫺ is an intracellular acidification, as a result of increasing H⫹ levels in the cytoplasm (20, 21, 28, 32, 33, 43). The activity of NHE plays
a major role in maintaining intracellular pH (pHi)
balance by extruding H⫹ and thus plays a critical role
in maintaining fluid secretion. In support of this concept, fluid secretion from mouse parotid acinar cells is
reduced by ⬃1⁄3 in NHE1-null transgenic animals (39).
In addition, the activity of specific NHE may also play
important roles in the transcellular movement of Na⫹
as well as in regulation of cell volume during secretagogue stimulation (55).
Genes encoding eight mammalian NHEs have been
identified (17, 37, 55). The protein products have been
demonstrated to play a role in pH homeostasis, cell
volume regulation, transepithelial Na⫹ and water
movement, together with roles in cell adhesion and
proliferation (55). The NHE1 isoform appears to have a
widespread distribution, including the exocrine pancreas, and is thought to play a housekeeping role by
maintaining pHi and cell volume levels. In contrast,
NHE2, NHE3, and NHE4 have more limited tissue
distribution and are thought to be involved in NaCl
absorption (10, 55). The NHE5 isoform has been found
to be expressed at high levels in the brain, whereas an
intracellular distribution of NHE6 expression in mitochondria has been reported (5, 38). Most recently, the
NHE7 isoform has been found to be expressed in the
trans-Golgi network (37), whereas NHE8 has recently
been reported to be expressed in kidney (17).
Although the activity of NHE has been documented
in pancreatic acinar cells (20, 21, 32, 33), the goal of
Address for reprint requests and other correspondence: D. I. Yule,
Dept. of Pharmacology and Physiology, School of Medicine and Dentistry, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
fluid secretion; exocrine glands; intracellular pH regulation;
intracellular calcium
PANCREATIC ACINAR CELLS’ PRIMARY
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Brown, David A., James E. Melvin, and David I. Yule.
Critical role for NHE1 in intracellular pH regulation in
pancreatic acinar cells. Am J Physiol Gastrointest Liver
Physiol 285: G804–G812, 2003. First published July 3, 2003;
10.1152/ajpgi.00150.2003.—The primary function of pancreatic acinar cells is to secrete digestive enzymes together with
a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na⫹/H⫹
exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux
of Na⫹ and intracellular pH (pHi) regulation. Multiple NHE
isoforms have been identified in pancreatic tissue, but little is
known about their individual functions in acinar cells. The
Na⫹/H⫹ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced
acid challenge, confirming a general role for NHE in pHi
regulation. The targeted disruption of the Nhe1 gene also
completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3⫺-containing and HCO3⫺-free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had
no effect on pHi recovery. In addition, NHE1 activity was
upregulated in response to muscarinic stimulation in wildtype mice but not in NHE1-deficient mice. Fluctuations in
pHi could potentially have major effects on Ca2⫹ signaling
following secretagogue stimulation; however, the targeted
disruption of Nhe1 was found to have no significant effect on
intracellular Ca2⫹ homeostasis. These data demonstrate that
NHE1 is the major regulator of pHi in both resting and
muscarinic agonist-stimulated pancreatic acinar cells.
ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
this study was to identify the particular NHE isoform
that is expressed and is physiologically important in
pancreatic acinar cells. In addition, since changes in
pHi can potentially impact the Ca2⫹-signaling machinery in nonexcitable cells (11, 31, 34, 35), we extended
our study to investigate the effects of disruption of pHi
regulation on Ca2⫹ signaling. Using transgenic knockout animals, we demonstrated that NHE1 is responsible for pHi recovery after an acid load and is upregulated during muscarinic stimulation. NHE1 is therefore a major regulator of pHi in both resting and
secreting cells. Loss of this pH regulation, however,
appears to have a minor impact on secretagogue-stimulated Ca2⫹ homeostasis.
METHODS
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sion chamber on the stage of a Nikon Diaphot 200 microscope
interfaced with an Axon Imaging Workbench system (Axon
Instruments, Foster City, CA). Experiments were performed
at room temperature. Cells were then alternately excited at
440 and 490 nm by using a DG-4 filter changer (Sutter
Instruments, Novato, CA) every 10 s, and emitted fluorescence was captured at 530 nm by using a Cooke Sensicam
(Cooke, Auburn Hills, MI) 12-bit frame transfer digital camera. A ratio of the fluorescence at 490 vs. 440 nm was
computed. pHi was then estimated by using an in situ calibration (52), where external pH was changed in the presence
of high K⫹ and the ionophore nigericin. The fluorescence
ratios computed for pH solutions over the physiological range
of 6.4–7.6 were linear. Recovery consisted of an initial nearlinear increase in pHi, followed by a slower recovery. The
linear portion was used to calculate the initial rate of pHi
recovery (pHi units/min). All data are representative of three
or more experimental runs. In individual experiments, multiple cells from an individual acini were imaged, and the
means ⫾ SD for all cells measured in an experimental run
are shown in each figure.
Fluorescence measurement of Ca2⫹ concentration. Pancreatic acinar cells were loaded with the Ca2⫹-sensitive dye
fura-2 AM (2 ␮M) by incubation for 30 min at room temperature. Fura-2-loaded cells were allowed to adhere to a glass
coverslip, which was perfused in a HEPES-buffered physiological saline solution that contained (in mM) 137 NaCl, 0.56
MgCl2, 4.7 KCl, 1 Na2HPO4, 10 HEPES, 5.5 glucose, and 1.26
CaCl2, pH 7.4, that was gravity fed. Imaging was performed
by using an inverted epifluorescence Nikon microscope with
a ⫻40 oil immersion objective lens (numerical aperture, 1.3).
Fura-2-loaded cells were excited alternately with light at 340
and 380 nm by using a monochrometer-based illumination
system, and the emission at 510 nm was captured by using a
high-speed, digital frame transfer charge-coupled device
camera (T.I.L.L. Photonics). Images were acquired every 500
ms with an exposure of 20 ms. Intracellular Ca2⫹ concentration ([Ca2⫹]i) was calculated from the fluorescence ratios by
using the equation of Grynkiewicz (18). The maximum and
minimum fluorescence ratios were obtained by imaging a
5-␮l droplet of physiological saline containing 1 and 0 mM
[Ca2⫹], respectively. All imaging experiments were performed at room temperature, essentially as previously described (15, 51). Traces are from a single cell, representative
of multiple individual cells from at least one imaged acini in
a particular experimental run, and n represents the number
of experimental runs.
Membrane preparation. The purified membrane preparation was prepared by mincing the pancreas in ice-cold homogenizing buffer followed by further homogenization and centrifugation, essentially as described previously for parotid
tissue (36). Aliquots were quickly frozen in liquid nitrogen
and stored in a ⫺85°C freezer until later use. Protein concentration was measured by using Bio-Rad protein assay
reagent (Bio-Rad, Hercules, CA).
Western blot analysis. Equal amounts of purified pancreatic plasma membrane proteins from either wild-type mice or
NHE-deficient mice (60–100 ␮g/lane) were resolved on 7.5%
SDS-PAGE and transferred to nitrocellulose membranes
(Bio-Rad) as previously described (15). Membranes were incubated with the indicated primary antibody and then detected with a horseradish peroxidase-linked secondary antibody (Pierce, Rockford, IL) and the Super Signal detection
system (Pierce) exposed on XAR film (Eastman Kodak, Rochester, NY).
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Materials. The acetoxymethyl ester form of 2⬘-7⬘bis(carboxyethyl)-5-carboxyfluorescein (BCECF-AM) and
5-(N-ethyl-N-isopropyl) amiloride (EIPA) were purchased
from Molecular Probes (Eugene, OR). Monoclonal ␣-NHE1
was obtained from Chemicon (Temecula, CA). Fura-2 AM
was purchased from TEFLABS (Austin, TX), and all other
chemicals were purchased from Sigma (St. Louis, MO).
HCO3⫺-free solutions contained (in mM) 135 NaCl, 5.4 KCl,
0.4 KH2PO4, 0.33 NaH2PO4, 0.8 MgSO4, 1.2 CaCl2, 10 glucose, and 20 HEPES, pH 7.4. HCO3⫺-containing solutions
contained (in mM) 110 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33
NaH2PO4, 0.8 MgSO4, 1.2 CaCl2, 10 glucose, 20 HEPES, and
25 NaHCO3, pH 7.4, with NaOH after 30 min of gassing with
95% O2-5% CO2 to equilibrate. NH4Cl solution was used to
induce an acid load, and it was prepared by replacing 30 mM
NaCl with 30 mM NH4Cl in HCO3⫺-free and HCO3⫺-containing solutions. The high-K⫹ solution used to calibrate ratios
into pH values contained (in mM) 120 KCl, 20 NaCl, 0.8
MgCl2, 20 HEPES, and 0.005 nigericin, and the pH was
adjusted to between 5.6 and 8.4.
Wild-type and null mutant animals. 129/SvJ-Black Swiss
mice were housed in microisolator cages in the University of
Rochester vivarium on a 12:12-h light-dark cycle, and they
were given access to laboratory chow and water ad libitum.
The targeted disruption of murine NHE isoforms NHE1,
NHE2, and NHE3 was carried out as described (9, 49, 50).
Heterozygous animals were used to establish breeding colonies. Offspring were tail clipped, and genotypes were determined by PCR or by Southern blotting.
Isolation of mouse pancreatic acinar cells. Single and small
groups of pancreatic acinar cells were isolated by collagenase
digestion of freshly dissected pancreata from wild-type or
NHE-deficient Black Swiss mice as previously described (15,
51, 56). Briefly, pancreata were removed and enzymatically
digested with 400 units of collagenase type IV (Sigma) in
minimum essential medium with 1% BSA and 1 mg/ml soybean trypsin inhibitor for 30 min and gently agitated while
being gassed with 95% O2-5% CO2. The cells were then
dispersed by trituration before being filtered through a
100-␮m nylon mesh. Filtered cells were centrifuged for 2 min
at 100 rpm and then resuspended in 2% BSA minimum
essential medium solution.
Fluorescence measurement of pHi. Pancreatic acinar cells
were loaded with pH-sensitive fluoroprobe by incubation for
30 min at room temperature with BCECF-AM (2 ␮M).
BCECF-loaded cells were gassed continuously with 95%
O2-5% CO2 as previously described (14, 36). BCECF-loaded
pancreatic acinar cells were allowed to settle and subsequently adhere to a coverslip forming the base of a superfu-
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ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
RESULTS
⫹
⫹
Fig. 1. 5-(N-ethyl-N-isopropyl) amiloride (EIPA) reversibly inhibits Na⫹/H⫹
exchanger (NHE) activity in wild-type
mouse pancreatic acini. 2⬘-7⬘-Bis(carboxyethyl)-5-carboxyfluorescein (BCECF)loaded mouse pancreatic acini from
wild-type animals were acid loaded by
the addition and subsequent removal
of 30 mM NH4Cl (NH3/NH4⫹ prepulse
technique) indicated by the closed
bars. A: acini from wild-type animals
rapidly recover from intracellular acidification toward the resting intracellular pH (pHi). Incubation of cells with
NHE inhibitor EIPA (10 ␮M) significantly reduced the rate of intracellular
recovery (3rd prepulse). The 4th prepulse shows that acini are capable of
recovering pHi following the washout
of EIPA, indicating that EIPA inhibition is reversible. B: overlay of traces
indicating the difference in kinetics between control pHi recovery and EIPAtreated pHi recovery. Each trace shown
is representative of 3 or more experiments.
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Na /H exchange inhibitor EIPA blocks pHi recovery. In rat acinar cells, recovery from acid load has
been shown to be mediated by NHE family proteins
(32, 33). To confirm that pHi recovery in the mouse
pancreas is accomplished by similar mechanisms, the
selective Na⫹/H⫹ exchange inhibitor EIPA was used.
NHE activity was assessed by acid loading the cells by
using the NH3/NH4⫹ prepulse technique (45, 46). A
representative pHi trace in HCO3⫺-free buffer in response to a NH4Cl pulse is shown in Fig. 1A. Resting
pHi under these conditions averaged 7.59 ⫾ 0.05 (n ⫽
12 experiments). The addition of 30 mM NH4Cl resulted in a rapid alkalinization of 0.53 ⫾ 0.01 pH units.
Subsequent removal of NH4Cl led to an intracellular
acidification below basal pH levels by 0.42 ⫾ 0.03
units, followed by a pHi recovery back to the resting
values. The initial rate of recovery was 0.18 ⫾ 0.04 pH
units/min (Fig. 4). Incubation of cells with 10 ␮M EIPA
resulted in complete inhibition of the recovery from
acidification following removal of NH4Cl (n ⫽ 5 experiments; Fig. 1A; initial recovery rates are summarized
in Fig. 4). This inhibition of NHE activity was completely reversible on removal of EIPA (Fig. 1A). Figure
1B, inset, shows an overlay of control recovery vs. the
use of 10 ␮M EIPA. One minute after removal of
NH4Cl, the pHi of the EIPA-treated cells continued to
acidify. These data suggest in a similar fashion to data
from parotid gland cells that the NHE family of proteins is responsible for pHi recovery in pancreatic acinar cells (14).
Loss of pHi regulation in acini isolated from NHE1null mutants. To identify which particular isoform of
the NHE family is responsible for pH recovery in pan-
creatic acini, we performed experiments using transgenic animals in which Nhe genes had been disrupted.
We initially focused on the NHE1 protein because the
expression of this protein has been reported in pancreatic tissue. As shown in Fig. 2A and Fig. 1, after an
NH4Cl pulse, wild-type cells recover from an acid challenge within minutes. In contrast, no recovery from an
acid load was observed in acini prepared from Nhe1⫺/⫺
mutant mice (Fig. 2B; initial recovery rates are summarized in Fig. 4). The inset confirms by Western blot
analysis that the NHE1 isoform is absent in pancreatic
acini from these transgenic animals. However, the
resting pHi (7.57 ⫾ 0.03; n ⫽ 10 experiments) and the
extent of alkalinization (0.55 ⫾ 0.1 pH units) and of
subsequent NH4Cl-induced acidification (0.42 ⫾ 0.06
pH units) was not significantly different in the knockout compared with wild-type. Essentially, the lack of
recovery of pHi after acidification in NHE1-deficient
cells mimicked the inhibition of pHi recovery by EIPA,
as demonstrated in Fig. 1.
These experiments were repeated in a more physiological, HCO3⫺-buffered solution. Under these conditions, basal pHi was significantly lower compared with
HCO3⫺-free solutions (7.18 ⫾ 0.04; n ⫽ 5 experiments),
as previously reported (32, 33). However, wild-type
cells recovered from an acid challenge with similar
kinetics to that observed in a HCO3⫺-free environment
(Fig. 2C). In a similar manner to experiments in
HCO3⫺-free solutions, acini prepared from NHE1-deficient animals showed no pHi recovery following an acid
challenge (Fig. 2D). These data indicate that a similar
mechanism, i.e., the activity of NHE1, is used by pancreatic acini to recover pHi after an acid load whether
in HCO3⫺-containing or HCO3⫺-free medium and sug-
ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
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gest that Na⫹/HCO3⫺ exchange is not a major mechanism for pHi regulation under these conditions.
The data in Fig. 2 suggest that NHE1 has an exclusive role in pHi recovery in response to an acid challenge in pancreatic acini. It is, however, formally possible that disruption of the Nhe1 gene could have
resulted in lowered levels of other NHE isoforms. To
address this issue, we investigated pHi recovery in
transgenic mice in which the Nhe3 or Nhe2 gene had
been disrupted, resulting in a null mutant animal.
Acini prepared from either of these animals retained
their ability to recover from an acid challenge in a
similar fashion to wild-type mice; indeed, the rates of
recovery were not significantly different from wild-type
animals (Fig. 3, A and B; initial recovery rates are
summarized in Fig. 4; n ⫽ 3 experiments each). Furthermore, when membranes prepared from NHE2-deficient animals were probed with NHE1 antiserum,
similar amounts of NHE1 protein were evident (Fig. 3,
inset). These data collectively suggest that disruption
of an Nhe gene per se does not appear to result in
decreased levels of other NHE family members. More
importantly, these results demonstrate that the NHE1
isoform is the dominant regulator of pHi in unstimulated mouse pancreatic acinar cells. This is consistent
with the role of NHE1 in other tissues, such as the parotid, lacrimal, and sublingual glands (14, 22, 36, 48).
Muscarinic stimulation of NHE1 activity. NHE activity is frequently increased or “upregulated” during
agonist stimulation (6, 22, 32). We therefore specifically investigated whether NHE1 activity, in addition
to functioning in unstimulated acini, was responsible
for the increased activity in pancreatic acinar cells
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during secretagogue stimulation. Experiments were
performed in which acini from wild-type animals were
incubated with 10 ␮M CCh during the period of recovery from acid load (Fig. 5A). Incubation with CCh
resulted in a significant increase in the rate of recovery
in the absence of HCO3⫺, which averaged 152 ⫾ 15%
faster for CCh-treated trials vs. paired controls in the
same cells (P ⫽ 0.03 by paired t-test; see Fig. 5, inset).
In addition, CCh treatment resulted in an alkaline
shift or “overshoot” above the initial resting rate of
0.16 ⫾ 0.03 pHi units (n ⫽ 3 experiments). A similar
overshoot was observed in HCO3⫺-containing media
(0.18 ⫾ 0.06 pHi units; Fig. 5C; n ⫽ 3 experiments). In
contrast, CCh did not cause any change in the rate of
pHi recovery in the Nhe1⫺/⫺ animals and there was no
overshoot in either the presence (Fig. 5D) or absence
(Fig. 5B) of HCO3⫺ (n ⫽ 3 experiments). These data
demonstrate that the activity of NHE is upregulated on
secretagogue stimulation in pancreatic acini. Furthermore, this affect can be attributed to the increased
activity of the NHE1 isoform, in particular during
muscarinic receptor agonist-induced fluid secretion.
Role of NHE1 in buffering the intracellular acid load
resulting from HCO3⫺ efflux. The results described
above demonstrate that NHE1 directly regulates the
recovery from an acid load in both stimulated and
unstimulated pancreatic acinar cells. Experiments
were then performed to determine whether the activity
of NHE1 was altered by agonist stimulation under
relatively physiological conditions in the absence of an
artificial acid load. Exposure of acini to low concentrations of CCh (100–500 nM) resulted in no discernible
changes in pHi in either wild-type or Nhe1⫺/⫺ cells.
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Fig. 2. NHE1-dependent pH recovery
in mouse pancreatic acini. BCECFloaded mouse pancreatic acini prepared as described in Fig. 1 legend
from wild-type and Nhe1⫺/⫺ animals.
Experiments were performed in both
the absence (A and B) and presence (C
and D) of HCO3⫺. A: acini from wildtype animals recover from intracellular acidification in the absence of
HCO3⫺. B: recovery from NH4Cl-induced intracellular acid load is inhibited completely in acini from Nhe1⫺/⫺
animals in the absence of HCO3⫺. C:
acini from wild-type animals recover
from intracellular acidification in the
presence of HCO3⫺. D: even in the presence of HCO3⫺, acini from Nhe1⫺/⫺ animals do not recover from an NH4Clinduced intracellular acid load. Each
trace shown is representative of 4 or
more experiments. B, inset: immunoblot analysis of pancreas membrane
proteins (100 ␮g/lane) using antiNHE1 antibodies. Membranes were
prepared from either wild-type (⫹/⫹)
or Nhe1⫺/⫺ animals as indicated. Arrow indicates location of NHE1 protein.
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ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
Fig. 3. Pancreatic acini from Nhe3⫺/⫺ and Nhe2⫺/⫺ mice retain NHE
activity. BCECF-loaded mouse pancreatic acini were prepared as
described in Fig. 1 legend from Nhe3⫺/⫺ or Nhe2⫺/⫺ animals. Acini
isolated from both Nhe3⫺/⫺ (A) and Nhe2⫺/⫺ (B) mice recover from an
intracellular acidification with kinetics similar to that seen in wildtype animals. Traces are representative of 6 or more experiments.
C: NHE1 levels are similar in wild type and Nhe2⫺/⫺ animals, which
suggests that disrupting one Nhe gene does not effect other NHE
proteins. Arrow indicates location of NHE1 protein.
These data suggest that any changes in pHi that occur
under these conditions are below the threshold for
detection or represent local changes. However, when
cells were stimulated with a higher concentration of
CCh (5–10 ␮M) a rapid alkalinization of 0.22 ⫾ 0.05
pHi units occurred (Fig. 6; n ⫽ 4 experiments), indicating that pHi is dynamically regulated during secretagogue-induced fluid secretion. This alkalinization was
presumably the result of upregulation of NHE1 because the effect was absent in Nhe1⫺/⫺ mice (Fig. 6).
Stimulation of Nhe1⫺/⫺ mice with CCh resulted in a
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Fig. 4. Summary of initial rates of recovery. Initial rates of recovery
were calculated as described in METHODS. Data show that initial rates
of pHi recovery are not significantly different in wild-type, Nhe2⫺/⫺,
and Nhe3⫺/⫺ pancreatic acini. In contrast, no recovery from acid load
occurs in either Nhe1⫺/⫺ acini or wild-type acini treated with EIPA.
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slow acidification, which after 3 min averaged 0.29 ⫾
0.04 pHi units (n ⫽ 3 experiments), presumably as a
result of HCO3⫺ loss from the cells via the basolateral
Cl⫺/HCO3⫺ exchanger or exit via the luminal Cl⫺ channel. Thus loss of NHE1 activity results in disruption of
the cellular regulation of pHi during the secretory
process.
Does loss of pHi regulation impact [Ca2⫹]i signaling?
The data presented above indicate that secretagogue
stimulation of pancreatic acinar cells results in an
upregulation of NHE1 activity. A critical signal in the
overall process that gives rise to fluid secretion during
muscarinic stimulation is an increase in the [Ca2⫹]i.
Because the cellular machinery that results in an increase in [Ca2⫹]i is a rich source of potential sites for
regulation by changes in pHi (11, 31, 34, 35), the
consequences of the loss of this pHi regulation during
Ca2⫹ signaling was investigated next. An experimental
paradigm was designed to initially assess any effect of
disordered pHi regulation on both a physiological, oscillatory signal and a peak and plateau response to a
higher concentration of agonist. In addition, since we
have shown a marked pHi change in response to the
high concentration of secretagogues, which is essentially nonreversible over the time course of the experiment in the Nhe1⫺/⫺ mice (Fig. 6), we assessed the
effects of this change in pHi on a subsequent oscillatory
response by restimulating with a low concentration of
CCh. Figure 7 shows wild-type (Fig. 7A) and Nhe1⫺/⫺
(Fig. 7B) pancreatic acinar cells responding to a low
and then subsequently a high concentration of CCh,
followed by a repeat exposure to a low concentration of
CCh. Acini from both wild-type and Nhe1⫺/⫺ animals
responded with very similar Ca2⫹-signaling profiles,
characteristic of the concentration of CCh used. When
the characteristics of the signals were analyzed in
detail, no statistical differences were observed in the
response to maximal CCh concentration. These experiments revealed that the initial peak used as a measure of maximal Ca2⫹ release {[Ca2⫹]i over basal was
ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
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335 ⫾ 36 nM (n ⫽ 6; 29 cells) vs. 379 ⫾ 54 nM (n ⫽ 4;
22 cells) for wild-type vs. Nhe1⫺/⫺, respectively} or the
plateau height measured after 5 min as an indicator of
depletion-activated Ca2⫹ entry {⌬[Ca2⫹]i was 133 ⫾ 18
nM (n ⫽ 6; 29 cells) vs. 121 ⫾ 11 nM (n ⫽ 4; 22 cells) for
wild-type vs. Nhe1⫺/⫺, respectively} were not different.
In a similar fashion, there were no obvious differences
in the initial peak height or oscillation frequency when
comparing the first or second response to 300 nM CCh
in the wild-type or Nhe1⫺/⫺ acini. For example, the
initial elevation over basal stimulated by the first application of agonist averaged a ⌬[Ca2⫹]i of 290 ⫾ 34 nM
(n ⫽ 12; 59 cells) vs. 356 ⫾ 42 nM (n ⫽ 8; 43 cells) for
wild-type vs. Nhe1⫺/⫺, respectively, and the average
oscillation frequency was 4.32 ⫾ 0.24 vs. 4.7 ⫾ 0.31
oscillations/min for wild-type vs. Nhe1⫺/⫺ acini, respectively. These parameters, although decreased on second application of 300 nM CCh, did not change relatively. For example, the initial ⌬[Ca2⫹]i was 55 ⫾ 9%
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Fig. 6. Effects of muscarinic stimulation on resting pH levels. In the
absence of extracellular HCO3⫺, 5 ␮M CCh addition to wild-type
BCECF-loaded acini induced an intracellular alkalization (top trace,
filled symbols). In the Nhe1⫺/⫺ acini (bottom trace, open symbols), 10
␮M CCh failed to produce the same alkalinization and in fact resulted in a significant acidification. Each trace shown is representative of 3 or more experiments.
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Fig. 5. Upregulation of NHE1 activity during muscarinic stimulation. Experiments were performed as described
previously, i.e., application of 10 ␮M CCh (open bar) immediately following the NH4Cl prepulse. Each wild-type
experiment was part of a paired experiment as shown in the inset. A: application of CCh in HCO3⫺-free solutions
in wild-type cells resulted in upregulation of NHE activity because after pHi recovery an intracellular alkalinization relative to the initial resting pHi (represented by the dashed line) was evident. B: evidence of increased NHE
activity in HCO3⫺-free solutions is abolished in CCh-stimulated acini prepared from Nhe1⫺/⫺ mice. C: application
of CCh in HCO3⫺-containing solutions retained the increased activity of NHE and intracellular alkalinization
relative to the resting pHi. D: result of CCh treatment of Nhe1⫺/⫺ acini in HCO3⫺-containing solutions was identical
to HCO3⫺-free data.
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ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
(n ⫽ 4; 17 cells) of the first response in wild-type and
40 ⫾ 5% in the Nhe1⫺/⫺ acini (n ⫽ 3; 18 cells), whereas
the oscillation frequency was 90 ⫾ 4% of the first
response vs. 106 ⫾ 8% for wild-type vs. Nhe1⫺/⫺. Together, these data indicate that although NHE1 activity is critically important in pHi regulation in pancreatic acini, disruption of this regulation does not appear
to exert a major effect on [Ca2⫹]i-signaling events.
DISCUSSION
Expression of a sodium-dependent proton exchanger
that is regulated during secretion in pancreatic acinar
cells has been demonstrated in a number of studies (21,
23, 32, 33). However, the molecular identity of this
exchanger had not, to this point in time, been elucidated. At least eight genes encode members of the
NHE family of proteins, and multiple members of this
family have been identified in exocrine cells (1, 14, 17,
30, 39, 47). In particular, although the presence or
absence of other isoforms has not been determined,
mouse pancreatic cells have been reported to express
NHE1 and NHE4 based on their profile of inhibition by
various agents and by immunocytochemistry (1, 47). In
addition to the predicted localization to the basolateral
membrane of acinar and ductal cells, the NHE1 and
NHE4 isoforms have also been reported to be expressed intracellularly on zymogen granule membranes (1, 47). A role has been proposed for these
intracellular exchangers in regulated exocytotic secretion. Other related exocrine glands such as salivary
gland acinar cells express NHE1, NHE2, and NHE3 in
a manner that appears to be species and/or gland
specific (14, 19, 25, 39).
In this study, we have shown that NHE1 accounts
for a majority, if not all, of Na⫹/H⫹ exchange activity in
mouse pancreatic acinar cells under conditions of imposed acid load. Moreover, this observation holds under both HCO3⫺-free conditions where this activity is
isolated and under more physiological conditions in the
presence of HCO3⫺ where Cl⫺/HCO3⫺ exchange activity
could contribute to the acid loading of the cell and
AJP-Gastrointest Liver Physiol • VOL
where Na⫹-HCO3⫺ cotransport could potentially play a
role in recovery from acid challenge. Since mouse pancreatic acinar cells from Nhe1⫺/⫺ mice were incapable
of pHi recovery from an acid load, it follows that other
NHE isoforms expressed cannot substitute for NHE1
activity, presumably because of localization to an intracellular compartment. Furthermore, these data indicate that Na⫹-HCO3⫺ cotransport appears to play
little if any role in recovery from acid load. In addition,
we demonstrate that the activity of this particular
isoform is increased during muscarinic secretagogue
stimulation both under secretory conditions and on
recovery from an acid load. These data are in agreement with the idea that the ubiquitous expression of
NHE1 functions as the “housekeeping” isoform and
more specifically is consistent with reports from both
sublingual and parotid acinar cells demonstrating an
important role for NHE1 in secretagogue-stimulated
fluid secretion (14, 36).
The mechanism responsible for upregulation of NHE
activity by secretagogues was not addressed in this
study. However, an abundant literature on epithelial
cell types indicates that stimulation of NHE1 activity
is mimicked acutely by maneuvers that result in elevations of [Ca2⫹]i but not by activation of protein kinase C (26, 27, 55). This is indicated as upregulation
and can be mimicked by Ca2⫹ ionophore treatment but
is not duplicated by diacylglycerol analogs. NHE activation by Ca2⫹ is thought to occur either by the direct
interaction of Ca2⫹ or by the rapid, high-affinity binding of Ca2⫹-calmodulin to region A of the exchanger
(54). Although additional mechanisms such as cell
shrinkage or Cl⫺ depletion (44) could contribute to the
regulation of NHE1 in pancreatic acinar cells, regulation by changes in [Ca2⫹]i would be entirely consistent
with intracellular events known to be stimulated by
CCh treatment in this cell type.
The machinery responsible for initiating, propagating, and clearing an increase in [Ca2⫹]i on stimulation
by pancreatic secretagogues is a rich potential source
of loci for regulation by changes in pHi. For example,
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Fig. 7. Disruption of the NHE1 protein does not affect
Ca2⫹ signaling. Fura-2 loaded mouse pancreatic acini
from wild-type and Nhe1⫺/⫺ animals were stimulated
with either a low (300 nM) or high (5 ␮M) concentration
of CCh, which in the Nhe1⫺/⫺ acini resulted in a sustained acidification after removal of the agonist. After
removal of CCh, the acini were stimulated by a second
challenge with 300 nM CCh. A: 300 nM CCh treatment
caused repeated oscillations in wild-type pancreatic
acini. Higher concentrations of CCh caused an increase
in the initial peak of the intracellular Ca2⫹ concentration ([Ca2⫹]i) response and resulted in a sustained
plateau where [Ca2⫹]i levels remained elevated during
agonist application. A second application of 300 nM
CCh again results in an oscillatory Ca2⫹ signal, of
somewhat reduced magnitude. The trace is representative of 4 experiments. B: identical paradigm applied to
Nhe1⫺/⫺ pancreatic acini resulted in [Ca2⫹]i signals
with similar characteristics to wild type. The trace is
representative of 5 experiments.
ROLE OF NHE1 IN PANCREATIC ACINAR CELL PHi REGULATION
We thank Drs. Ha-Van Nguyen, Keith Nehrke, and Trevor
Shuttleworth for helpful discussion during the course of this study.
DISCLOSURES
Grants from the National Institutes of Health to J. E. Melvin
(RO1-DE-08721) and D. I. Yule (DK-54568) supported this study.
D. A. Brown was supported by National Institute of Dental Research
Training Grant T32-DE-07202.
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