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Am J Physiol Gastrointest Liver Physiol 293: G1288-G1299, 2007. First published October 4, 2007; doi:10.1152/ajpgi.00234.2007
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MUCOSAL BIOLOGY

Fenofibrate inhibits intestinal Cl– secretion by blocking basolateral KCNQ1 K+ channels

Poonam J. Bajwa,1 Abderrahmane Alioua,2 Jimmy W. Lee,1 Daniel S. Straus,1 Ligia Toro,2 and Christian Lytle1

1Division of Biomedical Sciences, University of California, Riverside; and Department of Anesthesiology, Division of Molecular Medicine, 2David Geffen School of Medicine, University of California, Los Angeles, California

Submitted 23 May 2007 ; accepted in final form 26 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fibrates are peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current (Isc) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl– secretion by isolated jejunum and colon without affecting electroneutral fluxes of 22Na+ or 86Rb+ (K+) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 µM fenofibrate inhibited cAMP-stimulated Isc within 5 min. In T84 cells, fenofibrate rapidly inhibited ~80% the Cl– secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca2+). Both fenofibrate and clofibrate inhibited cAMP-stimulated Isc with an IC50 ~1 µM, whereas other PPAR{alpha} activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K+ channels (putatively KCNQ1/KCNE3) without affecting Ca2+-stimulated K+ channel activity, whereas clofibrate inhibits both K+ pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl– channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 µM fenofibrate rapidly inhibits K+ currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 β-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl– secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.

clofibrate; peroxisome proliferator-activated receptor-{alpha}; Kv7.1; KvLQT1; KCNE3; MiRP2; secretory diarrhea; mouse intestine; human T84 cells; antidiarrheal agents

THE SECRETION OF CL– AND FLUID by intestinal epithelial cells is regulated by a complex interplay of neurocrine, paracrine, and inflammatory signals that provide for optimal luminal fluidity, convective mixing, mucus transit, and flushing in defense against pathogenic intruders (3). The importance of this secretion and its regulation to intestinal function is demonstrated by human diseases and mouse models in which it is congenitally absent (cystic fibrosis) or persistently stimulated (secretory diarrhea). Chloride secretion requires the concerted operation of apical membrane Cl– channels and basolateral membrane Na-K-2Cl cotransporters (NKCC1), Na+-K+-ATPase pumps, and one or more K+ channels. Signals that raise intracellular cAMP (VIP, PGE2, and forskolin) or cGMP (STa toxin) elicit a sustained increase in Cl– secretion by concurrently activating apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channels and basolateral small-conductance K+ channels (39). By contrast, agonists that elevate intracellular Ca2+ (acetylcholine and carbachol) evoke a transient response, mainly by activating basolateral intermediate-conductance K+ channels that hyperpolarize the cell membrane and thereby amplify the driving force for apical Cl– exit through constitutively open CFTR channels (5, 11). Basolateral cotransporters harness the combined inward chemical gradients of Na+ and Cl– to accumulate Cl– within the cell and to replenish Cl– ions lost during secretion. The cotransported cations are then recycled across the basolateral membrane, Na+ via the Na+-K+-ATPase, and K+ through regulated channels. Energetic requirements for sustained Cl– secretion are met by basolateral Na+-K+-ATPase activity and conductive K+ recycling.

Both the small intestine and the colon have an enormous capacity to secrete Cl– along with fluid when stimulated. Unbridled secretion can rapidly overwhelm the absorptive capacity of the intestinal surface cells and produce massive diarrhea, dehydration, and death within hours. A number of enteric pathogens, including certain strains of Vibrio cholerae and Escherichia coli, can colonize the small intestine and elaborate enterotoxins that promote the production of intracellular cAMP (cholera toxin) or cGMP (heat stable enterotoxin or STa; Ref. 55), which in turn drives persistent CFTR Cl– channel activity and isosmotic anion secretion (20, 28). Infectious secretory diarrhea remains one of the leading causes of morbidity and mortality worldwide, particularly among malnourished children in developing countries where inadequate clean water and poor sanitation persist (36).

Fibrate drugs (fenofibrate, clofibrate, and gemfibrozil) have long been in widespread clinical use for the treatment of primary hypercholesterolemia, mixed dyslipidemia, and hypertriglyceridemia (34). After oral ingestion, fenofibrate and clofibrate are completely absorbed and promptly hydrolyzed by plasma and cellular esterases to their pharmacologically active metabolites, fenofibric acid and clofibric acid (56). The effects of fibrates on lipid metabolism (fatty acid transport, synthesis, and β-oxidation) and inflammation are generally attributed to their action as synthetic ligands of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}; Ref. 37). PPARs are nuclear hormone receptors that function as transcriptional regulators of key metabolic pathways (7, 66). PPAR{alpha}, one of three known PPAR isoforms ({alpha}, β/{delta}, and {gamma}), is most prominently expressed in tissues that engage actively in mitochondrial β-oxidation of fatty acids (6), including mouse colonic epithelial cells (40) and human intestinal HT29 (40) and T84 cells (50). The possibility that PPAR{alpha} might influence intestinal epithelial cell function prompted us to evaluate the acute and chronic effects of fibrate drugs on intestinal transport. We report here that fenofibrate promptly and persistently inhibits intestinal electrogenic Cl– secretion without affecting jejunal Na+-sugar absorption, at concentrations ~30-fold lower than those required to activate PPAR{alpha}, through a nongenomic mechanism. We demonstrate that fenofibrate inhibits the cAMP-dependent mode of intestinal Cl– secretion by selectively blocking basolateral K+ recycling through cAMP-stimulated KCNQ1/KCNE3 K+ channel complexes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Human intestinal cell lines (T84 and HT29/cl.19A) were obtained from Dr. K. Barrett (University of California, San Diego, CA). T84 cells were cultured in DMEM and Ham's F-12 media (1:1) supplemented with 5% newborn calf serum (Biomedia), 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO2-95% air as described previously (17). Experiments were performed on cells between passages 25–42. Cells were maintained in T-75 tissue culture flasks until 80–90% confluent and then were subcultured onto 12 mm Snapwell inserts (Corning, Corning, New York) at a density of 2 x 105 cells/well. Measurements of short circuit current (Isc) were made after 10–14 days when monolayer resistance exceeded 1,000 {Omega}/cm2. HT29/cl.19A cells were cultured in McCoy's 5A medium plus 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Mice. Female CD1 mice (Charles River) were fed ad libitum a standard powered diet or the same diet supplemented with 0.05% wt/wt fenofibrate (Sigma, St. Louis, MO), corresponding to 50 mg·kg·–1·day–1 (19) for 6 days. All animal protocols were approved by the University of California, Riverside, Institutional Animal Care and Use Committee.

Electrical measurements. The Isc and electrical resistance (RT) across isolated intestinal mucosa was measured using a conventional Ussing chamber technique (27). A segment of mid-jejunum (~25 cm from cecum) or "early" distal colon (extending 2 cm proximal from the peritoneal border) was isolated from control or fenofibrate-treated mice and rinsed in ice-cold Tyrode's solution comprised of (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 1.25 CaCl2, 12 glucose, and 10 Na-HEPES (pH 7.4). The intestinal wall was cut open along the mesentery, and was partially stripped of serosal and muscle layers to obtain a mucosa-submucosa preparation. Tissues were mounted on small pins across an oblong aperture (2.8 x 11 mm, exposed surface area 0.33 cm2, Physiologic Instruments, P2304) and incubated in an Ussing chamber (Physiologic Instruments; EM-CSYS-2). For experiments on colon, both sides of the mucosa were bathed in Tyrode's solution; for jejunum, glucose in the mucosal solution was replaced with equimolar mannitol to preclude electrogenic Na+-glucose absorption. Chambers were maintained at 37°C by heated water jackets and continuously mixed by oxygen gas lift. Indomethacin (1 µM) was included in the serosal bath to suppress endogenous prostanoid generation and basal Cl– secretion. The transmucosal potential difference (Vt) was measured through 3 M KCl agar bridges connected to a pair of calomel electrodes and monitored with a voltage clamp amplifier (Physiological Instruments; VCC-MC2). Vt was maintained at a command voltage of 0 mV, with compensation for solution resistance, by applying an Isc through a pair of Ag/AgCl electrodes kept in contact with the mucosal and serosal solutions through 3 M KCl agar bridges. Transmucosal conductance (Gt) was calculated using Ohm's law from the change in current evoked by a 2-mV bipolar pulse every 20 s. Output from the voltage clamp was recorded on an Apple computer through an eight-bit A/D converter (Biopac Systems; MP30). The same setup was used for measuring electrogenic Cl– secretion in T84 cells grown on Snapwell permeable supports. Paired epithelial monolayers were mounted in Ussing chambers and allowed to equilibrate for 15 min in Parson's solution containing (in mM) 110 NaCl, 4 KCl, 2 NaPO4, 12 glucose, 1 MgSO4, 1.25 CaCl2, and 25 NaHCO3 and maintained at pH 7.4 by continuous bubbling with 5% CO2-95% O2.. To assess the acute effect of fenofibrate on electrogenic Cl– secretion, the drug was added to both serosal and mucosal reservoirs of the chamber to a final concentration of 100 µM (from a 100 mM stock solution in DMSO) 30 min before stimulation with forskolin (20 µM). Pilot experiments indicated that vehicle DMSO at 0.2% was without effect.

To evaluate the effects of fenofibrate on intestinal electrogenic Na+-coupled glucose absorption, adjacent segments of the proximal jejunum (~10 cm from the pylorus) were stripped of serosa and muscularis and mounted in paired Ussing chambers. Mucosal sheets were incubated for 30 min in Parson's solution containing 12 mM glucose (serosal reservoir) or mannitol (luminal reservoir) and 1 µM indomethacin. One of the paired chambers included fenofibrate at 100 µM. Electrogenic Cl– secretion was recorded as the increase in Isc evoked by serosal forskolin (10 µM). Once Isc reached a steady plateau (~8 min), luminal glucose (20 mM) and serosal mannitol (20 mM) were simultaneously added and the increment in Isc was recorded as Na+-coupled glucose absorption.

Whole cell patch-clamp analysis. The effect of fenofibrate on KCNQ1/KCNE3 K+ channel complex activity was evaluated by patch-clamp analysis of human embryonic kidney (HEK) 293T cells transiently transfected with human KCNQ1 or KCNQ1 along with its auxiliary subunit KCNE3 (MiRP2). Neither channel protein is endogenous to HEK-293 cells (53). Human (h) KCNQ1 and hKCNE3 subcloned in pRAT plasmid were kindly provided by Dr. S. A. N. Goldstein (Yale University School of Medicine, New Haven, CT), and pGFP-N2 was from Clontech. HEK-293T cells in 35-mm culture dishes were transiently cotransfected at 70–80% confluence with 2 µg hKCNQ1 ± 2 µg hKCNE3 along with 0.1 µg pGFP using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Two to 3 days posttransfection, cells were mechanically dissociated, seeded on glass coverslips precoated with 0.1 mg/ml poly-D-lysine, and maintained under standard culture conditions for ~2 h before use. GFP-positive cells were studied within 2–10 h of plating. Patch-clamp experiments were performed in the standard whole cell recording configuration at room temperature using an Axopatch 200A amplifier (Axon Instruments). Borosilicate glass electrodes were pulled and fire-polished to tip resistances of 2–3 M{Omega} when filled with a pipette solution containing (in mM) 140 K-methanesulfonate, 4.8 MgATP, 1.2 KH2PO4, 10 EGTA, 0.1 CaCl2, 2 MgCl2, 10 HEPES (pH 7.4), and 5 glucose and 0.1 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP; Calbiochem). The bath solution contained (in mM) 135 Na-methanesulfonate, 5 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES (pH 7.4), and 10 glucose. Series resistances were electronically compensated by ~75%. Recorded membrane currents were filtered at one-fifth the sampling frequency, which was 10 kHz. Data acquisition and analysis were performed with custom-made software.

Transmucosal 22Na and 86Rb fluxes. Measurements of mucosal-to-serosal (Jm->s) and serosal-to-mucosal (Js->m) ion flux were performed using a sample and replace method described previously (2). Mucosal sheets isolated from the distal colon of control and fenofibrate-treated mice were mounting in Ussing chambers and allowed to equilibrate for 15 min in HCO3– buffered Parson's solution containing (in mM) 137 Na+, 4 K+, 116.5 Cl–, 25 HCO3–, 2 PO42–, 1.25 Ca2+, 1 Mg2+, 1 SO42–, and 12 glucose, and they were bubbled with 5%CO2-95%O2 to maintain pH 7.4. Along with indomethacin (1 µM), amiloride was included to inhibit electrogenic Na+ absorption. Vt was clamped at 0 V, and 3 µCi of 22Na (GE Healthcare) were added to the mucosal reservoir. After an initial preincubation period of 45 min, Jm->s was measured in two sequential 15-min intervals. Pilot experiments indicated that the accumulation of 22Na in the serosal reservoir remained proportional to time over the 15-min interval. At 0 and 15 min, samples of the serosal solution were removed and immediately replaced with an equal volume of prewarmed serosal solution. Both hemi-chambers were then emptied, rinsed to remove residual isotope, and refilled with fresh prewarmed media. 22Na was then added to the serosal solution, and Js->m was measured over two additional 15-min intervals. Net ion flux (Jnet; expressed as µeq·cm–2·h–1) was calculated as the difference between the average values of Jm->s and Js->m obtained with the same tissue. A similar procedure was used to measure transmucosal K+ fluxes, using 86Rb (GE Healthcare) as a surrogate for K+, except that the concentration of Na+ in the medium was reduced to 50 mM by substitution with N-methyl-D-glucamine+ (NMDG). Throughout each flux period, Isc and RT were monitored. The activity of 22Na+ and 86Rb+ in samples was measured with a Beckman gamma counter.

Permeabilized T84 cell monolayers. Basolateral membrane K+ conductance was measured by permeabilizing the apical membrane with the pore-forming antibiotic amphotericin B (20 µM; Sigma) while imposing a serosal to mucosal K+ gradient, as described previously (35, 52). T84 cells grown to confluence on Snapwell inserts were mounted in an Ussing chamber with a mucosal solution containing predominantly K+ (143 mM K-gluconate) and a serosal solution containing predominantly NMDG (137 NMDG-gluconate/5 mM K-gluconate). Both solutions also contained (in mM) 1.25 CaCl2, 0.4 MgSO4, 0.43 KH2PO4, 0.35 K2HPO4, 5.6 glucose, and 10 HEPES at pH 7.4, and they were continuously bubbled with O2. Under these conditions, Isc and Gtm largely reflect K+ electrodiffusion through basolateral channels (35). Changes in Isc and Gtm evoked by cAMP (20 µM forskolin) and Ca2+ (100 µM carbachol) were measured 30 min after adding amphotericin B.

Apical membrane Cl– conductance was measured by permeabilizing the basolateral membrane with nystatin while imposing a mucosal to serosal Cl– gradient, as described previously (60). T84 cell monolayers were mounted in Ussing chambers with a mucosal solution containing (in mM): 120 NaCl, 25 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, 3.3 KH2PO4, 0.8 K2HPO4, 10 glucose, and they were bubbled with 5% CO2-95% O2. (pH 7.4). In the serosal solution, sodium gluconate replaced NaCl, and CaCl2 was increased to 4 mM to compensate for buffering by gluconate. Nystatin (Sigma) was added to the serosal solution to a final concentration of 180 µg/ml from a freshly prepared 90 mg/ml DMSO stock solution. Under these conditions, the transmucosal Isc mainly reflects apical Cl– channel activity (60). The effects of cAMP and Ca2+ stimulation were measured 20 min after permeabilization by adding 20 µM forskolin and 100 µM carbachol, respectively, to the serosal bath.

Statistics. Data are means ± SE. Statistical significance was calculated by t-test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Fenofibrate inhibits intestinal Cl– secretion in vivo. Ten-week-old CD-1 female mice were fed a standard control diet or the same diet formulated with 0.05% wt/wt fenofibrate for 6 days. This dose of fenofibrate (~50 mg·kg·–1·day–1) corresponds to that used in previous studies of PPAR{alpha} signaling in mice (19). Mice treated with fenofibrate for 6 days exhibited no signs of external pathology or intestinal obstruction. Mucosal sheets were dissected from the distal colon of six control and nine fenofibrate-treated mice and preincubated in an Ussing chamber without fenofibrate for 1 h. Figure 1 illustrates a representative tracing of short-circuit current (A), along with the aggregate results from all six experiments (B). In colon isolated from fenofibrate-treated mice, the Cl– secretory responses to prostaglandin E2, to the Ca2+-mobilizing muscarinic agonist carbachol, and to the adenylate cyclase activator forskolin were all reduced, although the inhibitory effect of fenofibrate reached statistical significance only with forskolin stimulation.


Figure 1
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  		Fig. 1. Effect of dietary fenofibrate (0.05% wt/wt for 6 days) on mouse distal colon short-circuit current (Isc). All tissues were preincubated for 1 h in a medium lacking fenofibrate. All measurements of Isc were carried out in a medium lacking fenofibrate. A: representative tracing of Isc from single mouse. B: composite results of Isc responses in replicate experiments on 6 control mice (open bars) and 9 fenofibrate-treated mice (shaded bars). *P < 0.01. Secretagogues were added to the serosal bath in the sequence: 5 µM PGE2, 100 µM carbachol, and 10 µM forskolin.

 
To evaluate the possibility that fenofibrate also affects the intestinal transport of Na+ and K+, we measured bidirectional isotope fluxes across muscle-stripped early distal colon. A bicarbonate-buffered Parson's solution was used to preserve constitutive 22Na+ movements via coupled Na/H exchange (NHE3) and Cl/HCO3 exchange (SLC26A3), the principal mode of electroneutral NaCl absorption by the small intestine and proximal colon (4). Amiloride (1 µM) was included to eliminate any possible contribution from electrogenic Na+ absorption, although preliminary experiments indicated that this mode of absorption was negligible in the early distal colon of mice maintained on a normal salt diet, as reported previously (45). In control tissues, we observed a small net absorption of Na+, comparable to previously reported values (25). Treatment of mice with oral fenofibrate for 6 days had no significant effect on the movement of 22Na+ in either direction across early distal colon isolated from these mice (Fig. 2A ). A similar procedure was used to measure K+ (86Rb+) fluxes across the early distal colon. In these experiments, the concentration of Na+ on both sides of the mucosa was reduced to 50 mM to minimize the inhibitory effect of mucosal Na+ on colonic K+ absorption (23). Under these conditions, treatment of mice with fenofibrate had no significant effect on the fluxes of 86Rb across segments of colon isolated from these mice (Fig. 2B).


Figure 2
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  		Fig. 2. Effect of dietary fenofibrate (FF; 0.05% wt/wt for 6 days) on amiloride-insensitive transmucosal fluxes of 22Na+ (A) and 86Rb+ (B) in unstimulated mouse distal colon. Js->m, serosal-to-mucosal flux; Jm->s, mucosal-to-serosal flux, Jnet, net flux. Data are means ± SE for 8 control (open bars) and 6 fenofibrate-treated mice (A; shaded bars) and 6 control (open bars) and 6 fenofibrate-treated mice (B; closed bars). *P < 0.05.

 
Because the transcriptional responses to PPAR{alpha} activation typically become apparent over a time scale of hours to days, we were surprised to find that fenofibrate inhibited intestinal Cl– secretion within just minutes. Pretreatment of isolated mouse distal colon with fenofibrate for just 20 min reduced the secretory current by 58.7 ± 17.8% (P = 0.022; Fig. 3).


Figure 3
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  		Fig. 3. Effect of fenofibrate on forskolin-stimulated Isc and glucose-stimulated Isc. A: paired muscle-stripped segments of mouse distal colon (2 cm from peritoneal border), terminal ileum (4 cm from cecum), and jejunum (10–15 cm from pylorus) were mounted in Ussing chambers and then treated with fenofibrate (100 µM bilaterally; shaded bars) or vehicle (open bars) for 30 min. After a stable baseline Isc was reached, the response to serosal forskolin (10 µM) was recorded ({Delta} Isc). Data are means ± SE; the number of replicate experiments on different mice is indicated at the top of each bar. B: effect of fenofibrate on electrogenic Na+-glucose absorption. Segments of muscle-stripped mouse jejunum (10–15 cm from pylorus) were mounted in Ussing chambers lacking luminal glucose (mannitol substitution) and pretreated with 100 µM fenofibrate for 30 min. After the response to forskolin was recorded, the effect of adding 20 mM luminal glucose plus 20 mM serosal mannitol was measured. *P < 0.05.

 
Because intestinal Cl– transport is regulated by a variety of mechanisms that have specific segmental and spatial distributions, we investigated whether other regions of the intestine were likewise affected by fenofibrate. Treatment of muscle-stripped colon, ileum, and jejunum with 100 µM fenofibrate for 20 min reduced the forskolin-induced Isc by 58.7 ± 17.8, 64.6 ± 10.2, and 48.3 ± 18.3%, respectively (Fig. 3). Importantly, fenofibrate did not affect the Isc response to luminal glucose in the forskolin-stimulated jejunum (Fig. 3B), even though it did inhibit the component of Isc evoked by forskolin in the same set of tissues. Thus, the antisecretory effect is not associated with significant impairment of electrogenic Na+-coupled glucose absorption under conditions simulating toxigenic secretory diarrhea (cAMP hyperstimulation). Collectively these results suggest that fenofibrate selectively inhibits cAMP-stimulated Cl– secretion in both small intestine and colon through a rapid PPAR{alpha}-independent mechanism.

Fenofibrate inhibits Cl– secretion by cultured intestinal epithelial cells. To distinguish whether fenofibrate acts directly on the intestinal epithelial cells, or secondarily through enteric neurons or other mucosal factors, we examined its effect on human T84 colonic adenocarcinoma cells, a well-established experimental model of epithelial Cl– secretion (18). Confluent monolayers of T84 cells cultured on permeable supports were mounted in Ussing chambers and pretreated with 100 µM fenofibrate for 20 min. The Cl– secretory currents evoked by sequential addition of forskolin and carbachol, or to E. coli heat-stable enterotoxin (STa), were then measured in the continued presence of fenofibrate. In replicate experiments, fenofibrate inhibited the sustained responses to the cAMP-dependent secretogogue forskolin 82.3 ± 11.3% and to the cGMP agonist STa toxin 91.9 ± 21.7%, whereas it had no effect on the subsequent burst of Cl– secretion evoked by the Ca2+-dependent secretagogue carbachol (Fig. 4). Additional experiments examined the effect of fenofibrate on the Cl– secretory response to VIP, a neuropeptide implicated in the regulation of intestinal Cl– secretion and in the pathogenesis of toxigenic secretory diarrhea (9, 49). In intestinal crypt cells, VIP can stimulate cAMP-dependent Cl– secretion by binding to basolateral receptors coupled to adenylate cyclase (49). In control T84 cells, serosal application of 50 nM VIP increased Isc by 49.5 ± 2.0 µA/cm2 (n = 3) but had no measurable effect in paired cells treated with 100 µM fenofibrate ({Delta}Isc = –1.7 ± 0.3 µA/cm2; n = 3) or with 50 µM chromanol-293B ({Delta} Isc = –1.6 ± 1.7 µA/cm2; n = 3).


Figure 4
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  		Fig. 4. Effect of fenofibrate on electrogenic Cl– secretion by human intestinal T84 cells. T84 cells grown on permeable supports were pretreated with fenofibrate (100 µM bilaterally; shaded bars) or vehicle (open bars) for 30 min. Data indicate Isc responses upon addition of 20 µM forskolin (n = 5 for controls; n = 6 or fenofibrate), 100 µM carbachol (n = 3, control and feno), or 100 U/ml apical STa toxin (n = 3, control and fenofibrate). *P < 0.01.

 
A similar pattern of inhibition was observed in another model of intestinal Cl– secretion, the HT29 Cl-19A cell. In control HT29 cells, 100 µM fenofibrate inhibited the Isc response to forskolin by 57% (control: 137 ± 8 µA/cm2; fenofibrate: 59 ± 20 µA/cm2; n = 4). In contrast, fenofibrate had no significant effect on the subsequent Isc response to carbachol (control: 113 ± 24 µA/cm2; fenofibrate: 113 ± 50 µA/cm2; n = 4).

Two observations indicated that fenofibrate inhibits Cl– secretion at a step distal to the generation of cAMP by adenylate cyclase. First, bypassing the cAMP generation step by adding the cell-permeant cAMP analog 8-CPT-cAMP failed to overcome the inhibitory effect of fenofibrate: in control T84 cells, addition of 70 µM 8-CPT-cAMP increased Isc by 67.1 ± 4.3 µA/cm2, whereas in fenofibrate-treated cells (100 µM for 30 min) the response was only 18.3 ± 3.9 µA/cm2 (n = 3). Second, in fenofibrate-treated T84 cells, forskolin evoked only a weak response, and subsequent addition of 8-CPT-cAMP produced no further increase in Isc.

Prolonged (5 day) treatment of T84 cells in culture with 100 µM fenofibrate inhibited the forskolin-evoked Isc to a similar degree (95.1 ± 5.6%, n = 3) as acute treatment (82.3 ± 11.3%, n = 5), without significantly affecting the carbachol-evoked Isc. Additional experiments confirmed that the inhibitory effect of fenofibrate was rapid in onset. Brief pretreatment (3 min) of T84 cells with 100 µM fenofibrate inhibited the subsequent Isc response to forskolin by 67% (control: {Delta} Isc = 78.6 ± 7.3 µA/cm2; fenofibrate: {Delta} Isc = 25.6 ± 1.0 µA/cm2; n = 3). Although the inhibitory effect of fenofibrate become apparent within minutes, its reversal after washout required several hours. This was initially evident in intestinal segments isolated from mice that had been fed fenofibrate: 1 h after mucosal sheets had been isolated and rinsed free of extracellular fenofibrate, they continued to exhibit blunted responses to cAMP-dependent secretagogues (Fig. 1). A similar phenomenon was observed in T84 cells (Fig. 5). Cells were pretreated with 100 µM fenofibrate for 30 min and then rinsed repeatedly to remove extracellular fenofibrate. Two hours after fenofibrate washout, the cells remained almost entirely refractory to stimulation by forskolin, with partial (~60%) recovery after 24 h (Fig. 5). The response to carbachol was unaffected by fenofibrate. The observed persistence of the inhibitory effect of fenofibrate might be explained by diffusion (ionic) trapping. Fenofibrate is an uncharged hydrophobic molecule that crosses cell membranes rapidly by nonionic diffusion. Once inside the cell, fenofibrate is promptly converted by cytoplasmic esterases into the anion fenofibric acid, which may then combine to form a dimer (51), rendering it membrane impermeable.


Figure 5
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  		Fig. 5. Inhibitory effect of fenofibrate persists long after removal of extracellular fenofibrate. T84 cells were treated for 30 min with 100 µM fenofibrate and were then incubated in culture medium lacking fenofibrate for either 2 or 24 h. Isc was then measured in a medium lacking fenofibrate. Each symbol indicates the Isc response to forskolin (Fk, filled bars) or carbachol (open bars) obtained with 3 culture supports expressed as % of the value observed with paired cells never exposed to fenofibrate (means ± SE).

 
Fenofibrate inhibited the forskolin-stimulated component of Isc in T84 cells with an IC50 of 1 µM (Fig. 6). This is ~30-fold lower than the EC50 of fenofibrate for activation of human PPAR{alpha} (66). The structurally related PPAR{alpha} agonist clofibrate inhibited the secretory response over a similar range of concentrations (1–10 µM), consistent with a previously reported value of 6.7 ± 1.1 µM (16); this IC50 is an order of magnitude lower than the EC50 of clofibrate for activation of human PPAR{alpha} (55 µM). In contrast, pretreatment of cells for 24 h with 100 µM Wy-14,643 (pirinixic acid), which selectively activates PPAR{alpha} with an EC50 of 5 µM (22), had no effect on the Cl– secretory response to forskolin (Fig. 6). The PPAR{alpha} agonist gemfibrozil, applied at 100 µM for 24 h, was also without effect (Fig. 6). Thus, intestinal Cl– secretion is strongly inhibited by a subset of fibrate drugs with structural features shared by fenofibrate and clofibrate. The rapid time course for this effect, pharmacological potency of fenofibrate and clofibrate, and inactivity of Wy-14,643 and gemfibrozil are not consistent with the effect being mediated by PPAR{alpha}. A significant component (~20%) of the forskolin-stimulated Isc was insensitive to 100 µM fenofibrate (Fig. 6). In contrast, clofibrate (100 µM) inhibited the forskolin-evoked Isc completely (Fig. 6), confirming the results of Devor et al. (16).


Figure 6
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  		Fig. 6. Concentration dependence of PPAR{alpha} agonists on forskolin-stimulated Isc in human intestinal T84 cells. T84 cells were incubated for 30 min in Ussing chambers containing various concentrations of fenofibrate, clofibrate, gemfibrozil, and Wy-14,643 in both serosal and mucosal reservoirs. Data indicate Isc response to 20 µM forskolin in 6 culture supports (fenofibrate) or 3 culture supports (all other drugs), expressed as a percentage of its paired control. Curve represents fit of fenofibrate data to a logistic model with an IC50 of 1.0 µM and a slope (Hill coefficient) of 3.9.

 
Target of fenofibrate inhibition. The cAMP-stimulated mode of Cl– secretion requires the concerted operation of four transport processes: apical Cl– exit (CFTR), compensatory basolateral Cl– entry (Na-K-2Cl cotransporter-1), regenerative basolateral K+ recycling (K+ channels), and primary active transport (Na+-K+-ATPase). Presumably fenofibrate suppresses Cl– secretion by inhibiting one or more of these essential pathways. To evaluate the effect of fenofibrate on apical Cl– exit, the conductance of T84 cell monolayers was measured after permeabilizing the basolateral membrane with nystatin and after a large mucosal-to-serosal Cl– concentration gradient was imposed (14, 60). After Isc and conductance reached stable levels (~10 min), the effects of agonists and inhibitors were evaluated. In control monolayers, addition of serosal forskolin promptly evoked a large increase in conductance and inward current consistent with electrodiffusive Cl– flow. This conductance was entirely refractory to a concentration of bumetanide (50 µM) that prevents Na-K-2Cl cotransport (Fig. 7), confirming adequate permeabilization of the basolateral membrane by nystatin. A major component of this conductance appeared to reflect CFTR Cl– channel activity because it was stimulated by cAMP (forskolin) and inhibited by CFTRinh-172 (25 µM), a thiazolidinone inhibitor of CFTR with a Ki ~0.3 µM (43). In three replicate experiments, fenofibrate had no significant effect on this Cl– conductance or its response to forskolin (Fig. 7). As expected, paired measurements of Isc (Cl– current) yielded equivalent results; we found no difference in the forskolin-stimulated Isc between control and fenofibrate-treated monolayers (240 ± 35 vs. 270 ± 20 µA/cm2 for control vs fenofibrate; n = 4; not significant). These results exclude the possibility that fenofibrate inhibits intestinal Cl– secretion by interfering with the generation of cAMP or with the activation of CFTR or with the regulated insertion/retrieval of CFTR channels at the apical membrane in response to forskolin.


Figure 7
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  		Fig. 7. Effect of fenofibrate on apical conductance in basolaterally-permeablized T84 cell monolayers. Left: representative tracings from cells pretreated for 30 min with 100 µM fenofibrate (open circles) or vehicle (filled circles). At t = –20 min, cells were exposed to serosal ionophore (nystatin) and a steep mucosal-to-serosal Cl– gradient. Addition of 20 µM serosal forskolin evoked a large increase in apical Cl– conductance that was unaffected by 50 µM serosal bumetanide yet partially inhibited by 25 µM apical CFTRinh-172. Right: composite results obtained with 3 culture supports. *P < 0.01, significant inhibition with CFTRinh-172.

 
An alternative possibility is that fenofibrate inhibits basolateral K+ recycling. To evaluate this hypothesis, we used the cationophore amphotericin-B to selectively permeabilize the apical membrane in the presence of a mucosal to serosal K+ gradient. Under these conditions, transepithelial Isc mainly reflects electrodiffusive K+ movement through basolateral membrane channels (52). This K+ current increased after addition of serosal forskolin, consistent with the expected activation of basolateral cAMP-dependent K+ channels (Fig. 8), and decreased after adding serosal chromanol-293B, a known inhibitor of these channels (42). This current was also inhibited by 100 µM serosal glibenclamide, which at this high concentration is known to inhibit a variety of K+ channels and Cl– channels (54). By contrast, in cells pretreated for 20 min with 100 µM fenofibrate, the K+ current was largely insensitive to forskolin, chromanol-293B and glibenclamide. In four replicate experiments, pretreatment with fenofibrate or chromanol-293B did not affect the basolateral K+ current under unstimulated conditions (Fig. 8B). Addition of forskolin evoked a basolateral K+ current that was inhibited 75.4 ± 14.8% by fenofibrate (Fig. 8C) and 83.5 ± 16.5% by chromanol-293B (n = 3). Subsequent addition of the Ca2+ agonist carbachol triggered a large transient basolateral K+ current that was not affected by fenofibrate (Fig. 8D). These data suggest that fenofibrate, like chromanol-293B, inhibits intestinal Cl– secretion by selectively blocking basolateral K+ recycling via cAMP-stimulated channels.


Figure 8
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  		Fig. 8. Effect of fenofibrate on basolateral K+ conductance in apically permeabilized T84 cell monolayers. A: representative recordings from cells pretreated for 30 min with 100 µM fenofibrate (gray trace) or vehicle (black trace). At t = –20 min, cells were exposed to mucosal ionophore (amphotericin-B) and a mucosal-to-serosal K+ gradient. In control cells, basolateral K+ conductance, recorded as Isc, was increased by 20 µM serosal forskolin and inhibited by 100 µM serosal glibenclamide and 50 µM serosal chromanol-293B. B: baseline Isc in unstimulated apically permeabilized T84 cells is not significantly affected by fenofibrate or chromanol-293B (n ≥ 9 culture supports). C: Isc in apically permeabilized T84 cells is stimulated by forskolin (cAMP), and this component is inhibited by fenofibrate (n ≥ 9 culture supports). *P < 0.01. D: Isc in apically permeabilized T84 cells is stimulated by carbachol (Ca2+), and this component is not inhibited by fenofibrate (n = 5 culture supports).

 
In an earlier study using T84 cells (16), Devor et al. noted that 60 µM clofibrate fully inhibits both cAMP-activated and Ca2+-activated basolateral K+ currents. This prompted us to compare the effects of fenofibrate and clofibrate on these two modes of K+ conductance. A series of experiments with apically permeabilized T84 monolayers (Fig. 9A) confirmed that 100 µM fenofibrate exerts little if any significant effect (Fig. 9C) on the K+ currents evoked by either carbachol or chlorzoxazone, a structural analog of 1-ethyl-2-benzimidazolone (1-EBIO) that directly activates Ca2+-stimulated K+ channels (61). By contrast, 100 µM clofibrate strongly inhibited these K+ currents (Fig. 9, B and C), confirming previous findings (16). Moreover, clofibrate even inhibited the K+ current evoked by chlorzoxazone in fenofibrate-treated cells (Fig. 9A). Interestingly, the inhibitory effect of clofibrate on the Ca2+-stimulated K+ current became apparent almost immediately (Fig. 9, A and B), whereas the effect of fenofibrate on the cAMP-stimulated mode of Cl– secretion emerged after a brief delay.


Figure 9
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  		Fig. 9. Differential effect of fenofibrate and clofibrate on Ca2+-stimulated basolateral K+ conductance in apically permeabilized T84 cell monolayers. At t = –20 min, cells were exposed to mucosal amphotericin-B and a mucosal-to-serosal K+ gradient. Representative recordings from cells pretreated for 17 min with vehicle (black trace) or 100 µM fibrate drug (gray trace): fenofibrate (A); clofibrate (Clofib; B). Chlzx, chlorzoxazone. C: aggregate data on carbachol (Carb)-induced {Delta} Isc in cells pretreated with vehicle (Control), or 100 µM fenofibrate or 100 µM clofibrate. Each circle represents data from a different culture support. Only clofibrate inhibited to a significant extent (*P < 0.01).

 
The cAMP-stimulated mode of sustained intestinal Cl– secretion depends energetically on basolateral K+ recycling. Recent electrophysiological, pharmacological, and gene knockout studies have suggested that KCNQ1/KCNE3 channel complexes contribute importantly to this K+ current (47, 53, 64). To directly assess whether fenofibrate inhibits these channels, we transfected HEK-293T cells with human KCNQ1 {alpha}-subunit with or without its KCNE3 β-subunit and characterized the resulting K+ currents by whole cell patch-clamp analysis. Expression of KCNQ1 alone produced K+ currents activated by depolarization (Fig. 10, A and B). Co-expression with KCNE3 altered the functional characteristics of KCNQ1: currents were 2.5- to 3.0-fold larger at potentials greater than –40 mV, exhibited an accelerated rate of activation, and became active at potentials more negative than –60 mV (Fig. 10, A and B), as reported previously (48, 53). Because the cells in both groups exhibited similar capacitance (15 ± 0.6 pF for KCNQ1 alone and 17 ± 1.5 pF for KCNQ1+KCNE3), the observed differences in current amplitudes were not likely attributable to differences in cell size. The currents associated with KCNQ1 alone (Fig. 10C) and with the KCNQ1/KCNE3 channel complex (Fig. 10D) were inhibited by 100 µM fenofibrate. The inhibition was rapid in onset and essentially complete within ~5 min (Fig. 10, C and D). Washout experiments confirmed that the inhibitory effect of fenofibrate on KCNQ1/KCNE3 channel activity, like its effect on intestinal (Fig. 1) and T84 cell Cl– secretion (Fig. 5), was persistent: after extensive perfusion of the bath with a medium lacking fenofibrate, the current remained fully inhibited. In a preliminary experiment, the concentration of fenofibrate in the bath was increased stepwise from 0.1 to 100 µM in 10-fold increments; 50% of the KCNQ1/KCNE3 current was inhibited with ~1 µM; this compares favorably with the IC50 found for inhibition of forskolin-stimulated Isc in human T84 cells (Fig. 6).


Figure 10
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  		Fig. 10. Fenofibrate inhibits KCNQ1 K+ channels. A: whole cell K+ current traces in HEK-293T cells transfected with hKCNQ1 alone (left) or hKCNQ1 and hKCNE3 (right). currents were recorded after stimulation with 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP; 100 µM) during a series of 2-s voltage steps from –70 to +70 mV, followed by a repolarization to –30 mV. B: current voltage relation in cells expressing hKCNQ1 alone or hKCNQ1 and hKCNE3 (n = 3). C: time course of fenofibrate inhibition of whole cell K+ current in 8-CPT-cAMP stimulated cells transfected with hKCNQ1 alone. Cells were pulsed from a holding potential of –70 mV to +40 mV for 500 ms every 2 s. Decay of current amplitude after perfusion with fenofibrate was fit to a double exponential function with time constants of 0.17 min and 2.83 min. Inset: representative whole-cell current traces recorded from cells in the absence (control) or presence (+FF) of 100 µM fenofibrate. D: same analysis on cells cotransfected with hKCNQ1 and hKCNE3. Decay of current amplitude was fit to a double exponential function with time constants of 0.23 and 1.3 min. Similar results were obtained in 3 replicate experiments.

 

   DISCUSSION
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The identification of selective inhibitors of cAMP-dependent anion secretion by the intestinal crypts remains an important step in the development of drugs that mitigate fluid and electrolyte losses in toxigenic secretory diarrhea. We demonstrate here that fenofibrate exerts a prompt and protracted antisecretory effect on both the small intestine and colon through a nongenomic mechanism that involves an inhibition of cAMP-activated basolateral KCNQ1/KCNE3 K+ channels. The effect was evident in mice fed fenofibrate for 6 days and in cultured human intestinal cells (T84 and HT29) exposed to fenofibrate for 2–20 min.

The clinical utility of fenofibrate as a lipid-lowering agent is attributed to its action as a synthetic ligand for PPAR{alpha}, a nuclear receptor that regulates the expression of key proteins involved in fatty acid transport and β-oxidation. Oral administration of fenofibrate to mice at the doses used in our experiments has been shown to activate PPAR{alpha} in intestinal epithelial cells (40, 50). However, three lines of evidence argue against a role for this receptor in the antisecretory effect described here. First, the effect of fenofibrate on intestinal Cl– secretion becomes apparent within 2 min; this rapid onset of action is incompatible with gene regulation and de novo protein synthesis. Second, fenofibrate inhibits human T84 cell Cl– secretion at concentrations ~30-fold lower than those required to activate human PPAR{alpha} (24). Third, whereas fenofibrate inhibits T84 cell Cl– secretion, other PPAR agonists with greater affinity for PPAR{alpha} (Wy-14,643 and gemfibrozil) do not, even after prolonged exposure (24 h).

In both intestinal mucosa and T84 cells, Cl– secretion remained refractory to cAMP stimulation even hours after fenofibrate was removed from the extracellular medium. Likewise, K+ currents associated with KCNQ1/KCNE3 expression in HEK-293T cells remained inhibited after extensive washout of extracellular fenofibrate. The observed persistence of the inhibitory effect of fenofibrate might reflect intracellular retention of the metabolite fenofibric acid through the mechanism of diffusion (ionic) trapping. After ingestion, fenofibrate is rapidly absorbed from the intestinal lumen and hydrolyzed by plasma and intracellular esterases into the anion fenofibric acid (8), which may then dimerize through intermolecular hydrogen bonding (51). In addition to slow washout, diffusion trapping may cause accumulation of the bulky ionized dimer within the intracellular compartment to concentrations exceeding those in the extracellular space; thus, the IC50 of the drug acting at its putative intracellular inhibitory locus could be greater than the value of 1 µM determined by extracellular titration.

Sustained Cl– secretion depends energetically on an electrodiffusive loss of K+ across the basolateral membrane to preserve the electrical impetus for apical Cl– exit (26, 65). The molecular identity of the channels involved in basolateral K+ recycling remains incompletely defined (29). Based on the macroscopic behavior of the basolateral K+ current, two operationally distinct pathways can be distinguished (46). One type (KCa) provides for conductive basolateral K+ recycling during cholinergic (Ca2+)-stimulated Cl– secretion. KCa exhibits an inwardly rectifying intermediate conductance (13); is activated by the benzimidazone derivatives 1-EBIO (10), DCEBIO (30), and chlorzoxazone (60); and is inhibited by charybdotoxin and tetraethylammonium (13). The KCa current appears to involve a K+ channel designated KCa3.1 (SK4, KCNN4) that is activated by Ca2+, phosphatidylinositol-3 phosphate (62), and 1-EBIO (32). Knockout of this KCa3.1 channel in mice results in a selective impairment of the Ca2+-evoked mode of intestinal Cl– secretion and drier stools (21, 47).

An alternate pathway for basolateral K+ recycling is activated during cAMP-stimulated Cl– secretion. KcAMP exhibits a low conductance (1–3 pS) and is inhibited by chromanol-293B (38, 42, 65), barium (65), and zinc (31). An important participant in KcAMP appears to be the K+ channel KCNQ1 (Kv7.1, KvLQT1) and its auxiliary subunit KCNE3 (MiRP2). Assembly with KCNE3 gives KCNQ1 a linear current-voltage relationship and a 10-fold greater sensitivity to chromanol-293B (53). Both KCNQ1 and KCNE3 proteins colocalize along the lateral margin of the Cl–-secreting epithelial cells that inhabit the intestinal crypts (12, 41, 42, 53). Genetic ablation of KCNQ1 reduces jejunal cAMP-dependent Cl– secretion by ~50% (64). Acute inhibition of KCNQ1 with chromanol-293B reduces cAMP-dependent Cl– secretion in the colon ~75% (42, 44, 47) and in T84 cells ~55% (15). Our results indicate that fenofibrate, like chromanol 293B, inhibits cAMP-dependent intestinal Cl– secretion and KCNQ1/KCNE3 channels at micromolar concentrations. While this supports a prominent role for KCNQ1/KCNE3 in KcAMP, the involvement of other K+ channels remains possible. Supporting this possibility are recent reports that fenofibrate, at the concentrations employed in our study, can inhibit macroscopic K+ currents ascribed to Kv (58) and to KATP potassium channels (59) in hamster insulinoma cells.

Our results, together with those of Devor et al. (16), reveal important differences in the inhibitory selectivity of fenofibrate and clofibrate for KcAMP and KCa. In apically permeabilized T84 cells, 100 µM fenofibrate inhibits only KcAMP (Figs. 7, 8), whereas 100 µM clofibrate inhibits both KcAMP and KCa (Fig. 8; Ref.16). This could explain why a substantial component of the forskolin-stimulated Isc (~20%) in T84 cells persists in the presence of fenofibrate or chromanol-293B but not in the presence of clofibrate. Presumably, after inhibition of KcAMP with fenofibrate or chromanol-293B, ongoing KCa provides for enough basolateral K+ recycling to energize a reduced rate of apical anion exit. Our results suggest that structural differences between fenofibrate and clofibrate confer some degree of selectivity between the KcAMP and KCa conductance pathways; these structural features may offer clues in the development of K+ channel inhibitors with improved specificity.

Fenofibrate and chromanol-293B inhibit intestinal Cl– secretion with equal potency (IC50 = 0.5–2.0 µM) and to a similar extent (42, 44, 65). Nevertheless, fenofibrate and chromanol-293B exhibit important differences. First, chromanol-293B is not entirely selective for KCNQ1/KCNE3 channel complexes; Bachmann et al. (1) have reported that chromanol-293B also inhibits CFTR Cl– channels with an IC50 of 20–30 µM. Our measurements of apical Cl– conductance in T84 cells detected no inhibition of CFTR channel function by fenofibrate even at concentrations two orders of magnitude above its IC50 for Cl– secretion. Second, chromanol-293B is known to prolong the cardiac action potential duration and is predicted to be arrythmogenic in the setting of high sympathetic tone (57); indeed, mutations in KCNQ1 underlie the prolonged cardiac action potentials observed in patients with long-QT syndrome type-1 (33). Our observation that fenofibrate inhibits K+ currents associated with expression of human KCNQ1 alone prompts speculation that fenofibrate might also be arrythmogenic, although no prolongation of the QT interval has been reported in patients on fenofibrate therapy (34). In future studies, we plan to evaluate the effect of fenofibrate on the cardiac action potential, including the possibility that the association of KCNQ1 with the KCNE1 subunit in cardiac myocytes renders the channel complex less susceptible to fenofibrate. Finally, inhibition of KCNQ1 with chromanol-293B, or genetic ablation of this channel in mice, is associated with a modest (33%) reduction in electrogenic Na+-coupled glucose absorption by the unstimulated jejunum (64); conversely, we detected no such effect of fenofibrate on electrogenic Na+-glucose absorption, even under conditions (forskolin) favoring the participation of KCNQ1 in basolateral K+ recycling.

Our observation that fenofibrate exerts a nongenomic antisecretory effect has pharmacological implications. Fenofibrate inhibits cAMP-dependent Cl– secretion in human intestinal T84 cells at concentrations ~30-fold lower than those required to activate human PPAR{alpha}. Thus, the clinical use of these drugs as lipid-lowering agents may include antisecretory side effects on intestinal crypts and other Cl– secreting exocrine glands. Constipation is not a common side effect of fenofibrate, perhaps because the Ca2+-dependent mode of intestinal fluid secretion, which appears necessary for proper stool hydration (21), would be spared and/or because cAMP-dependent fluid secretion in not completely inhibited.

The jejunum is the principal site of intestinal fluid secretion and the primary target of diarrheagenic enterotoxins (e.g., cholera toxin and STa). Our evidence that fenofibrate inhibits cAMP- and cGMP-stimulated Cl– secretion in both small and large intestine, and in human intestinal cells, suggests this drug as a potentially useful therapeutic agent in human secretory diarrheal diseases. Both fenofibrate and clofibrate are well-characterized and economical Food and Drug Administration-approved drugs with no major adverse effects when used long term (34, 63). Moreover, because fenofibrate does not impair jejunal Na+-coupled glucose absorption, it may offer added benefit as an adjunct to conventional oral rehydration therapy.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This study was supported by the Crohn's and Colitis Foundation of America (to D. Straus and C. Lytle), Collaboration Grants from the Division of Biomedical Sciences, University of California, Riverside (to D. S. Straus and C. Lytle), American Heart Association National 0435084N (to A. Alioua), and National Heart, Lung, and Blood Institute Grant HL-054970 (to L. Toro).


   ACKNOWLEDGMENTS
 
We thank Dr. D. R. Halm (Wright State University) for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Lytle, Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121 (e-mail: christian.lytle{at}ucr.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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