NSP4 elicits age-dependent diarrhea and
Ca2+mediated
I
influx into intestinal
crypts of CF mice
Andrew P.
Morris1,
Jason K.
Scott1,
Judith M.
Ball2,
Carl Q.-Y.
Zeng2,
Wanda K.
O'Neal3, and
Mary K.
Estes2
1 Departments of Integrative
Biology and Internal Medicine-Gastroenterology, University of Texas
at Houston Health Science Center, and
2 Divisions of Molecular
Virology and Medicine-Gastroenterology and
3 Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, Texas
77030
 |
ABSTRACT |
Homologous disruption of the murine gene
encoding the cystic fibrosis (CF) transmembrane conductance regulator
(CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying
this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of
mortality. We investigated whether infectious mammalian rotavirus, the
recently characterized rotaviral enterotoxin protein NSP4, or its
active NSP4114-135 peptide,
can overcome these gastrointestinal complications in CF
(CFTRm3Bay null mutation) mice.
All three agents elicited diarrhea when administered to wild-type
(CFTR+/+), heterozygous
(CFTR+/
), or homozygous
(CFTR
/
) 7- to
14-day-old mouse pups but were ineffective when given to older mice.
The diarrheal response was accompanied by non-age-dependent intracellular Ca2+ mobilization
within both small and large intestinal crypt epithelia. Significantly,
NSP4 elicited cellular I
influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed
to evoke influx in the
CFTR
/
background.
This unique plasma membrane halide permeability pathway was age
dependent, being observed only in mouse pup crypts, and was abolished
by either the removal of bath Ca2+
or the transport inhibitor DIDS. These findings indicate that NSP4 or
its active peptide may induce diarrhea in neonatal mice through the
activation of an age- and
Ca2+-dependent plasma membrane
anion permeability distinct from CFTR. Furthermore, these results
highlight the potential for developing synthetic analogs of
NSP4114-135 to counteract
chronic constipation/obstructive bowel syndrome in CF patients.
cystic fibrosis transmembrane conductance regulator; halide
permeability; NSP4 enterotoxin; rotavirus
 |
INTRODUCTION |
THE MOST COMMONLY REPORTED gastrointestinal
manifestation in cystic fibrosis (CF) is malabsorption due to
pancreatic insufficiency, caused by occlusion of the pancreatic ducts
by abnormally viscous mucus secretions. This alteration in intraluminal
fluid and mucus content extends to other solid organs and hollow
viscera of the gut, creating the clinical conditions of meconium ileus
at birth and distal intestinal obstruction syndrome, chronic
constipation with acquired megacolon, and rectal prolapse in older
individuals (1).
Underlying these gastrointestinal complications is the basic defect in
CF, which is caused by mutations in the gene that encodes the CF
transmembrane conductance regulator (CFTR; Ref. 43). The most commonly
reported mutation in CF, accounting for the genetic defect in
60-70% of the chromosomes of CF individuals, is a three-base-pair
deletion that removes phenylalanine at position 508 (
F508) of the CFTR protein (54). CF patients homozygous for this mutation exhibit the severe clinical phenotype of lung disease, pancreatic insufficiency, and predisposition to
gastrointestinal obstruction (48). Electrophysiological analysis of the
gut mucosa isolated from these individuals demonstrates a lack of
cAMP-dependent fluid secretion (6, 37, 53). Reduced fluid transport,
which is the primary cellular defect of CF epithelia, is believed to significantly enhance the pathology of mucus plugging in the gut.
Over the past six years, mouse models for CF that duplicate the
pathophysiological phenotype of CF in the human intestine have been
produced with the use of gene-targeting techniques. Transgenic
CFTR-deficient animals fail to exhibit CFTR-mediated fluid secretion
and present with gastrointestinal disease (15). In this study, we
utilized the CFTRm3Bay null
mutation mouse as a model for CF to investigate whether infectious
rotavirus or the novel rotaviral enterotoxin NSP4 (3) can cause
diarrhea. These mice fail to express functional CFTR protein because of
multiple stop codons engineered within exon 3 of the murine CFTR genome
(23).
Rotaviruses are the leading cause of severe gastroenteritis in infants
and young animals (25). We have recently shown that NSP4,
a rotavirus nonstructural protein (16), can cause diarrhea in young
mice (3). Furthermore, electrophysiological analyses of intact
intestinal mucosa from mice revealed that NSP4 mobilizes Ca2+ to mimic the secretory
effects of the cholinergic agonist carbachol (CCh) in potentiating
cAMP-dependent fluid secretion (3). We therefore asked whether NSP4
could evoke a diarrheal response in CFTR-depleted mice that exhibit no
functional cAMP-dependent secretory pathway. Here, we demonstrate that
NSP4 injected intraperitoneally into CFTR-deficient
(CFTR
/
) mouse pups
induces an age-dependent diarrhea. Hence, CFTR-mediated changes in
intestinal fluid transport were not directly involved in NSP4-elicited
diarrhea. However, NSP4 was found to mobilize Ca2+ in the crypt epithelia of
both small and large intestine from wild-type
(CFTR+/+) as well as
CFTR
/
pup and adult
mice. These ex vivo data correlated with our in vitro findings in human
gastrointestinal cell lines, in which NSP4 was shown to mobilize
intracellular Ca2+ concentration
([Ca2+]i)
via phospholipase C (PLC) activation and inositol 1,4,5-trisphosphate (IP3) production (13). To
investigate in greater detail the relationship between NSP4-induced
epithelial cell Ca2+ mobilization
and our previously reported age-dependent effects of NSP4 on murine
intestinal mucosa Cl
secretory current generation (3), we measured plasma membrane halide
permeability changes ex vivo within the epithelial cells of isolated
distal colon crypts. We found that NSP4 elicited age-dependent I
influx into mouse pup
crypts, inhibited by either the removal of bath
Ca2+ or the transport inhibitor
DIDS. The fact that this
Ca2+-dependent plasma membrane
halide permeability pathway was not CFTR may provide a possible
explanation as to why
CFTR
/
mouse pups
exposed to NSP4 develop age-dependent diarrhea.
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MATERIALS AND METHODS |
Reagents.
Fura 2-AM and
6-methyoxy-N-(3-sulfopropyl)
quinolinum (SPQ) were purchased from Molecular Probes (Eugene,
OR). All other reagents, including DIDS, were purchased from Sigma (St.
Louis, MO). NSP4 protein was fast-performance liquid
chromatography- and affinity-purified from Sf9
insect cells infected with a recombinant baculovirus pAC461-G10
expressing simian rotavirus gene 10 (encoding NSP4), as described
previously (13). The
NSP4114-135 peptide was
synthesized at the University of Pittsburgh protein core laboratory and
characterized as previously described (3).
Experimentally induced rotaviral infection or administration of
NSP4 protein and NSP4114-135 peptide to
mouse pups.
The simian rotavirus strain SA11 clone 3 (18) was used to infect
neonatal CD-1 or
CFTR
/
(23) mouse
pups between the ages of 6 and 8 days. The genotype of the
CFTR
/
mice was
determined by polyacrylamide gel analysis of PCR products generated
from DNA extracted from the tails of 1- to 2-day-old mice with the use
of primers, as described previously (23). Either 10 or 20 diarrheal
dose 50 of SA11 was administered in 50 µl of medium 199 by intragastric gavage. Alternatively, purified NSP4 protein or
NSP4114-135 was administered
to C57B
CFTR
/
/CFTR+/+
or CD-1 CFTR+/+ mouse pups in a
final volume of 50 µl of PBS by intraperitoneal injection. The doses
used were 0.5 nmol of NSP4 protein or 100 nmol of
NSP4114-135 peptide because
these doses were shown to be effective at inducing diarrhea in CD-1
CFTR+/+ mouse pups (3).
Diarrheal activity measurements.
To determine the presence of diarrhea following protein or peptide
treatment, each mouse pup was examined every 1-2 h for the first 8 h and at 24 h after inoculation by gently pressing the abdomen. After
virus inoculation, mouse pups were monitored twice a day for 4 days.
Diarrhea was noted and scored from 1 to 4, with a score of 1 reflecting
loose yellow stool and a score of 4 indicating completely liquid stool.
A score of 2 (mucous with liquid stool, some loose but solid stool) and
above was considered diarrhea. The scoring was performed on coded
animals by a single person.
Crypt isolation and dye loading.
Epithelial crypts from
CFTR
/
mice (aged
8-12 days) were isolated from 3-cm segments of the mid to distal
small intestine, 6 cm proximal to Bauhin's valve, and from the distal
colon. After euthanasia by an overdose of ether and cervical
dislocation, the entire small intestine and colon from the mouse were
removed and flushed with ice-cold physiological saline. Individual
intestinal segments were then mounted onto Perspex paddles and were
immersed in Ca2+-free standard
Krebs buffered saline (in mmol/l: 107 NaCl, 4.5 KCl, 0.2 NaH2PO4,
1.8 Na2HPO4,
10 glucose, and 10 EDTA), and continuously gassed with 5%
CO2-95%
O2 at 37°C for 10-20 min.
The crypts were then separated from the overlying mucosa by mechanical
vibration for 30 s into ice-cold KCl HEPES saline (in mmol/l: 100 potassium gluconate, 20 NaCl, 1.25 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 5 sodium pyruvate, and 0.1% BSA, pH 7.4), resembling the intracellular medium. Suspended crypts were then deposited (1,200 rpm for 1 min) onto
poly-L-lysine-coated microscope
coverslips (0 oz) with the use of a Shandon Cytopsin cell preparation
system (13). The coverslips with crypts were then attached with vacuum
grease to the base of customized perfusion wells and loaded in the dark with either Ca2+-sensing or
Cl
-sensing dye.
Fura 2-AM loading.
Isolated crypts were incubated with 10 µM fura 2-AM at room
temperature for 15-20 min. The coverslips were then mounted on an
inverted Nikon microscope and superfused with standard HEPES-buffered extracellular saline (in mmol/l: 140 NaCl, 4.7 KCl, 1.13 MgCl2, 10 HEPES, 10 glucose, and 1 CaCl2, pH 7.4) for 5 min before imaging.
SPQ loading.
Isolated crypts were loaded with SPQ at room temperature by a 6-min
exposure to hyposmotic (80 mosM
Cl
or
NO
3) HEPES-buffered extracellular
saline containing 5 mM SPQ. After loading, the crypts were allowed to recover in isosmotic (140 mosM
Cl
or
NO
3) HEPES-buffered extracellular
saline for 10-15 min. This period was used when performing anion
channel inhibitor studies to preload the crypts so that the reported
spectral interference of 0.5 mM DIDS with SPQ fluorescence was
minimized (61). DIDS binds irreversibly to cellular membranes;
therefore, crypts were loaded and the bathing saline was changed before
experimentation. The solution temperature was maintained at 37°C
throughout these studies by prewarming the extracellular solutions and
by water-jacketing the oil-immersion lens of the inverted microscope.
NSP4/NSP4114-135 addition to
isolated crypts.
Small (100 µl) volumes of either the NSP4 protein or
NSP4114-135 peptide were
superfused onto the isolated crypts during regular bath flow by
N2 pressure injection with the use
of a Picospritzer. Low-resistance glass pipettes were filled with
either compound dissolved in HEPES-buffered extracellular saline, and
the tip was maneuvered close to the isolated crypt. Peptide or protein was released directly into the vicinity of the crypt for 40 s before
being washed away by the bath flow.
[Ca2+]i
imaging.
All experiments were carried out with the aid of a high-resolution
camera imaging system as described previously (13). In brief, light
emitted from fura 2-loaded cells at 510 nm was captured by an
intensified video camera after exposure to both 340- and 380-nm
excitation light. The camera signal was then digitally encoded and
processed with image analysis software (IMAGE1/FL, Universal Imaging,
Media, PA). The background-subtracted images were ratioed on a
pixel-by-pixel basis to yield a bitmap field. Calibration of the fura 2 dye fluorescence was carried out with the use of the ionophore
ionomycin under Ca2+-free and
Ca2+-saturating conditions as
described previously, and the
[Ca2+]i
was calculated according to the Grynkiewicz equation (21). The
[Ca2+]i
values of individual field pixels obtained by this procedure were color
coded and displayed on an RGB monitor before being stored on the hard
disk. Six collection areas were chosen along the longitudinal axis
(base to neck) of the crypt for spatial and time-dependent analysis of
[Ca2+]i.
The averaged ratio signal obtained from each of the six areas was
digitally saved as a log file. The collected values from the six areas
imaged within a single experiment were averaged together to give an
experimental observation of one (n = 1). Values obtained from similarly placed collection areas along the
longitudinal axis of different crypts were also averaged to see if any
spatial localization to the Ca2+
signal existed. All protocols were completed within 1 h after crypt isolation.
Intracellular I
imaging.
The same high-resolution camera imaging system described above was
utilized for these studies. Excitation for SPQ fluorescence was
provided by a barrier filter centered at 365 ± 10 nm, reflected into the microscope objective by a 400-nm dichroic mirror. A neutral density filter (1.5 OD) was included in the light path to minimize photobleaching. The fluorescence emission from the SPQ-loaded cells
passed through a 450 ± 25-nm barrier filter before being detected
by the intensifier/camera. Fluorescent images were acquired at 0.8-s
intervals and averaged over eight frames to yield a 540 × 480 field of mean pixel intensities. Six collection areas were then chosen
within the longitudinal axis (base to midregions) of the crypt for
spatial and time-dependent analysis of cellular SPQ fluorescence. SPQ
does not exhibit excitation or emission shifts on anion interaction.
Thus ratiometric imaging is not possible, and dye fluorescence is
affected by volume changes within the cell (58). Both cAMP- and
Ca2+-mobilizing agonists have been
reported to induce shrinkage without compensatory volume recovery in
the lower to midregion of rat distal colonic crypts over a 5- to 30-min
time course (11). To determine the extent of this effect over the time
period of SPQ quenching (1-3 min), colonic crypts isolated from
the distal colon of the mouse were loaded with the AM form of calcein,
a dye inert to Ca2+ and pH changes
in cells within the physiological range (8, 9). Cell volume measured
over the first 30 s of agonist addition to the bath did not change
appreciably (<2%) within the lower/midcrypt region (data not shown).
These regions were utilized for all studies. Furthermore, to minimize
possible volume effects on dye fluorescence, we chose an experimental
design in which SPQ fluorescence quench by halide influx was employed
rather than dequenching following halide efflux. This ensured that
agonist-induced cell shrinkage, if present, would act to decrease
rather than to potentiate changes in plasma membrane halide
permeability. Thus our reported values of SPQ quench may modestly
underestimate agonist-evoked halide influx. The collected values from
the six areas imaged within a single experiment were averaged together
to give an experimental observation of one
(n = 1).
Calculation of Stern-Volmer constants for halide quenching of SPQ.
SPQ fluorescence is quenched collisionally by halide anions with
different potency. Nonphysiological anions such as thiocyanate (SCN
),
Br
,
F
, and
I
quench strongly in free
solution with an efficacy greater than Cl
(Cl
< Br
< SCN
< I
), whereas
NO
3 does not quench at all (27). The
Stern-Volmer equation that describes this interaction is
Fo/Fanion = 1 + Kq[anion],
where Fo is the fluorescence in
the absence of anion, Fanion is
the fluorescence in the presence of a given anion concentration,
Kq (slope) is the
Stern-Volmer quenching constant (in
M
1), and
[anion] is the
concentration of halide utilized (27). Stern-Volmer quenching constants
for Cl
,
Br
, and
I
anions were calculated
from linear curves obtained over a concentration range of 0-150 mM
halide (see RESULTS). The
near-linear Stern-Volmer quench curve for
I
(Fig.
1) and the fact that plasma membrane
I
permeability changes
occur largely through conductive pathways (see DISCUSSION
and Refs. 36 and 63) led us to choose this anion to quantify agonist
effects in isolated crypts.

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Fig. 1.
Stern-Volmer plot for quenching of
6-methoxy-N-(3-sulfopropyl)quinolinum
(SPQ) fluorescence by Cl ,
Br , and
I . Isolated crypts from
distal colon of wild-type cystic fibrosis transmembrane conductance
regulator (CFTR+/+) and
heterogeneous (CFTR+/ )
mice were loaded with SPQ. Intracellular SPQ quench by different
intracellular halide concentrations was induced with dual ionophore
technique. Fo and F represent
background and dye leak subtracted values of fluorescence in absence
and in presence of quenching halide, respectively. Values are means ± SD.
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To calibrate SPQ fluorescence in cells, the double ionophore technique,
with the use of high-K+ solutions
containing nigericin (5 µM) and tributyltin (10 µM), was employed
(7). In these experiments, isolated crypts were loaded with SPQ and
hyposmotic NO
3 (80 mM
KNO3) potassium saline and then
were transferred to isosmotic NO
3 (140 mM KNO3) potassium saline
containing the above ionophores. Fo/Fanion
values were obtained by perfusing the crypts with varying concentrations of test halide
(Cl
,
Br
, or
I
) plus ionophore
followed by SCN
in the
presence of valinomycin (5 µM) to completely quench the intracellular
SPQ signal and obtain basal fluorescence values. Dye leakage was
~17% of the total cellular signal over 60 min (0.0125 ± 0.008 fluorescent units/s, n = 60).
Fo and
Fanion values were corrected for
this signal on a per-experiment basis by interpolation from the
fluorescence intensity, measured in cells bathed in
Cl
-free medium at the onset
of the experiment and at the end of the experiment or by back
extrapolation from the rate of change measured during the
SCN
fluorescence minimal
quench signal (i.e., Fo
Fex and
Fanion
Fex, where
Fex = background fluorescence at
the time of measurement). Both methods gave comparable values. All
experimental protocols were completed within 15-20 min. The
Stern-Volmer quench constants for halide quenching of SPQ in pup and
adult crypts (see RESULTS) were
utilized to calculate the halide influx rates in the standard experimental procedure according to the following equation:
JI
=(Fo/Kq · F2) · (
F/
t),
in which
F/
t is the rate of
fluorescence change measured at time
0. In addition to this method, running averages of
halide influx at 1.6-s intervals during the first 40 s of the fluorescence quench were calculated.
Standard experimental procedure for measuring agonist-stimulated
I
influx.
SPQ-loaded crypts were perfused with HEPES-buffered
NaNO3 containing extracellular
saline, and, after establishment of a stable fluorescence signal, bath
NO
3 was replaced with I
(140 mosM). The quenched
SPQ fluorescent signal (NO
3 does not
quench SPQ nor compete with
I
) was recorded over
predefined measurement windows along the base to midcrypt axis. Basal
rates of I
influx
(JI
)
were then calculated with the use of the Stern-Volmer quench constants
for I
obtained for either
mouse pup or adult crypts (see Calculation of Stern-Volmer
constants for halide quenching of SPQ and
RESULTS). Cl
was then reintroduced,
and I
was simultaneously
removed from the bath. A single agonist was administered during this
recovery phase (forskolin, FSK) or after establishment of a new stable
fluorescence signal (CCh or NSP4) when bath
I
was reintroduced in
Cl
-free saline. The
resulting agonist-stimulated SPQ quench curve was recorded, and
JI
agonist
was calculated. Agonist-induced changes in
I
influx rate were then
calculated from pooled values of
JI
agonist and
JI
basal
as
JI
= (JI
agonist
JI
basal). In addition to this approach, mean ± SD rates of fluorescence change at 1.6-s intervals over the first 40 s of fluorescence quench
were graphically displayed in histogram format as change in
agonist-induced quench rate,
RI
= (
F/
tagonist
F/
tbasal).
This later approach allowed us to determine latency and to record
changes in dye quench unrelated to initial (time
0) changes in quench rate. All SPQ-loaded crypts were
superfused with saline at the same bath perfusion rate (4 ml/min);
solution exchange in the experimental chamber (200 µl) occurred
within 3 s.
Statistical analysis.
To determine statistical significance of differences between
observations within an experiment, the paired Student's
t-test was used. For statistical
differences between experiments, the unpaired Student's
t-test was used. Between 6 and 12 separate experimental observations pooled from different dye loadings
were routinely collected for each experimental condition. The Fisher's exact test was used to estimate the probability of difference for
diarrheal activity measurements within CF and non-CF groups in which
the tabulated frequency of occurrence was too small for
2 analysis.
 |
RESULTS |
Effect of orally administered rotavirus and injected viral NSP4
peptide(s) in mouse pups.
Inoculation of CFTR
/
mice with infectious SA11cl3 rotavirus caused diarrhea in four of five
mice within 24-48 h (Table 1). When
CFTR+/+ or
CFTR+/
mouse pups from the
same C57B1/J6 background were infected with the SA11 virus, ~78% of
the mice exhibited diarrhea (Table 1). Intraperitoneal injection of
intact NSP4 protein or
NSP4114-135 was similarly
found to elicit diarrhea in
CFTR+/+ or
CFTR+/
mouse pups (66 and
70% responding, respectively).
CFTR
/
mouse pups
inoculated with these reagents exhibited diarrhea in 50% and 43% of
these mice, respectively (Table 1). Diarrheal content was graded as
equally severe as that encountered in
CFTR+/+ mice (3). Although the
incidence of diarrhea caused by NSP4 protein or
NSP4114-135 appeared lower in
the CF mouse studies, there was no significant difference between
CFTR+/+,
CFTR+/
, and
CFTR
/
littermates
(Fisher's exact test for 2 × 2 contingency tables; Ref.
32). All three reagents (SA11 virus,
NSP4 protein, or
NSP4114-135) were effective
diarrheal agents. In contrast, when infectious SA11cl3 virus was given
to either adult CFTR+/+,
CFTR+/
, or
CFTR
/
mice, none
exhibited diarrhea (Table 1).
NSP4 peptide mobilizes
[Ca2+]i
levels in epithelial cells isolated from the small and large bowel of
both
CFTR+/+
and CFTR
/
mice.
Rotaviral protein production has been correlated with changes in
[Ca2+]i
homeostasis (39) and with
Ca2+-dependent cytotoxicity during
viral infection in cultured cells (33, 34). With the use of the human
colonic epithelial cell line HT-29 clone 19A, which expresses
plasmalemmal proteins found in both small and large intestinal cells,
we recently demonstrated that exogenously added NSP4 and
NSP4114-135 peptide causes [Ca2+]i
mobilization by PLC-mediated IP3
production (13). We therefore investigated whether the diarrheal
effects of NSP4 and
NSP4114-135 peptide in vivo
were also associated with native mucosal intestinal cell
[Ca2+]i
mobilization. Fluorescence video microscopy was used to study the
effects of exogenous NSP4 addition on
[Ca2+]i
in intestinal crypts isolated from either small or large bowel of 8- to
13-day-old mouse pups.
Small intestinal crypts.
Crypts from the small intestine were chosen in preference to villi,
which dissociated during isolation. Addition of 100 nM NSP4 protein, a
dose shown to promote near-maximal
[Ca2+]i
rises in vitro (13), elicited rapid rises in
[Ca2+]i
that lasted 2-5 min in
CFTR+/+,
CFTR+/
, and
CFTR
/
small
intestinal crypts. Resting
[Ca2+]i
values of 126 ± 28 nM
(CFTR
/
,
n = 13) and 110 ± 63 nM
(CFTR+/+ and
CFTR+/
,
n = 14) increased to peak values of
205 ± 54 nM and 260 ± 116 nM, respectively (Table
2). A representative example (Fig.
2, A-C)
shows consecutive images taken before, during, and after NSP4 addition
to an epithelial crypt isolated from a
CFTR
/
mouse. No
significant difference was seen between mouse CFTR genotypes with
respect to basal, peak, or net
Ca2+ values
(P > 0.01, Student's
t-test; Table 2). These results indicated that NSP4 was an effective
Ca2+-mobilizing agonist in the
small intestine and that Ca2+
homeostasis, both basal and agonist-stimulated, is unaffected by the
CFTR gene knockout. Not all experimental crypts responded; 27 of 108 (25%) gave
[Ca2+]i
rises. Increasing the dose of NSP4 from 100 to 500 nM and above did not
increase the number of NSP4-responsive crypts (3 of 18 responded = 17%; data not shown).
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Table 2.
Summary of the effects of 100 nM NSP4 and 100 µM CCh on
[Ca2+]i levels in either
CFTR / or
CFTR+/+,+/ small and large
intestine mouse crypts
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Fig. 2.
Consecutive pseudocolor images of retroviral enterotoxin protein
NSP4-induced intracellular Ca2+
concentration
([Ca2+]i)
rise in an isolated CFTR-deficient
(CFTR / ) small
intestinal mouse crypt, loaded with fura 2. A: resting
Ca2+ level of ~120 nM before
agonist stimulation. B: peak
Ca2+ level approaching 220 nM
immediately after addition of 100 nM NSP4.
C: gradual return of crypt to resting
Ca2+ levels ~1.5 min after
addition of NSP4.
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Colonic crypts.
When isolated crypts from the distal colon of
CFTR+/+,
CFTR+/
, and
CFTR
/
mice were
superfused with 100 nM NSP4 protein,
[Ca2+]i
rises similar to those obtained from small intestinal crypts were
recorded (P > 0.1, Student's
t-test; Table 2). Peak NSP4-induced [Ca2+]i
values were similar in all three genetic backgrounds, although basal
values were slightly, but not significantly, higher in
CFTR
/
mice when all
samples were pooled (P > 0.01, Student's t-test; Table 2). In our in
vitro studies, we found that the NSP4-induced [Ca2+]i
mobilization was susceptible to predepletion by other agonists (remaining unresponsive for tens of minutes; Ref. 13). In contrast, NSP4-induced
[Ca2+]i
release in both ex vivo colonic and ileal mucosal cell preparations was
not desensitized by repetitive agonist administration
(n = 15). In the
CFTR
/
mouse crypt
preparation shown in Fig. 3, identical
[Ca2+]i
rises were seen for consecutive NSP4 or CCh followed by NSP4 challenge.
Ca2+ homeostasis in the isolated
crypt therefore appears to be more tightly regulated than in colonic
cell lines.

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Fig. 3.
A representative example of a single crypt. Average
[Ca2+]i
values from 6 measurement areas along length of an isolated
CFTR / colonic mouse
crypt, showing effect of 100 nM NSP4
(A), 500 µM carbachol (CCh,
B), and 100 nM NSP4
(C). SD values are shown as error
bars.
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|
To address why the NSP4 response rate of both small and large
intestinal crypts was not >25%, we quantified the responsiveness of
isolated crypts to the cholinergic agonist CCh. The addition of 100 µM CCh to the bath elicited
[Ca2+]i
rises in a similar percentage of isolated crypts from both small and
large intestine (33% responded; Table 2). Lower, more physiological
doses of CCh would likely produce identical success rates for both
agonists. These findings suggested that a more general cellular
phenomenon, other than a lack of NSP4 receptors, was accountable for
this effect. Because both
M3-muscarinic receptors and the
cellular response to NSP4 have been shown to be sensitive to protease
digestion (13), true in vivo NSP4 potency may have been masked.
Spatial analysis of the
Ca2+-mobilizing effects of NSP4
revealed no regional differences along the longitudinal axis of either colonic or small intestinal crypts. Mean resting values for base, mid,
and neck regions (separated by ~25 µm) were 112 ± 142, 100 ± 25, and 102 ± 39 nM, rising to 250 ± 99, 229 ± 97, and 215 ± 79 nM, respectively (n = 29). The lack of localization-dependent effects on
[Ca2+]i
indicates that, regardless of the cell maturity, the membranes of
epithelial cells within the crypt remained responsive to NSP4. An
example of this spatial homogeneity is shown in Fig.
4. In Fig. 4, a sustained component to
NSP4-induced
[Ca2+]i
mobilization is observed that disappeared on removal of NSP4 from the
bath. Nominally Ca2+-free
conditions did not affect the Ca2+
response to NSP4 protein (n = 12, data
not shown), confirming that the major source of the
Ca2+ rise was from intracellular
stores. However, under extracellular Ca2+-free conditions, no sustained
component was observed, indicating that
Ca2+ influx also occurred. The
effects of NSP4 ex vivo reproduced those recorded in vitro, which have
been linked to the intracellular production of
IP3 (13).

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Fig. 4.
[Ca2+]i
measurements taken from 3 different regions separated by ~15 µm
along longitudinal axis of a
CFTR / small
intestinal crypt, showing magnitude of NSP4
[Ca2+]i
rise at crypt base (A), midregion of
crypt (B), and near neck of crypt
(C). Agonist (100 nM NSP4) was
washed out of bath ~10 min after addition.
|
|
NSP4 peptide mobilizes
[Ca2+]i
levels in crypt epithelial cells from both pup and adult mice.
Isolated colonic crypts from either normal
(CFTR+/+) C57B or BALB/c mice
were loaded with fura 2 and exposed to 100 nM NSP4. In the example
shown (BALB/c, Table 3), NSP4 mobilized
Ca2+ in crypts from both 7- to
12-day- and >25-day-old mice. Although the mean number
of BALB/c crypt responses was higher than that observed in the C57B
background (data not shown), there was no age dependency to the
magnitude of the NSP4-induced
[Ca2+]i
rise (values were not statistically significant, unpaired
t-test, P > 0.05, n = 13; Table 3). When CCh (100 µM)
was applied after a NSP4-induced
Ca2+ mobilization, 75% of pup
crypts and 80% of adult crypts responded with a second
[Ca2+]i
rise (n = 13). In this instance, the
net agonist-induced
[Ca2+]i
rise was significantly smaller in adult crypts
(P < 0.001, unpaired
t-test; Table 3). When CCh alone was
applied to crypts, a similar age-dependent trend in
Ca2+ mobilization was observed,
which failed to be significant because of the great variability in the
magnitude of the peak
[Ca2+]i
values (P > 0.05; Table 3). These
results indicated that events distal to NSP4-elicited
[Ca2+]i
mobilization must regulate the age- and
Ca2+-dependent
Cl
secretory activity of
NSP4 observed in the ex vivo mouse intestine (3).
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|
Table 3.
Summary of the effects of 100 nM NSP4 and 100 µM CCh
on [Ca2+]i levels in pup and adult
BALB/c mouse colonic crypts
|
|
Calculation of Stern-Volmer constants for
Cl
,
Br
, and
I
in adult and pup mouse crypts.
To separate carrier-mediated anion transport events from conductive
anion transport in SPQ-loaded crypts, the halide
I
was used as a surrogate
anion for Cl
.
I
is not a substrate for
either the
Na+-K+-2Cl
cotransporter or
Cl
/HCO
exchanger (36, 63). This choice was not, however, without technical
drawbacks, because the Stern-Volmer relationships for Br
,
I
, and
SCN
in free solution are
reported to become positively nonlinear at >15 mM halide (24).
Nonlinear increases in
Kq above this concentration suggest that, in addition to dynamic collision
interactions, static interactions occur (i.e., those leading to
nonfluorescent halide-SPQ complex formation; Ref. 28). Irrespective of
the exact nature of this quenching (assumed to be static buffering), the dye would become noncalibrated at high halide concentrations, compromising I
usefulness
as a surrogate anion for conductive
Cl
influx. The Stern-Volmer
relationship for Cl
on the
other hand has been demonstrated to remain linear when measured both in
free solution and cells and shown to be the product of the dynamic
collision-quench rate constant
Kq (24).
Stern-Volmer relationships for
Cl
,
I
, and
Br
were constructed in
SPQ-loaded crypts with the use of the dual ionophore technique (Ref. 7;
see MATERIALS AND METHODS). We found
that apparent Stern-Volmer quench constants for both
Br
and
I
, calculated from halide
concentrations of either <15 mM or <150 mM, exhibited modest
<15% and slight <5% nonlinear increases in slope, respectively.
This deviation was not large enough to accurately determine an
additional buffering coefficient (28). Thus
Kq values for
Br
and
I
were estimated by linear
curve fitting and found to be 38.5 M
1 and 51.4 M
1,
respectively.1
The Kq value for
Cl
was found to be 21.5 M
1 (measurements made in
both pup and adult crypts, n = 8 per
halide; Fig. 1), reflecting very well the ratio of SPQ collisional
quench (1:1.7:1.9) reported for colonocytes (45). These values were slightly lower than those calculated in the rabbit crypts (45) and
higher than those estimated in a variety of cancer cell lines (which
range between 12 M
2 and 18 M
2 depending on cell type;
Ref. 58). Because we failed to measure changes in tubular geometry
(shrinkage) associated with agonist stimulation when confining our
measurements to basal crypt areas, we did not resort to more
theoretical measurements of the Stern-Volmer constant (22). With the
use of these values, 50% of cellular SPQ quench would be achieved at
26, 19.6, and 47 mM Br
,
I
, and
Cl
, respectively.
Calculation of basal and agonist-stimulated halide permeability.
The dynamic Kq
values determined above were used to calculate influx rates for the
respective anions that were dependent on plasma membrane permeability
and not on alterations in SPQ quench rate. Basal influx was in the
order I
> Cl
> Br
(Table
4) for
CFTR+/+,
CFTR+/
, and
CFTR
/
pup crypts. An
age-dependent but not significant (P > 0.05) trend in basal influx was seen, with adult values being
somewhat higher than those in pups. The lack of CFTR was associated
with a statistically significant (P < 0.001) decrease in basal halide influx rate
(Jhalide
) in CFTR
/
mice (Table
4).
With the calculated
Kq value for
I
, we tabulated changes in
agonist-evoked I
influx
rate
(
JI
agonist)
in response to FSK (10 µM), CCh (500 µM), and NSP4 peptide (500 nM) in all three CFTR genomic backgrounds (Table
5). Time-based histograms of changes in
agonist-induced quench rate
[
RI
= (
F/
tagonist
F/
tbasal)]
are also shown (Figs. 5-7).
FSK-stimulated I
influx into crypt
cells was age independent and required CFTR.
FSK, as predicted from previous studies (10, 45), induced significant
I
influx in both
CFTR+/+ and
CFTR+/
mouse pup and adult
crypts;
JI
agonist increased 3.7- to 4-fold over
JI
basal and were statistically significant (P < 0.001, n = 5; Table 5). Age-dependent differences in the magnitude of
JI
were absent (P > 0.05, n = 5; Table 5). Time-dependent
analysis revealed that the peak FSK-induced quenching rate
(RI
agonist) occurred 8-10 s after bath
I
exchange (Fig.
5A).

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Fig. 5.
Change in SPQ fluorescence quench rate in mouse distal colon crypts
elicited by cAMP-mobilizing agonist forskolin (FSK, 10 µM).
A: values obtained from adult and pup
CFTR+/+ and
CFTR+/ mouse crypts,
showing a lack of any age dependency.
B: values obtained from pooled
CFTR+/+ and
CFTR+/ and
CFTR / pup crypts,
demonstrating that effects of FSK on SPQ quench rate were dependent on
CFTR expression. Values are means ± SD.
FU, fluorescence unit.
|
|
In contrast, when crypts isolated from
CFTR
/
mouse pup
littermates were exposed to FSK, much lower
JI
agonist and
JI
values were obtained. In this instance,
JI
agonist
failed to be significantly different from basal
JI
values (P > 0.05) and both
JI
and
RI
values were significantly lower than corresponding values obtained from
CFTR+/+ and
CFTR+/
mouse pup crypts
(
JI
CFTR
/
= 0.03 ± 0.02 M
3/s vs.
JI
CFTR+/+ and
CFTR+/
= 1.4 ± 0.46 M
3/s,
P < 0.001, n = 5; Table 5 and Fig.
5B). The absence of CFTR was
correlated with >95% inhibition of FSK-stimulated
I
influx into isolated
mouse pup crypts. In all genotypes, FSK stimulated changes in influx in
~90% of crypts tested.
CCh-stimulated I
influx into crypt
cells was age independent and required CFTR expression.
CCh (100 µM)
JI
agonist
values were twofold greater than
JI
basal values in wild-type and heterozygous CF genotypes and were
significantly different (P < 0.001, n = 5; Table 5). The resulting
JI
values from both groups displayed no age dependency
(
JI
pup = 0.62 ± 0.30 M
3/s
vs.
JI
adult = 0.69 ± 0.24 M
3/s,
n = 5, P > 0.05; Table 5). These changes
approximated to 51 and 40% of the values elicited by FSK in pup and
adult crypts, respectively (Table 5). CCh-elicited
RI
changes were greatest 8-10 s after bath
I
exchange, with a latency
similar to that recorded for FSK (Fig. 6A). CCh
was therefore a potent age-independent stimulator of plasma membrane
I
influx in CFTR-containing
crypts, causing identical but smaller changes in
I
influx rate compared with
FSK.

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Fig. 6.
Change in SPQ fluorescence quench rate in mouse distal colon crypts
elicited by Ca2+-mobilizing
agonist CCh (100 µM). A: values
obtained from pooled adult and pup
CFTR+/+ and
CFTR+/ mouse crypts,
showing a lack of any age dependency.
B: values obtained from pooled
CFTR+/+ and
CFTR+/ and
CFTR / pup crypts,
demonstrating that effects of CCh on SPQ quench rate were delayed and
significantly smaller in magnitude when CFTR was absent from crypt.
Values are means ± SD.
|
|
Like FSK, CCh failed to significantly increase the plasma membrane
I
influx rate in
CFTR
/
mouse pup
crypts.
JI
agonist and
JI
basal
values failed to be statistically different
(P < 0.05, Student's
t-test,
n = 5; Table 5). The
JI
recorded in CF mouse pup crypts was significantly smaller than
CFTR+/+ and
CFTR+/
littermate values
(CFTR
/
JI
agonist = 0.11 ± 0.08 M
3/s vs.
CFTR+/+ and
CFTR+/
JI
= 0.62 ± 0.3 M
3/s,
P < 0.001, n = 5; Table 5). When
RI
for CCh was plotted by histogram analysis in the
CFTR
/
background,
quite different kinetics were observed. Both the onset and subsequent
peak in
RI
trailed wild-type and heterozygous values by 4 and 18 s, respectively (Fig. 6B). This delay was not well
defined by the single exponential fitted at time
0 during initial rate analysis
(JI
). Thus time-based histograms of rate change are shown. Peak values of
CCh-induced
RI
recorded from CFTR
/
mouse pup crypts approximated to 60% of values recorded from wild-type
and heterozygous genotypes. In all genotypes, CCh stimulated changes in
~65% of crypts tested. We therefore concluded that CCh, like FSK,
requires CFTR to exert its major effects on plasma membrane
I
influx. Rate analysis
revealed, however, that a slower component of SPQ quench rate was also
present when CCh, but not FSK, was added to crypts from
CFTR
/
pups.
NSP4-stimulated I
influx into
crypt cells was age dependent and largely independent of CFTR
expression.
NSP4 protein (100 nM) was found to elicit changes in plasma membrane
I
influx in crypts isolated
from wild-type and heterozygous mouse CFTR genotypes. However, unlike
either FSK or CCh, the effects of NSP4 were clearly age dependent
(Table 5). In the pup crypt, NSP4 stimulated plasma membrane
I
influx by 2.7-fold,
whereas, in the adult crypt, NSP4-stimulated I
influx was
<1.1-fold. Corresponding P values
for
JI
agonist vs.
JI
basal
were significantly different for pup
(P < 0.001) but not adult
(P > 0.05) crypts. Net changes in
NSP4-elicited influx rate were as follows:
JI
pup = 0.67 ± 0.42 M
3/s
vs.
JI
adult = 0.06 ± 0.02 M
3/s, and were
significantly different (P < 0.001, n = 5; Table 5). When the NSP4-induced
change in I
influx rate was
displayed in histogram format, peak changes in influx rate had a lag of
10 s after introduction of
I
into the bath, similar to
that recorded for both FSK and CCh (Fig.
7A).

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Fig. 7.
Change in SPQ fluorescence quench rate in mouse distal colon crypts
elicited by NSP4 (100 nM). A: values
obtained form pooled adult and pup
CFTR+/+ and
CFTR+/ mouse crypts,
demonstrating that NSP4 effects were age dependent.
B: values obtained from pooled
CFTR+/+ and
CFTR+/ and
CFTR / pup crypts,
demonstrating that effects of NSP4 on SPQ quench rate were independent
of CFTR expression. Values are means ± SD.
|
|
Significantly, when NSP4 protein (100 nM) was tested on
CFTR
/
mouse pup
crypts, a threefold increase in
I
influx rate was recorded.
JI
agonist was statistically different from
JI
basal (P < 0.001) and was not
statistically different from
JI
agonist values obtained in wild-type and heterozygous CFTR backgrounds (P > 0.05; Table 5). The net change
in NSP4-induced I
influx
rate approximated to 64% of
CFTR+/+ and
CFTR+/
littermate values
(
JI
CFTR
/
= 0.43 ± 0.1 compared with
JI
CFTR+/+ and
CFTR+/
= 0.67 ± 0.42). The
RI
values failed to exhibit either slow onset or delayed peak (as observed
for CCh) in the
CFTR
/
genotype (see
above) but instead peaked 10 s after
I
introduction into the
bath, paralleling NSP4, FSK, and CCh effects in CFTR-expressing crypts
(Fig. 7B). In all genotypes, NSP4
stimulated changes in
I
influx in
~70% of crypts tested, a response rate twice that of NSP4-induced
Ca2+ mobilization. Thus
NSP4-evoked plasma membrane
I
influx was age dependent,
occurring selectively in mouse pup crypts, and was largely independent
of CFTR expression. In crypts from wild-type and heterozygous CFTR
pups, the NSP4-induced I
influx rates were larger but not significantly different (1.56-fold, n = 5; Table 5). Thus CFTR expression
may additionally be utilized by NSP4 to promote greater overall changes
in plasma membrane halide permeability.
NSP4-induced I
influx into pup
crypt cells was dependent on
[Ca2+]i
mobilization.
To investigate the relationship between NSP4-induced
Ca2+ mobilization and plasma
membrane I
influx,
JI
basal,
JI
agonist, and
JI
were measured in CFTR+/+ and
CFTR+/
mouse pup crypts
loaded and bathed in Ca2+-free
HEPES-buffered saline (nominally
Ca2+-free plus 1 mM EGTA). Basal
I
influx rate was
not appreciably lower in this instance than in crypts bathed with
standard (1 mM CaCl2)
extracellular saline (JI
basal
Ca2+-free = 0.32 ± 0.08 M
3/s vs.
JI
basal
Ca2+-containing = 0.4 ± 0.14 M
3/s, values were not
significantly different, P > 0.05, n = 4 and 15, respectively; Table 5).
However, in all crypts the absence of bath
Ca2+ completely inhibited
NSP4-mediated I
influx
(
JI
Ca2+-free =
0.02 ± 0.06 M
3/s vs.
JI
Ca2+-containing = 0.67 ± 0.42 M
3/s,
P < 0.001, n = 4 and 5, respectively; Table 5).
These findings demonstrated that NSP4-stimulated plasma membrane
I
influx was dependent on
[Ca2+]i mobilization.
NSP4 protein-induced I
influx in
mouse pup crypt cells was inhibited by DIDS.
The effectiveness of NSP4, but not CCh, at eliciting significant
I
influx into SPQ-loaded
CFTR
/
mouse pups
raised the question as to the nature of this novel conductive pathway.
To begin to characterize this pathway, crypts were preexposed to 0.5 mM
DIDS and then washed before experimentation. DIDS inhibits a variety of
Cl
channels identified in
epithelial cells, including the outward-rectifying Cl
channel, the
swelling-activated Cl
conductance channel, and the
Ca2+-sensitive
Cl
channel, but does not
affect cAMP-stimulated CFTR
Cl
channels (reviewed in
Ref. 19). DIDS also inhibits a variety of organic osmoltye exchange
mechanisms (52) and the
Cl
/HCO
3
exchanger (63). Basal I
influx rate in the presence of DIDS was lower than controls but was not
significantly different
(JI
basal DIDS = 0.29 ± 0.13 M
3/s
vs.
JI
basal
control = 0.4 ± 0.14 M
3/s,
P > 0.05, n = 5; Table 5). This
general halide transport inhibitor completely abolished the
NSP4-elicited increase in plasma membrane
I
influx in all crypts
(JI
agonist DIDS = 0.34 ± 0.14 M
3/s vs.
JI
agonist
control = 01.07 ± 0.32 M
3/s,
P < 0.001, n = 4), and the net change in
I
influx rate reflected
this difference
(
JI
DIDS = 0.05 ± 0.06 vs.
JI
control = 0.67 ± 0.24, P < 0.001, n = 4; Table 5). Thus the
ontogenically regulated NSP4-activated plasma membrane
I
influx pathway present in
mouse pup crypts was DIDS sensitive.
 |
DISCUSSION |
Relationship between the diarrhea effects of infectious rotavirus
and NSP4.
The induction of diarrhea by NSP4 protein and
NSP4114-135 peptide compared
with active rotavirus indicates that the enterotoxic effects of NSP4
may account for the initial diarrheal phase of rotaviral infection (3).
During this period, diarrhea occurs without significant ultrastructural
damage to the small intestinal mucosa (38, 57). This contrasts with
diarrhea seen during the later stages of rotaviral infection in some
animal species, in which villus blunting has been suggested to
contribute to diarrhea by reducing small intestinal absorptive area
(5). This later histopathological feature of rotaviral infection is not
seen in all animal models; in the mouse, significant villus blunting is
rare (38, 49). Therefore, loss of absorptive area is likely to be
auxiliary to the underlying pathophysiological basis of diarrhea in
this model. Supporting this hypothesis, we have shown that NSP4 and its
active NSP4114-135 peptide fail to cause significant histological damage to the mucosa when injected intraluminally or interperitoneally into wild-type mice (unpublished observations and Ref. 3).
Correlation between
[Ca2+]i
mobilization and fluid transport in the gut.
Addition of Ca2+-mobilizing
agonists such as CCh to intestinal mucosal sheets from either
CFTR
/
mice or CF
patients fails to evoke a
Cl
secretory
current
(6, 37, 53). This lack of response suggests that the cellular effects
of Ca2+ mobilization on the
Cl
secretory current in the
gastrointestinal tract occur secondary to cAMP-dependent activation of
CFTR channels and are limited to the upregulation of basolateral
membrane ion transporters and K+
channels (51, 60). Results from NSP4-induced
[Ca2+]i
mobilization in both native small and large intestinal crypts (Fig. 2,
A-C,
Fig. 3, and Table 2) support the hypothesis that the enterotoxic
activity of NSP4 in normal mucosa may be partly due to the secondary
effects of increased cellular
Cl
uptake at the
basolateral plasma membrane and cellular hyperpolarization. These
facilitating but not controlling mechanisms for intracellular Cl
conduction across the
luminal membrane do not explain all of our observations. Two important
questions relating to the role of NSP4-induced
[Ca2+]i
mobilization, namely the age-dependent effects of NSP4 on
Cl
secretion and diarrhea
and the diarrheal effects of NSP4 in CF mice, remained unanswered.
NSP4-Ca2+
signaling does not directly regulate age-dependent
Cl
secretion.
If changes in
[Ca2+]i
levels alone were solely responsible for
Cl
secretion and diarrhea
in mouse pups, then Ca2+
mobilization by NSP4 would be expected to be cell differentiation and/or crypt age dependent. Our studies found, however, that NSP4 mobilized
[Ca2+]i
equally well in all crypt regions and was therefore independent of
cellular maturity (Fig. 4). These findings contrast with the reported
effects of both purinergic and muscarinic agonists on [Ca2+]i
in intestinal crypts, which show greater responsiveness in the basal
region, where Cl
secretion
is believed to occur (29, 47). The more widespread [Ca2+]i
mobilization elicited by NSP4 would argue that antiabsorptive as well
as prosecretory effects may be present in rotaviral-infected intestinal
mucosa (see An antiabsorptive model for NSP4-induced [Ca2+]i mobilization). Furthermore, the
effects of NSP4 on
[Ca2+]i
mobilization were neither strain dependent nor related to the age of
the animals from which the crypts were isolated (Table 3). These
findings therefore indicated that NSP4-mediated
Ca2+ mobilization cannot directly
account for the age dependency to Cl
secretion and therefore
secretory diarrhea reported by us in neonatal mice (3).
NSP4-Ca2+
signaling activates an age-dependent anion permeability in normal and
CF mice.
Both CCh and NSP4 have been shown to utilize PLC-dependent
IP3 signaling to elevate
[Ca2+]i
(13). We therefore examined how NSP4, but not CCh, induces diarrhea in
CF mouse pups. The SPQ fluorescence imaging technique has been used
previously to measure basal and agonist-stimulated Cl
permeability changes in
both freshly isolated crypts (22) and primary cultures of rabbit (10,
45, 46) and human (45) distal colon.
Cl
transport measurements
made with SPQ have demonstrated the absence of CFTR from epithelial
cells of CF patients (41, 42, 50), and modeling studies have shown that
the SPQ technique provides equivalent information to that obtained from
either electrophysiological or isotopic flux studies (59).
Whereas both FSK and CCh elicited age-independent increases in
I
influx that are dependent
on the presence of CFTR (Table 5, Fig. 5, and Fig. 6,
A and
B), the effects of NSP4 were age
dependent and largely CFTR independent (Table 5). Both CCh and NSP4
protein were more effective at causing plasma membrane permeability
changes than causing generalized
[Ca2+]i
mobilization. We believe this reflects both our technical limitations at isolating crypts with endogenous levels of receptor activity (see
RESULTS) and the fact that
Ca2+-mobilizing agonists can
couple efficiently with plasma membrane anion channels through
localized
[Ca2+]i
rises at the level of the plasma membrane that often do not provoke a
generalized
[Ca2+]i
mobilization (35).
The lack of an age dependency for CFTR-dependent
I
permeability changes
corroborated Cl
transport
studies made in weaning and adult rat and pig colon but differed from
the age-dependent effects of cGMP or Escherichia coli heat-stable enterotoxin (STa) in primary cultures
of weaning and adult rabbit colon. In these instances agonist responses
were greater in crypts isolated from older mice (10). Differences may,
however, reflect the fact that
Cl
was used as a quencher
in the latter studies, and rates may represent both conductive and
nonconductive components of plasma membrane Cl
permeability. In addition, STa effects
on transmucosal Cl
secretion and crypt Cl
permeability are absent in the CF mice (20). Thus NSP4 utilizes a
different plasma membrane permeability pathway in distal colon crypts
from that utilized by STa or its mammalian counterparts, guanylin or
uroguanylin, and other nitroso compounds of nitric oxide-signaling pathways.
The age dependency to NSP4-elicited changes in plasma membrane
I
influx suggests that
NSP4-elicited diarrhea in CF mouse pups (Table 1) may be brought about
by the activation of a novel cellular permeability pathway. The fact
that neither FSK nor CCh promoted similar changes in CF mice, and that
NSP4-induced I
permeation
was both Ca2+ dependent and
blocked by DIDS (Table 5), supported the hypothesis that we are
reporting on a different permeability pathway. NSP4-induced I
influx into
CFTR
/
mouse pup
crypts was ~60% of that elicited in the wild-type and heterozygous
background. This difference may possibly explain the higher, but not
significantly different, incidence of diarrhea reported for infectious
rotavirus, NSP4 protein, and
NSP4114-135 peptide in
CFTR+/+ and
CFTR+/
mice. NSP4-induced
[Ca2+]i
mobilization would be expected to facilitate both CFTR-dependent and
CFTR-independent anion permeation by upregulating
Ca2+-dependent secondary active
transport mechanisms at the basolateral membrane.
Our findings would also provide a functional explanation for the recent
observations of Angel and colleagues (2), who demonstrated that CF mice
and their non-CF siblings exhibit diarrhea after rotaviral infection.
The age- and Ca2+-dependent
changes in intestinal epithelial cell plasma membrane anion
permeability evoked by NSP4, shown in this study to be CFTR independent, may contribute to (or constitute) the acute phase of the
rotaviral diarrheal response. This cellular phenomena would precede
immune/morphological changes occurring within the mucosa after viral infection.
Candidate anion conductances for NSP4-induced age-dependent
permeability changes in the mouse pup crypt.
Although the plasma membrane halide permeability changes promoted by
NSP4 were clearly Ca2+ dependent
and could be inhibited pharmacologically, our investigations have yet
to confirm the biophysical nature of the conductance. Furthermore, the
SPQ technique does not allow us to determine the plasma membrane
location of the NSP4-elicited
I
influx. Future studies
utilizing alternate methods should address these
questions. Even so, at this point, it is of interest to consider
candidate conductances that may participate in this phenomena.
Direct activation of apical plasma membrane
Ca2+-sensitive
Cl
channels by classical
PLC-dependent Ca2+-mobilizing
secretagogues, although clearly demonstrated in gastrointestinal cell
lines and epithelial cells from other organs, e.g., the lungs (19), has
not been shown in native intestinal epithelia. However, both
enteroendocrine and macrophage/lymphocyte populations of the gut are
known to secrete cryptdin proteins, some of which are believed to act
themselves as anion conductances (30), whereas others, notably the
defensins, are proposed to act at nanomolar concentrations to stimulate
Ca2+-dependent
Cl
secretion (4, 31). We
therefore propose that NSP4 either 1) itself creates a conductance in
the apical plasma membrane, 2)
activates a dormant Ca2+-activated
anion channel within this membrane with biophysical properties similar
to those recorded in vitro (reviewed in Ref. 19) through its
stimulatory effects on PLC-induced
[Ca2+]i
mobilization (13), or 3) acts as a
regulatory protein at the level of
Ca2+- or anion-channel signaling
to elicit Ca2+-dependent
permeability changes across the cellular plasma membrane. CCh failed to
promote similar changes in the initial rate of
I
influx into CF mouse pup
crypts as NSP4 but elicited a response with a delayed onset, consisting
of ~20% of wild-type and heterozygous CFTR values
(Table 5 and Fig. 6B). Because
supramaximal concentrations of CCh were used, these differences do not
reflect dose dependency and argue against the presence of an
ontogenically regulated conductance that can be stimulated by all
Ca2+-mobilizing agonists. The
delayed onset changes in I
permeability promoted by CCh (Fig.
6B) are unlikely to be localized to
the apical membrane because it is clear that CCh fails to elicit a
Cl
secretory response in CF
mucosal sheets (6, 37, 53).
A possibility exists that NSP4 may activate a native cell counterpart
of osmotically regulated Cl
channels. These conductances, although
not directly activated by Ca2+,
are indirectly Ca2+ sensitive and
have been shown to participate in regulatory volume decreases (RVD) in
intestinal epithelia (12). These channels, believed to be localized on
the basolateral plasma, participate in the maintenance of fluid and
osmolyte absorption. They are functionally expressed in the surface and
neck regions of the crypt as well as the small intestinal villi, areas
known to exhibit RVD (11) and have been shown to be impaired in
CFTR
/
mice (55). Our
SPQ fluorescent measurements were made, however, in lower crypt
regions, which do not undergo RVD. Thus they are poor candidates for
the NSP4 response.
Volume changes in response to
Ca2+-mediated secretagogues have
been reported in CF mouse small intestinal crypts (56), suggesting that
additional conductive pathways exist. The slow onset response observed
for I
influx after CCh
challenge in CFTR
/
mouse crypts may reflect this alternate pathway (Fig.
6B). However, the timing of the
effects of NSP4 in the
CFTR
/
mouse crypt
was very different (Fig. 7B),
mimicking the profile observed for activation of apical membrane CFTR
in wild-type crypts by all agonists (Figs.
5A,
6A, and
7A). We interpret these results to
suggest that the effects of NSP4 on any basolateral membrane permeability, if present, are likely to be secondary phenomena and
unrelated to the primary effects of NSP4 on the apical plasma membrane
I
influx.
A prosecretory model for NSP4-induced
[Ca2+]i
mobilization.
A characteristic property of rotavirus infection is that it causes
age-dependent gastroenteritis in neonatal mammals (25). In the neonatal
mouse, increases in small and large intestinal luminal
Na+,
Cl
, and water content have
been correlated with diarrheal activity during rotaviral infection
(49). Previously, we have reported (3) that NSP4 protein addition to
mouse pup small intestinal mucosal sheets mimics the transepithelial
Cl
secretory effects of
CCh, an endogenous Ca2+-mobilizing
agonist, but that this secretory response was lost in adult tissues.
Further strengthening our hypothesis that enterocytes express an
ontogenically regulated,
Ca2+-activated ionic conductance
that underlies the diarrheal response, we now report that NSP4 induces
Ca2+-dependent, DIDS-inhibitable
I
influx in
CFTR
/
mouse pup
crypts. This phenomenon did not occur in adult crypts. The primary
effect of NSP4 may therefore be to activate an apical plasma membrane
Ca2+-dependent
Cl
conductance in mouse pup
enterocytes with biophysical properties similar to those described in
vitro (19). The secondary effect of NSP4, like that of CCh, may be to
facilitate CFTR-mediated fluid transport by upregulating
Ca2+-sensitive basolateral
Cl
uptake and cellular
hyperpolarization. Combined, both may account for the
Ca2+-dependent fluid secretory
response of normal and CF mucosa and may delineate a secretory
component to rotaviral diarrhea.
An antiabsorptive model for NSP4-induced
[Ca2+]i
mobilization.
NSP4 mobilized Ca2+ in all regions
of the crypt from both the small intestine and colon. We have also
found that NSP4 is equally effective at mobilizing
Ca2+ in dispersed villus
epithelial cells from the small intestine (data not shown). Basal
electroneutral NaCl absorption into the villus has been shown to be
inhibited by Ca2+-dependent
processes, including PLC-
, calmodulin, and protein kinase C
activation (14, 17, 26). Thus another predicted secondary effect of
NSP4-induced Ca2+ mobilization is
downregulation of this absorptive process. Released NSP4 protein, if it
encounters luminal receptors on neighboring cells (3), would be
expected to promote similar antiabsorptive changes. NSP4-induced
Ca2+ mobilization would not,
however, be expected to affect absorptive Na+-coupled glucose-solute
transport (62), which is the cellular basis of oral rehydration therapy
given during bacterial and viral gastroenteritis (40).
We have shown that the nonstructural protein of rotavirus NSP4 and its
peptide both elevate intestinal cell
[Ca2+]i
levels and induce diarrhea in neonatal mice with either wild-type or
CFTR-deficient genotypes. Ca2+
mobilization may effect a diarrheal response in these cells through a
variety of cellular mechanisms, including the activation of an
ontogenically regulated,
Ca2+-sensitive,
Cl
-conductive pathway in
small intestinal crypts and
Ca2+-inhibitable
Na+-absorptive processes in small
intestinal villi and crypts. Fluid loss by any of these
mechanisms would occur independently of CFTR function. The diarrheal
properties of NSP4 indicate that molecules based on the active domain
of NSP4 may prove useful for the treatment of neonatal meconium ileus
syndrome or the less severe clinical condition of constipation, which
is prevalent in CF patients (44). The low dose (nanomolar)
effectiveness of the NSP4 peptide and protein (3, 13), coupled with the
fact that attenuated NSP4 proteins with differing diarrheal activities
have been recently identified by us (64), suggests that further
molecular characterization of the active domain of NSP4 may help design
chemical structures with therapeutic value.
 |
ACKNOWLEDGEMENTS |
We thank Sharon Krater for help in breeding and genotyping the CF
mice. We also thank A. Beaudet, F. Murad, R. O'Neal, and J. Sellin for
critical evaluation of this manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-30144 (M. K. Estes), Texas Advanced
Technology Program Grant 004949-062 (M. K. Estes and A. P. Morris), and Cystic Fibrosis Foundation Postdoctoral Fellowship F806
(W. K. O'Neal).
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. §1734 solely to indicate this fact.
1
When utilizing
I
and
NO
3-containing saline, it could be
argued that highly membrane-permeable oxidation species (e.g.,
IO
3,
IO
5) may explain this additional
buffering component. There is no clear way of establishing an
equilibrium concentration of these anions in our solutions. However,
this does not invalidate this well-established fluorescent procedure
because matched controls are always performed (i.e., ± agonist) and
increases in Kq
would serve to underestimate influx.
Address for reprint requests and other correspondence: A. P. Morris,
Depts. of Integrative Biology and Internal
Medicine-Gastroenterology, Medical School, Univ. of Texas at
Houston, Houston, TX 77030 (E-mail:
amorris{at}girch1.med.uth.tmc.edu).
Received 1 February 1999; accepted in final form 12 May 1999.
 |
REFERENCES |
1.
Argons, G. A.,
W. R. Corse,
R. I. Markowitz,
E. S. Suarez,
and
D. R. Perry.
Gastrointestinal manifestations of cystic fibrosis: radiologic-pathologic correlation.
Radiographics
16:
871-893,
1996[Abstract].
2.
Angel, J.,
B. Tang,
N. Feng,
H. B. Greenberg,
and
D. Bass.
Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea.
J. Infect. Dis.
177:
455-458,
1998[Medline].
3.
Ball, J. M.,
P. Tian,
C. Q. Zeng,
A. P. Morris,
and
M. K. Estes.
Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.
Science
272:
101-104,
1996[Abstract].
4.
Bateman, A.,
R. J. MacLeod,
P. Lembessis,
J. Hu,
F. Esch,
and
S. Solomon.
The isolation and characterization of a novel corticostatin/defensin-like peptide from the kidney.
J. Biol. Chem.
271:
10654-10659,
1996[Abstract/Free Full Text].
5.
Barnes, G. L.
Viral infections.
In: Pediatric Gastrointestinal Disease, edited by A. W. Walker,
P. R. Durie,
J. R. Hamilton,
J. A. Walker-Smith,
and J. B. Watkins. St. Louis, MO: Mosby Year Book, 1996, chapt. 27, p. 654-663.
6.
Berschneider, H. M.,
M. R. Knowles,
R. G. Azizkhan,
R. C. Boucher,
N. A. Tobey,
R. C. Orlando,
and
D. W. Powell.
Altered intestinal chloride transport in cystic fibrosis.
FASEB J.
2:
2625-2629,
1988[Abstract/Free Full Text].
7.
Chao, A. C.,
J. H. Widdicombe,
and
A. S. Verkman.
Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator.
J. Membr. Biol.
113:
193-202,
1990[Medline].
8.
Chiu, V. C.,
and
D. H. Haynes.
High and low affinity Ca2+ binding to the sarcoplasmic reticulum: use of a high-affinity fluorescent calcium indicator.
Biophys. J.
18:
3-22,
1977[Abstract].
9.
De Clerck, L. S.,
C. H. Bridts,
A. M. Mertens,
M. M. Moens,
and
W. J. Stevens.
Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cell and the effect of fluorochromes on cellular function.
J. Immunol. Methods
172:
115-124,
1994[Medline].
10.
Desai, G. N.,
J. Sahi,
P. M. Reddy,
J. Venkatasubramanian,
D. Vidyasagar,
and
M. C. Rao.
Chloride transport in primary cultures of rabbit colonocytes at different stages of development.
Gastroenterology
111:
1541-1550,
1996[Medline].
11.
Diener, M.
Segmental differences along the crypt axis in the response of cell volume to secretagogues or hypotonic medium in the rat colon.
Pflügers Arch.
426:
462-464,
1994[Medline].
12.
Diener, M.,
and
V. Gartmann.
Effect of somatostatin on cell volume, Cl currents, and transepithelial Cl transport in rat distal colon.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G1043-G1052,
1994[Abstract/Free Full Text].
13.
Dong, Y.,
C. Q. Zeng,
J. M. Ball,
M. K. Estes,
and
A. P. Morris.
The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production.
Proc. Natl. Acad. Sci. USA
412:
3960-3965,
1997.
14.
Donowitz, M.,
M. E. Cohen,
M. Gould,
and
G. W. G. Sharp.
Elevated intracellular Ca2+ acts through protein kinase C to regulate rabbit ileal NaCl absorption.
J. Clin. Invest.
83:
1953-1962,
1989[Medline].
15.
Dorin, J. R.
Development of mouse models for cystic fibrosis.
J. Inherit. Metab. Dis.
18:
495-500,
1995[Medline].
16.
Dubois-Dalcq, M.,
K. V. Holmes,
and
B. Rentier.
Assembly of rotaviruses.
In: Assembly of Enveloped RNA Viruses, edited by D. W. Kingsbury. New York: Springer-Verlag, 1986, chapt. 10, p. 171-182.
17.
Emmer, E.,
R. P. Rood,
J. H. Wesolek,
M. E. Cohen,
R. S. Braithwaite,
G. W. G. Sharp,
H. Murer,
and
M. Donowitz.
Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter.
J. Membr. Biol.
108:
207-215,
1989[Medline].
18.
Estes, M. K.,
E. L. Palmer,
and
J. F. Obijeski.
Rotaviruses: a review.
Curr. Top. Microbiol. Immunol.
105:
123-184,
1983[Medline].
19.
Frizzel, R. A.,
and
A. P. Morris.
Chloride conductances of salt-secreting epithelial cells.
In: Current Topics in Membranes
Chloride Channels, edited by W. B. Guggino. San Diego, CA: Academic, 1994, chapt. 6, p. 173-214, 1994.
20.
Goldstein, J. L.,
J. Sahi,
M. Bhuva,
T. J. Layden,
and
M. C. Rao.
Eschericha coli heat-stable enterotoxin-mediated colonic Cl
secretion is absent in cystic fibrosis.
Gastroenterology
107:
950-956,
1994[Medline].
21.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
22.
Halm, D. R.,
K. L. Kirk,
and
K. C. Sathiakumar.
Stimulation of Cl permeability in colonic crypts of Lieberkuhn measured with a fluorescent indicator.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G423-G431,
1993[Abstract/Free Full Text].
23.
Hasty, P.,
W. K. O'Neal,
K. Q. Liu,
A. P. Morris,
Z. Bebok,
G. B. Shumyatsky,
T. Jilling,
E. J. Sorscher,
A. Bradley,
and
A. L. Beaudet.
Severe phenotype in mice with termination mutation in exon 2 of cystic fibrosis gene.
Somat. Cell Mol. Genet.
21:
177-187,
1995[Medline].
24.
Illsley, N. P.,
and
A. S. Verkman.
Membrane chloride transport measured using a chloride-sensitive fluorescent probe.
Biochemistry
26:
1215-1219,
1987[Medline].
25.
Kapikian, A. Z.,
and
R. M. Chanock.
Rotaviruses.
In: Virology, edited by B. N. Fields,
D. M. Knipe,
and P. M. Howley. New York: Raven, 1996, chapt. 55, p. 1657-1708.
26.
Khurana, S.,
S. Kreydiyyeh,
A. Aronzon,
W. A. Hoogerwerf,
S. G. Rhee,
M. Donowitz,
and
M. E. Cohen.
Asymmetric signal transduction in polarized ileal Na(+)absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2-PLC and translocates PLC
1 only to the brush border.
Biochem. J.
313:
509-518,
1996[Medline].
27.
Krapf, R.,
N. P. Illsley,
H. C. Tseng,
and
A. S. Verkman.
Structure-activity relationships of chloride-sensitive fluorescent indicators for biological application.
Anal. Biochem.
169:
142-150,
1988[Medline].
28.
Lakowicz, J. R.
Quenching of fluorescence.
In: Principles of Fluorescence Spectroscopy. New York: Plenum, 1983, chapt. 9, p. 257-289.
29.
Leipziger, J.,
D. Kerstan,
R. Nitschke,
and
R. Greger.
ATP increases [Ca2+]i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa.
Pflügers Arch.
434:
77-83,
1997[Medline].
30.
Lencer, W. I.,
G. Cheung,
G. R. Strohmeier,
M. G. Currie,
A. J. Ouellette,
M. E. Selsted,
and
J. L. Madara.
Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3.
Proc. Natl. Acad. Sci. USA
94:
8585-8589,
1997[Abstract/Free Full Text].
31.
MacLeod, R. J.,
J. R. Hamilton,
A. Bateman,
D. Belcourt,
J. Hu,
H. P. Bennett,
and
S. Solomon.
Corticostatic peptides cause nifedipine-sensitive volume reduction in jejunal villus enterocytes.
Proc. Natl. Acad. Sci. USA
88:
552-556,
1991[Abstract].
32.
Mathews, D. E.,
and
V. T. Farewell.
Fisher test for 2 × 2 contingency tables.
In: Using and Understanding Medical Statistics (3rd ed.). New York: Karger, 1996, chapt. 3, p. 19-37.
33.
Michelangeli, F.,
F. Liprandi,
M. E. Chemello,
M. Ciarlet,
and
M. C. Ruiz.
Selective depletion of stored calcium by thapsigargin blocks rotavirus maturation but not the cytopathic effect.
J. Virol.
69:
3838-3847,
1995[Abstract].
34.
Michelangeli, F.,
M. C. Ruiz,
J. R. del Castillo,
J. E. Ludert,
and
F. Liprandi.
Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells.
Virology
181:
520-527,
1991[Medline].
35.
Morris, A. P.,
K. L. Kirk,
and
R. A. Frizzell.
Simultaneous analysis of cell Ca2+ and Ca2+-stimulated chloride conductance in colonic epithelial cells.
Cell Regul.
1:
951-963,
1990[Medline].
36.
O'Grady, S. M.,
H. C. Palfrey,
and
M. Field.
Characteristics and functions of Na-K-Cl cotransport in epithelial tissues.
Am. J. Physiol.
253 (Cell Physiol. 22):
C177-C192,
1987[Abstract/Free Full Text].
37.
O'Loughlin, E. V.,
D. M. Hunt,
K. J. Gaskin,
D. Stiel,
I. M. Bruzuszcak,
H. C. Martin,
C. Bambach,
and
R. Smith.
Abnormal epithelial transport in cystic fibrosis jejunum.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G758-G763,
1991[Abstract/Free Full Text].
38.
Osborne, M. P.,
S. J. Haddon,
A. J. Spencer,
J. Collins,
W. G. Starkey,
T. S. Wallis,
G. J. Clarke,
K. J. Worton,
D. C. Candy,
and
J. Stephen.
An electron microscopic investigation of time-related changes in the intestine of neonatal mice infected with murine rotavirus.
J. Pediatr. Gastroenterol. Nutr.
7:
236-248,
1988[Medline].
39.
Poruchynsky, M. S.,
D. R. Maass,
and
P. H. Atkinson.
Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER.
J. Cell Biol.
114:
651-656,
1991[Abstract].
40.
Rabalais, G. P.
Recent advances in the prevention and treatment of diarrheal diseases.
Curr. Opin. Infect. Dis.
9:
210-213,
1996.
41.
Ram, S. J.,
and
K. L. Kirk.
Cl
permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl
permeability in cystic fibrosis.
Proc. Natl. Acad. Sci. USA
86:
10166-10170,
1989[Abstract].
42.
Rich, D. P.,
M. P. Anderson,
R. J. Gregory,
S. H. Cheng,
S. Paul,
D. M. Jefferson,
J. D. McCann,
K. W. Klinger,
A. E. Smith,
and
M. J. Welsh.
Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.
Nature
347:
358-363,
1990[Medline].
43.
Riordan, J. R.,
J. M. Rommens,
B. S. Kerem,
N. Alon,
R. Rozmahel,
Z. Grzelczak,
J. Zielenski,
S. Lok,
N. Plavsic,
J.-L. Chou,
M. L. Drumm,
M. C. Iannuzzi,
F. S. Collins,
and
L.-C. Tsui.
Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.
Science
245:
1066-1073,
1989[Medline].
44.
Rubenstein, S.,
R. Moss,
and
N. Lewiston.
Constipation and meconium ileus equivalent in patients with cystic fibrosis.
Pediatrics
78:
473-479,
1986[Abstract].
45.
Sahi, J.,
J. L. Goldstein,
T. J. Layden,
and
M. C. Rao.
Cyclic AMP- and phorbol ester-regulated Cl
permeabilities in primary cultures of human and rabbit colonocytes.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G846-G855,
1994[Abstract/Free Full Text].
46.
Sahi, J.,
M. P. Wiggins,
G. B. Gibori,
T. J. Layden,
and
M. C. Rao.
Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes.
J. Cell. Physiol.
168:
276-283,
1996[Medline].
47.
Satoh, Y.,
Y. Habara,
K. Ono,
and
T. Kanno.
Carbamylcholine- and catecholamine-induced intracellular calcium dynamics of epithelial cells in mouse ileal crypts.
Gastroenterology
108:
1345-1356,
1995[Medline].
48.
Shalon, L. B.,
and
J. W. Adelson.
Cystic fibrosis. Gastrointestinal complications and gene therapy.
Pediatr. Clin. North Am.
43:
157-196,
1996[Medline].
49.
Starkey, W. G.,
J. Collins,
D. C. A. Candy,
A. J. Spencer,
M. P. Osborne,
and
J. Stephen.
Transport of water and electrolytes by rotavirus-infected mouse intestine: a time course study.
J. Pediatr. Gastroenterol. Nutr.
11:
254-260,
1990[Medline].
50.
Stern, M.,
F. M. Munkonge,
N. J. Caplen,
F. Sorgi,
L. Huang,
D. M. Geddes,
and
E. W. Alton.
Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects.
Gene Ther.
2:
766-774,
1995[Medline].
51.
Strabel, D.,
and
M. Diener.
Evidence against direct activation of chloride secretion by carbachol in the rat distal colon.
Eur. J. Pharmacol.
274:
181-191,
1995[Medline].
52.
Stutzin, A.,
A. L. Eguiguren,
L. P. Cid,
and
F. V. Sepulveda.
Modulation by extracellular Cl
of volume-activated organic osmolyte and halide permeabilities in HeLa cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C999-C1007,
1997[Abstract/Free Full Text].
53.
Taylor, C. J.,
P. S. Baxter,
J. Hardcastle,
and
P. T. Hardcastle.
Failure to induce secretion in jejunal biopsies from children with cystic fibrosis.
Gut
29:
957-962,
1988[Abstract].
54.
Tsui, L. C.
The cystic fibrosis transmembrane conductance regulator gene.
Am. J. Respir. Crit. Care Med.
151:
S47-S53,
1995[Medline].
55.
Valverde, M. A.,
J. A. O'Brien,
F. V. Sepulveda,
R. A. Ratcliff,
M. J. Evans,
and
W. H. Colledge.
Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice.
Proc. Natl. Acad. Sci. USA
92:
9038-9041,
1995[Abstract].
56.
Valverde, M. A.,
J. A. O'Brien,
F. V. Sepulveda,
R. Ratcliff,
M. J. Evans,
and
W. H. Colledge.
Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca(2+) mediated secretagogue-induced volume decrease in small-intestinal crypts.
Pflügers Arch.
425:
434-438,
1993[Medline].
57.
Vellenga, L.,
H. J. Egberts,
T. Wensing,
J. E. van Dijk,
J. M. V. M. Mouwen,
and
H. J. Breukink.
Intestinal permeability in pigs during rotavirus infection.
Am. J. Vet. Res.
53:
1180-1183,
1992[Medline].
58.
Verkman, A. S.
Development and biological applications of chloride-sensitive fluorescent indicators.
Am. J. Physiol.
259 (Cell Physiol. 28):
C375-C388,
1990[Abstract/Free Full Text].
59.
Verkman, A. S.,
A. C. Chao,
and
T. Hartmann.
Hormonal regulation of Cl transport in polar airway epithelia measured by a fluorescent indicator.
Am. J. Physiol.
262 (Cell Physiol. 31):
C23-C31,
1992[Abstract/Free Full Text].
60.
Welsh, M. J.
Cystic fibrosis.
In: Molecular Biology of Membrane Transport Disorders, edited by S. G. Schultz,
T. E. Andreoli,
A. M. Brown,
D. M. Fambrough,
J. F. Hoffman,
and M. J. Welsh. New York: Plenum, 1996, p. 605-619.
61.
Woll, E.,
M. Gschwentner,
J. Furst,
S. Hofer,
G. Buemberger,
A. Jungwirth,
J. Frick,
P. Deetjen,
and
M. Paulmichl.
Fluorescence-optical measurements of chloride movements in cells using the membrane-permeable dye diH-MEQ.
Pflügers Arch.
432:
486-493,
1996[Medline].
62.
Wright, E. M.
The intestinal Na+/glucose cotransporter.
Annu. Rev. Physiol.
55:
575-589,
1993[Medline].
63.
Yao, B.,
D. L. Hogan,
K. Bukhave,
M. A. Koss,
and
J. I. Isenberg.
Bicarbonate transport by rabbit duodenum in vitro: effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate.
Gastroenterology
104:
732-740,
1993[Medline].
64.
Zhang, M.,
C. Q.-Y. Zeng,
Y. Dong,
J. M. Ball,
L. J. Saif,
A. P. Morris,
and
M. K. Estes.
Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence.
J. Virol.
72:
3666-3672,
1997[Abstract/Free Full Text].
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