Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling

Paul A. Rufo,1,5,* Patricia W. Lin,2,5,* Adriana Andrade,7,{dagger} Lianwei Jiang,3,6,8 Lucia Rameh,4 Charles Flexner,7 Seth L. Alper,3,6,8 and Wayne I. Lencer1,5,8

1GI Cell Biology, Combined Program in Pediatric Gastroenterology and Nutrition, 2Division of Newborn Medicine, Children's Hospital, 3Molecular and Vascular Medicine and Renal Units and 4Division of Signal Transduction, Beth Israel Deaconess Medical Center, and Departments of 5Pediatrics and 6Medicine, Harvard Medical School, Boston, Massachusetts 02115; 7Division of Clinical Pharmacology, Johns Hopkins University, Baltimore, Maryland 21205; and 8Harvard Digestive Diseases Center, Boston, Massachusetts 02115

Submitted 22 August 2003 ; accepted in final form 24 December 2003


    ABSTRACT
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 ABSTRACT
 METHODS
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 DISCUSSION
 REFERENCES
 
Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl- secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca2+-dependent Cl- conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca2+ transients via activation of a long-lived, store-operated Ca2+ entry pathway. The enhanced intracellular Ca2+ signal is associated with uncoupling of the Cl- conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca2+ signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects.

nelfinavir; clotrimazole; barium


ASPARTYL PROTEASE INHIBITORS (APIs) are important components of most highly active antiretroviral therapy (HAART) regimens used in the treatment of human immunodeficiency virus (HIV)-1 infection. These agents decrease plasma HIV viral load, increase peripheral CD4+ T lymphocyte counts, delay clinical progression, and extend life expectancy (17). However, the clinical utility of these agents has been limited by serious side effects, which include lipodystrophy, insulin resistance, and diarrhea (14). The mechanistic bases of these dose-limiting side effects of API treatments remain unexplained.

In these studies, we examine the effects of the API nelfinavir on the intestine and on intestinal epithelial cells. Intestinal fluid secretion in the human depends on the closely regulated transport of Cl- ions by epithelial cells lining the intestinal crypt. Crypt epithelia utilize the basolateral membrane Na+-K+-ATPase and Na+- and K+-coupled cotransporter NKCC1 to accumulate intracellular Cl- above its electrochemical equilibrium potential. The regulated opening of apical membrane Cl- channels in that setting results in a net secretion of Cl- ions into the intestinal lumen. Coordinated opening of basolateral K+ channels to maintain an inside-negative membrane potential sustains the Cl- secretory response by enhancing both the electrical gradient favoring electrogenic apical Cl- exit and the chemical gradient favoring Na+- and K+-coupled Cl- uptake by basolateral NKCC1. Water and Na+ are thought to follow Cl- passively into the intestinal lumen to effect net fluid secretion.

Neural, endocrine, paracrine, and autocrine mechanisms tightly regulate intestinal fluid secretion in the human via agonists that utilize either cyclic nucleotides or Ca2+ as second messengers. Agonists that depend on adenosine 3',5'-cyclic monophosphate (cAMP) to initiate Cl- secretion activate the apical membrane Cl- channel CFTR (cystic fibrosis transmembrane receptor) and the basolateral membrane K+ channel KCNQ1/KCNE3 (2, 10, 32, 42). Agonists that utilize Ca2+ as a second messenger activate the apical membrane Ca2+-activated Cl- conductance and the basolateral membrane K+ channel IK1 (KCNN4) (22, 24, 25, 47).

Muscarinic innervation of intestinal crypts regulates Cl- secretion through local release of acetylcholine. The secretory response induced in the crypt epithelial cell requires an elevation of intracellular Ca2+ that initially activates an apical membrane Ca2+-sensitive Cl- conductance. However, coordinate generation of inositol 3,4,5,6-tetrakisphosphate (IP4) and phosphorylation of the MAP kinase intermediates extracellular signal-regulated kinase (ERK) and p38 rapidly downregulate this Ca2+-sensitive Cl- conductance to keep muscarinically induced Cl- secretory responses short-lived (2, 7, 23, 2931).

In the current study, we have found that the API nelfinavir induces a secretory form of diarrhea in HIV-1-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl- secretion in the human intestinal cell line T84 through the prolongation of a long-lived, storeoperated Ca2+ entry pathway. The resulting prolonged period of increased intracellular Ca2+ correlates with uncoupling of the apical membrane Ca2+-dependent Cl- conductance from effects of the downregulatory signals IP4, phosphorylated ERK (pERK), and phospho-p38, all present at normal levels. We propose that this prolonged, store-operated Ca2+ influx provokes in intestinal epithelia the enhanced Cl- secretion and consequent secretory diarrhea observed clinically in patients treated with APIs.


    METHODS
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 DISCUSSION
 REFERENCES
 
Clinical studies. Eight HIV-infected subjects (ages 21–54) with diarrhea (stool output >=300 g/24 h) for longer than 1 mo while on nelfinavir-containing regimens were admitted for 48 h and received a controlled diet (~4,200 calories/day: 50% carbohydrates, 12% protein, and 38% fat). Subjects remained on their prescribed daily dose of nelfinavir (5 subjects received 1,250 mg twice daily, and 3 three subjects received 750 mg 3 times daily). Antidiarrheal medications were discontinued 3 days before and during hospitalization. Subjects receiving APIs other than nelfinavir or with any condition known to cause diarrhea were excluded from study participation. Stool output was collected in preweighed containers and stored at 4°C. Specimens were weighed, homogenized, and centrifuged for 10 min at 2,000 rpm. Fecal supernatants were analyzed for [Na+] and [K+] by ion-selective electrodes (Hitachi 917; Boehringer Mannheim), and pH and osmotic content were measured. Fecal osmolar gap was defined as the difference between predicted ([Na+ + K+] x 2) and estimated fecal osmolality (290 mosmol/kgH2O) (15). Osmotic diarrhea was defined as a fecal osmolar gap of >60 mosmol/kgH2O with stool [Cl-] <= 15 meq/l and [Na+] <= 30 meq/l and a stool pH >= 6.0. Secretory diarrhea was defined as a fecal osmolar gap <=50 mosmol/kgH2O, pH >= 6.0, with stool [Na+] >= 60 meq/l and stool [Cl-] >= 30 meq/l. This study was approved by the IRB at Johns Hopkins University, and all subjects provided informed consent before participation.

Materials. Nelfinavir (Agouron Pharmaceuticals, La Jolla, CA), saquinavir (Roche Pharmaceuticals, Nutley, NJ), indinavir (Merck, West Point, PA), and ritonavir (Abbot Laboratories, North Chicago, IL) were used without excipients as kindly provided by the manufacturers. Stock solutions (20 mM) were stored at 4°C in equal parts of ethanol and DMSO. Cells were pretreated for 30 min with nelfinavir (or other API) unless otherwise stated. Anti-pERK (New England Biolabs, Beverly, MA) and anti-phospho-p38 antibodies (Cell Signaling, Beverly, MA) were used at 1:1,000 dilution. [3H]inositol was obtained from PerkinElmer (Boston, MA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Short-circuit measurement in intact monolayers. Short-circuit current (Isc) and transepithelial resistance were measured in confluent T84 cell monolayers grown on 0.33-cm2, 3-µm-pore polycarbonate filter inserts (Costar, Cambridge MA) in symmetrical baths of Hanks' balanced salt solution (HBSS) containing 0.05% BSA at 37°C as previously described (40, 41). Carbachol (CCh) was used at 100 µM and forskolin at 10 µM. We routinely observe a variability of 10–15 µA/cm2 in maximal Isc elicited by the muscarinic agonist CCh in T84 monolayers due to cell culture and plating of T84 cells on filter inserts from sequential passages, consistent with previous studies (28, 48).

Short-circuit current measurement in semipermeabilized monolayers. T84 cell monolayers (grown on 0.33-cm2 inserts) were incubated in the presence or absence of nelfinavir in buffers containing K+ or Cl- as the sole permeant ions (Table 1). Basolateral membrane K+ conductances, measured as short-circuit current [I(bl)K], were studied in cells permeabilized apically with 20 µM amphotericin B, in the presence of asymmetrical buffers that imposed a basolaterally directed sevenfold K+ gradient (apical solution 4, basal solution 5; see Table 1) as previously described (41). Transmembrane potential was clamped at 0 mV, and I(bl)K was measured before and after stimulation with CCh. Apical Cl- conductances, measured as short-circuit current [I(ap)Cl], were studied in cells permeabilized basolaterally with 100 µM amphotericin B, in the presence of symmetric high-Cl- buffer (solution 1) with transmembrane potential clamped at +10 mV (apical) as previously described (34). I(ap)Cl was measured before and after thapsigargin stimulation. Anion selectivity was measured in asymmetrical nelfinavir-containing buffers that imposed an apically directed ~20-fold gradient of either I- (basal solution 6, apical solution 7) or Cl- (basal solution 2, apical solution 3) as the sole permeant ions. Transepithelial currents were measured during 1-s voltage clamp periods ranging from -80 to +80 mV and normalized to baseline Isc at rest as described (34). Baseline current-voltage (I-V) curves obtained in the absence of agonist were subtracted from those measured after agonist treatment to calculate agonist-induced currents.


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Table 1. Composition of solutions used for electrophysiology studies

 

Immunoblots of pERK. T84 cell monolayers (grown on collagencoated 5-cm2 filters) were preincubated in the presence or absence of nelfinavir (0.4 to 40 µM) at 37°C for 30 min. Five minutes after the subsequent addition of CCh, cells were transferred to ice-cold PBS. Total cell lysates were prepared by scraping cells into lysis buffer (1 mM NaF, 1 mM sodium vanadate, 1% Triton X-100, one protease inhibitor MiniTab with EDTA; Hoffman-La Roche, Nutley, NJ) and then clarified by centrifugation. Lysates were analyzed for pERK by SDS-PAGE and immunoblot. Equal protein loads were confirmed by Ponceau stain.

Immunoblots of phospho-p38. T84 cell monolayers (grown on collagen-coated 5-cm2 filters) were preincubated in the presence or absence of 30 µM nelfinavir for 30 min at 37°C. After exposure to CCh (100 µM) for the indicated intervals (between 0 and 15 min), monolayers were transferred into ice-cold lysis buffer (1 mM NaF, 1 mM sodium vanadate, 1% Triton X-100, one protease inhibitor MiniTab with EDTA; Hoffman-La Roche). Cell lysates were clarified by centrifugation, and phophorylated p38 was assayed by immunoblot.

IP4 measurements. T84 cell monolayers (grown on collagen-coated 45-cm2 filters) were labeled for 24 h in inositol-free DMEM containing 5% fetal calf serum and 5 µCi/ml [3H]inositol. To ensure that cells were studied at steady state, we treated monolayers in the presence or absence of nelfinavir (40 µM) during the final 2.5 h of labeling with [3H]inositol. Three minutes after addition of CCh, inserts were transferred into ice-cold PBS, lysed in 10% trichloroacetic acid by repeated freeze-thaw cycles, and clarified by centrifugation. Total cell lipids were then extracted in H2O-saturated ether, dried overnight, and analyzed by HPLC as previously described (39).

Intracellular Ca2+ measurements. T84 cells cultured at subconfluent density on collagen-coated 5-cm2 coverslips were incubated at 37°C in growth medium containing 2 µM fura 2-AM (Molecular Probes, Eugene, OR) for 30 min, washed, and mounted in a modified Leiden chamber. T84 clusters containing >20 fura 2-stained cells at the cluster periphery were selected, and the intracellular Ca2+ concentration ([Ca2+]i) was measured in all stained cells within a single cluster by fura 2 fluorescence ratio imaging at 20°C in room air, as described previously (41).

Statistical methods. Unless otherwise indicated, data were tested for statistical significance by ANOVA (StatView; SAS, Cary, NC). P < 0.05 was chosen to denote statistical significance.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nelfinavir induces secretory diarrhea through selective potentiation of signaling by muscarinic and other Ca2+-dependent agonists. Stool samples collected from eight HIV-infected individuals on chronic nelfinavir therapy exhibited fecal electrolyte concentrations ([Na+] and [Cl-]) consistent with a secretory process. Stool pH and the fecal supernatant osmotic gap were consistent with a secretory process in seven subjects (Table 2). These data show that HIV-1-infected patients on chronic nelfinavir therapy have a secretory diarrhea.


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Table 2. Stool output in HIV-infected patients treated with nelfinavir

 

To examine the cellular mechanisms underlying this secretory defect, we utilized the human intestinal T84 cell line. T84 cells model regulated Cl- secretion in the human intestine (1). Exposure of resting T84 cells to nelfinavir (30 µM) produced no detectable effect on Cl- secretion (Isc) or transepithelial resistance. Nelfinavir-pretreated monolayers subsequently exposed to CCh showed a three- to fourfold increase in peak Cl- secretory response (Fig. 1A) and a prolonged duration of Cl- secretion (30 min after CCh exposure, Isc was 3.5 ± 0.3 µA/cm2 in control and 9.1 ± 0.83 µA/cm2 in nelfinavir-pretreated monolayers, means ± SE, n = 7 experiments, P <= 0.001). Nelfinavir pretreatment did not alter the decrease in transepithelial resistance observed after subsequent treatment with CCh (598 ± 74 and 723 ± 133 {Omega}·cm2, means ± SE, in control and nelfinavir-treated T84 cell monolayers, respectively). Nelfinavir had no effect on Cl- secretion elicited by the cAMP-dependent agonists vasoactive intestinal peptide (VIP; 5 nM) (Fig. 1, B and E) or adenosine (10 µM; see below). Stimulation of Isc by nelfinavir (at all tested concentrations) was observed only after CCh treatment (Fig. 1C). The EC50 value for nelfinavir was ~4–8 µM after 30-min preincubations approximating the peak plasma concentration (4 mg/l or 6 µM) measured in humans treated with 1,250 mg of nelfinavir twice daily (17). Nelfinavir (30 µM) did not change the EC50 value of CCh, and the CCh-potentiating effect of nelfinavir was not reversed after 24 h (nelfinavir increased the peak response to CCh by 180%). Nelfinavir also potentiated the action of two other Ca2+-dependent secretagogues (Fig. 1D): thapsigargin (5 µM) and the bile acid taurodeoxycholate (500 µM). The effects of nelfinavir on agonist-stimulated Isc in T84 cells are summarized in Fig. 1E. The structurally related HIV APIs saquinavir and indinavir similarly potentiated muscarinic Cl- secretion in T84 cells (Fig. 2). Nelfinavir pretreatment of the human intestinal cell line HT29 C119a also potentiated CCh-activated Isc (data not shown). Thus nelfinavir and other APIs produce long-lasting stimulatory effects on Isc induced by Ca2+ agonists in two human colonocyte cell lines.



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Fig. 1. Nelfinavir acts synergistically with muscarinic agonists to potentiate Cl- secretion. A: time course of 100 µM carbachol (CCh)-induced shortcircuit current (Isc) in intact T84 cell monolayers pretreated for 90 min with ({diamondsuit}) or without ({square}) nelfinavir (30 µM). Data are representative of 6 independent experiments. B: time course of 5 nM vasoactive intestinal peptide (VIP)-induced Isc in monolayers pretreated with ({diamondsuit}) or without ({square}) nelfinavir. Data are representative of 3 independent experiments. C: nelfinavir concentration-response curve for monolayers stimulated with CCh ({square}) and VIP ({bullet}). Data are means ± SE; n = 3 experiments. D: time courses of Isc induced by thapsigargin in cells pretreated with ({diamondsuit}) or without ({square}) nelfinavir. E: agonist-induced Isc in T84 monolayers pretreated with (light shaded bars) or without (dark shaded bars) nelfinavir. Data are means ± SE; n >= 3 experiments for each agonist. *P < 0.05. TDC, tauro-deoxycholate; Thaps, thapsigargin.

 


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Fig. 2. Human immunodeficiency virus (HIV)-1 protease inhibitors potentiate Cl- secretion elicited by the muscarinic agonist CCh. Time courses of CCh-induced Isc are shown in intact T84 monolayers pretreated ({diamondsuit}) or untreated ({square}) with 100 µM saquinavir (A) or indinavir (B). Data for both inhibitors are representative of 2 independent experiments.

 

Nelfinavir potentiates an apical Ca2+-dependent Cl- conductance and uncouples it from downregulatory signals. We assessed the relative contributions of basolateral and apical conductances to nelfinavir-stimulated Isc by studying selectively permeabilized monolayers. To test stimulation of basolateral K+ channels, we permeabilized selectively the apical membranes of T84 cells with the ionophore amphotericin B, and the monolayer was exposed to an apical-to-basolateral K+ gradient with K+ as the sole permeant ion, as previously described (41). After achievement of steady state, CCh was added and basolateral K+ conductance was measured as the short-circuit current I(bl)K. These apically permeabilized monolayers exhibited similar basolateral K+ conductances after muscarinic stimulation in the presence or absence of nelfinavir (Fig. 3, A and B). Thus nelfinavir has no effect on basolateral K+ conductance activated by CCh stimulation (believed to be mediated by the Ca2+-dependent K+ channel IK1/KCNN4) (12). Nonetheless, Cl- secretion elicited by nelfinavir requires basolateral K+ conductance and is blocked fully by high concentrations of the K+ channel inhibitor clotrimazole (Fig. 4).



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Fig. 3. Nelfinavir does not inhibit basolateral K+ conductance in T84 cells. A: time course of CCh-induced basolateral membrane K+ conductance [I(bl)K] in apically permeabilized T84 monolayers pretreated with ({diamondsuit}) or without ({square}) nelfinavir. B: peak increase in I(bl)K above baseline [{Delta}I(bl)K]. Data are means ± SE; n = 3 experiments. NS, nonsignificant.

 


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Fig. 4. Nelfinavir-induced Cl- secretion is inhibited by the K+ channel blocker clotrimazole. CCh-induced Cl- secretion in control and nelfinavirtreated intact cell monolayers in the absence (solid bars) or presence (shaded bars) of 30 µM clotrimazole. Data are means; n = 2 experiments. CLT, clotrimazole.

 

Nelfinavir could also act by increasing the apical membrane Ca2+-dependent Cl- conductance or possibly (though less likely, in view of Fig. 1B) through activation of the apical cAMP-dependent CFTR. We therefore permeabilized selectively the basolateral membrane of T84 cell monolayers with amphotericin B. Cells were then studied in symmetrical solutions containing Cl- as the only permeant ion, and apical Cl- conductance, measured as I(ap)Cl, was measured as previously described (41). Basolateral permeabilization precludes the use of the muscarinic agonist CCh in studies assessing apical Ca2+-activated Cl- conductances. Thus we used the endoplasmic Ca2+-ATPase pump inhibitor thapsigargin in these experiments. These studies showed that thapsigargin-induced I(ap)Cl in basolaterally permeabilized T84 cell monolayers was significantly higher in cells pretreated with nelfinavir than in control cells (Fig. 5, A and B).



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Fig. 5. Nelfinavir increases T84 transepithelial Cl- secretion by potentiation of a Ca2+-dependent apical Cl- conductance. A: time course of 5 µM thapsigargin-induced apical membrane current [I(ap)Cl] in basolaterally permeabilized T84 monolayers pretreated with ({square}) or without ({diamondsuit}) nelfinavir and studied in Cl- solutions. B: increase in I(ap)Cl measured 15 min after exposure to 5 µM thapsigargin [{Delta}I(ap)Cl]. Data are means ± SE; n = 7 experiments. P < 0.05. The voltage dependence of 5 µM thapsigargin-induced (C) or 10 µM forskolin-induced (D) apical membrane currents [I(ap)anion] in basolaterally permeabilized T84 monolayers were studied as described in METHODS in media containing either I- ({blacksquare}) or Cl- ({square}) as sole permeant anions. Data are means ± SE; n = 3 experiments. E: sensitivity of Cl- secretion to inhibition by dithiothreitol (DTT) in monolayers treated with CCh alone, CCh in the presence of nelfinavir (Nelf), or forskolin. Data are means ± SE; n = 3–7 experiments.

 

To characterize further this nelfinavir-enhanced apical Cl- conductance, we compared apical Cl- and I- conductances of nelfinavir-pretreated, basolaterally permeabilized T84 cells before and after stimulation with thapsigargin or with the cAMP agonist forskolin. Thapsigargin-stimulated apical membrane current measured across nelfinavir-pretreated T84 monolayers in asymmetric I- solutions [I(ap)I] was greater than thapsigargin-stimulated apical membrane current measured in asymmetric Cl- solutions [I(ap)Cl]. This result was consistent with activation of an apical membrane Ca2+-gated Cl- conductance (although the reversal potential of the thapsigargin-stimulated currents suggested either substantial gluconate permeability of the anion conductance or substantial contribution of stimulated cation conductance) (Fig. 5C). In contrast, forskolin stimulated I(ap)Cl to a greater degree than I(ap)I in nelfinavir-pretreated monolayers, consistent with activation of CFTR (Fig. 5D). The nelfinavir-potentiated CCh-induced apical anion conductance also displayed sensitivity to inhibition by dithiothreitol (DTT; 2 mM), in contrast to the forskolin-induced conductance (Fig. 5E). Thus the nelfinavir-enhanced apical membrane Cl- conductance resembles the Ca2+-activated Cl- conductance CaCC, rather than the cAMP-gated CFTR (18).

The transient elevation of Isc that typifies muscarinically induced Cl- secretion in T84 cells is generally attributed to rapid downregulation of apical membrane Cl- channels by the parallel synthesis of IP4 and the phosphorylation of the MAP kinase intermediates ERK and p38 (2931). These downregulators of apical Ca2+-dependent Cl- conductance render it refractory to further stimulation by Ca2+-dependent agonists for up to 30 min after withdrawal of muscarinic activation (26). Downregulation of basolateral membrane K+ channels by transactivation of the EGF receptor also follows muscarinic activation in T84 cells and also contributes to the transient nature of CCh-elevated Isc. However, basolateral K+ conductance is unaltered in nelfinavir-pretreated cells as shown in Fig. 3, A and B. We therefore tested whether nelfinavir potentiation of apical Cl- conductance might be explained by inhibition of the muscarinic activation of IP4 synthesis or by inhibition of phosphorylation of ERK and p38.

We first tested whether nelfinavir had any effect on the refractory period to Ca2+-dependent agonists observed after muscarinic activation. Control monolayers stimulated with CCh exhibited a typical increase in Isc followed by a rapid return to baseline. There followed a period refractory to subsequent treatment with thapsigargin (Fig. 6A). In contrast, nelfinavir-pretreated monolayers failed to exhibit such a refractory period after muscarinic stimulation (Fig. 6A). The nelfinavir-pretreated cells displayed normal sensitivity to thapsigargin exposure only 15 min after the initial exposure to CCh. This response resembles that of cells exposed to thapsigargin without pretreatment with nelfinavir or CCh (Fig. 6A). The mean results from three independent studies (Fig. 6B) show that nelfinavir abrogates the refractory period seen normally in T84 cells after stimulation with CCh. Despite abolition of the post-CCh-refractory period of T84 cells by nelfinavir pretreatment, nelfinavir had no detectable effect on CCh-induced levels of pERK (Fig. 6C), phospho-p38 (Fig. 6D), or IP4 (Fig. 6, E–H). These data demonstrate that the Ca2+-activated Cl- conductance in nelfinavir-pretreated cells is functionally un-coupled from normal levels of these physiological downregulatory signals.



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Fig. 6. Nelfinavir uncouples Ca2+-activated Isc from downregulation by phosphorylated ERK (pERK), phospho-p38, and inositol 3,4,5,6-tetrakisphosphate (IP4). A: time course of Isc in intact T84 monolayers treated without CCh ({bullet}) or with CCh in the presence ({diamondsuit}) or absence ({square}) of nelfinavir. Thapsigargin was applied to all monolayers at 45 min. B: peak {Delta}Isc induced by thapsigargin. Data are means ± SE; n = 3 experiments. C: Western blot of total cell extracts for pERK prepared from intact T84 cell monolayers pretreated with or without the indicated concentrations of nelfinavir and subsequently exposed or not exposed to 100 µM CCh for 5 min. Data are representative of 2 independent experiments. D: Western blot of total cell extracts for phospho-p38 prepared from T84 cell monolayers pretreated without (-Nelfin) or with (+Nelfin) nelfinavir studied at baseline (lane 2) and after addition of CCh for 0, 0.5, 1, 5, and 15 min (lanes 2–7). Lane 1 was loaded with phospho-p38. The gel was divided and vertically offset for visual clarity. E–H: HPLC analyses of [3H]IP4 in total cell extracts prepared from control (E and G) or nelfinavir-pretreated monolayers (F and H) and subsequently exposed (G and H) or not to CCh (E and F). IP4 controls were run in parallel (not shown). IP4 levels were similar in control (14,268 dpm) and nelfinavir-pretreated (15,191 dpm) T84 cell monolayers. Data are representative of 2 independent experiments.

 

Nelfinavir potentiates cytosolic [Ca2+]i signaling. Because the effects of nelfinavir pretreatment were observed only in cells exposed to Ca2+-dependent agonists, we examined the effect of nelfinavir on intracellular Ca2+ signaling in fura 2-loaded T84 cells. Nelfinavir itself had no detectable effect on [Ca2+]i in resting cells (not shown). After muscarinic stimulation, however, Ca2+ transients in nelfinavir-pretreated cells were increased in magnitude and duration compared with those observed in cells not exposed to nelfinavir (Fig. 7A). The peak increase in [Ca2+]i induced by CCh in nelfinavir-pretreated cells was 138 ± 10 nM (n = 9) vs. 56 ± 4 nM (n = 4; means ± SE) in cells unexposed to nelfinavir. In contrast, nelfinavir pretreatment had no detectable effect on intracellular Ca2+ transients induced by CCh in a Ca2+-free bath (increase in [Ca2+]i: 43 ± 7 vs. 45 ± 5 nM, respectively, n = 3; mean ± SE) (Fig. 7B). Thus the enhanced [Ca2+]i response induced by CCh in nelfinavir-pretreated cells was entirely dependent on influx of extracellular Ca2+. The enhanced muscarinic Cl- secretion observed in nelfinavir-pretreated monolayers was inhibited by the Ca2+-permeable cation channel inhibitor SKF-96365 (50 µM) (Fig. 8, n = 3, P < 0.05). In contrast, the L-type Ca2+ channel blockers verapamil (25 µM) and nifedipine (1 µM) were without apparent effect on nelfinavir-induced secretory responses (not shown).



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Fig. 7. Nelfinavir enhances plasma membrane Ca2+ influx induced by CCh. A: CCh (added at filled arrows) elicited intracellular Ca2+ concentration ([Ca2+]i) transients in clusters of T84 cells pretreated with ({bullet} and {square}) or without ({blacktriangleup}, right-displaced trace) nelfinavir. The increased [Ca2+]i in nelfinavir-pretreated cells was reversed ({bullet}) by 3 mM BaCl2 (added at open arrow). Each trace is representative of 4–5 cell clusters imaged on 2–4 separate coverslips. B: CCh-induced [Ca2+]i transients in T84 cells in nominally Ca2+-free medium pretreated with ({circ}) or without ({blacktriangleup}) nelfinavir. Each trace is representative of 3 cell clusters imaged on 3 coverslips.

 


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Fig. 8. Nelfinavir-induced Isc is blocked by the Ca2+-permeable cation blocker SKF-96365 (SKF). CCh-induced Cl- secretion in control and nelfinavir-pretreated intact T84 monolayers is shown in the absence (solid bars) or presence (shaded bars) of SKF-96365. Data are means ± SE; n = 3 experiments. *P < 0.05.

 

Basolateral addition of Ba2+ (3 mM) to T84 monolayers inhibited the enhancement of the muscarine-induced Isc observed in nelfinavir-pretreated monolayers (Fig. 9A), whereas apical Ba2+ had no effect (not shown). As expected, basolateral application of Ba2+ to untreated monolayers failed to inhibit normal CCh-induced Cl- secretion (Fig. 9A; summarized in Fig. 9B). Moreover, basolateral Ba2+ did not inhibit the CCh-triggered increase in basolateral K+ conductance in apically permeabilized T84 cells pretreated with nelfinavir (Fig. 9, C and D) confirming directly that Ba2+ does not affect IK1 in this experimental system. The inhibitory effect of Ba2+ is also not due to inhibition of the K+ channel KCNQ1/KCNE3, because the chromanol inhibitor of this channel, 293B, similarly had no effect on the potentiation of the CCh-induced Isc in intact monolayers pretreated with nelfinavir (not shown). Thus the inhibition by Ba2+ of the enhanced Ca2+ transient in nelfinavir-pretreated T84 cells did not appear secondary to inhibition of either the cAMP-regulated K+ channel KCNQ1/KCNE3 or the Ca2+-gated K+ channel IK1.



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Fig. 9. Nelfinavir activates a Ba2+-sensitive plasma membrane Ca2+ entry pathway not normally induced by muscarinic agonists. A: representative time course of CCh-induced Isc in intact T84 monolayers pretreated with nelfinavir alone ({diamondsuit}), with nelfinavir and Ba2+ (3 mM, {diamond}), with Ba2+ alone ({blacktriangleup}), or with medium ({square}). B: summary of peak Isc values in experiments similar to those in A. Ba2+-treated cells are shown in shaded bars. Data are means ± SE; n = 3 experiments. C: representative time courses of I(bl)K in apically permeabilized T84 monolayers pretreated with nelfinavir and activated by CCh in the absence ({diamond}) or presence ({bullet}) of 3 mM Ba2+. D: summary of peak I(bl)K values in experiments similar to those in C. Data are means ± SE; n = 3 experiments. E: representative time courses of CCh-induced Isc in nelfinavir-pretreated intact T84 monolayers. Thapsigargin was applied to all monolayers 12 min after addition of CCh. In 1 set of monolayers ({blacktriangleup}), Ba2+ was added after the thapsigargin response had peaked. F: summary of inhibition by Ba2+ of peak thapsigargin-induced {Delta}Isc during the refractory period following CCh treatment. Data are means ± SE; n = 3 experiments.

 

We then considered the possibility that Ba2+ blocks a CCh-activated Ca2+ entry pathway in nelfinavir-pretreated cells. As shown in Fig. 7A, CCh induced [Ca2+]i transients in nelfinavir-pretreated, fura 2-loaded T84 cells that exceeded both in magnitude and duration those observed in cells unexposed to nelfinavir. Addition of Ba2+ to the bath rapidly reduced [Ca2+]i toward baseline levels (Fig. 7A). Thus Ba2+ exposure of nelfinavir-pretreated T84 cells inhibits in parallel the nelfinavir-potentiated Isc across monolayers and the nelfinavir-potentiated [Ca2+]i transient recorded on coverslips, without detectable inhibition of basolateral K+ conductance. This correlation suggested that Ba2+ might also inhibit the nelfinavir-associated escape from the refractory period that follows muscarinic activation. Indeed, Ba2+ inhibited fully the Isc induced by thapsigargin added soon after muscarinic activation (Fig. 9, E and F). These data suggest that nelfinavir pretreatment potentiates Ca2+ entry in CCh-stimulated T84 cell monolayers via a mechanism that can be inhibited by basolateral exposure to Ba2+. However, in T84 cells not pretreated with nelfinavir, Ba2+ has no effect on CCh-activated Isc or on the CCh-induced [Ca2+]i transient. Thus nelfinavir-elicited, Ba2+-sensitive, CCh-activated Ca2+ entry is not part of the normal response to muscarinic stimulation in untreated T84 cells.

We tested the role of intracellular Ca2+ stores in the regulation of the nelfinavir-elicited Ca2+ entry pathway. Intracellular Ca2+ stores of T84 cells grown on coverslips were depleted by muscarinic stimulation in nominally Ca2+-free bath. In the absence of extracellular Ca2+, CCh induced small [Ca2+]i transients with indistinguishable peak [Ca2+]i values 134 ± 58 nM above baseline in nelfinavir-pretreated cells and 108 ± 38 nM in untreated cells (n = 4, mean ± SE) that rapidly returned to baseline levels (Fig. 10, A and B). Readdition of 2.7 mM extracellular Ca2+ in the continued presence of CCh rapidly increased [Ca2+]i to peak values of 229 ± 31 and 183 ± 27 nM above baseline in nelfinavir-pretreated and untreated cells, respectively (n = 4, means ± SE) (Fig. 10, A and B). However, the rate of the subsequent decline in [Ca2+]i in nelfinavir-pretreated cells was much slower than that in untreated cells. [Ca2+]i in the absence of nelfinavir fell 78 ± 4% (mean ± SE) from peak values within 8 min after bath Ca2+ readdition (Fig. 10, A and C). In contrast, [Ca2+]i in nelfinavir-pretreated cells decreased only 30 ± 3% from peak levels during the same period (P < 0.0005; Fig. 10, B and C). Thus influx-dependent elevation of [Ca2+]i following bath Ca2+ readdition to CCh-stimulated T84 cells was prolonged by nelfinavir pretreatment.



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Fig. 10. Nelfinavir potentiates store-regulated Ca2+ entry into T84 cells. A: representative time course of changes in T84 cell [Ca2+]i during transition from 1.3 mM Ca2+ to a nominally Ca2+-free bath, followed by exposure to CCh (100 µM), then bath Ca2+ readdition (2.7 mM). B: [Ca2+]i time course in similarly treated T84 cells in the presence of nelfinavir (30 µM, with 30-min pretreatment). C: percent return of [Ca2+]i toward the pre-Ca2+ readdition baseline value (%recovery) 8 min after bath Ca2+ readdition to CCh-stimulated cells untreated (-) or pretreated (+) with nelfinavir. Data are means ± SD; n = 4 coverslips. P < 0.0001. D: time course of changes in T84 cell [Ca2+]i during transition from 1.3 mM Ca2+ to a nominally Ca2+-free bath, followed by exposure to thapsigargin (5 µM), then bath Ca2+ readdition (2.7 mM). E: [Ca2+]i time course in similarly treated T84 cells in the presence of nelfinavir (30 µM, with 30-min pretreatment). F: percent return of [Ca2+]i toward the pre-Ca2+ readdition baseline value (%recovery) 15 min after bath Ca2+ readdition to thapsigargin-stimulated cells untreated or pretreated with nelfinavir. Data are means ± SD: n = 4 coverslips. P < 0.0005.

 

After depletion of intracellular Ca2+ stores by thapsigargin exposure of T84 cells in a nominally Ca2+-free medium, readdition of bath Ca2+ induced an elevation of [Ca2+]i larger than that observed after CCh stimulation, with a peak value of 402 ± 11 nM above baseline (n = 4, mean ± SE; Fig. 10D). This store depletion-activated Ca2+ influx was larger still in nelfinavir-pretreated cells, with a peak value of 816 ± 125 nM above baseline (n = 4, mean ± SE, P < 0.05; Fig. 10E). Moreover, the rate of subsequent [Ca2+]i decline in nelfinavir-pretreated cells was again much slower than that in untreated cells. Whereas 15 min after bath Ca2+ readdition, [Ca2+]i had declined 52 ± 6% from peak values in untreated cells, this decline was only 26 ± 4% in nelfinavir-pretreated cells (means ± SE, P < 0.0005; Fig. 10F). Thus nelfinavir pretreatment enhanced both the magnitude and duration of thapsigargin-induced store depletion-activated Ca2+ influx in T84 cells. These data suggest activation by nelfinavir of a store-operated plasmalemmal Ca2+ entry pathway.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These studies provide the first mechanistic insights into the pathophysiology of API-induced diarrhea in HIV-infected patients. Nelfinavir potentiates muscarinic Cl- secretion in intestinal epithelial cells by recruitment of a slowly inactivating basolateral Ca2+ entry pathway that can be further activated by depletion of intracellular Ca2+ stores and is inhibited by basolateral exposure to Ba2+. This additional Ca2+ uptake pathway differs from that activated by CCh in the normal muscarinic signal transduction cascade, because Ba2+ has no detectable effect on CCh-activated I(ap)Cl in untreated cells. The enhanced [Ca2+]i signal induced by nelfinavir may cause or contribute to the uncoupling of a CaCC-like apical Cl- conductance from normal downregulation by IP4, pERK, and phospho-p38. The resulting prolonged activation of apical Cl- conductance contributes to (and may suffice to explain) the enhanced Cl- secretory response. This mechanism of action is distinct from that recently proposed for the chemotherapeutic agent flavopiridol, which elicits a secretory response in T84 cells via inhibition of the downregulatory signals affecting the apical Ca2+-activated Cl- conductance (27).

On the basis of these results, we propose that nelfinavir acts in vivo directly on the intestinal mucosa to enhance the activity of muscarinic and other Ca2+-dependent agonists by recruiting an additional store-operated plasmalemmal Ca2+ entry pathway to potentiate an otherwise normal secretory response. Such a mechanism of action on intestinal Cl- secretion should initiate a secretory form of diarrhea, a prediction confirmed by our clinical studies in hospitalized HIV-infected adults. At peak in vivo plasma concentrations of 6 µM for nelfinavir, the Cl- secretory response would be near the ED50 for nelfinavir's in vitro effect on T84 cells. Thus small differences among individual patient plasma nelfinavir concentrations due to differences in drug metabolism or excretion may have large effects on Cl- secretion and diarrhea severity.

Other tested APIs also potentiate the muscarinically induced Cl- secretory response in T84 cells. Furthermore, nelfinavir effectively potentiates the Cl- secretory responses elicited by a wide range of Ca2+-dependent agonists, including those induced by bile acids often present in the human colon. Thus the secretory diarrhea described in up to 30% of API-treated patients may be the result of enhanced secretion by normal neurocrine and paracrine secretory regulators, triggered by subclinical degrees of bile acid malabsorption as well as other genetic, dietary, or environmental factors.

The apical membrane conductance potentiated by nelfinavir in T84 cells exhibits several properties characteristic of the CLCA family of Ca2+-activated Cl- conductances, including Ca2+ dependence, a preference for I- over Cl-, and sensitivity to inhibition by DTT. Although the molecular identity of the intestinal crypt cell Ca2+-activated Cl- conductance remains uncertain, members of the CLCA gene family have been proposed as candidates (3, 19). This apical Ca2+-activated Cl- conductance is thus a therapeutic target, but specific inhibitors for it and for the cloned CLCA channels remain unidentified. Chlorotoxin, active against the Ca2+-activated Cl- conductance of gliomas and astrocytes, appears inactive in T84 cells (33). Our results also show that nelfinavir effectively uncouples the apical Cl- conductance from downregulation by the intracellular mediators IP4, pERK, and phospho-p38 but has no apparent effect on basolateral K+ conductance after muscarinic activation. Thus nelfinavir may elevate levels of intracellular antagonists of these downregulatory signals to potentiate the physiological agonists of Ca2+-dependent intestinal Cl- secretion.

The reversal potential of thapsigargin-activated apical membrane currents in nelfinavir-pretreated cells (Fig. 5C) suggests that nelfinavir may potentiate in parallel an apical membrane Ca2+-actived cation conductance and an apical Ca2+-activated Cl- conductance, as suggested also by our previous studies in the absence of nelfinavir (34). Merlin et al. (34) also showed that gluconate permeability of the thapsigargin-induced T84 cell apical membrane conductance is minimal. Similarly, CCh-induced increase in Isc across intact, nelfinavir-pretreated T84 cell monolayers is abrogated in nominally Cl--free, symmetrical sodium gluconate solutions (not shown). Activation of apical cation currents is consistent with previous reports of nonspecific cation currents in T84 cells (4, 8, 11, 45, 46).

We have previously shown that the imidazole antifungal clotrimazole and its des-imidazolyl metabolite block intestinal Cl- secretion by inhibition of both the cAMP-activated K+ conductance (likely mediated by KCNQ1/KCNE3) and the Ca2+-gated K+ conductance likely mediated by IK1 (KCNN4) (41). Blockade of the appropriate K+ channel(s) fully inhibits Cl- secretion induced by either cAMP or Ca2+-dependent agonists in vitro and by cAMP-dependent agonists in vivo. Basolateral K+ channel activity is also required for nelfinavir-stimulated Cl- secretion by T84 cells and is blocked fully by 30 µM clotrimazole, although at this concentration clotrimazole is nonspecific. At the more specific concentration of 1 µM, clotrimazole had no effect on CCh-induced Cl- Isc in cells pretreated with nelfinavir or (as previously reported) without nelfinavir (not shown). Clotrimazole has been administered in humans at doses sufficient to block IK1 with minimal toxicity (5, 6). Thus clotrimazole or other more specific IK1 blockers (16, 44) may be useful for treatment of the secretory diarrhea induced by nelfinavir and other APIs used in the treatment of HIV.

Direct blockade of the nelfinavir-recruited Ca2+ entry pathway, however, might affect more selectively the adverse effects of APIs on regulation of intestinal Cl- secretion. Such blockade would allow for specific inhibition of API-induced potentiation of Ca2+-dependent Cl- secretory responses without affecting the normal muscarine-induced secretory response. Thus molecular identification of this Ca2+ entry pathway might facilitate development of specific inhibitors of this pathway as well as APIs that do not increase its activity.

One family of plasmalemmal Ca2+-permeable cation channels is the transient receptor potential (TRP) superfamily (9, 36). TRPV6 (ECaC2/CaT1) and TRPV5 (ECaC1/CaT2) are the most extensively studied TRP channels of the intestine. ECaC2/CaT1 has been localized by in situ hybridization to surface enterocytes of the rat (37) but appears to be absent from human colon (21, 38). ECaC1/CaT2 has been immunolocalized to the apical membrane of villous tip enterocytes in rabbit duodenum (20) and in transverse and distal colon of the human (21, 38). In addition, TRPV6 overexpressed in some cultured cells confers increased store-operated cation channel activities. However, the pathway recruited by nelfinavir in T84 cells may represent a basolateral pathway of intestinal crypt cells.

This novel pathway is notable for the ability of Ba2+ to block the nelfinavir-associated enhancement of apical Cl- secretion as well as for the enhanced magnitude and prolonged duration of muscarinically induced [Ca2+]i elevation. In contrast, Ba2+ has no effect on the muscarinically induced Isc in cells not treated with nelfinavir. Thus the inhibitory effect of Ba2+ on nelfinavir-potentiated Cl- secretion may represent a Ba2+ block of the nelfinavir-induced Ca2+ entry pathway. It is also possible that Ba2+ may permeate the nelfinavir-induced Ca2+ entry pathway. Once inside the cell, Ba2+ might then block a nelfinavir-induced, Ca2+-dependent reversal of the normal inactivation processes for the apical membrane Ca2+-activated Cl- conductance. Thus the molecular identity of the nelfinavir target(s) remains to be determined.

The effects of nelfinavir on Ca2+ signaling in intestinal epithelial cells may similarly apply to other cell types affected by API-based therapeutics. If so, altered Ca2+ signaling in adipocytes, myocytes, or hepatocytes may contribute to other API-associated dose-limiting side effects including lipodystrophy and insulin resistance (13, 43).


    ACKNOWLEDGMENTS
 
We thank C. Sears for assistance in the design and analysis of clinical studies and Jason Borawski for help in completing the basic studies.

GRANTS

This work was supported by National Institutes of Health (NIH) Grants DK-48106 and DK-57827 (to W. I. Lencer), DK-51056 and CA-86207 (to S. L. Alper), Clinical Investigator Award DK0–2729 (to P. A. Rufo), NIH Training Grant HD-07466 (P. W. Lin), and DK-34854 of the Harvard Digestive Diseases Center (to W. I. Lencer and S. L. Alper). P. A. Rufo was a Pfizer Fellow in the Clinical Investigator Training Program of the Harvard-MIT Division of Health Sciences and Technology and the Beth Israel Deaconess Medical Center. Human studies were completed with the assistance of the General Clinical Research Center at Johns Hopkins Hospital (RR-00035).

Present address of P. W. Lin: Emory University School of Medicine, Department of Pediatrics, 2040 Ridgewood Drive, Atlanta, GA 30322.

Present address of A. Andrade: Division of Infectious Diseases, Johns Hopkins University, Baltimore, MD 21205.


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. I. Lencer, GI Cell Biology, Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail: wayne.lencer{at}childrens.harvard.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.

* P. A. Rufo and P. W. Lin contributed equally to this work. Back

{dagger} A. Andrade was lead investigator for the clinical study. Back


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Barrett KE. Positive and negative regulation of chloride secretion in T84 cells. Am J Physiol Cell Physiol 265: C859-C868, 1993.[Abstract/Free Full Text]

2. Barrett KE and Keely SJ. Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 62: 535-572, 2000.[CrossRef][ISI][Medline]

3. Bertelsen LS, Eckmann L, and Barrett KE. Prolonged interferon-{gamma} exposure decreases ion transport, NKCC1, and Na+-K+-ATPase expression in human intestinal xenografts in vivo. Am J Physiol Gastrointest Liver Physiol 286: G157-G165, 2004.[Abstract/Free Full Text]

4. Braun AP and Schulman H. A non-selective cation current activated via the multifunctional Ca2+-calmodulin-dependent protein kinase in human epithelial cells. J Physiol 488: 37-55, 1995.[Abstract]

5. Brugnara C, Armsby CC, Sakamoto M, Rifai N, Alper S, and Platt O. Oral administration of clotrimazole and blockade of human erythrocyte Ca++-activated K+ channels: the imidazole ring is not required for inhibitory activity. J Pharmacol Exp Ther 273: 266-272, 1995.[Abstract]

6. Brugnara C, Gee B, Armsby CC, Kurth S, Sakamoto M, Rifai N, Alper SL, and Platt OS. Therapy with oral clotrimazole induces inhibition of the gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest 97: 1227-1234, 1996.[Abstract/Free Full Text]

7. Carew MA, Yang X, Schultz C, and Shears SB. myo-Inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line. J Biol Chem 275: 26906-26913, 2000.[Abstract/Free Full Text]

8. Champigny G, Verrier B, and Lazdunski M. A voltage, calcium, and ATP sensitive non selective cation channel in human colonic tumor cells. Biochem Biophys Res Commun 176: 1196-1203, 1991.[ISI][Medline]

9. Clapham D. TRP channels as celluar sensors. Nature 426: 517-524, 2003.[CrossRef][ISI][Medline]

10. Cuthbert AW, Hickman ME, Thorn P, and MacVinish LJ. Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone. Am J Physiol Cell Physiol 277: C111-C120, 1999.[Abstract/Free Full Text]

11. Devor DC and Duffey ME. Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study. Am J Physiol Cell Physiol 263: C780-C787, 1992.[Abstract/Free Full Text]

12. Devor DC, Singh AK, Frizzell RA, and Bridges RJ. Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel. Am J Physiol Lung Cell Mol Physiol 271: L775-L784, 1996.[Abstract/Free Full Text]

13. Dowell P, Flexner C, Kwiterovich PO, and Lane MD. Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors. J Biol Chem 275: 41325-41332, 2000.[Abstract/Free Full Text]

14. Duran S, Spire B, Raffi F, Walter V, Bouhour D, Journot V, Cailleton V, Leport C, and Moatti JP. Self-reported symptoms after initiation of a protease inhibitor in HIV-infected patients and their impact of adherence to HAART. HIV Clinical Trials 2: 38-45, 2001.[Medline]

15. Eherer AJ and Fordtran JS. Fecal osmotic gap and pH in experimental diarrhea of various causes. Gastroenterology 103: 545-551, 1992.[ISI][Medline]

16. Fanger CM, Rauer H, Neben AL, Miller MJ, Rauer H, Wulff H, Rosa JC, Ganelin CR, Chandy KG, and Cahalan MD. Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels. J Biol Chem 276: 12249-12256, 2001.[Abstract/Free Full Text]

17. Flexner C. HIV1 protease inhibitors. N Engl J Med 338: 1281-1292, 1998.[Free Full Text]

18. Fuller CM, Ji HL, Tousson A, Elble RC, Pauli BU, and Benos DJ. Ca2+-activated Cl- channels: a newly emerging anion transport family. Pflügers Arch 443: S107-S110, 2001.[CrossRef][ISI][Medline]

19. Gaspar KJ, Racette KJ, Gordon JR, Loewen ME, and Forsyth GW. Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes. Physiol Genomics 3: 101-111, 2000.[Abstract/Free Full Text]

20. Hoenderop JG, Hartog A, Stuiver M, Doucet A, Willems PH, and Bindels RJ. Localization of the epithelial Ca2+ channel in rabbit kidney and intestine. J Am Soc Nephrol 11: 1171-1178, 2000.[Abstract/Free Full Text]

21. Hoenderop JG, Vennekens R, Muller D, Prenen J, Droogmans G, Bindels RJ, and Nilius B. Function and expression of the epithelial Ca2+ channel family: comparison of mammalian ECaC1 and 2. J Physiol 537: 747-761, 2001.[Abstract/Free Full Text]

22. Huber SM, Tschop J, Braun GS, Nagel W, and Horster MF. Bradykinin-stimulated Cl- secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels. Pflügers Arch 438: 53-60, 1999.[CrossRef][ISI][Medline]

23. Ismailov II, Fuller CM, Berdiev BK, Shlyonsky VG, Benos DJ, and Barrett KE. A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels. Proc Natl Acad Sci USA 93: 10505-10509, 1996.[Abstract/Free Full Text]

24. Jensen BS, Strobaek D, Olesen SP, and Christophersen P. The Ca2+-activated K+ channel of intermediate conductance: a molecular target for novel treatments? Curr Drug Targets 2: 401-422, 2001.[ISI][Medline]

25. Jensen BS, Stroebaek D, Christophersen P, Jorgensen TD, Hansen C, Silahtaroglu A, Olesen SP, and Ahring PK. Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275: C848-C856, 1998.[Abstract]

26. Kachintorn U, Vajanaphanich M, Barrett KE, and Traynor-Kaplan AE. Elevation of inositol tetrakisphosphate parallels inhibition of Ca2+-dependent Cl- secretion in T84 cells. Am J Physiol Cell Physiol 264: C671-C676, 1993.[Abstract/Free Full Text]

27. Kahn ME, Senderowicz A, Sausville EA, and Barrett KE. Possible mechanisms of diarrheal side effects associated with the use of a novel chemotherapeutic agent, flavopiridol. Clin Cancer Res 7: 343-349, 2001.[Abstract/Free Full Text]

28. Keely SJ and Barrett KE. ErbB2 and ErbB3 receptors mediate inhibition of calcium-dependent chloride secretion in colonic epithelial cells. J Biol Chem 274: 33449-33454, 1999.[Abstract/Free Full Text]

29. Keely SJ and Barrett KE. p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells. Am J Physiol Cell Physiol 284: C339-C348, 2003.[Abstract/Free Full Text]

30. Keely SJ, Calandrella SO, and Barrett KE. Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells is mediated by intracellular Ca2+, PYK-2, and p60src. J Biol Chem 275: 12619-12625, 2000.[Abstract/Free Full Text]

31. Keely SJ, Uribe JM, and Barrett KE. Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion. J Biol Chem 273: 27111-27117, 1998.[Abstract/Free Full Text]

32. Kunzelman K, Hubner M, Schreiber R, Levy-Holzman R, Garty H, Bleich M, Warth R, Slavik M, von Hahn T, and Greger R. Cloning and function of the rat colonic epithelial K+ channel KVLQT1. J Membr Biol 179: 155-164, 2001.[CrossRef][ISI][Medline]

33. Maertens C, Wei L, Tytgat J, Droogmans G, and Nilius B. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels. Br J Pharmacol 129: 791-801, 2000.[Abstract/Free Full Text]

34. Merlin D, Jiang L, Strohmeier GR, Nusrat A, Alper SL, Lencer WI, and Madara JL. Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells. Am J Physiol Cell Physiol 275: C484-C495, 1998.[Abstract/Free Full Text]

36. Montell C, Birnbaumer L, and Flockerzi V. The TRP channels, a remarkably functional family. Cell 108: 595-598, 2002.[ISI][Medline]

37. Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, Brown EM, and Hediger MA. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 274: 22739-22746, 1999.[Abstract/Free Full Text]

38. Peng JB, Chen XZ, Berger UV, Weremowicz S, Morton CC, Vassilev PM, Brown EM, and Hediger MA. Human calcium transport protein CaT1. Biochem Biophys Res Commun 278: 326-332, 2000.[CrossRef][ISI][Medline]

39. Rameh LE and Cantley LC. The role of phosphoinositide 3-kinase lipid products in cell function. J Biol Chem 274: 8347-8350, 1999.[Free Full Text]

40. Rufo PA, Jiang L, Moe SJ, Brugnara C, Alper SL, and Lencer WI. The antifungal antibiotic, clotriamzole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells. J Clin Invest 98: 2066-2075, 1996.[Abstract/Free Full Text]

41. Rufo PA, Merlin D, Riegler M, Ferguson-Maltzman MH, Dickinson BL, Brugnara C, Alper SL, and Lencer WI. The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera. J Clin Invest 100: 3111-3120, 1997.[Abstract/Free Full Text]

42. Schroeder BC, Waldegger S, Fehr S, Bleich M, Warth R, Greger R, and Jentsch TJ. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 403: 196-199, 2000.[CrossRef][ISI][Medline]

43. Shi H, Halvorsen YD, Ellis PN, Wilkison WO, and Zemel MB. Role of intracellular calcium in human adipocyte differentiation. Physiol Genomics 3: 75-82, 2000.[Abstract/Free Full Text]

44. Stocker JW, DeFranceschi L, McNaughton-Smith GA, Corrocher R, Beuzard Y, and Brugnara C. ICA-17043, a novel Gardos channel blocker, prevents sickled red blood cell dehydration in vitro and in vivo in SAD mice. Blood 101: 2412-2418, 2003.[Abstract/Free Full Text]

45. Tabcharani JA and Hanrahan JW. On the activation of outwardly rectifying anion channels in excised patches. Am J Physiol Gastrointest Liver Physiol 261: G992-G999, 1991.[Abstract/Free Full Text]

46. Vaca L and Kunze DL. Anion and cation permeability of a large conductance anion channel in the T84 human colonic cell line. J Membr Biol 130: 241-249, 1992.[ISI][Medline]

47. Warth R, Hamm K, Bleich M, Kunzelman K, von Hahn T, Schreiber R, Ullrich E, Mengel M, Trautman N, Kindle P, Schwab A, and Greger R. Molecular and functional characterization of the small Ca2+-regulated K+ channel (rSK4) of colonic crypts. Pflügers Arch 438: 437-444, 1999.[CrossRef][ISI][Medline]

48. Zund G, Madara JL, Dzus AL, Awtrey CS, and Colgan SP. Interleukin-4 and interleukin-13 differentially regulate epithelial chloride secretion. J Biol Chem 271: 7460-7464, 1996.[Abstract/Free Full Text]





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