pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line

Matthew E. Loewen,1 Lane K. Bekar,2 Wolfgang Walz,2 George W. Forsyth,1 and Sherif E. Gabriel3

1Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4; 2Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5; and 3Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599-7020

Submitted 15 January 2004 ; accepted in final form 18 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The effects of CLCA protein expression on the regulation of Cl conductance by intracellular Ca2+ and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl conductance by pCLCA1 protein expression. Cl transport was measured as 36Cl efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of 36Cl efflux and amplitude of currents in patch-clamp studies after the addition of the Ca2+ ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca2+-sensitive Cl conductance could be detected in 14-day-postpassage cells. In contrast to Ca2+-sensitive Cl conductance, endogenous cAMP-dependent Cl conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl conductance were retained through differentiation in Caco-2 cells, whereas Ca2+-dependent Cl conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl channels to Ca2+ and cAMP agonists but apparently lacks inherent Cl channel activity under growth conditions where endogenous channels are not expressed.

cystic fibrosis transmembrane conductance regulator; cyclic adenosine 5'-monophosphate; ionomycin; calcium


TRANSPORT OF Cl across the apical membrane is a defining property of secretory epithelial cells. This apical Cl conductance is activated in small intestinal and tracheal tissues by increases in local concentration of cAMP or Ca2+. The cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) is thought to be the principal Cl channel responsible for this Cl conductance. The CFTR was identified by reverse genetics as the protein directly involved with the secretory defects of cystic fibrosis (20). However, members of the Ca2+-dependent CLCA protein family were discovered when small intestinal (8) and tracheal (4) expression libraries were screened with monoclonal antibody that inhibited conductive Cl transport. Related murine and human CLCA proteins with reported Cl conductance activity have also been cloned (7, 10, 11, 19).

The role of the CLCA proteins in the apical Cl conductance that drives intestinal fluid secretion has been investigated by heterologous expression in cell lines from nonepithelial sources that have low levels of endogenous Cl channel activity. Expression of CLCA proteins in HEK-293 and NIH/3T3 cell lines caused the appearance of an enhanced Ca2+-dependent Cl channel activity in these cell lines (8, 11). The Cl conductance appearing on CLCA expression in HEK-293 and NIH/3T3 cells was activated by treatment of cells with Ca2+ ionophores, but not by A kinase activation (8). If cAMP-dependent CFTR is the most significant Cl conductor in intestinal epithelium, it is curious that an antibody that inhibits Cl conductance should identify a Ca2+-dependent Cl channel.

Functional expression of CLCA proteins in simple, undifferentiated cells may give limited information about the physiological potential of this protein (15), because the protein normally functions in differentiated epithelial cells. This study was initiated to determine how pCLCA1 may affect the regulation of Cl conductance in a true secretory epithelial cell line. Caco-2 cells are of intestinal epithelial origin, and they differentiate in culture to produce enterocyte-like monolayers complete with tight junction formation and surface mucus secretion. Caco-2 cells normally express CFTR and have an endogenous cAMP-dependent Cl conductance (22). We previously reported the enhancement of endogenous cAMP-dependent Cl conductance in Caco-2 cells expressing pCLCA1 (14). This cell type-specific, functional response to pCLCA1 transfection could reflect a need for a basal Na+-K+-2Cl cotransporter and K+ conductance to demonstrate the activity of a cAMP-dependent Cl conductance mediated by pCLCA1 (14). However, it is also possible that Cl conductance effects attributed to pCLCA1 expression may be due to increased responsiveness of existing conductance channels to normal agonists, either Ca2+ or cAMP. In this study, we present evidence from differentiating Caco-2 cells that distinguishes between these alternative explanations for pCLCA1 effects on Cl conductance.

Differentiation of maturing epithelial cell cultures during in vitro growth is accompanied by the loss of Ca2+-dependent Cl conductance as they polarize and form tight junctions (1, 5, 23). We confirm that Caco-2 cells lose Ca2+-dependent Cl conductance during differentiation, and we exploit this phenomenon to show that pCLCA1 may modulate the Cl conductance attributed to other ion channels but is not sufficient, by itself, to conduct Cl.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials.

Tissue culture media and G418 antibiotic were purchased from GIBCO Life Technologies. Peptide synthesis, conjugation, and polyclonal antibody production were carried out by Sigma Genosys. Sigma/Aldrich was the source of molecular biology-grade chemicals, including ionomycin, A-23187, forskolin, and IBMX. 36Cl was purchased from New England Nuclear.

Cell lines.

Caco-2 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, MEM nonessential amino acids (0.1 mM), 1% MEM vitamin solution, and 1 mM sodium pyruvate (complete DMEM). Stable transfections of Caco-2 cells with pcDNA3 plasmid, with and without the pCLCA1 cDNA, were carried out as described previously (15). After electroporation, cells were plated at 2 x 105 cells per well in 24-well plates. G418 (2.5 mg/ml) was added to wells 24 h later to select for successful transfectants. Cells were switched to maintenance medium (500 µg/ml of G418 in complete DMEM) 7 days after transfection. For pCLCA1 and pcDNA3 control transfections, cells from three separate wells showing good growth under selection pressure and randomly designated lines 1, 2, and 3 were passaged and used subsequently to test for clonal selection effects.

Production of transient transfectants.

Recipient Caco-2 cells were plated at low density (2.5 x 105 cells per 75-ml flask) in complete DMEM, grown for 24 h, and then transfected with the pIRES2-EGFP vector (Invitrogen) containing a polycistronic construct of pCLCA1 and enhanced green fluorescent protein (EGFP). For transfections, 2 µg of vector DNA were mixed with 10 µl of Lipofectamine 2000 (Invitrogen) in 800 µl of DMEM 20 min before addition to the flask containing 5 ml of complete DMEM without antibiotic. The transfection medium was replaced with fresh medium after 12 h, and cells were grown for a further 48 h before they were plated on 35-mm dishes. Cells showing EGFP fluorescence were patched ~10 h after transfer to 35-mm dishes.

RT-PCR and Western blot analysis.

The conditions for RT-PCR have been described previously (16). We used an antisense primer uniquely specific for pCLCA1 in the RT reaction (5'-TTTAGTCGACCATATCTAGTTGTTTAGATTG-3') with nested antisense primer (5'-CAGGTTGGTCTTATCGACAG-3') and a sense primer (5'-GTGAACACGCCACGCAGAAG-3') to generate a 518-bp cDNA in PCR. The PCR negative control was produced by omission of the antisense primer from the RT reaction.

For protein comparisons by Western blot analysis, cells were washed with PBS, removed from plates by the addition of 1 ml of PBS containing 10 mM EDTA, and suspended by gentle pipetting. Washed cells were collected by centrifugation and resuspended in 40 mM Tris buffer, pH 9.0, containing 1 mM PMSF, 0.1 µM pepstatin, and leupeptin (10 µg/ml). Cell protein was separated by SDS-PAGE and transferred to nitrocellulose membranes by wet cell electrophoresis. Membranes were blocked with 5% nonfat milk dissolved in Tris-buffered saline containing 0.1% polyoxyethylenesorbitan monolaurate (TTBS). They were incubated with diluted polyclonal rabbit antiserum prepared to a keyhole limpet hemocyanin conjugate of the 17-mer peptide CKEKNHNKEAPNDQNQK, corresponding to deduced amino acid sequence residues 250–266 in pCLCA1. Secondary mouse anti-rabbit alkaline phosphatase conjugate (Sigma) was diluted in TTBS and equilibrated with the nitrocellulose membrane before exposure to alkaline phosphatase substrate.

Flow cytometry.

Stable pCLCA1- and control-transfected Caco-2 cells were grown in flasks for 24 h or 14 days after passage. Medium was removed from flasks and replaced by PBS containing 10 mM EDTA. Cells were dislodged, filtered through 30-µm nylon mesh, and suspended at a density of 106 cells/ml in PBS + 3% BSA. Diluted preimmune or anti-pCLCA1 serum was incubated with cell suspensions for 30 min at 4°C. Cells were washed three times and suspended in diluted fluorescein-conjugated goat anti-rabbit Fab secondary antibody for a second 30-min incubation. The first of three washes contained 50 µg/ml propidium iodide. Washed cells were analyzed for fluorescence by flow cytometry using photomultiplier gates optimized for discrimination between emission of fluorescein and propidium iodide.

Cl efflux.

Stable pCLCA1- and control-transfected Caco-2 cells were plated at a density of 5 x 105 in 35-mm culture dishes and grown in DMEM supplemented with 2 mM glutamine, 10% fetal calf serum, and G418 (500 µg/ml). After 24 h or 14 days of growth, the confluent monolayers were loaded with 36Cl by removal of growth medium and incubation for 2 h with loading buffer containing 4 mM KCl, 2 mM MgCl2, 1 mM KH2PO4, 1 mM CaCl2, 5 mM glucose, 10 mM HEPES, pH 7.5, and 140 mM NaCl plus 2 µCi/ml 36Cl. Extracellular 36Cl was removed by rapid washing of cells five times with 1 ml of efflux buffer (loading buffer without 36Cl) before time 0. Agonists (10 µM ionomycin or 10 µM forskolin + 2 mM IBMX) were added to the efflux buffers from time 0 to the end of the timed efflux. 36Cl release from the cells was determined by consecutive addition and removal of 1 ml of efflux medium. Residual cell 36Cl at the end of the efflux was determined by washing cells from the plates with 1.0 mM EDTA and liquid scintillation counting. The rate constant for Cl release was calculated as follows: 0.5[ln{(cell [Cl]t1)/(cell [Cl]t2)}], where t1 and t2 are any two sequential time points during the course of an efflux assay.

Short-circuit current measurements.

Differentiated monolayers of Caco-2 cells expressing pCLCA1 or control cells transfected with the pcDNA3 vector were mounted in Ussing chambers and equilibrated for 20 min in standard Krebs bicarbonate-Ringer solution with 10 mM basal glucose and 10 mM apical mannitol before the addition of agonists. Cl gradients were established across monolayers of permeabilized cells by replacement of Cl with gluconate and Na+ and K+ with Tris. The high-Cl solution contained 147 mM Tris chloride, 30 mM mannitol, 5 mM TES, 2 mM CaCl2, and 1 mM MgCl2, pH 7.4. The low-Cl solution contained 130 mM Tris base, 130 mM D-gluconic acid, 17 mM Tris chloride, 30 mM mannitol, 2 mM CaCl2, and 1 mM MgCl2, pH 7.4.

Patch-clamp whole cell current.

Transfected cells were identified using an Axiovert 10 inverted microscope (Zeiss) equipped with a mercury lamp and excitation and emission filters of 425 and 540 nm, respectively. Transfected cells were patched using a computer-controlled micromanipulator (model MP-285, Sutter Instruments) and an audio monitor (model AM8, Grass-Telefactor, West Warwick, RI). After a seal (>1 G{Omega}) was obtained, capacitance compensation was carried out before whole cell access. Subsequent to whole cell access, all cells were dialyzed for 1 min before recording.

Whole cell currents were acquired with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at 10 kHz and filtered at 2 kHz by a low-pass Bessel filter with Clampex 8 and analyzed with Clampfit 8 (Axon Instruments). Cells were held at 0 mV with voltage pulses applied for 800 ms from –100 to +100 mV in 20-mV increments. Current differences were normalized by a whole cell capacitance recorder from an integrating 10-mV hyperpolarizing pulse. The pipette offset was adjusted on immersion into the bath solution. A single-cell bath solution change (Perfusion Fast Step SF-77B perfusion system, Warner Instruments, Hamden, CT) was used to add agonists and change bath Cl concentration.

The pipette solution for intracellular dialysis contained 110 mM CsCl, 1 mM MgCl2, 3 mM MgATP, 5 mM TES, 1 mM EGTA, and 0.38 mM CaCl2, with pH adjusted to 7.4 by addition of CsOH (~9.2 mM Cs). The high-Cl bath solution contained 135 mM CsCl, 3 mM MgCl2, 5 mM TES, 2 mM CaCl2, and 90 mM mannitol, with pH adjusted to 7.4 with addition of ~2.7 mM CsOH. The low-Cl bath solution contained 40 mM CsCl, 3 mM MgCl2, 5 mM TES, 2 mM CaCl2, and 280 mM mannitol, with pH adjusted to 7.4 by the addition of ~2.2 mM CsOH. Calculated junction potential corrections for these solutions (–2 mV) were less than the error for recorded reversal potentials (Erev), so no corrections for junction potentials were made.

Bath solution for recording with A-23187 agonist contained 135 mM N-methyl-D-glucamine HCl, 3 mM MgCl2, 5 mM TES, 2 mM CaCl2, and 80 mM D-mannitol (pH 7.4). The pipette solution for intracellular dialysis was 90 mM D-gluconic acid, 90 mM Tris, 40 mM Tris·HCl, 5 mM TES, 1 mM sodium pyruvate, 1 mM EGTA, 2 mM MgCl2, 0.1 mM CaCl2, 1 mM MgATP, and 0.1 mM Na2GTP (pH 7.4). Corrections were applied for junction potentials.

Statistical methods.

Two-way repeated-measures ANOVA was used to compare overall and treatment over time or voltage interactions. Fisher's least significant difference method for pairwise multiple comparisons was used as a post-ANOVA test to determine statistical significance at individual points. A paired t-test was used to compare groups of paired data.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported agonist effects on Cl efflux rates from Caco-2 cells plated at a density of 5 x 105 per 3.5-cm plate and grown for 24 h after passage (8, 15). However, Ca2+-dependent Cl conductance has been shown to decrease significantly in fully differentiated epithelial cells (1, 23). For this reason, this study compares 24-h undifferentiated Caco-2 cultures with 14-day differentiated cultures for effects of pCLCA1 expression on Ca2+- and cAMP-dependent Cl efflux.

pCLCA1 enhances endogenous Ca2+-dependent Cl efflux in 1-day cultures but shows no Cl conductance activity in mature cells.

The effect of pCLCA1 expression and Ca2+ ionophore addition on the rate of release of 36Cl from Caco-2 cells is shown in Fig. 1. Ca2+-sensitive Cl transport was present in cells assayed 24 h after passage. The effect of the Ca2+ ionophore A-23187 on the rate of 36Cl efflux from these cells occurred immediately, as observed previously in NIH/3T3 cells. pCLCA1 transfection increased the rate of Ca2+-sensitive Cl efflux from these freshly passaged cells. In contrast, the addition of Ca2+ ionophore to mature differentiated Caco-2 cells had no effect on the rate of 36Cl release from these cells. Mature differentiated cells expressing pCLCA1 also failed to increase the rate of Cl release on the addition of Ca2+ ionophore. We conclude that heterologous pCLCA1 expression was not sufficient to confer Ca2+-dependent Cl efflux to mature Caco-2 cells lacking endogenous Ca2+-dependent Cl efflux activity.



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Fig. 1. Loss of Ca2+-activated Cl efflux in 14-day-old cells expressing pCLCA1. A: effect of 10 µM A-23187 on rates of Cl efflux from pCLCA1-transfected and vector-control-transfected Caco-2 cells grown 1 or 14 days after passage. A-23187 was added to pCLCA1-transfected ({bullet}) or vector-control-transfected ({circ}) cells. There were no additions to pCLCA1-transfected ({blacktriangledown}) or vector-control-transfected ({triangledown}) cells. *P < 0.05 compared with A-23187-stimulated control-transfected cells. For day 1, P = 0.031 for overall pCLCA1 effect on A-23187-dependent rate of Cl efflux (compared with agonist-stimulated vector control) and P = 0.004 for gene x time interaction. For day 14, P = 0.002 for gene x time interaction. A-23187 and pCLCA1 transfection significantly decreased Cl efflux rate constant 2 min after A-23187 addition (P = 0.009, by post-ANOVA) compared with stimulated vector control. Values are means ± SE; n = 9. B: peak A-23187-induced Cl efflux rate constant for cells grown 1 and 14 days after passage. pCLCA1 effect on maximum Cl efflux rates after A-23187 is shown on days 1 (P = 0.009) and 14 (P = 0.009). Values are means ± SE; n = 9. *P < 0.05 vs. control.

 
pCLCA1 enhancement of endogenous cAMP-dependent Cl efflux is maintained through differentiation and polarization.

The disappearance of Ca2+-dependent Cl conductance from mature Caco-2 cells raised questions about the effects of differentiation and tight junction formation on other Cl conductance in these cells. We previously showed a cAMP-dependent Cl efflux that is enhanced by expression of pCLCA1 in freshly plated Caco-2 cells (14) or NIH/3T3 cells expressing CFTR (16). This response to cAMP and pCLCA1 transfection in freshly plated cells is shown in Fig. 2 compared with the rates of 36Cl efflux observed in mature, confluent Caco-2 monolayers. Within the first 2 min of agonist addition, there was no immediate effect of pCLCA1 transfection on the rate of PKA-dependent 36Cl efflux. However, there was a delayed effect of PKA agonists to increase the rate of Cl efflux in freshly plated and mature Caco-2 cells expressing pCLCA1. pCLCA1-dependent increases in Cl efflux rates appeared at 4 min (freshly plated cells) or 6 min (mature differentiated monolayers) after forskolin and IBMX addition compared with control transfected cells. It is significant that, in contrast to the Ca2+ effect, cAMP-dependent Cl conductance was not lost from mature cells. In addition, the ability of pCLCA1 expression to modulate that cAMP-dependent Cl conductance was present in freshly plated and mature monolayers. These functional effects of pCLCA1 expression in mature Caco-2 cells are interpreted as evidence for protein expression, despite the absence of a Ca2+-dependent conductance.



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Fig. 2. Stimulation of cAMP-dependent Cl efflux by pCLCA1 transfection is retained in 14-day-old cells. Effect of forskolin and IBMX addition on Cl efflux rates is shown for pCLCA1-transfected or vector-control-transfected cells grown 1 or 14 days after passage. Forskolin (10 µM) and IBMX (2 mM) were added to pCLCA1 transfected ({bullet}) or vector-control-transfected ({circ}) cells. There were no additions to pCLCA1-transfected ({blacktriangledown}) or vector-control-transfected ({triangledown}) cells. *Significant effects of pCLCA1 transfection on Cl efflux rate from cells with activated A kinase (P < 0.05). For day 1, P = 0.012 for overall pCLCA1 effect on forskolin-IBMX-dependent rate of Cl efflux compared with vector agonist control and P = 0.004 for gene x time interaction. For day 14, P = 0.035 for overall pCLCA1 effect on forskolin-IBMX-dependent rate of Cl efflux and P = 0.006 for gene x time interaction. Values are means ± SE; n = 8.

 
Presence of cAMP- but not Ca2+-dependent Cl conductance in polarized Caco-2 cells in Ussing chambers.

The Cl efflux rate constants reported in Fig. 1 represent the rate of release of isotopically labeled Cl from these cells. Release rate constants should reflect permeability of the cytoplasmic membrane to Cl. However, release rates measured in the presence of unlabeled 140 mM extracellular Cl are not definitive measurements of a Cl conductance uniport. Membrane leakage, K+-Cl cotransport, and exchange of intracellular 36Cl for extracellular 35Cl or bicarbonate will contribute to release rates. In contrast, short-circuit current (Isc) measured across polarized epithelial cell layers reflects active ion transport processes that define net ion conductance. The conductive nature of the enhanced anion transport response to forskolin and IBMX in cells transfected with pCLCA1 was tested by measuring transepithelial electrical parameters in confluent Caco-2 monolayers mounted in Ussing chambers. The movement of negative ions to the lumen is expressed as a positive Isc. The addition of forskolin and IBMX caused a significantly greater increase in Isc in cells transfected with pCLCA1 than in control cells transfected only with the pcDNA3 expression vector (Fig. 3A; P < 0.001 for overall gene effect and gene x time interaction). This finding confirms a conductive component in Cl transport rates reported in Fig. 2.



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Fig. 3. Agonist-induced short-circuit current (Isc) changes ({Delta}Isc) in transfected Caco-2 cells. pCLCA1- and vector-control-transfected Caco-2 cells were grown for 14 days on semipermeable membranes before they were mounted in Ussing chambers. A: changes in Isc in response to agonists forskolin (10 µM) and IBMX (2 mM). Isc response to forskolin and IBMX was larger in pCLCA1-transfected cells (P < 0.001, overall gene effect) than in vector control cells. B: changes in Isc in response to 10 µM A-23187. Overall gene effect for A-23187 on Isc was not significant (P = 0.257) with a power of 0.087. Values are means ± SE; n = 10.

 
CLCA ion channels were identified by activation of Cl conductance on exposure to Ca2+ ionophores (7, 10, 11, 19). Isc increments in response to treatment of Caco-2 cell monolayers with 10 µM A-23187 are shown in Fig. 3B. There was no evidence for a Ca2+-dependent Cl conductance in mature confluent monolayers of control pcDNA3-transfected Caco-2 cells. Transfection with pCLCA1 failed to confer Ca2+-dependent Cl conductance to these mature polarized cell monolayers (P = 0.25 for overall gene effect and P = 0.992 for gene x time interaction). Hence, the Isc response of mature polarized cells in Ussing chamber studies confirms the retention of the cAMP effect and the loss of Ca2+ effect on 36Cl efflux in mature 14-day monolayer Caco-2 cultures.

Isc associated with Cl conductance could be elevated by an increase in the permeability of the apical membrane of the polarized cells to Cl or by an increase in the driving force for Cl release (cell hyperpolarization). If pCLCA1 expression affects Cl channel activity, its primary effect should be on the former process, i.e., membrane permeability to Cl. The permeability of the apical membrane to Cl was determined by measuring Isc changes in response to imposed Cl gradients after the basolateral membrane of polarized cells was made permeable to Na+ and K+ (electrically isolated) by treatment with nystatin. The nature of the cAMP-dependent increase in Cl conductance in pCLCA1-transfected cells was determined by measuring Isc changes in response to an imposed transmembrane Cl gradient: 153 vs. 21 mM Cl. Isc changes caused by A kinase activation in pCLCA1- and pcDNA3-transfected cells are shown in Fig. 4A. Isc changes in response to A kinase activation were significantly larger in Caco-2 cells expressing pCLCA1 than in control cells transfected with pcDNA3. These increments in Cl permeability were independent of the direction of the imposed Cl gradient, as predicted for a truly electrically isolated apical membrane (P = 0.001 for basal-to-apical gradient and P = 0.023 for apical-to-basal gradient).



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Fig. 4. Agonist-induced Isc changes across electrically isolated apical membrane of transfected Caco-2 cells. Changes in Isc in response to imposed apical-to-basal or basal-to-apical Cl gradients are shown. A: changes induced by addition of 10 µM forskolin and 2 mM IBMX. B: changes induced by the addition of 10 µM A-23187. *P < 0.05 (paired t-test). Values are means ± SE; n = 9.

 
In contrast to the findings with A kinase activation, there was no significant Ca2+-dependent increase in apical membrane permeability (Fig. 4B). Neither the control nor the pCLCA1-transfected cells responded to A-23187 treatment with significant increments in Isc (P = 0.91 for basal-to-apical gradient and P = 0.66 for apical-to-basal gradient). These findings support an effect of A kinase activation on the apical permeability of mature differentiated Caco-2 cells. They also confirm a functional effect of expressed pCLCA1 in mature cells lacking Ca2+-dependent Cl conductance.

pCLCA1 expression is maintained throughout differentiation/maturation.

Some CLCA gene products, including bCLCA1 and hCLCA1, are reported to code for proteins that function as Cl channels that are regulated by changes in free cytosolic Ca2+ concentration (4, 10). The loss of a Ca2+-dependent Cl conductance in mature, differentiated Caco-2 cells transfected with pCLCA1 raises questions about the effect of cell differentiation on expression of the pCLCA1 gene product. The constitutive cytomegalovirus promoter in the pcDNA3-pCLCA1 construct reduces the probability that pCLCA1 expression should be affected by cell maturation. Persistent functional effects of pCLCA1 expression on cAMP-dependent Cl efflux and Isc changes in transfected cells are direct evidence for the continued expression of pCLCA1 in mature, differentiated Caco-2 cells. Additional evidence can be obtained by measuring specific pCLCA1 mRNA and protein expression.

RT-PCR using primers specific for pCLCA1 confirmed expression of pCLCA1 mRNA in cells grown for 1 and 14 days after passage (Fig. 5A). Western blot analysis of whole cell protein from 24-h and 14-day-transfected Caco-2 cells showed comparable levels of a ~58-kDa processed NH2-terminal fragment of pCLCA1 protein at these two growth stages (Fig. 5B). Alkaline phosphatase-conjugated mouse anti-rabbit IgG did not bind to duplicate blots when preimmune rabbit serum was substituted for immune antiserum as primary antibody (not shown). Persistent pCLCA1 antigen expression from 24 h after passage to 14 days was consistent with the retention of pCLCA1-dependent enhancement of cAMP effects on Cl conductance but did not correlate with age-related loss of Ca2+-dependent Cl conductance.



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Fig. 5. Expression of pCLCA1 in transfected Caco-2 cells. A: RT-PCR of pCLCA1 mRNA in transfected cells 1 and 14 days after passage. PCR was run for 26 cycles (45 s at 52°C, 1 min at 72°C, 45 s at 94°C) using template reverse transcribed from 100 cells. PCR positive control contained 0.5 pg of pCLCA1 in pcDNA3 vector. B: Western blot of whole cell protein from control and pCLCA1-transfected Caco-2 cells grown 1 or 14 days after transfection; 60 µg of protein were applied to each lane for SDS-PAGE and blotting to nitrocellulose.

 
Cell surface expression of pCLCA1 may be a significant prerequisite for interaction with ion channel proteins located in the plasma membrane. Cell surface localization of pCLCA1 epitope was investigated using rabbit anti-pCLCA1 antiserum, with detection by flow cytometry using FITC-conjugated goat anti-rabbit Fab serum. Freshly plated and mature monolayers of Caco-2 cells were dispersed by mechanical disruption after exposure to EDTA and exposed to the impermeant propidium iodide marker. Trypsin treatment was avoided to protect any extracellular pCLCA1 epitope. Fluorescence energy gates on the flow cytometer were set to distinguish between emission energy of fluorescein and propidium iodide. Mechanical disruption of confluent monolayers with adherent tight junctions caused significant plasma membrane damage, as indicated by the proportion of cells with propidium iodide fluorescence (Fig. 6). Of the cells with intact plasma membrane (cells excluding propidium iodide), the peptide epitope corresponding to amino acids 250–266 of pCLCA1 was detected on 30% of intact 14-day-cultured cells vs. 20% of intact 24-h-cultured cells (Table 1). Stable pCLCA1 transfection approximately doubled the percentage of cells expressing surface CLCA epitope compared with control pcDNA3-transfected Caco-2 cells. These results confirm the presence of pCLCA1 epitope on the surface of mature differentiated cells that show cAMP-dependent effects of transfection but have lost Ca2+-dependent Cl conductance.



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Fig. 6. Effect of Caco-2 cell maturation on surface expression of CLCA epitope. Surface-expressed pCLCA1 epitope was protected by mechanical removal of permanently pCLCA1-transfected cells from plates without trypsin. Cells were incubated with primary antiserum to a peptide with an amino acid sequence of 250–266 of pCLCA1. Secondary antibody was labeled with FITC. Photomultiplier gains for flow cytometry were set to differentiate damaged vs. intact cells by exclusion of propidium iodide (50 µg/ml for 5 min followed by 3 washes) and cells with FITC fluorescence; 10,000 events were collected for analysis.

 

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Table 1. Identification of surface pCLCA1 epitope on intact Caco-2 cells with fluorescein-labeled antibody

 
pCLCA1 effects on Cl efflux and whole cell current are not clonally specific.

Caco-2 cell populations may not be totally uniform in Cl channel expression, activity, or susceptibility to regulatory agonists. Random clonal selection during the passage of permanently transfected cells could give a pCLCA1-transfected cell line with a larger-than-average content of endogenous apical Cl channel targets available for activation by pCLCA1 expression and agonist addition. This point was investigated by examining the effects of agonist on two additional permanently transfected Caco-2 cell lines (2 more pcDNA3 controls and 2 more pCLCA1 test lines). The percent change in Cl efflux rate induced by pCLCA1 expression compared with a paired pcDNA control is shown in Fig. 7. In three independently selected cell lines plated for 24 h, the pCLCA1-transfected cells had an enhanced rate of 36Cl release in response to C or A kinase agonist addition in cells. The cAMP response persisted to maturation in all three lines, but the Ca2+-dependent response to pCLCA1 transfection disappeared in three of three cell lines tested.



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Fig. 7. Effect of Caco-2 differentiation on Cl efflux response to agonist addition in independent pCLCA1-transfected cell lines (1, 2, and 3). Responses were measured in 3 independent, randomly identified, pCLCA1-transfected cell lines and compared with 3 more randomly identified, pcDNA3 control-transfected cell lines. A: Cl efflux response to forskolin + IBMX on days 1 and 14 in 3 independent pCLCA1-transfected cell lines. Cl efflux rates are expressed as percentage of agonist-stimulated efflux rate of 3 independent control cell lines (controls set as 100%) randomly paired to pCLCA1-expressing lines. B: Cl efflux response to A-23187 on days 1 and 14 in 3 independent pCLCA1-transfected cell lines. Cl efflux rates are expressed as percentage of agonist-stimulated efflux rate as in A. Values are means ± SE; n = 8.

 
The issue of clonal selection in permanent transfections can also be addressed by producing transient transfections. The pIRES2-EGFP vector was used to permit independent bicistronic expression of pCLCA1 and EGFP with identification of successful transfectants by fluorescence microscopy. Transient transfections were carried out on freshly passaged cells comparable to 24-h cells at the time of patch-clamp studies. Caco-2 cells expressing pCLCA1 showed an enhanced whole cell conductance response to A kinase activation compared with cells transfected only with the pIRES2-EGFP vector (Fig. 8A). Erev measurements confirmed the involvement of Cl in the observed conductance (Fig. 8B). Measured Erev in the 145 mM Cl external bath solution was –1.78 ± 0.85 and –4.43 ± 1.05 mV for cells expressing pCLCA1 and control transfected cells, respectively. These measured values were close to the calculated Erev of –6.4 mV for Cl at 21°C. When the bath solution was switched to 40 mM Cl, the measured Erev of 12.22 ± 1.28 mV for pIRES2-EGFP-containing pCLCA1 and 12.64 ± 1.46 mV for control pIRES2-EGFP shifted toward the calculated Erev of 20.33 mV at 21°C.



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Fig. 8. Normalized whole cell current response to the addition of forskolin and IBMX in transiently transfected Caco-2 cells. Control cells were transfected with pIRES2-EGFP vector ({circ}) and pCLCA1 test cells with pIRES2-EGFP containing pCLCA1 ({bullet}). A: internal 112 mM Cl and external 145 mM Cl. P = 0.002 for gene x voltage interaction. B: corresponding representative trace of data in A. C: low-Cl shift: 112 mM internal Cl and 50 mM external Cl. P = 0.011 for gene x voltage interaction. *Significantly higher in cells transfected with pCLCA1 (P > 0.005, by post-ANOVA). Values are means ± SE; n = 8.

 
Stimulation of pCLCA1-transfected Caco-2 cells with A-23187 produced significantly larger increments in normalized current than in control pIRES2-EGFP cells (Fig. 9). Measured Erev values (–26 ± 3.1 and –24 ± 2.1 mV) did not significantly differ from each other and were close to the calculated Cl Erev of –31 mV for the bath and pipette solutions.



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Fig. 9. Normalized whole cell current response to the addition of A-23187 in transiently transfected Caco-2 cells. A: control cells were transfected with pIRES-EGFP vector ({circ}) and pCLCA1 test cells with pIRES2-EGFP containing pCLCA1 ({bullet}). P = 0.0022 for overall gene effect. P < 0.001 for gene x voltage interaction. *Significantly higher in cells transfected with pCLCA1 (P < 0.05, by post-ANOVA). Values are means ± SE; n = 10. B: corresponding representative trace of data in A.

 
These observations after transient transfection support the data collected from permanently transfected cells (Fig. 7) and exclude clonal selection as a basis for the enhanced Cl channel responsiveness to activation associated with pCLCA1 expression. They also confirm an increase in Ca2+-dependent Cl conductance across the cytoplasmic membrane of freshly passaged Caco-2 cells expressing pCLCA1.


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The loss of Ca2+-dependent Cl conductance in differentiated secretory epithelial tissues is relatively well recognized (1, 23, 27, 28). Exploitation of this phenomenon to investigate association of pCLCA1 with Ca2+-dependent Cl conductance is complicated by a need for independent verification of the developmental effect by different analytic techniques. Cl efflux studies are suited to comparing exogenous protein expression effects and agonist effects in newly passaged and mature differentiated cell cultures, but Cl efflux from the cell need not be electrogenic. Isc values from Ussing chambers are good measurements of conductance effects, but they are not amenable to studies on freshly plated cells that have maximal levels of Ca2+-dependent Cl conductance activity. The current-voltage dependence of whole cell conductance can be measured by patch clamp, but this technique is difficult to apply to confluent differentiated cells. Hence, it seems best to use each of these techniques where it is most applicable to search for a consistent answer to the question of Ca2+-dependent Cl conductance activity associated with pCLCA1 expression.

Cl efflux and Ussing chamber measurements confirm the loss of Ca2+-dependent Cl conductance from mature Caco-2 cells. Simple interpretations of the inability of pCLCA1 transfection to correct this loss could be 1) failure to express the pCLCA1 protein in mature Caco-2 cultures, 2) failure of proper pCLCA1 insertion into the cytoplasmic membrane, 3) loss of sensitivity of differentiated Caco-2 cells to Ca2+ ionophore effects of A-23187, 4) the presence of an inhibitor of Ca2+-dependent Cl conductance in differentiated Caco-2 cells, or 5) inability of pCLCA1 to confer Ca2+-dependent Cl conductance in the absence of some accessory protein.

Parallel measurements on alternate functions, namely, cAMP-dependent Cl conductance, provide Cl efflux and Ussing chamber evidence that functional, membrane-located effects of pCLCA1 expression are retained as cells differentiate and Ca2+ -dependent Cl conductance disappears. RT-PCR data and Western blot analysis confirm that Caco-2 cells maintain exogenous pCLCA1 expression while losing Ca2+-dependent Cl conductance.

Quantitative extracellular pCLCA1 epitope expression measurements required dispersion of confluent monolayers for flow cytometry without protease assistance. Despite the loss of significant numbers of cells to mechanical dispersion forces, the similarities in surface expression of the pCLCA1 epitope between freshly plated and 14-day-old Caco-2 cultures suggest that pCLCA1 expression is independent of the stages of cell differentiation. There was a small endogenous background of antibody binding in control-transfected Caco-2 cells, assumed to be due to hCLCA1 expression. The 17-mer peptide antigen used for antibody production (pCLCA1 amino acids 250–266) has the following differences in hCLCA1: K2 -> T, K4 -> Q, and D13 -> K. The lack of coincidence of pCLCA1 epitope expression and Ca2+-dependent Cl conductance supports the independence of these two processes.

Some property of well-differentiated Caco-2 cells could interfere with the access of the Ca2+ ionophore to the cells and prevent the Ca2+ signaling effect. The issue of A-23187 access to the membranes of mature Caco-2 cells has been addressed by others (21, 25). Direct effects of A-23187 on Ca2+ flux and indirect effects of A-23187 on signaling responses to changes in intracellular Ca2+ concentration were maintained in differentiated Caco-2 cells grown on permeable supports identical to our culture conditions.

The use of independent permanently transfected cell lines and the measurements of whole cell current in transiently transfected Caco-2 cells confirm the presence of an increased activity of Ca2+-dependent Cl conductance after pCLCA1 transfection. Disappearance of this activity on cell differentiation was also confirmed. Hence, the combined results of measurements using a range of independent techniques are consistent with interpretation 4 or 5 (see above), i.e., the presence of an endogenous inhibitor or an inability of pCLCA1 to confer Ca2+-dependent Cl conductance in the absence of some accessory protein.

Inhibition of Ca2+-dependent Cl conductance by some substance found in mature differentiated Caco-2 cells could account for the disappearance of endogenous conductance from these cells and the fact that it cannot be restored by transfection with pCLCA1. Inositol 3,4,5,6-tetrakisphosphate is reported to inhibit Ca2+-dependent Cl conductance in epithelial cells (2, 12, 26). However, sustained activation of phospholipase C is required to produce inhibitory concentrations of inositol 3,4,5,6-tetrakisphosphate (24). There is no agonist present in these mature cultures to constitutively activate phospholipase C. Annexin IV and epidermal growth factor may also participate in inhibition of Ca2+-dependent Cl conductance (26), but no manipulations have been carried out to elevate the concentrations of these substances. Although known inhibitors of Ca2+-dependent Cl conductance are unlikely to be responsible for the loss of the conductance response, it is difficult to rule out the presence of some unknown inhibitor until the molecular identity of the endogenous channel is clarified.

It is also noteworthy that the electrical signature of Cl currents in pCLCA1-transfected cells is variable, depending on the cell type or the agonist involved in channel activation. The Ca2+-dependent Cl currents seen in pCLCA1-transfected NIH/3T3 cells were outwardly rectifying (15), but linear current-voltage relations of the cAMP-activated conductance channel in whole cell patch clamp of pCLCA1-transfected Caco-2 cells are consistent with current passing through a nonrectifying endogenous Cl channel (14).

The identity of accessory proteins that may be necessary for pCLCA1 expression to generate Cl conductance is speculative at this time. HEK-293 and NIH/3T3 cell lines, chosen for low backgrounds of endogenous Cl conductance, have been used to functionally characterize exogenously expressed Ca2+-dependent CLCA Cl channels (6, 10, 15). Low levels of endogenous Cl channel activity in these cells have not been widely acknowledged, but the addition of Ca2+ ionophore to the untransfected HEK-293 and NIH/3T3 cells produced a small endogenous conductance of low cation-anion selectivity, as indicated by Erev measurements (15, 27). Such a channel could be one possible target for activation on expression of suitable regulatory proteins. A precedent has been reported describing interaction of a K+ channel subunit with mCLCA1 (9).

The recent localization of the bestrophin proteins to the basolateral plasma membrane of the retinal pigment epithelium (RPE) by Marmorstein et al. (17) and implication of these proteins in CaCC activity by Qu et al. (18) suggest that the molecular identity of the CaCC may reside in bestrophins. In addition to expression in secretory areas of the small intestine and the trachea, pCLCA1 expression has also been localized to the RPE, where it has been postulated that it may function as a regulator of CaCC activity (16). Taken together, these findings may be evidence against an independent Cl channel activity for pCLCA1 and increase interest in pursuing possible functional or regulatory interactions between CLCA and other ion channel proteins.

We conclude that the disappearance of Ca2+-dependent Cl conductance from mature Caco-2 cells due to the accumulation of some inhibitory compound cannot be excluded. However, the difference in the conductance properties associated with pCLCA1 expression in different systems increases the probability that pCLCA1 may function chiefly as a modulator of endogenous Cl conductance channels. The Ca2+- and cAMP-dependent Cl conductances that are modulated by pCLCA1 expression are assumed to represent the activity of distinct ion channels, indicating a possible pleiotropic effect of pCLCA1 expression on the activity of apparently unrelated Cl channels with different regulatory and current-voltage signatures. Young secretory cells arising from stem cell populations in the ileal crypts (13) or tracheal submucosal glands (3) may be the best physiological counterpart of the pCLCA1-sensitive Ca2+-dependent Cl conductance in Caco-2 cells 24 h after passage. Differentiated Caco-2 cells that lack endogenous Ca2+-dependent Cl conductance may be better models for the mature senescent absorptive cells on the villus tip. The association of pCLCA1 with the actions of Ca2+ and cAMP in secretory epithelial cells shows that it may be fruitful to study the role of this protein as a modulating element in normal ion transport and in pathophysiological conditions involving abnormal epithelial fluid secretion.


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This research was supported by the Canadian Cystic Fibrosis Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. W. Forsyth, Veterinary Biomedical Sciences, Univ. of Saskatchewan, 52 Campus Dr., Saskatoon, SK, Canada S7N 5B4 (E-mail: george.forsyth{at}usask.ca).

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


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