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J. Biol. Chem., Vol. 275, Issue 35, 26906-26913, September 1, 2000

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myo-Inositol 3,4,5,6-Tetrakisphosphate Inhibits an Apical Calcium-activated Chloride Conductance in Polarized Monolayers of a Cystic Fibrosis Cell Line^*

Mark A. Carew^§, Xiaonian Yang, Carsten Schultz^¶, and Stephen B. Shears

From the  Inositide Signaling Section, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ^¶ Institut für Organische Chemie, Universität Bremen, UFT, 28359 Bremen, Germany

Received for publication, March 20, 2000, and in revised form, May 29, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) inhibit apical Ca²⁺-activated Cl conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P₄ levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P₄ levels were therefore specifically elevated, independently of receptor activation, by incubating cells with a cell-permeant bioactivable analogue, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester (Bt₂Ins (3,4,5,6)P₄/AM). The latter inhibited Ca²⁺-activated Cl secretion by 60%. We next used nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from electrochemically dominating ion movements through the apical membrane. Thus, we studied autonomous regulation of apical Cl channels in situ. The properties of Cl flux across the apical membrane were those expected of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold greater permeability of I over Cl. Following nystatin-treatment, we elevated intracellular levels of Ins(3,4,5,6)P₄ with either purinergic agonists or with Bt₂Ins(3,4,5,6)P₄/AM. Both protocols inhibited Ca²⁺-activated Cl secretion (up to 70%). These studies provide the first demonstration that, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P₄-inhibited CaCC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The movement of Cl ions across secretory epithelia underpins the process of salt and fluid secretion (1-3). Some of the molecular aspects of the secretory process have been well characterized. Cl enters the cell across the basolateral membrane through a Na⁺/K⁺/2Cl co-transporter (Fig. 1). The CFTR¹ comprises an important conduit for Cl exit across the apical membrane (Fig. 1). However, considerable controversy and many questions surround attempts to understand the extent to which Ca²⁺-activated Cl conductance (CaCC)² both participate in the secretory process (Fig. 1), and might even be a focal point for its regulation. The resolution of these issues is of considerable biochemical and physiological significance, in no small part because this uncertainty impacts the relevance of proposals that epithelial salt and fluid secretion can be regulated by Ins(3,4,5,6)P₄ (4-8).

In order to understand the nature of the controversy concerning the contribution that CaCC makes to salt and fluid secretion, it is necessary to appreciate the electrochemical interdependence of the apical and basolateral membranes (Fig. 1). The primary regulation of CaCC activity does potentially provide a mechanism by which Cl secretion can be regulated. However, electrically uncompensated Cl efflux would depolarize the cell, inhibiting further Cl efflux. This does not occur during epithelial fluid secretion. A compensatory increase in the driving force for Cl efflux across the apical membrane comes from the hyperpolarization provided by the actions of Ca²⁺-dependent K⁺ channels in the basolateral membrane provides (Fig. 1). Equally, a stimulus that directly regulates K⁺ channel activity can secondarily affect Cl flux through CaCC. Indeed, when we first discovered that Ins(3,4,5,6)P₄ inhibited Cl secretion (4), Barrett (9) proposed that this resulted indirectly from Ins(3,4,5,6)P₄ primarily down-regulating K⁺ channel conductance. This possibility is not excluded by the demonstration that ¹²⁵I efflux through Cl channels was inhibited by Ins(3,4,5,6)P₄ in human colonic epithelial (T84) cell monolayers (10), since the latter study did not separately determine if Ins(3,4,5,6)P4 affected K⁺ efflux. Indeed, it has been emphasized that these assays of isotope efflux from monolayers may not reveal the primary effector sites of regulatory agents (11). The reduction in rate of ¹²⁵I efflux that followed the addition of Ins(3,4,5,6)P₄ (10) could have resulted indirectly from Ins(3,4,5,6)P₄ inhibiting K⁺ channel conductance. This interpretational difficulty underscores the need for different types of experiments that study if Ins(3,4,5,6)P₄ directly regulates CaCC in situ.

Another factor has prevented Ins(3,4,5,6)P₄ from being widely accepted as a physiologically relevant regulator of CaCC. The polarized cell monolayer is the paradigm for studying Cl secretion, which is a process that CaCC can only contribute to, if this Cl channel is expressed in the apical membrane (Fig. 1). Evidence has been published indicating that, when polarized, T84 cells do not contain CaCC in their apical membrane (12-15). Furthermore, it has been argued that CaCC also does not contribute to intestinal Cl secretion in vivo, since in the intestinal epithelium of human CF individuals, a Ca²⁺-activated, Cl secretory pathway was not observed (14, 16). Thus, the colonic epithelial T84 cell model has been criticized as not offering physiologically relevant information on the role of CaCC in regulating Ca²⁺-activated salt and fluid secretion in vivo. This is unfortunate because T84 cells were used for all of the previous experiments that have studied the influence of Ins(3,4,5,6)P₄ upon secretion (4, 6, 10, 17). There is, therefore, a need to study the mechanism of action of Ins(3,4,5,6)P₄ in cell that does not originate from intestinal epithelia.

In this report, we confront these problems by taking a new approach to the study of CaCC regulation by Ins(3,4,5,6)P₄. We selected a particular cell-type, namely, the human CFPAC-1 pancreatic ductal cell line (18), in which the effects of Ins(3,4,5,6)P₄ could be investigated in a less controversial context. For example, in some cells at least, Cl efflux that is activated by Ca²⁺ has been suggested to occur through CFTR and not CaCC (13). As CFPAC-1 cells were derived from an individual with cystic fibrosis (18), we could interpret effects of Ins(3,4,5,6)P₄ with the knowledge that we can exclude any Cl secretion occurring through CFTR (Fig. 1). The selection of CFPAC-1 cells for our study was also driven by their ability to form electrically resistive monolayers (19) so that electrogenic Cl transport can be studied in Ussing chambers (20). Finally, we have previously shown that non-polarized CFPAC-1 cells have an Ins(3,4,5,6)P₄-sensitive CaCC (7). Thus, we had the opportunity to now determine the physiological relevance of the latter effect to the control of Cl secretion from polarized CFPAC-1 monolayers.

In order to specifically study the actions of Ins(3,4,5,6)P₄, we delivered it into the interior of the cells in the polarized monolayer, by using the cell-permeant and bioactivable analogue, Bt₂Ins(3,4,5,6)P₄/AM (4). We went on to study the actions of Ins(3,4,5,6)P₄ under conditions in which we also isolated the apical membrane from the electrochemical influences of ion channels in the basolateral membrane (Fig. 1); this is essential if, in polarized monolayers, one is to study direct regulation of apical CaCC by Ins(3,4,5,6)P₄ in situ. The latter objective was achieved by using nystatin to selectively permeabilize the basolateral membranes to monovalent anions and cations (21). This perforation procedure deprives the basolateral membrane of its transmembrane electrical gradient which otherwise provides a major driving force for Cl exit across the apical membrane (13, 22). The results we have obtained represent the first unequivocal demonstration that Ins(3,4,5,6)P₄ is an active constituent of a second messenger system designed to carefully regulate the participation of apical CaCC in the important process of salt and fluid secretion.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Ion transport mechanisms in secretory epithelia. The schematic shows five ion transport mechanisms that can participate in epithelial salt and fluid secretion (adapted from Ref. 46). Cl enters the cell through the basolateral Na+/K+/2Cl co-transporter; the Na+ and K+ are recycled across the basolateral membrane by the Na+/K+ ATPase and Ca2+-activated K+ channels (KCA). At least one Cl transporter, CFTR, is in the apical membrane. There is some controversy concerning which secretory epithelia also contain apical CaCC. See text for further details.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Culture of CFPAC-1 Cells-- CFPAC-1 cells (CRL 1918, American Type Culture Collection, Manassas, VA) were cultured at 37 °C (5% CO₂, 95% air) in Iscove's modified Dulbecco's medium (Hyclone) supplemented with 10% (w/v) fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin.

Assay of Inositol Phosphate Turnover-- Cells were radiolabeled with 100 µCi/ml [³H]inositol (American Radiolabeled Chemicals, St. Louis, MO) for 4 days (medium was replaced on the third day) in 700 µl on 24-well plates. The cells were confluent by the end of this radiolabeling protocol. After labeling was completed, the culture medium was removed and the cells were washed twice in Krebs-Henseleit medium from which bicarbonate was omitted and 10 mM HEPES (pH 7.5) was added. Cells were then stabilized in 300 µl of the same buffer for 2 h, then 20 mM LiCl was added for an additional 20 min. Any subsequent additions to the cells are as described under "Results." Cells were quenched with 300 µl of 0.6 M ice-cold perchloric acid containing 0.2 mg/ml InsP_6. The acid-soluble extract was neutralized with 200 µl of 1 M K₂CO₃ containing 5 mM Na₂EDTA. Inositol phosphates were then separated by HPLC using a 250 × 4.6-mm Synchropak Q100 SAX column (Thompson Instruments, Chantilly, VA) eluted with the following gradient, generated by mixing water with Buffer B (2 M (NH₄)H₂PO₄, pH 3.35, with H₃PO₄): 0-5 min, 0% B; 5-240 min, 0-65% B. The flow rate was 0.5 ml/min. Typically, the eluate was mixed on-line with 3 volumes of Monoflow-4 scintillant (National Diagnostics, Atlanta, GA), and ³H-labeled inositol phosphates were detected using a Radiometric D515 Flow Scintillation Analyzer (Packard Instrument Co., Meriden, CT). Levels of ³H-labeled inositol phosphates were normalized against [³H]InsP₆, which was not affected by any of the experimental manipulations employed in this study.

In some experiments, the HPLC eluate was divided into 1-ml fractions, and 150-µl aliquots were taken and manually mixed with scintillant so as to identify the [³H]Ins(3,4,5,6)P₄/[³H]Ins(1,4,5,6)P₄ peak. The proportion that was [³H]Ins(3,4,5,6)P₄ was evaluated in the following manner: 150-µl aliquots of this peak (240 to 580 disintegrations/min) were mixed with 120 µl of buffer containing 2 M HEPES (pH 8.0 with KOH), 0.7 mM EDTA, 8.7 mM MgSO₄, 6.7 mM ATP, 13.3 mM phosphocreatine, 3 units of phosphocreatine kinase. Next was added 22.5 µl of purified (100 µg/ml) Ins(3,4,5,6)P₄ 1-kinase (23) in buffer containing 20 mM Tris-HCl (pH 8.0), 2 mM MgCl₂, 1 µg/ml leupeptin. This final mixture (final pH 6.7) was incubated for 3 h at 37 °C. Assays were acid-quenched, neutralized, and then the InsP₄ and InsP₅ were separated by HPLC, as described above. The amount of [³H]InsP₅ that was formed was used to estimate the proportion of [³H]Ins(3,4,5,6)P₄ in the original [³H]Ins(3,4,5,6)P₄/[³H]Ins(1,4,5,6)P₄ peak. This assay was calibrated with a standard of [³H]Ins(3,4,5,6)P₄ prepared from [³H]inositol-labeled chick erythrocytes (24). Extracts from non-radiolabeled cells were "spiked" with the [³H]Ins(3,4,5,6)P₄ standard and chromatographed by HPLC. Aliquots of the fractions that contained [³H]Ins(3,4,5,6)P₄ were incubated with the 1-kinase, exactly as described above, and 90 ± 1% (n = 5) of the substrate was phosphorylated to [³H]InsP₅. Control experiments showed that the addition of twice as much enzyme, or extending the assay time to 4 h, did not increase the yield of [³H]InsP₅ (data not shown). Thus, the non-metabolized InsP₄ represents a slight contamination of the [³H]Ins(3,4,5,6)P₄ standards with [³H]Ins(1,4,5,6)P₄ (see also Ref. 24).

Bioelectric Measurements-- Cells were detached from culture flasks by incubation with 0.25% (w/v) trypsin, 0.02% (w/v) EDTA for 3-4 min. Cells were then resuspended in serum-containing medium and centrifuged at 500 × g for 3 min to remove trypsin. The supernatant was discarded and the cells were resuspended at a concentration of 1.5 × 10⁶/ml. Aliquots of 0.2 ml of cells were then seeded into the circular wells (area = 0.45 cm²) of permeable supports previously sterilized by UV irradiation. Permeable supports were made by gluing (Silastic^TM sealant, Dow Corning, Midland, MI) a Sylgard^TM ring to filters composed of mixed cellulose esters (pore size 0.45 µm, catalog number HAWP 02500, Millipore Corp., Bedford, MA). Seeded supports were floated on culture medium and incubated for 4 or 5 days before being mounted in modified Snapwell holders in Ussing chambers (EasyMount System, Physiologics Instruments, San Diego, CA). Both sides of the monolayer were bathed, at 37 °C, in Krebs-Henseleit solution containing 118 mM NaCl, 11.1 mM glucose, 4.7 mM KCl, 2.5 mM CaCl₂ 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 35 mM NaHCO₃; the medium was gassed with 95% O₂, 5% CO₂ to maintain a pH of 7.5. Where indicated, either the apical or basolateral solution was modified by the substitution of 80 mM Na gluconate for 80 mM NaCl, in order to generate a Cl gradient (128 mM to 48 mM Cl) between the two chambers of the Ussing apparatus. In these experiments, total Ca²⁺ was increased to 4 mM to compensate for Ca²⁺ chelation by gluconate (22). Current-passing and voltage-sensing Ag/AgCl pipette electrodes were filled with 3% agarose, 3 M KCl. Current-passing electrodes were connected directly to the head-stage of a voltage-clamp amplifier (EC-825, Warner Instrument Corp. Hamden, CT). Voltage-sensing electrodes were connected to the head-stage via 3% agarose, 3 M KCl bridges and calomel electrodes. The potential difference between electrodes was corrected and the resistance of the bathing solution was nullified before mounting the filters. The resistance across the monolayers was 290 ± 12 ohms/cm² (n = 90).

After being mounted in the Ussing chambers, the epithelial monolayers were continuously voltage-clamped (i.e. short-circuited) to zero transepithelial potential difference. The magnitude and the polarity of the applied short-circuit current (I_SC) is a measure of the extent and direction of the electrogenic ion flux across the epithelial layer (20). A positive change in I_SC indicates an increase in net anion flux from the basolateral to apical chambers. Conversely, a negative change in I_SC indicates an increase in net anion flux from the apical to basolateral chambers. I_SC was recorded, via a PowerLab/800 interface (AD Instruments Inc., Milford, MA), on a PC running Chart v3.4. Data were further processed using Graphpad Prism (Graphpad Software, San Diego CA); the zero time was set as indicated in the figures, then individual plots were each baseline subtracted using as a reference point the average value of I_SC between 1 and 2 min. Composite, average plots were then constructed from all individual I_SC traces in a dataset.

Other Materials-- Bt₂Ins(3,4,5,6)P₄/AM and Bt₂Ins(1,4,5,6)P₄/AM were synthesized as described previously (4;25). Stock solutions were prepared in dried dimethyl sulfoxide containing 5% (v/v) pluronic F-127; AM esters were added to the apical chamber by diluting the vehicle 250-fold in the Krebs-Henseleit solution. Ionomycin and BHQ were from Calbiochem (San Diego, CA). All other chemicals were from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Receptor-mediated Accumulation of Inositol Phosphates in CFPAC-1 Cells-- Berridge and colleagues (26, 27) were the first to show that receptor-activated PLC activity in intact cells can be assayed by recording changes in levels of Ins(1,4,5)P₃ and all of its downstream metabolites (InsP_n, where n = 1-4, but excluding Ins(3,4,5,6)P₄, see Fig. 2). Lithium was used to inhibit InsP dephosphorylation to inositol, thereby "trapping" InsP_n (26). These changes in levels of InsP_n were normalized against InsP₆, which was unaffected by receptor activation (data not shown). UTP, a purinergic agonist, was used to activate the CFPAC-1 cells (Fig. 3A). A dose of 100 µM UTP was chosen since, in CFPAC-1 cells, this provides maximal activation of receptor-coupled PLC, Ca²⁺ mobilization, and Cl secretion (19, 28, 29); data not shown). There was a biphasic effect of UTP upon InsP_n (Fig. 3A); their levels initially increased relatively rapidly, during the 1-2 min immediately following the addition of UTP. After that time, the rate of generation of InsP_n declined substantially (Fig. 3A). The levels of InsP_n did not decay significantly during this time course, due to the presence of the lithium trap (see above and Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Schematic illustrating the assay of receptor-dependent changes in inositol phosphate levels in cells. The schematic shows how receptor-coupled PLC activity hydrolyzes phosphatidylinositol 4,5-P2 to Ins(1,4,5)P3. The latter is converted to a number of downstream metabolites, which are "trapped" by using lithium to inhibit the InsP1 phosphatase (as indicated in the figure). Thus, changes in levels of InsPn (where n = 1-4, except that Ins(3,4,5,6)P4 is excluded, see below) represent an assay of PLC activity in situ (26, 47). Changes in Ins(3,4,5,6)P4 are recorded separately, because this polyphosphate belongs, with InsP5, in a distinct metabolic pool (32); this is why these two polyphosphates are depicted in an inset to the main figure. See Ref. 23 for a description of the mechanisms by which alterations in PLC activity are coupled to changes in the net rate of dephosphorylation of InsP5 to Ins(3,4,5,6)P4.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   The effect of UTP upon inositol phosphate levels in CFPAC-1 cells. [3H]Inositol-labeled CFPAC-1 cells were treated with either 100 µM UTP or vehicle and the ensuing changes in levels of inositol phosphates were determined by HPLC (see "Experimental Procedures"). Panel A shows the total increase in levels of [3H]Ins(1,3,4,5)P4 + [3H]Ins(1,3,4,6)P4 + [3H]Ins(1,4,5)P3 + [3H]Ins(1,3,4)P3 + [3H]InsP2 + [3H]InsP1. The increase in levels of all of these inositol phosphates (i.e. total counts/min of [3H]InsPn) was normalized against counts/min of [3H]InsP6. Basal levels of this parameter were 3.3 ± 0.3. The increases in levels of inositol phosphates that are depicted in panel A represent an assay of PLC activity in situ (26, 47). Data are means and standard errors from three experiments. Panel B depicts the separate, fold increase in [3H]Ins(3,4,5,6)P4 levels in the same experiments (see text for details). The [3H]Ins(3,4,5,6)P4 pool is 60-fold smaller than the concentration of its [3H]InsP5 precursor pool, so the latter was not statistically reduced during UTP activation (data not shown). Basal levels of [3H]Ins(3,4,5,6)P4/[3H]InsP6 were 0.071 ± 0.026.

Agonist-dependent accumulation of Ins(1,4,5)P₃ and its metabolites is invariably accompanied by an increase in the net rate of dephosphorylation of InsP₅ to Ins(3,4,5,6)P₄ (Fig. 2 and Ref. 5). The mechanisms that couple together these two signaling events are described elsewhere (23). We used a two-step procedure to analyze receptor-coupled increases in Ins(3,4,5,6)P₄ levels in CFPAC-1 cells. HPLC was first used to separate an Ins(3,4,5,6)P₄/Ins(1,4,5,6)P₄ peak from all the other inositol phosphates. Ins(3,4,5,6)P₄ and Ins(1,4,5,6)P₄ are stereoisomers that cannot be resolved from each other by HPLC. Thus, aliquots of this mixture of isomers were then incubated with the stereoselective Ins(3,4,5,6)P₄ 1-kinase (23), and the amount of InsP₅ that was formed was taken as proportional to the amount of Ins(3,4,5,6)P₄ that was in the original sample (see "Experimental Procedures"). Using these methods we made the surprising observation that, after UTP was added, Ins(3,4,5,6)P₄ responded relatively acutely. Levels of this particular isomer rose 3-fold within 1 min (Fig. 3B). This was unexpected because the consensus that has emerged from previous experiments is that activation of cell-surface receptors is accompanied by relatively slow rates of elevations in Ins(3,4,5,6)P₄ levels (30-32). Thus, in CFPAC-1 cells, an elevation in Ins(3,4,5,6)P₄ levels is a major response of inositol phosphates to purinergic activation. The Ins(3,4,5,6)P₄ concentration went on to be nearly 4-fold elevated after 10 min (Fig. 3B), and remained at this level for at least 30 min (data not shown).

Receptor-activated Cl Secretion in CFPAC-1 Cells-- Ussing chambers were used to record electrogenic ion transport across a polarized monolayer of CFPAC-1 cells grown on a permeable support. The parameter which records this ion transport is I_SC, which becomes more positive in value following an increased net anion flux from the basolateral to apical chambers of the Ussing apparatus. The addition of 10 µM amiloride to the apical chamber had no effect on I_SC in CFPAC-1 cells (data not shown, and Ref. 19), excluding the possibility that Na⁺ absorption might contribute to the I_SC response. A maximally effective dose of 100 µM UTP (19, 28, 29), when added to the apical membrane, elicited a transient increase in I_SC that peaked at 4.31 ± 0.78 µA/cm² above basal levels after approximately 80 s (Fig. 4). The I_SC then decayed, but not to baseline values; a sustained phase (approximately 14% of the peak response) was maintained for at least 600 s (Fig. 4). A similar response to purinergic activation of CFPAC-1 cells has been described in a previous report (19). The fact that this change in anion flux is primarily carried by Cl ions is confirmed by ion substitution experiments, as well as by the use of ion transport inhibitors. For example, in CFPAC-1 cells the purinergic-dependent increase in I_SC (Fig. 4) was almost completely inhibited when (i) extracellular Cl was removed (19), or (ii) the Cl channels in the apical membrane were inhibited by either 100 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ref. 19 and data not shown) or by 100 µM niflumic acid (Fig. 4). Thus, for the remainder of this study, we consider I_SC and Cl current (I_Cl) to be equivalent.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of UTP and niflumic acid on ISC. ICl across polarized monolayers of CFPAC-1 cells was measured in Ussing chambers (see "Experimental Procedures"). Immediately before the addition of 100 µM UTP to the apical chamber (indicated by the arrow), the absolute value of ISC was 4.0 ± 1.2 uA/cm2 (n = 8). The value of ISC was averaged and set to zero immediately prior to the zero time point, as indicated under "Experimental Procedures." The lower trace represents a mean of four experiments where 100 µM niflumic acid was added to the apical chamber 5 min prior to the addition of UTP. Niflumic acid was absent from the experiments in the upper trace, which represents a mean of eight experiments. The accompanying schematic illustrates the [Cl] in the apical and basolateral chambers, and the polarity of the UTP-activated Cl flux in these experiments (basolateral to apical) through CaCC in the apical membrane and the basolateral Na+/K+/2Cl co-transporter. Also shown is the site of niflumic acid action (i.e. CaCC).

Bt2Ins(3,4,5,6)P₄/AM Inhibits Ca²⁺-activated Cl Secretion in Polarized Monolayers of CFPAC-1 Cells-- To study the effect of Ins(3,4,5,6)P₄ upon Ca²⁺-activated Cl secretion, we followed the protocol that we developed in our earlier work with T84 cells (4). In the latter study, intracellular levels of Ins(3,4,5,6)P₄ were elevated from 0.8 to 3 µM by addition to the extracellular medium of 200 µM of a cell-permeant, bioactivable derivative, namely, Bt₂Ins(3,4,5,6)P₄/AM (4). Cl secretion was specifically activated by mobilizing cellular Ca²⁺ stores through inhibition of sarcoendoplasmic reticulum Ca²⁺-ATPase by BHQ (Fig. 5). Compared with the pulse of Cl secretion that follows receptor activation (Fig. 4), BHQ promoted a more slowly developing secretory response that took >10 min to reach its peak value (3.16 ± 0.68 µA/cm², Fig. 5). On the other hand, BHQ produces a more sustained Ca²⁺ response than that elicited by cell-surface receptor-coupled agonists (33), so BHQ-activated Cl secretion was also more sustained (Fig. 5) than that promoted by UTP (Fig. 4). There was no effect of BHQ upon the cellular levels of inositol phosphates (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of Bt2Ins(3,4,5,6)P4/AM and Bt2Ins(1,4,5,6)P4/AM upon ICl. ICl across polarized monolayers of CFPAC-1 cells was measured in Ussing chambers as described under "Experimental Procedures." In the experiments described by panel A, the monolayers were pretreated for 30 min by addition to the apical chamber of either 200 µM Bt2Ins(3,4,5,6)P4/AM, or an equivalent amount of the vehicle (trace labeled "control") that was used to dissolve the cell-permeant inositol phosphate (see "Experimental Procedures"). In the experiments described by panel B, the monolayers were pretreated for 30 min by addition to the apical chamber of either 200 µM Bt2Ins(1,4,5,6)P4/AM, or an equivalent amount of the vehicle (trace labeled "control") that was used to dissolve the cell-permeant inositol phosphate. The value of ISC for each condition was averaged (n = 7 for panel A, n = 4 for panel B) and set to zero immediately prior to the zero time point, as indicated under "Experimental Procedures." At the end of the 30 min pretreatment period, 25 µM BHQ was added to both apical and basolateral chambers.

Bt₂Ins(3,4,5,6)P₄/AM reduced BHQ-activated Cl secretion by 59% (to a peak of 1.29 ± 0.31 µA/cm², p < 0.05 versus vehicle control). It has also been shown many times that Ins(3,4,5,6)P₄ has no effect on Ca²⁺ mobilization itself (see Ref. 7, and references therein). Another important control experiment is the demonstration that Bt₂Ins(1,4,5,6)P₄/AM had no effect on Cl secretion (Fig. 5). Thus, our data represent the first demonstration that Ins(3,4,5,6)P₄ inhibits Ca²⁺-activated Cl secretion from CFPAC-1 cells. The experimental challenge we next undertook has not previously been attempted in any cell type: can we obtain any evidence that Ins(3,4,5,6)P₄ inhibits apical CaCC in a polarized monolayer?

Characterization of Apically Located Ca²⁺-activated Cl Channels in CFPAC-1 Cells-- In order to study the direct regulation of apical CaCC in polarized monolayers, it was necessary to eliminate the electrochemical gradient across the basolateral membrane that normally increases the driving force for Cl exit through CaCC. We achieved this by selectively treating the basolateral membrane with 0.5 mg/ml nystatin, which forms pores that are specifically permeable to small, monovalent cations and anions (21). Following the addition of nystatin, I_Cl settled to a new steady-state value that was 9.0 ± 1.3 µA/cm² (n = 25) less than the value of I_Cl that prevailed before nystatin was added.

In our studies we followed criteria that were previously introduced to verify the success of the nystatin permeabilization procedure in murine tracheal epithelial cells (22). A Cl concentration gradient was imposed in the apical (128 mM) to basolateral (48 mM) direction (see "Experimental Procedures"). Under these conditions, Ca²⁺-activated Cl secretion from intact monolayers still proceeds in the basolateral to apical direction (data not shown). However, once the electrochemical driving force across the basolateral membrane was eliminated with nystatin, net flux of Cl ions follows their concentration gradient, so the polarity of Ca²⁺-activated Cl flux was reversed relative to that in intact monolayers. Thus, for example, mobilization of Ca²⁺ by ionomycin promoted a negative change in I_Cl (maximally  = 19.1 ± 3.2 µA/cm², Fig. 6, A and C). The appearance of this more negative current acts as an internal control that verifies the efficacy of the nystatin treatment in every experiment.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Activation of ICl by ionomycin in CFPAC-1 monolayers following perforation of the basolateral membrane by nystatin. Monolayers of CFPAC-1 cells were placed in the Ussing apparatus as described under "Experimental Procedures." In these experiments, the [Cl] in the apical and basolateral chambers was adjusted as described under "Experimental Procedures," so as to impose a Cl concentration gradient of 128 to 48 mM, in either the apical to basolateral direction (panel C, see accompanying schematic in panel A) or the apical to basolateral direction (panel D, see accompanying schematic in panel B). The basolateral membrane was selectively perforated by addition of nystatin (0.5 mg/ml) to the basolateral chamber for 15 min prior to the addition (indicated by the arrow) to the apical chamber of 1 µM ionomycin. The value of ICl for each experiment was averaged (n = 5 for panel C, n = 6 for panel D). In panels A and B, the horizontal arrows show the direction of Cl flux, and the "holes" in the basolateral membrane depict the nystatin pores, which are selectively permeable only to small, monovalent anions and cations.

Note also that the activation by ionomycin was biphasic, such that I_Cl decayed from its maximum value to attain a steady-state level of 10. 9 ± 1.5 µA/cm² (Fig. 6C). A biphasic response to ionomycin has also previously been observed in nystatin-permeabilized bovine pancreatic duct cells (34). This phenomenon may reflect ionomycin elevating intracellular [Ca²⁺] in a biphasic manner. In addition, this ionomycin treatment slightly (1.9 ± 0.5-fold, n = 3) increased cellular levels of Ins(3,4,5,6)P₄, which could also inhibit Cl current. Third, it is possible there may be some channel deactivation (28). The idea that this biphasic phenomenon reflects, in part at least, some inherent property of the channel itself, is supported by the monophasic nature of the response of I_Cl to ionomycin when we reversed the direction of the Cl concentration gradient (Fig. 6, B and D). This procedure also caused the polarity of I_Cl to reverse, because the direction of Cl flux now followed the basolateral to apical Cl concentration gradient (Fig. 6B). Thus, ionomycin caused I_Cl to become more positive in value (Fig. 6D). The maximum response of I_Cl ( = +6.3 ± 1.0 µA/cm², Fig. 6D) now represented a smaller absolute change from baseline, compared with the effect of ionomycin upon I_Cl when the Cl concentration gradient was apical to basolateral (Fig. 6 and see above). This inherent preference to conduct Cl into rather than out of the cell (i.e. outward rectification) is exactly the property displayed by CaCC during whole cell patch clamp analysis (35).

Another characteristic of CaCC in whole cell patch clamp analysis is that it typically conducts I with an approximately 2-fold preference over Cl (11, 34). In our nystatin-permeabilized monolayers, when all of the NaCl in the apical chamber was replaced by equimolar NaI, the maximum current stimulated by ionomycin ( = 44.6 ± 4.3 µA/cm², n = 7) was a little over 2-fold greater than I_Cl (see above, and Fig. 6).

Sustained Cl secretion across intact monolayers requires continued Cl entry into the cell across the basolateral Na⁺/K⁺/2Cl co-transporter (Fig. 1). The latter requirement is by-passed if the basolateral membrane is successfully permeabilized to small monovalent anions and cations with nystatin. Thus, as an additional control to verify the nystatin was performing as anticipated, we examined whether inhibition of the basolateral Na⁺/K⁺/2Cl co-transporter (Fig. 1) by furosemide would have any effect on apical Cl flux (Fig. 7). If the co-transporter were contributing to net Cl current, furosemide would have caused an upward deflection of I_Cl. In fact, no such effect was observed even after the furosemide had been present for over 5 min (Fig. 7). The latter result confirms the conclusion from our other experiments (see above), namely, that nystatin successfully permeabilizes the basolateral membrane to monovalent anions and cations. Note we also performed a positive control using a different experimental paradigm, so as to verify that the batch of furosemide we used was active. That is, in intact monolayers, inhibition of the co-transporter by furosemide inhibited receptor-activated Cl secretion by about 30% (Fig. 8).

View larger version (11K): [in this window] [in a new window]   Fig. 7.   The effect of furosemide upon ICl in nystatin-perforated monolayers. ICl across monolayers of CFPAC-1 cells measured in Ussing chambers, in experiments in which the basolateral membrane was selectively perforated to small, monovalent anions and cations by addition of nystatin (0.5 mg/ml) to the basolateral chamber for 15 min. Then, 1 µM ionomycin was added (zero time) to the apical chamber, and at 8 min 100 µM furosemide was added to the basolateral chamber.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   The effect of furosemide upon ICl in intact monolayers. ICl across intact monolayers of CFPAC-1 cells was measured in Ussing chambers. The value of ICl for each experiment was averaged (n = 3). The point of addition of 100 µM UTP to the apical chamber is indicated by the arrow. Five min prior to the addition of UTP, either 100 µM furosemide (lower trace) or vehicle (upper trace) was added to the basolateral chamber.

The effect of Ins(3,4,5,6)P₄ upon Apical CaCC-- Having characterized an apical CaCC in nystatin-perforated monolayers (see above), we next studied the effect of Ins(3,4,5,6)P₄ in this paradigm (Fig. 9). As discussed previously (see above and Ref. 4), intracellular levels of Ins(3,4,5,6)P₄ can be elevated approximately 4-fold by adding 200 µM Bt₂Ins(3,4,5,6)P₄/AM to the extracellular medium. When we added 100 µM Bt₂Ins(3,4,5,6)P₄/AM to the apical chamber, the peak response of I_Cl to ionomycin was reduced by 45% to 10.6 ± 2.7 µA/cm² (p < 0.05 compared with control cells, see Fig. 9). The inhibition of the peak response by 200 µM Bt₂Ins(3,4,5,6)P₄/AM was approximately 70% (max  = 5.9 ± 0.9 µA/cm², p < 0.01 compared with controls, see Fig. 9) and in addition, the sustained response of I_Cl to ionomycin (5.2 ± 0.8 µA/cm²) was approximately 50% inhibited (p < 0.05 compared with control cells). These data represent the first demonstration that Ins(3,4,5,6)P₄ inhibits an apical CaCC in polarized epithelial monolayers. This new observation represents a major step forward in consolidating the hypothesis that Ins(3,4,5,6)P₄ is a physiologically significant regulator of Cl secretion.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   The effect of Bt2Ins(3,4,5,6)P4/AM upon ionomycin-activated ICl in nystatin-perforated monolayers. ICl across monolayers of CFPAC-1 cells was measured in Ussing chambers, in experiments in which the basolateral membrane was selectively perforated to small, monovalent anions and cations by addition of nystatin (0.5 mg/ml) to the basolateral chamber for 15 min prior to the addition to the apical chamber of 1 µM ionomycin (indicated by the arrow). In addition, 30 min prior to ionomycin, the apical chamber received 0 µM Bt2Ins(3,4,5,6)P4/AM (trace A, n = 6, vehicle only), 100 µM Bt2Ins(3,4,5,6)P4/AM (trace B, n = 4), or 200 µM Bt2Ins(3,4,5,6)P4/AM (trace C, n = 5). Also shown is the effect of 100 µM niflumic acid, added to the apical chamber 5 min prior to ionomycin, in the absence of Bt2Ins(3,4,5,6)P4/AM (trace D, broken line, n = 3).

A second method for studying the effect of elevated Ins(3,4,5,6)P₄ levels upon CaCC is by purinergic activation with UTP (Fig. 3). In control experiments, when UTP was added to the intact apical membrane by itself, there was a substantial ( = 14.2 ± 1.6, µA/cm², Fig. 9A) but largely transient stimulation of I_Cl in nystatin-treated monolayers. We next added 100 µM UTP to the intact apical membrane approximately 5 min after the addition of ionomycin, i.e. when I_Cl had attained a steady-state level (Fig. 10). UTP elicited a brief augmentation of I_Cl ( = 1.33 ± 0.17 µA/cm²). This effect was small, presumably because ionomycin had depleted most, but not all, of the UTP-sensitive Ca²⁺ stores. Immediately thereafter, UTP caused the I_Cl to become more positive in value, which under these conditions reflects an inhibition of net Cl flux. When added after ionomycin, UTP caused the levels of [³H]Ins(3,4,5,6)P₄ to be elevated 2.5 ± 0.3-fold (n = 3) compared with controls where ionomycin alone was added. Five min after the addition of UTP, the value of I_Cl (7.2 ± 1.1 µA/cm²) was statistically different (p < 0.05, paired t test) from the value of I_Cl at the time point where UTP was added (I_Cl = 8.9 ± 1.0 µA/cm²). In contrast, in paired control experiments where UTP was not added, the value of I_Cl did not change significantly over the same 5-min period. Note that the value of the steady-state ionomycin-dependent I_Cl, that was attained after addition of UTP (Fig. 10), was intermediate between the two Ins(3,4,5,6)P₄-inhibited steady-state values of I_Cl depicted in Fig. 9.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   The effect of UTP upon ionomycin-activated ICl in nystatin-perforated monolayers. ICl across monolayers of CFPAC-1 cells was measured in Ussing chambers, in experiments in which the basolateral membrane was selectively perforated to small, monovalent anions and cations by addition of nystatin (0.5 mg/ml) to the basolateral chamber for 15 min. After this pretreatment, the following additions were made to the apical chamber (as indicated by the arrows): 100 µM UTP alone (n = 7, panel A) or 1 µM ionomycin (n = 6, panel B) followed by either 100 µM UTP (upper trace, panel B) or vehicle alone (lower trace, panel B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our current study makes a major advance by showing for the first time that Ins(3,4,5,6)P₄ inhibits the conductance of CaCC in the apical membrane, in a physiologically relevant context of a polarized monolayer. This ability to study regulation of CaCC in situ was made possible by our using nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from having any electrochemical influence over CaCC function at the apical membrane.

These results also provide new information relevant to understanding the physiological control of secretion by the exocrine pancreas. The elevations in Ins(3,4,5,6)P₄ levels that accompany PLC activation of pancreatic duct cells will constrain the ability of purinergic agonists to act as efficient secretagogues. Indeed, in the CFPAC-1 cells, the response of Ins(3,4,5,6)P₄ to receptor activation by UTP was unexpectedly acute (3-fold increase within 1 min of receptor activation) compared with the less than 0.3-fold/min increases in Ins(3,4,5,6)P₄ typically observed following PLC activation in other cell types (32, 36-38).

The apical CaCC that we studied in CFPAC-1 monolayers has similar properties to the Ca²⁺-activated Cl channel described in many whole cell patch clamp studies of non-polarized secretory cells (see Ref. 35 for a review). In agreement with these earlier studies, we found that CaCC has higher permeability to I compared with Cl, and ionic conductance was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and by niflumic acid. The characteristic outward rectification of patch-clamped calcium-activated Cl currents (i.e. the inherent preference to conduct Cl into the cell), was also observed in our experiments (Fig. 6). Our studies therefore emphasize that the Cl current that has been previously measured in individual, non-polarized cells is relevant to the polarized monolayer. This conclusion counters the argument that measurements of CaCC in non-polarized cells are not relevant to understanding the physiological control of Cl secretion (12, 13).

Our new findings also have potential therapeutic implications. The absence of functional CFTR in the CF condition greatly impairs salt and fluid secretion, leading to obstructions in the pancreatic duct and intestinal tract, plus defective mucus clearance and bacterial infections in the lung (39). As there is agreement there is CaCC in the apical membrane of airway epithelial cells, there is some hope that CF individuals will benefit from pharmacological up-regulation of the alternative Ca²⁺-activated pathway for salt and fluid secretion (40-42). The activation of PLC-coupled receptors that are present on the surface of the lung epithelium can be achieved by inhalation of aerosolized UTP (40, 41). This procedure mobilizes cellular Ca²⁺ pools and, hopefully, up-regulates the rate of Cl flux through CaCC (41). Unfortunately, this UTP therapy yields only a small and transient improvement in airway fluid secretion and pulmonary function (41), so its clinical benefit is still under study. This approach is likely to be constrained by metabolism of the agonist, as well as by desensitization and internalization of the purinergic receptor itself (43). An additional, but previously unidentified factor that lies downstream of Ins(1,4,5)P₃ has also been acknowledged to inhibit purinergic-dependent Cl flux from secretory epithelia (44). Our data indicate Ins(3,4,5,6)P₄ could be this factor. Antagonism of the synthesis and/or actions of Ins(3,4,5,6)P₄ could therefore be a useful adjunct to UTP therapy.

There is evidence that Ca²⁺-dependent activation of apically located Cl channels in polarized epithelia is most effectively activated by the mobilization of Ca²⁺ stores that are ipsilateral rather than contralateral to the apical membrane (45). Our new data now show that Ins(3,4,5,6)P₄ also acts in the apical region of the cell. Thus, the same domain of the cell can contain signaling apparatus that directs both stimulatory and inhibitory inputs into overall regulation of CaCC activity. This spatial characterization of Ins(3,4,5,6)P₄ action adds an additional facet to our understanding that this inositol phosphate is a general and important regulator of Cl secretion.

	ACKNOWLEDGEMENTS

We thank the following for helpful comments on this manuscript: Drs A. Traynor-Kaplan, M. W. Y. Ho, C. M. P. Ribeiro, A. M. Paradiso, and J. W. Putney, Jr. We also thank Dr. M. Rudolf for assistance in the preparation of the cell-permeant inositol phosphates.

	FOOTNOTES

^* 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.

^§ To whom correspondence should be addressed. Tel.: 919-541-2630; Fax: 919-541-0559; E-mail: Carew@niehs.nih.gov.

Published, JBC Papers in Press, June 6, 2000, DOI 10.1074/jbc.M002316200

² There is some question as to the existence of cell-specific mechanisms which dictate whether ion conductance through the different CaCCs is either activated directly by Ca²⁺, and/or activated by Ca²⁺ indirectly through the actions of calmodulin-dependent protein kinase type II. To reflect this uncertainty, in this study we utilize "Ca²⁺-activated Cl conductance (CaCC)" as generic terminology that does not distinguish activation by CaMKII from direct stimulation by Ca²⁺.

	ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane regulator (i.e. cAMP-activated Cl channel); BHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; CF, cystic fibrosis; CaCC, Ca²⁺-activated Cl conductance; Ins(3, 4,5,6)P₄, myo-inositol 3,4,5,6-tetrakisphosphate; Ins(1, 4,5,6)P₄, myo-inositol 1,4,5,6-tetrakisphosphate; Bt₂Ins(1, 4,5,6) P₄/AM, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester; Bt₂Ins(3, 4,5,6)P₄/AM, 1,2-di-O-butyl-myo-inositol 1,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester; I_Cl, Cl current; I_SC, short-circuit current; HPLC, high performance liquid chromatography; PLC, phospholipase C.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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