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J Biol Chem, Vol. 274, Issue 21, 14670-14677, May 21, 1999
From the We have demonstrated previously the regulation of
Cl HCO3 To date direct evidence in support of the two models is meager indeed.
Localization of CFTR in the luminal membrane of all CFTR-expressing
epithelia is well documented (14-16).
Cl A relationship between HCO3 The intimate relationship between CFTR expression and
HCO3 The SMG and pancreatic ducts were selected as model systems for several
reasons. The fraction of electroneutral
HCO3 We show here that stimulation of CFTR with cAMP increased
Cl Solutions--
The standard perfusate was termed solution A and
contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes (pH 7.4 with NaOH), and 10 glucose. The
HCO3 Culture of T84 Cells--
T84 cells were purchased from American
Type Culture Collection (ATCC CCL 248, Rockville, MD) and maintained in
a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's
medium supplemented with 5% fetal calf serum. The cells were plated on
a sterile 22 × 40-mm coverslip at a density of 2.5 × 105 cells/cm2 for intracellular pH measurements.
Animals and Preparation of Ducts--
A cystic fibrosis mouse
model in which the
Duct fragments from the mouse SMG were prepared by a slight
modification of our published procedure (16). Mice were sacrificed by
exposure to a methoxyflurane-saturated atmosphere and subsequent cervical dislocation, and the SMGs were removed to a cold pancreatic solution A (PSA). The composition of PSA was (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes (pH 7.4 with NaOH), 10 glucose, 10 pyruvate, 0.1% bovine serum albumin, and
0.02% soybean trypsin inhibitor. Each gland was cleaned by injection
of 5 ml of PSA and minced. The minced tissue was transferred to 8 ml of
PSA containing 2.5 mg of collagenase (CLS4, 254 units/mg; Worthington
Biochemicals) and digested for 8-10 min at 37 °C. The dissociated
cells were then washed twice with PSA, resuspended in 2 ml of PSA, and
kept on ice until use.
Microperfusion experiments were performed with microdissected
pancreatic ducts from WT and Intracellular pH (pHi) Measurements--
The
procedure of pHi measurement in T84 cells was
identical to that described in detail in our recent work (28). In the
case of SMG cells, the dissociated cells were loaded with BCECF by a
10-min incubation at room temperature in PSA containing 1 µM BCECF-AM. The cells were then washed with PSA and
plated on a polylysine-coated coverslip that was assembled into a
perfusion chamber. The chamber was placed on an inverted microscope,
and intralobular ducts were identified by morphology. The BCECF
fluorescence of 10-16 cells of a duct fragment was recorded at
excitation wavelengths of 440 and 490 nm. Fluorescence ratios of
490/440 were calibrated using the procedures described previously (28).
In the case of the perfused pancreatic duct BCECF loading was
accomplished by including 2.5 µM BCECF-AM in the luminal
perfusate for 10 min.
Changes in Cl Intracellular Cl
The results of multiple experiments with each cell type and under the
different conditions were analyzed using paired or nonpaired Student's
t test, as appropriate.
Regulation of AE Activity in T84 Cells--
We have described
previously the regulation of AE activity by CFTR in cells stably or
transiently transfected with CFTR (28). The purpose of the present work
was to determine whether such regulation exists in a cell line and in
native cells naturally expressing CFTR. The first set of experiments
was performed with the human colonic cell line T84, which can serve as
a suitable model system in future studies. This cell line has been used
in the past in several studies as a model system to characterize natively expressed CFTR (32, 33).
Fig. 1 shows representative experiments,
and Fig. 2 summarizes the results under
each experimental condition. DIDS-sensitive, Cl
The potential regulation or function of CFTR as a
HCO3 AE Activity in the SMG of WT and
Fig. 3d shows that removal of Cl [Cl AE Activity under Depolarized Conditions--
The effect of
membrane depolarization with Ba2+ on
Cl
In these experiments Ba-Hepes (0.5 M, pH 7.4) was added to
HCO3 Regulation of AE Activity by Forskolin--
The effect of
forskolin stimulation on AE activity of SMG duct from WT and
To determine the actual stimulation of AE by CFTR in SMG duct we
measured the effect of forskolin in ducts treated with 5 mM
Ba2+. Fig. 6 shows that
forskolin stimulation of SMG ducts from WT mice, but not from
To avoid the possibility of a nonspecific effect of Ba2+,
we tested the effect of high K+ on AE activity in SMG ducts
from WT mice. Fig. 7 shows that
depolarizing the membrane potential with 100 mM
K+ was as effective as 5 mM Ba2+ in
unmasking the maximal AE activity. As expected, the
Cl Membrane-specific Regulation of
Cl
To obtain a better estimate of the AE activity in each membrane and
under resting and stimulated conditions, we measured separately the
basolateral and luminal AE activity under depolarized conditions. The
left part of Fig. 8b shows the higher
rate (and extent) of pHi changes caused by
Cl In the present work we used the SMG and pancreatic ducts of WT and
In agreement with our previous work in the rat SMG duct (18), in the
present work we were able to demonstrate AE activity in the mouse SMG
duct. The evidence includes a DIDS-sensitive, Naout+-independent, electroneutral
Clin We had a particular interest in evaluating
HCO3 Low luminal HCO3 The major finding of the present work was extending the finding of
regulation of AE activity by CFTR to native CFTR-expressing tissues
such as the SMG and pancreas. Even though the increase in AE activity
after forskolin stimulation of SMG duct was not as prominent as that
observed in the transfected cell lines (28), it could be clearly
demonstrated even when the cells were not depolarized (Fig. 5). It is
important to note that the extent of stimulation of AE activity by CFTR
in SMG ducts may be significantly underestimated. Studies on the rat
SMG duct showed the presence of AE activity in both the BLM and LM
(18). Expression of CFTR in the LM of SMG ducts (16) indicates that
CFTR should stimulate AE activity present in the LM but not in the BLM
of SMG ducts. Hence, AE activity in the BLM, although lower than that
in the LM (18), increases the background against which the stimulation of AE activity by CFTR is evaluated. This reasoning is reinforced by
the finding that CFTR stimulates the luminal but not the basolateral AE
activity of the pancreatic duct. Notably, in the SMG duct, when the AE
activity of both membranes contributed to the measurement, stimulation
of CFTR increased AE activity by about 43% (Fig. 7). Under the same
depolarized conditions CFTR increased luminal AE activity of the
pancreatic duct by about 96% (Fig. 8).
The exclusive expression of CFTR in the luminal membrane of the
SMG duct and other CFTR-expressing cells indicates that also in these
tissues CFTR regulates the luminal AE. To date the protein responsible
for the luminal AE activity of CFTR-expressing cells has not been
identified. Good immunocytochemical evidence indicates that the
housekeeping AE2 is expressed exclusively in the basolateral membrane
of SMG duct and acinar cells (19). This excludes AE2 as the isoform
regulated by CFTR. It has been suggested that a variant of AE1 is
expressed alternatively in the basolateral or luminal membrane of
intercalated cells of collecting duct based on the metabolic state of
the animal (41). However, preliminary reverse transcriptase-polymerase
chain reaction analysis of the AE isoforms expressed in the cell lines
used in the present paper and in Ref. 28 indicates that these cells
express only AE2 and AE3 (not shown). Hence, it is possible that CFTR
regulates AE3 and that AE3 is the isoform expressed in the luminal
membrane of the SMG duct. Another alternative is that a protein other
than the known AE isoforms mediates the luminal AE activity. Using expression systems we are attempting to test the effect of CFTR on the
activity of each AE isoform.
Our findings provide new insight into the mechanisms of fluid and
electrolyte secretion by CFTR-expressing cells and into the
pathophysiology of cystic fibrosis. An important function of
CFTR-expressing cells is the secretion of
HCO3 The global role of CFTR indicates that its action is not likely to be
replaced solely by activation of luminal Cl We thank Dr. Kirk R. Thomas (University of
Utah, Salt Lake City) for kindly providing the *
This work was funded in part by National Institutes of
Health Grants DE12309 and DK38938 (to S. M.) and DK49835 (to
P. J. T.).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.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
Yonsei University College of Medicine, 134 Sinchon-Dong, Seoul 120-752, Korea. Tel.: 82-2-361-5210; Fax: 82-2-313-1894; E-mail:
mlee{at}yumc.yonsei.ac.kr.
The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator;
BLM, basolateral
membrane;
LM, luminal membrane;
AE, Cl
Cystic Fibrosis Transmembrane Conductance Regulator Regulates
Luminal Cl
/HCO3
Exchange in Mouse Submandibular and Pancreatic Ducts*
§,
, and Department of Pharmacology, Yonsei
University College of Medicine, Seoul 120-752, Korea, and the
¶ Department of Physiology and the Program in Molecular
Biophysics, University of Texas Southwestern Medical Center,
Dallas, Texas 75235
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/HCO3 exchange
activity by the cystic fibrosis transmembrane conductance regulator
(CFTR) in model systems of cells stably or transiently transfected with
CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E.,
Marino, C. R., Thomas, P. J., and Muallem, S. (1999)
J. Biol. Chem. 274, 3414-3421). In the present work
we examine the significance of this regulation in cells naturally
expressing CFTR. These include the human colonic T84 cell line and the
mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl/HCO3 exchange
activity independently of CFTR Cl channel activity.
Freshly isolated submandibular gland ducts from wild type mice showed
variable Cl/HCO3
exchange activity. Measurement of
[Cl]i revealed that this was
largely the result of variable steady-state
[Cl]i. Membrane depolarization
with 5 mM Ba2+ or 100 mM
K+ increased and stabilized
[Cl]i. Under depolarized
conditions wild type and 
F/F mice had comparable basal
Cl/HCO3 exchange
activity. Notably, stimulation with forskolin increased Cl/HCO3 exchange
activity in submandibular gland ducts from wild type but not F/F
mice. Microperfusion of the main pancreatic duct showed
Cl/HCO3 exchange
activity in both the basolateral and luminal membranes. Stimulation of
ducts from wild type animals with forskolin had no effect on
basolateral but markedly stimulated luminal
Cl/HCO3 exchange
activity. By contrast, forskolin had no effect on either basolateral or
luminal Cl/HCO3
exchange activity of ducts from F/F animals. We conclude that CFTR regulates luminal
Cl/HCO3 exchange
activity in CFTR-expressing cells, and we discuss the possible
physiological significance of these findings regarding cystic fibrosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
secretion is a primary
function of many
CFTR1-expressing cells
(1-3). Most of the HCO3 is secreted
by duct or duct-like cells to the lumen and thus requires transductal
HCO3 transport. Little is known about
the pathways mediating HCO3 entry in
the basolateral membrane (BLM). The best studies available to date are
in the pancreatic duct, in which Case and co-workers (4-6) provided
strong evidence that HCO3 influx is
mediated largely by a BLM
Na+-HCO3 cotransport.
HCO3 efflux across the luminal
membrane (LM) and its regulation are equally poorly understood. Most
models assume that the electroneutral portion of
HCO3 secretion is mediated by a
luminal Cl/HCO3
exchanger (AE, anion exchanger) (1-3, 6). This function is also
believed to mediate part of Cl absorption by the duct.
Cl is supplied to the duct lumen in the plasma-like
primary fluid secreted by acinar cells (1-3, 7). The pathophysiology
of cystic fibrosis indicates that CFTR plays a critical, but poorly defined, role in HCO3 secretion and
Cl absorption. In tissues such as the salivary glands, in
which acinar cells secrete the bulk of the fluid, CFTR is assumed to mediate the electrogenic part of Cl absorption (2). The
same role is attributed to CFTR in sweat glands (7) and intestinal
epithelia (8, 9). Recent work suggests that airway epithelia absorb
Na+ and Cl to produce a hypotonic airway
surface fluid (Ref. 10 and references within, but see Ref. 11 and
references within). Because Na+ and Cl
concentrations in airway surface liquid produced by cystic fibrosis airway epithelium are isotonic (10, 11), CFTR may mediate electrogenic
Cl absorption in airway epithelia (12, 13). In glands
like the pancreas, fluid secretion by acinar cells is limited, and the duct secretes the bulk of the fluid in pancreatic juice (1). In this
type of gland the limited supply of Cl secreted by acinar
cells led to the proposal that CFTR mediates Cl secretion
to the lumen of duct cells to fuel the
Cl/HCO3 exchanger (1).
However, a recent work showed that agonist- and cAMP-stimulated
HCO3 secretion in guinea pig
pancreatic duct is independent of luminal Cl (6). Hence,
the role of CFTR in ion transport by these tissues remains obscure.
/HCO3 exchange
activity was found in the BLM and LM of pancreatic (17) and
submandibular gland (SMG) ducts (18). In SMG ducts the AE isoform 2 (AE2) was localized in the BLM (19). The isoform(s) expressed in the LM
is not known.
secretion
and CFTR was documented in two cell lines and intestinal epithelia. In
a human airway epithelial cell line CFTR-dependent
HCO3 conductance (20, 21) was proposed
to be mediated by CFTR itself. By contrast, similar studies in a human
pancreatic duct cell line concluded that electrogenic Cl
and HCO3 secretions are mediated by
independent proteins (22). In duodenal epithelium basal and
acid-stimulated HCO3 secretions were
reduced or absent in CFTR 
/ mice (23). Surprisingly, in a recent
study Seidler et al. (24) showed that all forms of
HCO3 secretion stimulated by agonists
or agents that elevate cAMP, cGMP, and, in particular,
[Ca2+]i were impaired in the
intestinal epithelia of CFTR / mice. These studies suggest the
likely involvement of CFTR in the electrogenic component of
HCO3 secretion, which is particularly
prominent in the intestine (25, 26). However, a large fraction of
HCO3 secretion in tissues such as
salivary glands (2) and the rat and mouse pancreas (1) is mediated by
an electroneutral HCO3 transport
mechanism. The role of CFTR in this critical component of
HCO3 secretion is unknown.
secretion seen in intestinal (23,
24) and airway epithelia (27) raises the question of whether and how
CFTR modulates HCO3 secretion in other
CFTR-expressing tissues. In addition to its possible function as a
regulator of a HCO3 conductive
channel, CFTR may also regulate luminal
Cl/HCO3 exchange
activity. In a recent study (28) we used cells stably transfected with
CFTR and transient transfection of WT CFTR and several CFTR mutants to
demonstrate regulation of AE activity by CFTR. To evaluate the
physiological relevance of these findings, in the present work we
report the regulation of the luminal AE activity by CFTR in the mouse
SMG and pancreatic ducts.
secretion in these tissues is
relatively high (1, 2). Among all CFTR-expressing tissues, the
mechanism of fluid and electrolyte secretion is understood best in the
SMG (2). The SMG and pancreatic ducts express high levels of CFTR (15),
and the abundance of ducts in the SMG (2, 29) facilitates
experimentation. Although the mouse is not the ideal species to study
HCO3 secretion (1, 6, 30), it was
selected for the present work because of the availability of F/F
mice (31). To supplement the studies in the native ducts we also used
the human colonic cell line T84 because these cells have been used
extensively to study the properties of naturally occurring CFTR (32,
33).
/HCO3 exchange
activity in T84 cells. More importantly, stimulation of CFTR by cAMP
resulted in selective activation of luminal
Cl/HCO3 exchange
activity in SMG and pancreatic ducts of WT mice, which was absent in
ducts prepared from F/F mice. We conclude that CFTR regulates
luminal Cl/HCO3 exchange
activity of SMG and pancreatic ducts and probably other CFTR-expressing cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-buffered NaCl solution B
contained (in mM) 120 NaCl, 25 NaHCO3, 5 KCl, 1 MgCl2, 1 CaCl2, 5 Hepes (pH 7.4 with NaOH), and
10 glucose. The HCO3-buffered
Cl-free solution C contained (in mM) 120 Na+-gluconate, 25 NaHCO3, 5 K+-gluconate, 1 MgSO4, 9.3 hemicalcium
cyclamate, 5 Hepes (pH 7.4 with NaOH), and 10 glucose. To prepare
HCO3-buffered high KCl (100 mM K+) solution D, 95 mM NaCl in
solution B was replaced with 95 mM KCl. To prepare
HCO3-buffered, high K+
(100 mM K+), Cl-free solution E,
95 mM Na+-gluconate was replaced with 95 mM K+-gluconate in solution C. For calibration
of intracellular Cl, solution E was supplemented with 5 µM nigericin and 10 µM tributyltin cyanide.
The KSCN solution contained (in mM) 127 KSCN, 25 choline- HCO3, and 5 Hepes (pH 7.4 with 2 M Tris). The osmolarity of all solutions was adjusted to
310 mosM with the major salt prior to use.
F508 mutation was introduced in the mouse CFTR by
gene targeting in ES cells (31) was obtained from Dr. Kirk R. Thomas
(Eccles Institute of Human Genetics, HHMI, University of Utah School of
Medicine, Salt Lake City). The mice were maintained on a standard diet,
and genotyping was carried out on day 14 postpartum as described
previously (16).
F/F mice. The procedure for
preparation and perfusion of the main pancreatic duct was identical to
that used for perfusion of the rat pancreatic duct (17). The ducts were
dissected in PSA, cannulated, and perfused through the lumen and the
bath with solution A. After completion of BCECF loading the ducts were
perfused with HCO3-buffered solution B
for at least 10 min prior to manipulation of Cl gradients.
/HCO3
exchange activity were estimated from the initial rate of
pHi changes (T84 cells and pancreatic ducts) or
from the extent of pHi changes (SMG ducts). Initial rates of pHi changes were obtained from
the first derivative of the traces using a single exponential fit. The
extent of pHi changes was estimated by averaging
the pHi changes measured as a result of
Cl removal and addition. All results are given as
mean ± S.E. of the indicated number of experiments.
Measurement--
[Cl]i was
measured with the aid of the Cl-sensitive dye MQAE using
the procedure described before for
6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; 18) with minor
modifications. SMG cells were suspended in PSA containing 10 mM MQAE and incubated for 20 min at room temperature and 40 min at 0 °C before plating on coverslips. About 2 min after plating,
unattached cells and external MQAE were washed by starting the
perfusion with solution A. MQAE fluorescence was measured at an
excitation wavelength of 360 nm with the dichroic mirror and emission
cut-off filter set normally used to monitor Fura-2 fluorescence. At the
end of each experiment a two-point calibration procedure was performed.
To obtain the maximal fluorescence the cells were perfused with high
K+, Cl-free solution containing 5 µM nigericin and 10 µM tributyltin cyanide.
Incubation in a Cl-free solution without ionophores did
not result in complete depletion of intracellular Cl. To
obtain the minimal fluorescence the cells were then exposed to a
solution containing 127 mM KSCN. Significant dye leak, in particular after exposure to tributyltin cyanide, precluded a more
extensive in vivo calibration. A Stern-Volmer constant of 12.4 M1 reported before for rabbit SMG ducts
(34) was used to calculate [Cl]i.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
HCO3-dependent changes in
pHi indicate the expression of relatively modest
AE activity in T84 cells. Stimulation of the cells with 5 µM forskolin caused a reproducible reduction in
pHi. This reduction in
pHi was less pronounced than that observed in
NIH 3T3 and HEK 293 cells expressing high levels of CFTR. Removal and
addition of Cl to the incubation medium showed that
forskolin increased the rate of AE activity by about 2.2-fold or 0.051 pH unit/min. As was found in NIH 3T3 and HEK 293 cells expressing
CFTR (28), the AE activity stimulated by forskolin was not affected by
inhibition of CFTR-mediated Cl current with 0.1 mM N-phenylanthranilic acid (DPC in
Fig. 1a) or 0.1 mM glibenclamide
(Glib in Fig. 1b). On the other hand, the AE
activity was nearly abolished by 0.5 mM DIDS (Fig.
1b), as was found in 293 cells expressing modest levels of
CFTR (see Fig. 7b of Ref. 28).
View larger version (26K):
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Fig. 1.
AE activity in T84 cells. T84 cells
attached to coverslips and loaded with BCECF were bathed in a
HCO3 -buffered solution. As indicated
by the bars, Cl was removed and added to the
perfusing medium before and after stimulation with 5 µM
forskolin. The cells were also incubated with 0.1 mM
N-phenylanthranilic acid (DPC) (panel
a), 0.1 mM glibenclamide (Glib), and then
0.5 mM DIDS (panel b), or high
K+-containing medium (panel c) before and during
removal and addition of Cl. Upper deflection
in all traces indicates an increase in pHi. The
results of multiple experiments are summarized in Fig. 2.
View larger version (26K):
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Fig. 2.
Properties of AE activity in T84 cells.
The protocols of Fig. 1 were used to evaluate the effect of forskolin
on AE activity in the presence of normal or high K+ media
and the effect of DIDS on AE activity before and after stimulation with
forskolin. The inset plots the forskolin-stimulated AE
activity under normal and high K+ conditions. The figure
shows the mean ± S.E. of the indicated number of
experiments.
channel (20, 35-37) raised the
possibility that the rate and extent of
HCO3 influx during Cl
removal are underestimated because of the CFTR-dependent
efflux of HCO3 which entered the cells
through the anion exchanger. To test this possibility we measured the
effect of membrane depolarization on
HCO3 fluxes. In Figs. 1c
and 2, T84 cells were depolarized by raising the external
K+ concentration from 5 to 100 mM. This had a
minor effect on pHi. Membrane depolarization
nearly doubled the initial rate of
HCO3 influx observed upon
Cl removal (Figs. 1c and Fig. 2,
first and third bars from
left). Stimulation with forskolin of cells bathed in high
K+ medium increased AE activity by about 1.9-fold (compare
third and fourth bars of Fig. 2), similar to the
stimulation found in the presence of 5 mM external
K+ (first and second bars in Fig. 2).
However, in the presence of high external K+, forskolin
stimulation increased the absolute rate of
HCO3 influx by 0.082 pH/min, which
was approximately 1.6-fold higher than that found in normal
K+ medium (Fig. 2, inset). The simplest
interpretation of these results is that membrane depolarization
increased the steady-state level of intracellular Cl (see
below). If the internal Cl site of the AE was not
saturated with Cl present in the cells under normal
conditions, the increased [Cl]i
caused by membrane depolarization will increase the rate of
Clin/HCO3
out exchange. Another contributing factor can be
reduction in a potential HCO3
permeability under depolarized conditions. An additional implication of
the findings in Fig. 1c is that most of the
HCO3 fluxes induced by changes in
transcellular Cl gradients are caused by the
electroneutral AE activity.
F/F Mice--
A critical
aspect of the regulation of AE activity by CFTR is to determine whether
it occurs in native CFTR-expressing cells. We elected to study the
relationship between the two proteins in the mouse SMG and pancreatic
ducts because of the availability of the F/F mouse strain. The
rat and mouse SMG and pancreatic ducts express a high level of CFTR
protein in the luminal membrane (16) and
Cl/HCO3 exchange
activity in the basolateral and luminal membranes (17, 18). However, a
previous study in the perfused main duct of the mouse SMG suggested
very low, if any,
Cl/HCO3 exchange
activity in either membrane of this duct (30). We reevaluated these
findings by measuring
Cl/HCO3 exchange
activity in the intralobular duct of the mouse SMG. As illustrated in
Fig. 3, a and b, we
found high variability in Cl/HCO3 exchange
activity in isolated SMG ducts. The traces in Fig. 3, a and
b, represent the range of high and low AE activity in SMD ducts from WT mice, respectively. Fig. 3c shows AE activity
in a SMG duct fragment from a F/F mouse. The AE activity of ducts from mutant mice was less variable, as reflected in the averaged results (Fig. 3d). In a significant number of experiments it
was difficult to estimate accurately the initial rate of
HCO3 efflux. Therefore in these
experiments we elected to evaluate AE activity from the extent of
pHi changes caused by Cl removal,
which gave more reproducible results and allowed us to include all of
the experiments performed in the statistical analysis.
View larger version (23K):
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Fig. 3.
AE activity in the SMG duct of WT and
F/F mice. Ducts were
isolated from the SMG of WT (panels a and
b) or F/F (panel c) mice and
used to measure pHi as described under
"Experimental Procedures." All solutions were buffered with
HCO3. Where indicated by
bars, Cl was removed and added to the
incubation medium. Panel d shows the summary of the results
of multiple experiments performed with ducts from 30 WT and 15 F/F mice. The model in the figure shows the possible relationship
between the illustrated transporters to explain the results in
panel d.
increased
pHi by about 0.078 ± 0.006 pH unit in
ducts from WT animals. The same protocol was used to evaluate AE
activity in SMG ducts from F/F mice. Because of the regulation of
AE activity by CFTR demonstrated before (28), we expected to find the
same or lower AE activity in the SMG duct of F/F mice.
Surprisingly, AE activity in ducts isolated from SMG of F/F mice
was significantly higher than in ducts isolated from the SMG of WT mice
(Fig. 3, c and d).
]i in SMG Ducts of WT Mice--
A
potential explanation for the paradoxical findings above is illustrated
in the model in Fig. 3. If CFTR was at least partially active in
unstimulated cells from WT mice, and resting membrane potential was
similar in ducts from WT and F/F mice, the steady-state [Cl]i is expected to be variable
and lower in SMG from WT mice. To test this possibility we measured
[Cl]i in SMG ducts with the aid
of the Cl-sensitive dye MQAE. The results are summarized
in Table I. In 17 ducts from 7 mice
[Cl]i in unstimulated cells
averaged about 24 mM. After a 5-10 min stimulation with
forskolin there was a slight increase in steady-state
[Cl]i by about 1.5 mM, which did not reach statistical significance. On the
other hand, depolarizing the cells with 100 mM external K+ or 5 mM Ba2+ significantly
increased steady-state [Cl]i by
about 5 mM. Also under depolarized conditions forskolin had
no effect on steady-state [Cl]i.
The overall increase in [Cl]i by
membrane depolarization with high K+ and Ba2+
averaged 4.8 ± 0.3 mM. This can account for most (but
not all) of the increase in
Clin/HCO3
out exchange activity under depolarized
conditions shown below. Thus, depolarization increased
pHi by an additional 0.088 ± 0.007 pH
unit. With a buffer capacity at pHi 7.3 and in
the presence of HCO3 of about 72 mM H+ or
HCO3/pH unit (38), this amounts to
6.3 ± 0.5 mM base equivalents. Hence, the increase in
steady-state [Cl]i can account
for about 75% (4.8/6.3) of the increased HCO3 influx. The remaining portion can
be explained by reduced HCO3 leakage
under the depolarized conditions. The possible contribution of a
HCO3 leak pathway is consistent with
our previous reports of a Ba2+-sensitive and
agonist-regulated H+/HCO3
leak pathway in the luminal membrane of the SMG duct (18, 29, 38). Not
only did membrane depolarization increase
[Cl]i, but it also reduced the
variability of [Cl]i in
unstimulated cells (Table I), which facilitated evaluation of
Cl/HCO3 exchange
activity in cells from WT and F/F mice. Hence, in most subsequent
experiments Cl/HCO3
exchange activity was measured under depolarized conditions.
[Cl]i in control and depolarized submandibular duct
cells
-buffered solution B for at least 10 min before
exposure to high K+ medium and/or stimulation with 5 µM forskolin. At the end of each experiment fluorescence
was calibrated to estimate [Cl]i as detailed under
"Experimental Procedures."
/HCO3 exchange is
illustrated in Fig. 4. In SMG ducts from
WT mice, inhibition of K+ conductance is expected to
depolarize the membrane and reduce Cl efflux, increasing
and stabilizing steady-state
[Cl]i. As a consequence, the
maximal Cl/HCO3 exchange
activity of these cells could be measured. In SMG ducts from F/F
mice membrane depolarization is expected to have smaller, if any,
effect on the Cl-dependent changes on
pHi. Importantly, membrane depolarization minimizes the contribution of all Cl channels expressed
in these cells (39) to allow a better comparison between the
electroneutral Cl/HCO3
exchange activity of ducts from WT and F/F mice. The traces of an
individual experiment (Fig. 4, a and b) and the
averages obtained from multiple experiments (Fig. 4c) show
that this was indeed the case.
View larger version (23K):
[in a new window]
Fig. 4.
Effect of membrane depolarization on AE
activity in SMG ducts from WT and
F/F mice. As
indicated by the bars, ducts from WT (panel
a) or F/F (panel b) mice were
exposed to Cl-free medium in the absence or presence of 5 mM Ba2+. All solutions were buffered with 5 mM Hepes and 25 mM
CO2/HCO3. Panel
c shows the mean ± S.E. of the number of experiments
performed under each condition.
-buffered solutions in which
Cl was replaced by gluconate. These solutions were clear
for about 30 min, after which precipitates, probably of Ba-gluconate,
started to form. At this time old solutions were replaced with fresh
Ba2+-containing, Cl-free solutions. Exposing
SMG ducts bathed in a HCO3-buffered
medium to 5 mM Ba2+ caused a small and
reproducible reduction in pHi, the cause of
which was not investigated in the present work. Membrane depolarization
with Ba2+ increased the apparent AE activity by more than
2-fold in SMG ducts from WT mice. By contrast, Ba2+ had a
small, statistically insignificant effect on AE activity of SMG ducts
from F/F mice. Furthermore, in the presence of Ba2+,
the AE activity of SMG duct from WT mice tended to be higher than that
of ducts from F/F mice, although it did not reach statistical
significance (p = 0.083). Finally, the
Cl-dependent changes in
pHi were blocked completely by DIDS (Fig. 4,
a and b).
F/F
mice is shown in Fig. 5. Fold stimulation was determined from the extent of pHi changes
caused by Cl removal and addition before and after
forskolin stimulation of the same duct fragments. Even in the absence
of Ba2+, stimulation of SMG duct from WT mice with
forskolin increased AE activity by about 1.7-fold. By contrast, and as
expected, forskolin had no effect on the AE activity of ducts isolated
from the SMG of F/F mice. We noticed that multiple removals and
additions of Cl to the incubation medium resulted in a
slightly reduced AE activity in each successive round. Thus, the small,
frequently observed reduction in AE activity in forskolin-stimulated
ducts from F/F mice is probably the result of this artifact.
Because in most experiments a control test preceded an experimental
test, the effect of forskolin on AE activity in SMG duct from WT mice
is probably underestimated. Nevertheless, it was sufficiently large to
be highly statistically significant (Fig. 5c).
View larger version (23K):
[in a new window]
Fig. 5.
Effect of forskolin on AE activity in SMG
duct from WT and F/F
mice. The AE activity of SMG ducts from WT (panel
a) or F/F mice (panel b) was
measured before and after stimulation with 5 µM
forskolin. Panel c shows the mean ± S.E. of all
experiments performed. Note that forskolin stimulated AE activity of
SMG ducts from WT but not F/F mice.
F/F
mice, incubated in Ba2+-containing solutions increased AE
activity. In SMG ducts from WT mice, forskolin increased
pHi caused by Cl removal by 0.06 pH unit above that measured in the same unstimulated ducts, which
consists of an approximately 1.3-fold stimulation. However, when
pHi changes caused by AE activity are compared in forskolin-stimulated ducts from WT and F/F mice,
pHi changes in WT ducts are higher by about 0.12 pH unit, which is 1.9-fold above that measured in ducts from F/F
mice. This stimulation is comparable to that found in the absence of
Ba2+ as illustrated in Fig. 5.
View larger version (25K):
[in a new window]
Fig. 6.
Stimulation of AE activity by forskolin in
the presence of Ba2+. SMG ducts from WT
(panel a) or F/F (panel
b) mice were incubated in
HCO3-buffered solutions containing 5 mM Ba2+ throughout the experiment. As indicated
by the bars, the ducts were exposed to Cl-free
solutions before and after stimulation with 5 µM
forskolin. Panel c shows the summary of all experiments
performed in terms of mean ± S.E. Note that forskolin-stimulated
ducts from WT mice showed higher AE activity than nonstimulated ducts
from WT mice and forskolin-stimulated ducts from F/F mice.
-dependent pHi
changes were inhibited by DIDS (Fig. 7a). Stimulation of the
ducts with forskolin increased the AE activity of these ducts. In these
experiments we compared the AE activity measured in the first exposure
of all ducts to Cl-free medium (Fig. 7, b and
c). The forskolin-stimulated increases in
pHi in the presence of 100 mM
K+ or 5 mM Ba2+ were similar.
View larger version (25K):
[in a new window]
Fig. 7.
Effect of high K+ on
pHi changes in SMG duct from WT mice. SMG
ducts from WT mice were incubated in
HCO3 -buffered solutions. As indicated
by the bars, the ducts were exposed to Cl-free
solutions in the presence of 5 or 100 mM K+
before (panels a and b) or after
stimulation with 5 µM forskolin (panel
c). Panel d illustrates the mean ± S.E. of
the indicated number of experiments.
/HCO3 Exchange
by CFTR in Pancreatic Ducts--
CFTR-expressing cells are likely to
express more than one anion exchanger, the housekeeping AE2 in the
basolateral membrane and as yet unidentified AE isoform or other
exchanger protein involved in transcellular
HCO3 transport in the
luminal membrane. Indeed, AE activity was measured previously in both
membranes of the SMG (18) and pancreatic ducts (17). To extend our
findings to another native CFTR-expressing tissue and determine the
membrane localization of the AE activity regulated by CFTR, we measured
luminal and basolateral AE activity in the microperfused pancreatic
ducts of WT and F/F mice. Fig. 8
summarizes the results of multiple experiments. It was satisfying to
find that stimulation with forskolin exclusively increased the activity
of the luminal AE without affecting the basolateral AE in pancreatic
ducts from WT mice. Furthermore, such regulation was absent in ducts
from F/F animals. In the first protocol we measured AE activity
under polarized conditions (Fig. 8a). As we reported before
for the rat duct, luminal AE activity was higher than basolateral AE
activity in the mouse pancreatic duct (compare the rate and extent of
pHi changes caused by the Cl
removal from the bath and lumen in panels a and b
before forskolin stimulation). Stimulation with forskolin caused the
typical initial acidification, had no effect on basolateral AE
activity, and prominently increased luminal AE activity in ducts from
WT mice. Forskolin had no effect on luminal or basolateral AE activity
of ducts from F/F mice (data not shown).
View larger version (36K):
[in a new window]
Fig. 8.
CFTR stimulates the luminal but not the
basolateral AE activity in the pancreatic ducts. The main
pancreatic ducts of WT (panels a and
b) and F/F (panel c) mice were
cannulated, dissected, and perfused through the luminal and basolateral
sides with the HCO3-buffered solution
B. Where indicated by the bars, the luminal and basolateral
AE activities were measured by perfusing the bath and the lumen with a
HCO3-buffered, Cl-free
solution C (panel a). In panels b and
c the ducts were perfused with the high K+,
depolarizing solution D before exposure to high K+,
Cl-free solution E. In all experiments, as indicated by
the bars, the ducts were stimulated with 5 µM
forskolin. Forskolin was included in the luminal solution. Panel
d summarizes the results of multiple experiments with ducts from
WT and F/F mice.
removal and addition to the lumen (and thus the higher
AE activity) of pancreatic duct from WT mice. The right part
of the trace shows that stimulation with forskolin had no effect on the
basolateral AE activity but increased the initial rate (and extent) of
pHi changes because of removal of luminal
[Cl]out in the same duct. Both activities
were inhibited completely by 0.5 mM DIDS. By contrast,
forskolin had no effect on basolateral and luminal AE activity of ducts
from F/F mice (Fig. 8c). Hence, we can conclude that
CFTR regulates the luminal but not the basolateral AE activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F/F mice to study regulation of
Cl/HCO3 exchange
activity by CFTR. Along with our experience with these cells, the SMG
and pancreatic ducts offer other advantages as model systems. Both
ducts express high levels of CFTR (16) and basolateral and luminal AE
activity (17, 18). In addition, the SMG offers the availability of
several experimental preparations from microperfused ducts (2, 18) to
isolated single cells (2, 16); and most importantly, among all
CFTR-expressing tissues, the mechanisms of fluid and electrolyte
transport are understood best in the SMG (2).
-dependent
HCO3 influx and
Clout-dependent
HCO3 efflux. By contrast, a work with
the main SMG duct concluded low or no AE activity in the mouse duct
(30). This discrepancy may be the result of sufficiently high
Cl and/or HCO3
conductance in the previous study (30) which masked the AE activity.
conductance in native
CFTR-expressing cells and its possible regulation by CFTR. In a recent
work, Ishguro et al. (6) reported the intriguing finding
that luminal AE mediated HCO3
transport in resting but not cAMP-stimulated guinea pig pancreatic ducts. In addition, CFTR-dependent
HCO3 conductance was reported in
pancreatic duct cells (35) and in an airway epithelial cell line
expressing CFTR (20). This would imply that stimulation of CFTR should
dramatically increase the HCO3
conductance of the luminal membrane. In the present work we were unable
to obtain evidence in support of such a conclusion. Our results in the
mouse are in agreement with the finding that
HCO3 conductance is at least 6-fold
lower than that of Cl (20, 35) and the absence of
HCO3 conductance in sweat duct (40).
Hence, in the presence of physiological Cl and
HCO3 gradients it is not likely that
CFTR mediates or modulates the HCO3
conductance in the luminal membrane of the rat and mouse secretory epithelia. Accordingly, the electrogenic component of
HCO3 transport in T84 cells (Fig. 1)
and SMG ducts (Figs. 4 and 7) was rather small and not affected by
inhibitors of CFTR Cl channel activity (Fig. 1 and not
shown). Furthermore, stable and transient transfection of CFTR in NIH
3T3 and HEK 293 cells, respectively, did not increase
HCO3 conductance even when the cells
were stimulated with forskolin (compare the effect of high
K+ in Fig. 10 of Ref. 28 and Figs. 1c and 7 of
this paper).
conductance in the
mouse SMG and pancreatic duct cannot be extended to other species and
tissues. This is because, most likely, different mechanisms mediate
HCO3 transport in different tissues
and species. For example, the electrogenic component of
HCO3 transport is much higher in the
duodenum than in the colon (3) or the SMG (2). The pancreatic juice of
the guinea pig contains a much higher
HCO3 concentration than that of the
rat (1, 6). However, despite this variability we can safely conclude
that in tissues secreting HCO3 in a
mechanism similar to that of the mouse SMG or the pancreatic ducts
(a) HCO3 conductance is of
secondary importance in HCO3
secretion, and (b) regulation of luminal AE activity by CFTR may be the major mechanism by which CFTR regulates
HCO3 secretion. In this respect it
would be of particular significance to test the effect of CFTR
stimulation on luminal AE activity and
HCO3 conductance in guinea pig SMG and
pancreatic ducts.
(1-3, 7, 9, 12, 25-27).
HCO3 is a chaotropic anion that is
commonly used to dissolve, and thus, strip membranes of peripheral
proteins. Moreover, HCO3 regulates the
pH of biological fluids. Fluids secreted by CFTR-expressing cells are
rich in proteins, in particular mucins (1-3, 7, 25-27). Mucin
solubility may be increased in high
HCO3, high pH fluids (42). In the
special case of the exocrine pancreas, the high pH is also needed to
prevent premature activation of harmful digestive enzymes (1). Impaired
HCO3 secretion may cause precipitation
of mucins and thus play a major role in obstruction of almost all
ductal systems in cystic fibrosis (43). Every model of luminal
HCO3 secretion in CFTR-expressing
cells suggests that AE activity mediates the electroneutral portion of
HCO3 secretion (1-3, 7). By
demonstrating regulation of AE activity by an activated CFTR, our
findings indicate that CFTR directly regulates the entire process of
HCO3 secretion. Furthermore, the
suggested regulation of luminal Na+ channel by CFTR (44)
allows the regulation of Na+ absorption by CFTR. Several
CFTR-expressing cells, such as the SMG duct, while absorbing
Na+ secrete isotonic K+ (2). The luminal
pathway responsible for K+ efflux is believed to be an
inward rectifier K+ channel such as ROMK II in the kidney
(3). CFTR has also been implicated as a regulator of ROMK II (45).
Hence, CFTR must be viewed as a global regulator of epithelial fluid
and electrolyte transport through its ability to function as a
Cl channel (46) and regulate Cl absorption,
regulate the epithelial Na+ channel to regulate
Na+ absorption, regulate the luminal K+ channel
to regulate K+ secretion, and regulate AE activity to
regulate HCO3 secretion.
channels
through stimulation of selective P2 purinoceptors. Again, the finding that cholinergic stimulation of intestinal
HCO3 secretion is abolished in the
intestines of CFTR / mice (24) supports the global role of CFTR in
epithelial fluid and electrolyte secretion. Cholinergic stimulation is
expected to activate the luminal Ca2+-activated
Cl channel found in many epithelial cells (47).
Activation of this Cl channel was not sufficient to cause
normal HCO3 secretion in
cholinergically stimulated intestinal epithelium (24). Hence, although
stimulation of luminal purinoceptors may induce Cl
secretion, it is not likely to be sufficient in itself in alleviating the symptoms of cystic fibrosis. Thus, improved expression of CFTR in
the luminal membrane seems to be the best option in this endeavor.
ACKNOWLEDGEMENTS
F/F mice.
FOOTNOTES
Established investigator of the American Heart Association.
ABBREVIATIONS
/HCO3 (anion)
exchanger;
SMG, submandibular gland;
WT, wild type;
F, deletion
mutant of Phe-508 from WT CFTR;
PSA, pancreatic solution A;
BCECF-AM, 2'7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein acetoxymethyl ester;
MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium
bromide;
DIDS, 4,4'-diisothiocyanato-
stilbene-2,2'-disulfonate.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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T. Greeley, H. Shumaker, Z. Wang, C. W. Schweinfest, and M. Soleimani Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells Am J Physiol Gastrointest Liver Physiol, November 1, 2001; 281(5): G1301 - G1308. [Abstract] [Full Text] [PDF]
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H. Barriere, C. Poujeol, M. Tauc, J. M. Blasi, L. Counillon, and P. Poujeol CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts Am J Physiol Cell Physiol, September 1, 2001; 281(3): C810 - C824. [Abstract] [Full Text] [PDF]
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G. Chandy, M. Grabe, H.-P. H. Moore, and T. E. Machen Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells Am J Physiol Cell Physiol, September 1, 2001; 281(3): C908 - C921. [Abstract] [Full Text] [PDF]
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V. J. Wheat, H. Shumaker, C. Burnham, G. E. Shull, J. R. Yankaskas, and M. Soleimani CFTR induces the expression of DRA along with Cl-/HCO3- exchange activity in tracheal epithelial cells Am J Physiol Cell Physiol, July 1, 2000; 279(1): C62 - C71. [Abstract] [Full Text] [PDF]
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H.-V. Nguyen, G. E Shull, and J. E Melvin Muscarinic receptor-induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na+-H+ exchanger-1 deficient mice J. Physiol., February 15, 2000; 523(1): 139 - 146. [Abstract] [Full Text] [PDF]
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M. H. Akabas Cystic Fibrosis Transmembrane Conductance Regulator. STRUCTURE AND FUNCTION OF AN EPITHELIAL CHLORIDE CHANNEL J. Biol. Chem., February 11, 2000; 275(6): 3729 - 3732. [Full Text] [PDF]
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 J. H. Widdicombe Yet Another Role for the Cystic Fibrosis Transmembrane Conductance Regulator Am. J. Respir. Cell Mol. Biol., January 1, 2000; 22(1): 11 - 14. [Full Text]
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R. L. Evans, K. Park, R. J. Turner, G. E. Watson, H.-V. Nguyen, M. R. Dennett, A. R. Hand, M. Flagella, G. E. Shull, and J. E. Melvin Severe Impairment of Salivation in Na+/K+/2Cl- Cotransporter (NKCC1)-deficient Mice J. Biol. Chem., August 25, 2000; 275(35): 26720 - 26726. [Abstract] [Full Text] [PDF]
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X. Luo, J. Y. Choi, S. B. H. Ko, A. Pushkin, I. Kurtz, W. Ahn, M. G. Lee, and S. Muallem HCO3- Salvage Mechanisms in the Submandibular Gland Acinar and Duct Cells J. Biol. Chem., March 23, 2001; 276(13): 9808 - 9816. [Abstract] [Full Text] [PDF]
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W. Ahn, K. H. Kim, J. A. Lee, J. Y. Kim, J. Y. Choi, O. W. Moe, S. L. Milgram, S. Muallem, and M. G. Lee Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO3- Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct J. Biol. Chem., May 11, 2001; 276(20): 17236 - 17243. [Abstract] [Full Text] [PDF]
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