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(The FASEB Journal. 1999;13:913-922.)
© 1999 FASEB

Research Communications

Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea

WEI ZHANG, ISHRAT MANNAN, STEPHANIE SCHULZ, SCOTT J. PARKINSON, ALEXEY E. ALEKSEEV*, LUIS A. GOMEZ*, ANDRE TERZIC* and SCOTT A. WALDMAN1

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
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria that produce heat-stable enterotoxins (STs), a leading cause of secretory diarrhea, are a major cause of morbidity and mortality worldwide. ST stimulates guanylyl cyclase C (GCC) and accumulation of intracellular cyclic GMP ([cGMP]i), which opens the cystic fibrosis transmembrane conductance regulator (CFTR)-related chloride channel, triggering intestinal secretion. Although the signaling cascade mediating ST-induced diarrhea is well characterized, antisecretory therapy targeting this pathway has not been developed. 2-ChloroATP (2ClATP) and its cell-permeant precursor, 2-chloroadenosine (2ClAdo), disrupt ST-dependent signaling in intestinal cells. However, whether the ability to disrupt guanylyl cyclase signaling translates into effective antisecretory therapy remains untested. In this study, the efficacy of 2ClAdo to prevent ST-induced water secretion by human intestinal cells was examined. In Caco-2 human intestinal cells, ST increased [cGMP]i, induced a chloride current, and stimulated net basolateral-to-apical water secretion. This effect on chloride current and water secretion was mimicked by the cell-permeant analog of cGMP, 8-bromo-cGMP. Treatment of Caco-2 cells with 2ClAdo prevented ST-induced increases in [cGMP]i, chloride current and water secretion. Inhibition of the downstream consequences of ST-GCC interaction reflects proximal disruption of cGMP production because 8-bromo-cGMP stimulated chloride current and water secretion in 2ClAdo-treated cells. Thus, this study demonstrates that disruption of guanylyl cyclase signaling is an effective strategy for antisecretory therapy and provides the basis for developing mechanism-based treatments for enterotoxigenic diarrhea.—Zhang, W., Mannan, I., Schulz, S., Parkinson, S. J., Alekseev, A. E., Gomez, L. A., Terzic, A., Waldman, S. A. Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea.


Key Words: E. coli heat-stable enterotoxin • intestinal cell water secretion • cyclic GMP • 2-substituted adenine nucleotides • CFTR-mediated chloride current


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DIARRHEAL DISEASES ARE the fourth leading cause of morbidity and mortality and the leading cause of pediatric mortality worldwide (1 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
[14C]mannitol (50 mCi/mmol; 200 µCi/ml) was obtained from DuPont-NEN (Wilmington, Del.). [3H]H2O (5 mCi/ml) was obtained from Amersham (Arlington Heights, Ill.). 8-Bromo-cGMP was obtained from Sigma (St. Louis, Mo.). ST purified from Escherichia coli was a generous gift from Dr. D. C. Robertson, University of Idaho, Moscow (12 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 [{gamma}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 (basolateral->apical) and (apical->basolateral) directions. Net water flux was calculated as the arithmetic difference between the fluxes in basolateral->apical and apical->basolateral 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.



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  		Figure 1. Differentiation of Caco-2 cells in vitro. Caco-2 cells were grown in culture as outlined in Materials and Methods. On the indicated days, cells were harvested and alkaline phosphatase activity (A) and GCC mRNA (B) were quantified. A) Alkaline phosphatase activity in Caco-2 cells after increasing time in culture. Alkaline phosphatase was quantified as described in Materials and Methods employing p-nitrophenylphosphate as substrate. Values are means of duplicate determinations obtained in at least three experiments. Error bars represent SE; * P < 0.05. B) GCC mRNA in Caco-2 cells after increasing time in culture determined by quantitative RT-PCR. Quantitative RT-PCR was performed with total RNA extracted from cells obtained after the indicated number of days in culture, as described in Materials and Methods. Values are means ± SE of duplicate determinations obtained in at least three experiments. *P < 0.05.

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{Omega}). 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differentiation of Caco-2 cells and development of vectorial water transport
Caco-2 cells differentiate as polarized monolayers connected by junctional complexes, with brush border microvilli containing alkaline phosphatase and GCC. Here, the specific activity of alkaline phosphatase significantly (P<0.05) increased from 45 ± 5 to 100 ± 25 µmol phosphate produced/min/mg protein (n=3) in Caco-2 cells grown for 5 or 10 days and was stable after longer periods in culture (Fig. 1A ). 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.



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  		Figure 2. Vectorial water transport by Caco-2 cells as a function of days in culture. Water transport by Caco-2 cells was quantified as described in Materials and Methods with [3H]H2O. Water flux was determined in basolateral -> apical (secretion) and apical -> basolateral (absorption) directions. Net water flux was calculated as the arithmetic difference between the fluxes in basolateral to apical and apical to basolateral directions. Data represent the means ± SE of at least three experiments performed at least in triplicate.

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).



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  		Figure 3. ST induces Cl- current in Caco-2 cells. A) Time course of steady-state outward current recorded at the end of 1-s depolarizing rectangular pulses from the holding potential of -40 mV to the membrane potential of +10 mV. Numbers along the time course correspond to times at which the voltage-current relationships (presented in A1) were collected. A1, Voltage-current relationships obtained at the holding potential +40 mV in response to a ramp pulse from -50 to +90 mV. Numbers on the right of each curve correspond to the following conditions (A): 1, basal current; 2, ST 100 nM; 3, low-Cl- bath solution; 4, return to Cl--rich solution; 5, glyburide 100 µM. B) ST-induced current component obtained by subtracting currents in the absence of ST (control) from currents recorded in the presence of the enterotoxin (ST 100 nM). Subtraction was performed between corresponding currents recorded from a holding potential of -40 mV, by applying 10-mV voltage steps to impose a membrane potential ranging from -90 to +50 mV. Results of subtraction were flattened using linear spline. B1) Voltage-current relationships of ST- (filled circles) and 8-bromo-cGMP- (open squares) induced current components obtained after subtraction as described in for panel B. Values for each point defining the voltage-current relationships were taken at the end of 1-s testing pulses.

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.



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  		Figure 4. ST induces transcellular, but not paracellular, water transport by differentiated Caco-2 cell monolayers. Water and mannitol transport were measured in Caco-2 cells with [3H]H2O and [14C]mannitol, respectively, as described in Materials and Methods. A1) Effect of ST (200 nM) on peak secretory (basolateral -> apical) and absorptive (apical -> basolateral) transfer of [3H]H2O and [14C]mannitol by Caco-2 monolayers. Water flux and mannitol permeability were quantified in basolateral and apical directions over the 2-min interval during which transfer was maximum. Data represent the means ± SE of at least three experiments performed at least in triplicate. A2) ST induces transcellular, but not paracellular transport. Peak flux or permeability was measured in the absence or presence of ST (200 nM). Data represent the means ± SE of at least three experiments performed at least in triplicate, *P < 0.05. B) Time course of net water secretion induced by ST. Net water secretion was quantified at 2-min intervals before and after addition (arrow) of ST (200 nM) as described in Materials and Methods. C) ST-induced water secretion is chloride dependent. Peak water secretion was quantified as described above in the absence or presence of ST (200 nM). Where indicated, an equimolar concentration of SO4- replaced chloride in the Ringer solution. Results are the mean of at least three experiments performed in triplicate, *P < 0.05. D) 8-Bromo-cGMP mimics ST, inducing water secretion by Caco-2 cells. Net water secretion was quantified at 2-min intervals before and after (arrow) addition of 8-bromo-cGMP (2 mM) as described in Materials and Methods.

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 ).



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  		Figure 5. ST failed to induce Cl- current in 2ClAdo-treated Caco-2 cells. A) Original current records from a Caco-2 cell under control condition, after application of ST (100 nM), and 8-bromo-cGMP (5 mM). Under each condition, currents were recorded from a holding potential of -40 mV by applying 10-mV voltage steps to impose a membrane potential from -90 to +50 mV. A1) Voltage-current relationships of ST- (filled circles) and 8-bromo-cGMP- (open squares) induced current components obtained after subtraction (as described in Fig. 2 B). Values for relationships were taken at the end of 1-s testing pulses. B) Time course of steady-state outward current recorded at the end of a 1-s depolarizing rectangular pulse from the holding potential of -40 mV to the membrane potential of +10 mV. ST was applied at 100 nM, and 8-bromo-cGMP at 5 mM. C) Average total current values obtained at the end of 1-s depolarizing rectangular pulses at the membrane potential of +10 mV in untreated and 2ClAdo-treated Caco-2 cells. Bars represent mean ± SE (n=5).

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.



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  		Figure 6. 2ClAdo prevents Caco-2 cell water secretion and [cGMP]i accumulation induced by ST, but not water secretion induced by 8-bromo-cGMP. Water transport was quantified as described in Materials and Methods. In these studies, cells were preincubated in serum-free media containing the indicated concentrations of 2ClAdo (2 mM) for 24 h before water transport studies. A) Time course of net water secretion induced by ST. Net water secretion was quantified at 2-min intervals before and after (bar) addition of ST (200 nM) in cells pre-incubated with 2ClAdo (2 mM). B) Concentration-dependence of inhibition of ST-induced peak water secretion by 2ClAdo. ST-induced peak water secretion was quantified in cells pre-incubated with the indicated concentrations of 2ClAdo. Peak secretion after exposure to 2ClAdo was compared to that in the absence of 2ClAdo to yield percent inhibition. Results are means ± SE of at least three determinations. Inset, double-reciprocal plot analysis of data presented in panel B. C) Caco-2 cell [cGMP]i accumulation induced by ST. [Cyclic GMP]i accumulation was quantified as described in Materials and Methods (14) . Cells were preincubated with 1 mM 2ClAdo for 24 h before incubation with 200 nM ST. D) 8-Bromo-cGMP induces water secretion in Caco-2 cells pretreated with 2ClAdo. Net water secretion was quantified at 2-min intervals before and after (bar) the addition of 8-bromo-cGMP (2 mM). D) Comparison of net water flux induced by ST or 8-bromo-cGMP in control cells and those exposed to 2ClAdo. Net water flux was quantified in the absence or presence of ST (200 nM) or 8-bromo-cGMP (2 mM). Results are means ± SE of at least triplicate determinations.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date, therapeutic options to treat enterotoxigenic diarrhea have remained nonspecific and have not exploited the current understanding of the molecular mechanisms underlying intestinal secretion. This study is the first demonstration that targeted disruption of guanylyl cyclase signaling, the most proximal event in the signal sequence leading to diarrhea, can be an effective strategy to treat enterotoxin-induced secretion. Specifically, allosteric inhibition of guanylyl cyclase by 2-substituted nucleotides prevents the downstream consequences of ST-GCC interaction, including chloride and water transport, processes central to the development of intestinal secretion and diarrhea. Thus, this study illustrates the feasibility of translating the understanding of molecular mechanisms mediating transmembrane signaling into novel therapeutic strategies.

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
 
This research was supported by grants from the National Institutes of Health (HL59214, CA75123), American Cancer Society (EDT106), Targeted Diagnostics and Therapeutics, Inc., and American Heart Association.


   FOOTNOTES
 
2 Abbreviations: A, surface area of the cell monolayer; cDNA, complementary DNA; CFTR, cystic fibrosis transmembrane conductance regulator; 2ClAdo, 2-chloroadenosine; [2ClAdo]e, extracellular concentrations of 2ClAdo; [2ClAdo]i, intracellular concentrations of 2ClAdo; 2ClATP, 2-chloroATP; [2ClATP]i, intracellular concentration of 2-ClATP; [cGMP]i, intracellular concentrations of cGMP; CO, initial concentration of mannitol in the donor chamber; dC/dt, change in mannitol concentration in the receiver chamber; DEPC, diethylpyrocarbonate; GCC, guanylyl cyclase C; HEK 293 cells, 293 human embryonic kidney epithelial cells; Jw, water flux; Ki, concentration yielding half-maximum inhibition; mV, millivolt; Pm, mannitol permeability; R, initial radioactivity per unit volume of donor chamber; RT-PCR, reverse transcriptase-polymerase chain reaction; SE, standard error of the mean; ST, heat-stable enterotoxin; T, elapsed time; TCA, trichloroacetic acid; V, volume of receiver chamber; Vt1, volume of receiver chamber at the measuring time point; Vt2, volume of receiver chamber at the point of the last sample taken.

Received for publication October 2, 1998. Revision received December 21, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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