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
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ABSTRACT |
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nelfinavir; clotrimazole; barium
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.
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METHODS |
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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 1015 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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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.
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RESULTS |
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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
·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
48 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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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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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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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, EH). 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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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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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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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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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.
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DISCUSSION |
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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).
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ACKNOWLEDGMENTS |
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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 DK02729 (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.
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FOOTNOTES |
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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.
A. Andrade was lead investigator for the clinical study.
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