| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Research Communications |
Division of Clinical Pharmacology, Departments of Medicine and Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and
* Departments of Medicine and Pharmacology, Division of Cardiovascular Diseases, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55905, USA
1Correspondence: Scott A. Waldman, Division of Clinical Pharmacology, Departments of Medicine and Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 1100 Walnut Street, MOB 813, Philadelphia, PA 19107. E-mail: waldmans{at}jeflin.tju.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: E. coli heat-stable enterotoxin • intestinal cell water secretion • cyclic GMP • 2-substituted adenine nucleotides • CFTR-mediated chloride current
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3)
. One of the most common causes of diarrheal disease in
humans is bacteria that secrete heat-stable enterotoxins
(STs)2
(1
2
3)
. ST induces diarrhea by binding to
the extracellular domain of guanylyl cyclase C (GCC), located
exclusively in the brush border of intestinal epithelial cells (4,
5)
.
Toxin-receptor interaction activates the associated intracellular
guanylyl cyclase catalytic domain, resulting in accumulation of
[cGMP]i (6
7
8)
. This cyclic nucleotide
activates cGMP-dependent protein kinase II, which phosphorylates the
cystic fibrosis transmembrane conductance regulator (CFTR), inducing an
electrogenic chloride current that drives intestinal secretion (6,
9,
10)
. Despite the identification of the molecular events mediating
ST-induced diarrhea, therapeutic options remain nonspecific and limited
to rehydration, antimotility agents, and antimicrobials (11)
. Thus far,
the molecules and the interactions that mediate intestinal secretion
have not been exploited to develop targeted therapy against
enterotoxigenic diarrhea.
In this regard, 2-substituted nucleotides are unique candidates
for developing mechanism-based approaches to antisecretory therapy.
2-Substituted adenine nucleotides, including 2-chloroATP (2ClATP),
allosterically inhibit native and recombinant GCC, uncoupling ST
binding from guanylyl cyclase activation in cell-free systems (12,
13)
.
Although phosphorylation of the 2-substituted purine ring at the 5'
position is required for interruption of guanylyl cyclase signaling,
the highly charged phosphate groups render nucleotides
membrane-impermeable and therefore ineffective as intracellular
therapeutic agents (14)
. In contrast, the nucleoside analog
2-chloroadenosine (2ClAdo) is specifically transported into intestinal
cells and phosphorylated, resulting in accumulation of
[2ClATP]i (14)
. Treatment of human intestinal
cells with the cell-permeant analog 2ClAdo disrupts ST-induced guanylyl
cyclase signaling and electrogenic chloride current (14)
. However,
whether this strategy could be exploited to prevent intestinal
secretion underlying enterotoxigenic diarrhea remains undefined.
The first step in establishing the utility of nucleotide disruption of
guanylyl cyclase signaling to treat toxigenic diarrhea is to evaluate
the effects of 2ClAdo on ST-induced water secretion by intestinal
cells. Caco-2 human intestinal cells differentiate in vitro
and form intact epithelial monolayers that mimic the ability of the
intact intestine to secrete water in a vectorial fashion (15,
16)
. This
model has been employed previously to examine the effects of various
secretagogues and antisecretory agents on water transport in
vitro (17
18
19)
. This study examined the ability of 2ClAdo to
interrupt ST stimulation of chloride current and water secretion in
Caco-2 cells and provides the first evidence that disruption of
transmembrane signaling can be effective mechanism-based therapy for
enterotoxigenic diarrhea.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
13
14)
. All reagents
commercially obtained were of the highest analytical grade.
Cell culture
Caco-2 human intestinal epithelial cells (ATCC HTB37; American
Type Culture Collection, Rockville, Md.) are an established model of
intestinal transport (14)
. In these studies, Caco-2 cells were seeded
in polycarbonate membrane insert wells (0.6 cm2,
0.4-µm pore size, Millipore, Bedford, Mass.) at a density of 2 x 104 cells/insert (17
18
19)
. Cells for transport
studies were employed between passage numbers 35 and 55. Cells were
maintained at 37°C in F12/minimum essential medium (MEM) with 10%
fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin
and 1% nonessential amino acids in an atmosphere of 95%
O2/5% CO2.
Nucleic acid extraction
Total RNA was extracted from samples through the use of the
Gentra Systems RNA Extraction Kit (Gentra Systems, Minneapolis, Minn.).
Only samples exhibiting intact 28s and 18s ribosomal RNA were analyzed.
RNA preparations were stored in diethylpyrocarbonate (DEPC) -treated
water (RNase-free) at -80°C. To remove contaminating genomic DNA,
RNA was treated with 1 unit/µl of RQ1 RNase-free DNase (Promega,
Madison, Wis.) for 15 min at 37°C, followed by a 30-min incubation
with 1 µl of RNase inhibitor (Panvera, Madison, Wis.).
Quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) analyses
Quantitative RT-PCR analysis was performed as outlined
previously with modifications (20)
. Briefly, an internal standard for
the RT-PCR that contained a single base mutation in native human GCC
creating a novel BamHI restriction site (sense strand
8195'TGGATCT3'824
8195'TGGATCC3'824)
was generated by in vitro transcription (20)
. This mutant
RNA construct was added as an internal standard to RNA extracted from
Caco-2 cells. These mixtures were subjected to reverse transcription
employing a human GCC antisense primer (nucleotides 952–973)
complementary to the sequences contained within the standard and target
RNA. Reverse transcription of total RNA (1 µg) was performed with
0.25 units/µl of AMV reverse transcriptase XL (Panvera) containing 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM
each of dATP, dCTP, dGTP, and dTTP, 1 unit/µl RNase inhibitor, and 1
µM of antisense primer in a total volume of 20 µl (20)
. Thermal
cycling proceeded for 1 cycle at 58°C for 30 min, 99°C for 5 min,
and 4°C for 5 min The resultant cDNA, containing GCC cDNA and DNA
complimentary to the standard, was subjected to PCR in the same
reaction tube and included 2.5 units of TaKaRa Taq
polymerase (Panvera) in 50 µl of: 10 mM Tris-HCl, 50 mM KCl, 2.5 mM
MgCl2, and 0.2 µM of a GCC-specific sense
primer (nucleotides 711–733) (20)
end-labeled with the use of T4
polynucleotide kinase and [
32P]dATP.
Incubation and thermal cycling conditions were as follows: 95°C for 2
min, 1 cycle; 94°C for 30 s, 58°C for 30 s, 72°C for
90 s, 35 cycles; 72°C for 7 min, 1 cycle. After RT-PCR, samples
were stored at 4°C until analysis within 24 h of amplification.
Amplification products were treated with BamHI, separated by
agarose gel electrophoresis, visualized by ethidium bromide, and the
bands corresponding to amplification products for human GCC (263 bp)
and the standard (150 and 110 bp) excised and radiation quantified by
scintillation spectroscopy. In these experiments, decreasing numbers of
standard molecules were added to a constant (1 µg) amount of total
RNA extracted from target cells and subsequently analyzed by RT-PCR.
The ratio of radioactivity recovered in the standard vs. target
amplification products (y-axis) was plotted against the
number of standard molecules in the incubation (x-axis). The
number of standard molecules (x-axis) corresponding to a
ratio of 1.0 (y-axis) reflects the number of target
molecules in 1 µg of total RNA (20)
.
Water transport studies
Caco-2 cells grown to confluence as a monolayer on polycarbonate
membrane inserts were mounted between two chambers of a water transport
measurement device (Millipore) (17
18
19)
. The two chambers of the device
separated by the cell insert represented apical (mucosal) and
basolateral (serosal) chambers. A 5-cm hydrostatic pressure was applied
on the apical side and the apical bathing solution was mixed by air
agitation (17
18
19)
. A magnetic stirrer was employed to ensure adequate
mixing of the basolateral chamber. Each chamber received 5.5 ml of
Ringer solution containing the following (in mM): NaCl, 114; KCl, 5;
Na2HPO4,1.65;
NaH2PO3, 0.3;
CaCl2, 1.25; MgCl2, 1.1;
NaHCO3, 25; and glucose, 10. In studies examining
the dependency of ST-induced water transport on chloride, an equimolar
concentration of sulfate replaced chloride in the Ringer solution (21)
.
Ringer solution was gassed with 95% O2/5%
CO2 and maintained at 37°C. In studies
examining the effect of 2ClAdo on ST-induced water secretion cells were
preincubated in OPTI-MEM serum-free media (Life Technologies, Inc.)
containing the indicated concentrations of 2ClAdo for 24 h. Water
transport studies were initiated by adding
[3H]H2O (5.5 µl,
1.81 x 107 cpm) to apical or basolateral
(donor) chambers. [14C]Mannitol was employed to
examine paracellular, compared to transcellular, transport (17,
18)
.
Mannitol transport studies were initiated by adding
[14C]mannitol (10 µL, 5 x
105 cpm) to apical or basolateral chambers. At
2-min intervals, 1.0-ml aliquots of the buffer from the opposite
(receiver) chamber were removed and immediately replaced with an equal
amount of fresh buffer. Radioactivity in aliquots was quantified in a
Packard 1900 TR liquid scintillation counter (Downers Grove, Ill.).
Quantification of water flux
Water flux, Jw, expressed as
microliters per square centimeter per minute, was calculated as
follows: Jw =
[(Vt1)(Rt1)-(Vt2)(Rt2)]/[(A)(R)(T)]
(Equation 1), where Vt1 is the volume
of receiver chamber at the measuring time point,
Rt1 is the radioactivity per unit
volume of Vt1,
Vt2 is the volume of receiver chamber
at the point of the last sample taken,
Rt2 is the radioactivity per unit
volume of Vt2, A is the
surface area of the cell monolayer, R is the initial
radioactivity per unit volume of donor chamber, and T is the
elapsed time (22)
. Where indicated, paracellular transport by Caco-2
cell monolayers was examined with the use of mannitol (17,
18)
.
Mannitol permeability, Pm, was
calculated according to the following equation:
Pm =
[(V)(dC/dt)]/[(A)(Co)]
(Equation 2), where V is the volume of receiver
chamber, dC/dt is the change in mannitol
concentration in the receiver chamber, A is the surface area
of the cell monolayer, and CO is the initial
concentration of mannitol in the donor chamber (23)
. Water flux,
calculated as described by Equation 1, was determined in
(basolateralapical) and (apicalbasolateral) directions. Net water
flux was calculated as the arithmetic difference between the fluxes in
basolateralapical and apicalbasolateral directions. Peak
water flux represents the net water flux during the 2-min interval of
maximum net flux.
[Cyclic GMP]i accumulation in Caco-2 cells
Caco-2 cells were seeded in 24-well plates and grown for at
least 15 days to acquire fully differentiated enterocyte
characteristics (see Fig. 1
). Cells were incubated employing conditions
described in the studies of water transport, above, and previously
(14)
. Cells were pre-incubated with either vehicle or 1 mM 2ClAdo for
24 h before incubation with 200 nM ST for 10 min (14)
.
Intracellular nucleotides were extracted by trichloroacetic acid (TCA)
precipitation and cGMP quantified by radioimmunoassay (RIA) (14)
. TCA
pellets were employed for determination of protein content.
|
Electrophysiological recordings
The perforated mode of the whole-cell patch-clamp recording,
which limits dialysis of intracellular signaling molecules, was applied
to Caco-2 cells (14,
24)
. Membrane potential was controlled through the
electrical access obtained by membrane perforation induced by
amphotericin B in the localized area under the patch pipette (3–5
M
). The pipette solution supplemented by amphotericin B (200–240
mg/ml) contained (in mM): K+-gluconate, 140;
MgCl2, 5; EGTA, 1; and HEPES-KOH, 5 (pH 7.3). The
chloride-rich bath solution contained (in mM): NaCl, 136.5; KCl, 5.4;
CaCl2, 1.8; MgCl2, 0.5;
glucose, 5.5; and HEPES-NaOH, 5 (pH 7.4). In low-chloride solution,
chloride was replaced by methanesulfonate. Voltage-clamp recordings
were performed with a patch-clamp amplifier Axopatch 1-C (Axon
Instruments) and data were acquired and analyzed using the BioQuest
software (24
25
26)
.
Miscellaneous
Protein was quantified through the use of a commercial
colorimetric assay using bovine serum albumin as the standard (BCA,
Pierce, Rockford, Ill.) (12
13
14)
. Alkaline phosphatase was quantified
as described previously (27)
. Data are presented as means ±
SE of at least three experiments, comparisons reflect
application of Student's t test, and values of
P < 0.05 were taken as significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). GCC mRNA (target mRNA) was quantified by RT-PCR employing
GCC-specific primers and an exogenous standard RNA (mutant GCC) in all
reactions. Decreasing quantities of standard mRNA were added to fixed
quantities of Caco-2 cell total RNA and RT-PCR was performed. The
concentration of standard at which target and standard amplification
products are equivalent reflects the quantity of GCC (target) mRNA in
the amplification reaction (20)
. GCC mRNA increased significantly
(P<0.05) from 3.0 ± 2 x
107 to 3.5 ± 0.5 x
108 copies/µg total RNA (n=3) in
Caco-2 cells grown for 5 or 10 days and was stable after longer times
in culture (Fig. 1B)
(28)
.
Vectorial transport functions characteristic of mature enterocytes
develop in conjunction with the appearance of brush border enzymes
(15
16
17
18
19)
. Total (transcellular + paracellular) water fluxes in
apical basolateral and basolateral apical directions were
measured using [3H]water, and net water fluxes
(secretion or absorption) reflect the arithmetic difference of these
components (Fig. 2
). Water flux in the apical basolateral direction predominated in
Caco-2 cells after 3 days in culture, yielding a large net absorptive
flux (-147 ± 9.3 µl/cm2/min), reflecting
paracellular water movement between cells that have not yet developed
junctional complexes, driven by hydrostatic pressure. Caco-2 cell
monolayers exhibited significantly (P<0.05) reduced water
permeability and developed a small net secretory flux (basolateral
apical; 1.7 ± 1.8 µl/cm2/min) after 15
days, which was stable after longer times in culture (Fig. 2)
(15
16
17
18
19)
.
Studies examining the effects of ST on water transport employed cells
after 15 days in culture.
|
ST induces chloride current in Caco-2 cells
In Caco-2 cells, ST (100 nM) induced a gradual increase in outward
current (Fig. 3
A). At a membrane potential of +10 mV, the current increased
from 0.04 ± 0.02 to 0.19 ± 0.06 nA after a 20-min exposure
to the enterotoxin (mean values of five experiments; Fig. 3A
). The
ST-induced current was abolished by bathing Caco-2 cells in
low-chloride solution (Fig. 3A
). Removal of chloride from
the bathing solution abolished the effect of ST throughout the range of
imposed membrane potential values (traces 1–3 in Fig. 3A
1). Return of chloride to the
bathing medium restored the ST-induced outward current (Fig. 3A
and trace 4 in Fig. 3A
1). This current was sensitive to
the sulfonylurea glyburide (100 [gm]M; Fig. 3A
and trace 5
in Fig. 3A
1), a known inhibitor of the
chloride conductance associated with the cystic fibrosis transmembrane
conductance regulator (CFTR) (29)
. The reversal potential for the
ST-induced chloride current was estimated at -68 ± 2 mV (mean
value of three experiments), a value obtained as the intercept of the
current-voltage curves recorded in the presence and absence of chloride
in the bathing solution (Fig. 3A
1).
|
Figure 3B
provides the current-voltage relationship for the
ST-induced current. The ST-induced component of the total current was
obtained by subtracting current recorded in the absence of ST from
current recorded in the presence of ST (Fig. 3B
). Similarly,
the current-voltage relationship was also constructed for the
8-bromo-cGMP-induced component of the total current. Comparison of ST-
and 8-bromo-cGMP-induced currents revealed a similar current-voltage
relationship with a characteristic outward rectification and reversal
potential (Fig. 3B
1). Thus, cGMP is
the likely mediator of the ST effect on chloride conductance in Caco-2
cells (14)
.
ST induces chloride-dependent water secretion in Caco-2 cell
monolayers
ST (200 nM) doubled the basal secretory water flux from 2.8 ± 0.1 to 6.3 ± 1.2 µl/cm2/min
(n=3; Fig. 4
, A and B). The increase in net water secretion
reflected an increase in water flux in the basolateral apical
direction, without alteration of water flux in the apical
basolateral direction (Fig. 4A
1). In
contrast, ST did not alter the permeability of Caco-2 cell monolayers
to mannitol, a marker of paracellular transport (Fig. 4, A
1 and
A2). ST-induced current is
chloride-dependent in Caco-2 cells (Fig. 3A
). Similarly,
replacement of chloride with sulfate in apical and basolateral bathing
solutions abolished ST stimulation of net water secretion (Fig. 4C
). 8-Bromo-cGMP mimicked the effects of ST to induce
chloride-dependent current in Caco-2 cells (Fig. 3B
1). Similarly, 8-bromo-cGMP induced
net water secretion (basolateral apical) that was identical in time
course and intensity to that induced by ST (Fig. 4D
). Thus,
cGMP probably mediates ST stimulation of water secretion by Caco-2
cells.
|
2ClAdo prevents ST-induced chloride current
In Caco-2 cells treated with 1 mM 2ClAdo (20 h), ST (100 nM)
failed to induce chloride current (Fig. 5
, A and A1). On average, in
2ClAdo-treated cells clamped at +10 mV, the current was 0.05 ±
0.02 and 0.06 ± 0.01 nA in the absence and presence of ST.
However, in the same cells, 2ClAdo did not prevent 8-bromo-cGMP from
inducing a current (Fig. 5
, A and A1) that was
chloride-dependent (Fig. 5B
). On average, at +10 mV of
membrane potential, 5 mM 8-bromo-cGMP induced a current of 0.27 ±
0.07 (n=3) nA in 2ClAdo-treated cells, a value that was not
significantly different from 0.22 ± 0.04 nA (n=3)
obtained in untreated cells (Fig. 5C
).
|
2ClAdo prevents ST-induced water secretion
2ClAdo (2 mM; 20 h) inhibited net water secretion induced by
ST (200 nM) (Fig. 6
A) in a concentration-dependent fashion (Fig. 6B
)
with a Ki of 0.72 mM (Fig. 6B
, inset).
Similarly, 2ClAdo (1 mM; 20 h) reduced ST-induced
[cGMP]i accumulation >80% in Caco-2 cells
(Fig. 6C
). Although ST did not induce water secretion in
Caco-2 cells pretreated with 2ClAdo, 8-bromo-cGMP (2 mM) increased net
water secretion in those cells (Fig. 6D
). Indeed, the effect
of 8-bromo-cGMP on net water secretion was nearly identical in Caco-2
cells incubated in the presence and absence of 2ClAdo (Fig. 6E
). Thus, 2ClAdo interrupts ST-stimulated signaling,
preventing water secretion by Caco-2 cells. Interruption appears to be
at the level of cGMP production, rather than ion channel function,
since ST-induced [cGMP]i accumulation is
inhibited in Caco-2 cells incubated with 2ClAdo, whereas 8-bromo-cGMP
stimulates chloride conductance and water secretion in those cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The signaling cascade mediating ST-induced secretion is well
characterized. Binding of ST to GCC activates the intrinsic guanylyl
cyclase catalytic activity, resulting in
[cGMP]i accumulation (6
7
8)
. This cyclic
nucleotide activates membrane-associated cGMP-dependent protein kinase
II, promoting phosphorylation of the CFTR (9,
10)
. Chloride current
conducted by the CFTR is presumed to drive water transport into the
intestinal lumen, resulting in secretory diarrhea (6,
9)
. Indeed,
intestinal cells lacking functional CFTR do not develop electrolyte
fluxes, and animals lacking that functional protein do not develop
diarrhea induced by ST (30)
. The cell-permeant analog, 8-bromo-cGMP,
mimics the effects of ST, inducing phosphorylation of the CFTR,
intestinal cell chloride current in vitro, and intestinal
secretion in vivo supporting the model in which CFTR is the
downstream response element of GCC (7,
9,
10,
14)
. GCC is absolutely
required for ST induction of diarrhea, and animals from which
expression of this protein has been eliminated do not develop
intestinal secretion in response to that toxin (31,
32)
.
We previously demonstrated that 2-substituted nucleotides, including
2ClATP, inhibit native and recombinant GCC in cell-free systems (12,
13)
. Inhibition of ST-stimulated guanylyl cyclase by 2ClATP is
allosteric and phosphorylation of the 2-substituted purine ring at the
5' position is required (12,
13)
. However, the highly charged phosphate
groups of 2-substituted nucleotides render them impermeable to cells.
Recently, we developed a cell-permeant 2-substituted nucleoside analog,
2ClAdo, which can be transported into intestinal cells and
phosphorylated to 2ClATP (14)
. Caco-2 cells incubated with 2ClAdo
accumulate [2ClATP]i, which disrupts ST-induced
guanylyl cyclase signaling (14)
. These earlier studies suggested that
interruption of ST signaling by 2-substituted nucleotides might be
exploited to develop targeted therapy against enterotoxigenic diarrhea
employing 2ClAdo as a prodrug (14)
. However, models of ST-induced
secretion developed in vivo are particularly unsuited to
examine the therapeutic efficacy of prodrugs that require
time-dependent transport and metabolic conversion to the active moiety.
The pharmacokinetic barriers to maintaining
[2ClAdo]e (prodrug) that produce effective
[2ClATP]i (active drug) in enterocytes in
vivo limits examination of the utility of 2-substituted
nucleosides to prevent ST-induced diarrhea. The potential utility of
2-substituted nucleosides to interrupt ST signaling and prevent
intestinal fluid secretion can best be examined in vitro
where [2ClAdo]e and
[2ClATP]i can be controlled. Caco-2 human
intestinal cells form intact epithelial monolayers in vitro
that mimic the ability of the intact intestine to secrete water in a
vectorial fashion (15
16
17
18
19)
. Caco-2 monolayers expressing GCC and
exhibiting differentiated enterocyte-like transport characteristics
could serve as an ideal model in which to examine the hypothesis that
allosteric disruption of GCC signaling prevents ST-induced water
secretion.
These studies demonstrate, for the first time, that ST induces
transcellular water transport associated with CFTR-mediated chloride
current by Caco-2 cells. Incubating cells with 2ClAdo, which interrupts
activation of GCC (14)
, prevents ST induction of cGMP accumulation,
chloride current, and water secretion. Prevention of water secretion
reflects uncoupling of ligand binding and effector activation by GCC,
rather than inhibition of a process further downstream in the signal
sequence, since 8-bromo-cGMP restored water transport in Caco-2 cells
incubated with 2ClAdo. These studies provide evidence that allosteric
inhibition of GCC might be exploited to prevent intestinal secretion
and diarrhea induced by ST-producing organisms in humans.
Tight epithelia transfer water by a transcellular route, presumably
through water channels located in apical membranes (33
34
35)
. Such
epithelia, characteristic of renal proximal convoluted tubules, exhibit
dissociation of mannitol and water permeabilities in response to a
transepithelial osmotic gradient (18,
35)
. In contrast, leaky epithelia
transfer water by a paracellular route through intracellular junctions
(18)
. These epithelia, characterized by rat jejunum and cecum, exhibit
a strong association of mannitol and water permeabilities in response
to a transepithelial osmotic gradient (18)
. Previous studies
demonstrated that Caco-2 cell monolayers transported water through
paracellular, but not transcellular routes because transepithelial
osmotic gradients yielded coordinated increases in mannitol and water
permeabilities (18)
. In the present studies, ST increased water
permeability without increasing mannitol permeability, demonstrating
that Caco-2 cells form watertight monolayers. These studies suggest the
possibility that differentiated Caco-2 cells express water channels
mediating transcellular transport in their apical membranes (18,
33
34
35)
. Differences in the present, compared with previous, results
may reflect differences in Caco-2 cell subtypes employed or the method
of establishing transepithelial osmotic gradients. Alternatively, ST
may alter water channels through receptor-mediated cGMP-dependent
or -independent mechanisms.
In conclusion, these studies demonstrate that inhibition of GCC signaling in intact Caco-2 human intestinal cells by 2-ClAdo prevents ST-induced chloride current and water secretion. These studies establish a critical proof of principal that translates targeted disruption of signaling by a novel biochemical pathway into prevention of cellular pathophysiological consequences of enterotoxin exposure. They form the foundation for future studies exploiting allosteric inhibition of GCC as a novel mechanism-based strategy to treat bacterial enterotoxigenic diarrhea in animals and humans.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication October 2, 1998.
Revision received December 21, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  G. M. Pitari, T. Li, R. I. Baksh, and S. A. Waldman Exisulind and guanylyl cyclase C induce distinct antineoplastic signaling mechanisms in human colon cancer cells Mol. Cancer Ther., May 1, 2006; 5(5): 1190 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Pitari, L. V. Zingman, D. M. Hodgson, A. E. Alekseev, S. Kazerounian, M. Bienengraeber, G. Hajnoczky, A. Terzic, and S. A. Waldman Bacterial enterotoxins are associated with resistance to colon cancer PNAS, March 4, 2003; 100(5): 2695 - 2699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. K. O'Donnell, R. L. Sedlacek, A. K. Singh, and B. D. Schultz Inhibition of enterotoxin-induced porcine colonic secretion by diarylsulfonylureas in vitro Am J Physiol Gastrointest Liver Physiol, November 1, 2000; 279(5): G1104 - G1112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Lucas, G. M. Pitari, S. Kazerounian, I. Ruiz-Stewart, J. Park, S. Schulz, K. P. Chepenik, and S. A. Waldman Guanylyl Cyclases and Signaling by Cyclic GMP Pharmacol. Rev., September 1, 2000; 52(3): 375 - 414. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
