Outwardly rectifying Clminus channel in guinea pig small intestinal villus enterocytes: effect of inhibitors

Alan S. Monaghan1, Gerard M. Mintenig2, and Francisco V. Sepúlveda3,4

1 Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom; 2 Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Spain; 3 Departamento de Medicina Experimental, Facultad de Medicina, Universidad de Chile, Casilla 70058, Santiago-7; and 4 Centro de Estudios Científicos de Santiago, Casilla 16443, Santiago-9, Chile

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Previous studies in enterocytes isolated from the villus region of small intestinal epithelium have identified a macroscopic current carried by Cl-. In this work a single-channel patch-clamp study was carried out in the same cells, and a spontaneously active, outwardly rectifying Cl- channel was identified and proposed to underlie the whole cell current. The channel had conductances of 62 and 19 pS at 80 and -80 mV, respectively, in symmetrical Cl- solutions in excised patches. Similar activity was seen in cell-attached patches, but only outward currents could be discerned in this configuration. The activity of the channel, measured as open probability, was independent of intracellular calcium levels and voltage. The selectivity sequence for different anions was SCN- > I- > Br- > Cl- > F- > (gluconate, glutamate, SO<SUP>2−</SUP><SUB>4</SUB>). The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), verapamil, and 4-hydroxytamoxifen (but not by tamoxifen), with potencies similar to those observed for Cl- channels previously described in other cells. Inhibition by trinitrophenyladenosine 5'-triphosphate was also observed but only at depolarized potentials. At 50 mV the half-maximal inhibitory concentration was 18 nM. It is proposed that this channel plays a role in transepithelial Cl- transport and certain regulatory Cl- fluxes.

epithelium; chloride transport; trinitrophenyladenosine 5'-triphosphate

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

OUTWARDLY RECTIFYING Cl- channels (ORCCs) were first identified in respiratory epithelial cells (16, 56) and later found in many cell types, including renal and small intestinal epithelium, pancreas, colon, human skin fibroblasts, and lymphocytes (2, 5, 8, 10, 17, 20). Also, they have been described in several cell lines of diverse origin (29). ORCCs have been proposed to have important roles in cell homeostasis, volume regulation, and secretory and absorptive processes in epithelial cells (15, 38).

Most studies of Cl- channels in epithelia have been done using secretory epithelial cells (15); in contrast, very little is known about Cl- channels in absorptive epithelial cells. A 30-pS inwardly rectifying anion channel of basolateral membranes of mammalian urinary bladder cells with a high open probability (Po) in the physiological voltage range has been described (23). A linear 40-pS adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channel in the basolateral membrane of the thick ascending limb of the mouse kidney is proposed to be involved in the basolateral Cl- exit step of NaCl absorption (37). A similar role has been proposed for an ORCC (13/96 pS) present in the basolateral membrane of rabbit renal cortical collecting duct cells (11).

Little is known about how Cl- that has been absorbed across the apical membrane of absorptive enterocytes exits across the basolateral membrane. Whole cell patch-clamp experiments, using small intestinal villus enterocytes isolated from guinea pig, have shown the presence of an outwardly rectifying Cl- current (43). This spontaneously active current was proposed to be mediated by basolateral membrane Cl- channels, whose function would be to allow the basolateral exit of Cl- during NaCl absorption. The single-channel configurations of the patch-clamp technique have been used here to find and characterize basolaterally located Cl- channels in small intestinal villus enterocytes isolated from guinea pigs. An outwardly rectifying channel was observed that appeared to be spontaneously active in cell-attached patches. Its selectivity and sensitivity to pharmacological agents have been explored. On this basis it is proposed to underlie the macroscopic currents previously described in the same cells. We hypothesize that this channel mediates Cl-, and perhaps nutrient, exit across the enterocyte basolateral membrane.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation. Guinea pig villus enterocytes were isolated by methods developed previously (9), with only minor modifications. Adult male guinea pigs (weighing 250-400 g) were starved for 24 h and killed by cervical dislocation. The first 30-40 cm of the small intestine (after the duodenum) were immediately excised and placed into ice-cold saline. The intestinal lumen was rinsed through with three 30-ml volumes of the cold saline solution and then with an intracellular-like solution [solution I, composition (in mM): 7 K2SO4, 44 K2HPO4, 9 NaHCO3, 15 Na3 citrate, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 180 glucose, pH 7.4, with tris(hydroxymethyl)aminomethane]. The lumen was filled with solution I, clamped at both ends, and incubated at 37°C for 8 min. After this time, the intestinal lumen was emptied and refilled with a similar solution containing 1 mM dithiothreitol and 0.2 mM EDTA. The intestine was again incubated at 37°C for 3 min and then gently palpated for 3 min, and its contents were emptied into 20-30 ml of ice-cold Dulbecco's modified Eagle's medium (DMEM; Sigma, Poole, UK). The process was repeated and the contents were pooled with those from the first palpation. The collected contents were centrifuged at 50 g for 4 min, and the resulting cell pellet was resuspended in 10 ml of DMEM containing ~1,000 U of collagenase type 1A. The cells were incubated, with gentle agitation, at 37°C for 15 min and then diluted with an equal volume of ice-cold DMEM and filtered through nylon mesh (60- and then 30-µm pore size) to remove clumps of cells and aggregated debris. After centrifugation, cells were resuspended in 20 ml of ice-cold DMEM, plated out into 35-mm plastic Petri dishes, and left on ice for 1 h to settle. Cells isolated by the above procedure have been shown previously to be mainly of villus origin (9, 49). Villus cells were 15-20 µm in diameter and could be identified easily by their clear brush border and basolateral domains located at opposite poles, as shown in the micrograph (Fig. 1). Cell viability assessed by trypan blue exclusion was >95%. Viable cells viewed with phase-contrast optics were birefringent and hence easily identified even without the use of trypan blue.


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Fig. 1.   Phase-contrast micrograph of isolated villus enterocytes used in this study, illustrating the apparent basolateral approach employed to obtain all membrane patches used in the present work. Calibration bar, 15 µm.

Patch-clamp recordings. Single-channel currents were recorded at room temperature using the patch-clamp technique (22). Micropipettes were fabricated from thin-walled borosilicate (hard) glass tubing of external diameter 1.5 mm and internal diameter 1.17 mm (Clark Electromedical, Pangbourne, UK). Pipettes were pulled using a two-stage vertical pipette puller (PP-83, Narishige, Japan) and fire-polished on a microforge. When filled with the appropriate intracellular or extracellular solution they had resistances of 5-10 MOmega (2-3 MOmega in whole cell recording experiments). The petri dish containing the cells was continuously superfused with normal saline (see Table 1), and excess solution was removed using a peristaltic pump. Nonstandard solutions were applied using a local microperfusion device similar to one described previously (50). This device allowed the directing of a small jet of the desired solution at the cell or membrane patch without greatly altering the bulk solution of the chamber. Current measurements were carried out using an EPC 7 (List Electronic, Darmstadt, Germany) amplifier. Whole cell recordings were performed as described previously (44).

                              
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Table 1.   Composition of pipette and bath solutions

Enterocytes were viewed on an inverted microscope (Nikon Diaphot), equipped with phase-contrast optics, at a total magnification of ×400. Seals of 10-100 GOmega were routinely obtained. Excised inside-out patches were obtained from cell-attached patches either by simply pulling the patch pipette away from the cell or (for cells not attached to the bottom of the petri dish) by using the microperfusion to blow the cell away from the pipette tip. For recording in the outside-out or whole cell configuration, a short burst of strong suction was applied before excision to break the patch membrane. The reference (ground) electrode consisted of a silver-silver chloride pellet connected to the bath solution by an agar bridge filled with saline solution. Liquid junction potentials, which occurred as a result of bath solution changes during an experiment, were calculated (4), and current-voltage relations were corrected accordingly. Voltage-pulse protocols were generated using an IBM-AT microcomputer connected to the patch-clamp amplifier through a Cambridge Electronic Design 1401 laboratory interface (Cambridge, UK). Current and voltage signals from the patch-clamp amplifier were filtered at 10 kHz and stored on digital audiotapes using a modified digital audiotape recorder (Sony). Acquisition of signals into the computer was done off-line at a rate of 1-5 kHz after the signal was filtered at 0.5-1 kHz with an eight-pole Bessel filter. Acquisition and analysis of single-channel data were carried out using a patch-clamp analysis (PAT) or voltage-clamp analysis (VCAN) program. The software was kindly provided by Dr. John Dempster (University of Strathclyde, Glasgow, UK).

Unless otherwise indicated, all chemicals were from Sigma (UK). 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was a kind gift from Prof. R. Greger (Freiburg, Germany).

Errors, where given, are ±SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Membrane patches were obtained from what, under phase-contrast optics, looked like a smooth (basolateral) surface as opposed to a fuzzy, presumably brush border, membrane (see Fig. 1). The general morphology of the cells was reminiscent of what has been termed bilobulated or figure-eight cells for epithelial cells isolated from kidney proximal tubule and gallbladder, respectively (42, 52). These cells have been demonstrated to maintain in suspension both structural and functional polarity. Structural polarity at the level of the electron microscope has been demonstrated in the enterocytes used here (9), but no further functional tests for polarity have been performed.

An ORCC is active in cell-attached patches. Figure 2A shows traces recorded from a cell-attached patch on a guinea pig enterocyte with a pipette containing an NaCl-rich solution and normal saline in the bath (see Table 1 for composition of solutions). A single channel was seen when the patch membrane was depolarized, but no inward transitions were observed on hyperpolarization down to -120 mV. Average slope conductances were 96 ± 6 pS (n = 24) at 110 mV and 45 ± 4 pS (n = 17) at 50 mV (command potentials). Excision of patches into an NaCl-rich (see Table 1) bath solution gave the activity illustrated in Fig. 2B. The outward currents seen at depolarized potentials were very similar to those in cell-attached patches. However, in excised patches small inward currents were seen at hyperpolarizing voltages. Figure 2C shows that the current-voltage relation for this and other similar channels in both the cell-attached and inside-out configurations was strongly outwardly rectifying. The mean chord conductance in 16 patches was 62 ± 3 pS at 80 mV and 19 ± 1 pS at -80 mV. Excision of cell-attached patches containing outward currents, such as those in Fig. 2A, always gave rise to both outward and inward currents. The Cl- channel was seen in 97 of 162 patches successfully recorded. Twelve of these appeared to correspond to single channels.


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Fig. 2.   Outwardly rectifying Cl- currents in cell-attached and excised inside-out patches. A: current traces at indicated command voltages in cell-attached patch. Arrows indicate closed-current level. Patch pipette contained NaCl-rich solution, and bath contained normal saline. B: currents of the same channel type after excision (inside-out) into NaCl-rich solution. C: current-voltage (I-Vc) relation for Cl- currents recorded from cell-attached (bullet ) or excised inside-out (black-triangle) patches as described in A and B, respectively.

Comparison of current-voltage relations for the cell-attached and excised inside-out configurations (Fig. 2C) revealed that the cell-attached curve was shifted by ~15 mV to the right of the excised-patch relation. Thus, if Cl- is assumed to be the charge carrier, the intracellular anion should be 15 mV above equilibrium. If a membrane potential of -40 to -50 mV is assumed [hence a reversal potential (Erev) of -25 to -35 mV], an intracellular Cl- concentration ([Cl-]i) value of 37-55 mM can be calculated. [Cl-]i measured in Necturus enterocytes with ion-selective electrodes was ~30 mM and was above equilibrium by ~10 mV (18).

Experiments to determine the kinetics of the ORCC were very difficult to carry out because patches from the isolated villus enterocytes often did not stand up to the lengthy time periods at different voltages required for such analysis. Also, most patches contained multiple channels. Po was ~0.8 at positive potentials. A greater variation was observed at negative potentials, but on average Po remained potential independent (in 4 experiments Po values were 0.86 ± 0.09 and 0.76 ± 0.10 at 60 and -60 mV, respectively). Po did not depend on intracellular Ca2+, which varied between 1.3 mM and no added Ca2+ plus 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (not shown).

Selectivity of guinea pig enterocyte Cl- channel. Replacing Na+ in the pipette by the impermeant cation N-methyl-D-glucamine (NMG+) had no effect on the current-voltage relation. The slope conductance at 110 mV was 102 ± 12 pS (41 ± 5 pS at 50 mV) in five separate experiments; these values do not differ from those obtained with Na+-rich pipette solutions. When cell-attached patches were excised into either NMG-Cl- or NaCl-rich bath solutions, there was no difference in the current-voltage relations obtained, and in both cases the current reversed at 0 mV (data not shown).

Figure 3 illustrates an experiment in an excised inside-out patch in which the pipette solution was NaCl rich (see Table 1) and the bath solutions were Na+ rich but contained different anions. Traces of currents measured while the potential was held at 40 or 0 mV are illustrated in Fig. 3. When Cl--rich solutions bathed both sides of the membrane, outward currents could be seen at 40 mV and no currents could be detected at 0 mV. Outward currents were seen at both voltages when SO<SUP>2−</SUP><SUB>4</SUB> or gluconate was the main anion in the intracellular medium. When SCN- was used as the main intracellular anion, inward currents were seen at 0 mV and outward currents (reduced in size compared with the equivalent situation in symmetrical Cl-) were seen at 40 mV. Only outward currents could be measured accurately at a range of voltages when either SO<SUP>2−</SUP><SUB>4</SUB> or gluconate was used as the main intracellular anion in inside-out patches (not shown).


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Fig. 3.   Anion selectivity of the outwardly rectifying Cl- channel (ORCC) of guinea pig enterocytes. Current traces were recorded from an excised inside-out patch with an NaCl-rich pipette solution at indicated voltages and bathing solutions containing indicated main anion. Arrows indicate zero-current level.

The permeability of the channel for different anions can be expressed as a ratio of the Cl- permeability, i.e., Panion/PCl, and was calculated using the Goldman-Hodgkin-Katz (GHK) equation. Concentration values were used instead of activities because, for monovalent salts, activity coefficients are all very similar. Table 2 shows the Erev values obtained for the different anions relative to the Erev obtained with a Cl--rich bath solution. Also shown in Table 2 are the Panion/PCl values calculated for the different anions.

                              
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Table 2.   Anion selectivity of the outwardly rectifying Cl- channel

Glutamic acid can permeate the enterocyte ORCC. Figure 4C illustrates current-voltage relations obtained from an excised inside-out patch in which the pipette solution was NMG-Cl rich and the bath solutions were as indicated. When the main intracellular anion was switched from Cl- to glutamate, the inward currents were much smaller and the Erev shifted in the negative direction. Thus, although glutamate permeated the channel much less easily than Cl-, it appears that the channel does nevertheless allow glutamate to traverse it.


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Fig. 4.   Activity of ORCCs in outside-out patch configuration: effect of replacement with glutamic acid. Traces are from an outside-out patch with a pipette containing corresponding NaCl-rich solution (see Table 1). A: traces at 80 and 0 mV where bath solution was NaCl rich. B: traces at 80 and 0 mV where bath solution was 500 mM N-methyl-D-glucamine (NMG) glutamate. C: current-voltage relations for the outside-out patch in A (down-triangle) and B (square ). Data for NMGCl-rich (open circle ) and 500 mM NMG glutamate (triangle ) solution replacements in an inside-out patch are also shown.

To get a more accurate measure of the channel permeability to glutamate, we decided it was more practical to look at the effect of substituting glutamate for Cl- at the extracellular side of the patch membrane. Thus the effects were on the larger, clearer outward currents. The Cl- channel was therefore recorded in outside-out patches. The mean chord conductance in four separate experiments was 70 ± 3 pS at +80 mV and 19 ± 1 pS at -60 mV. These values are similar to those derived from experiments on inside-out patches. Figure 4A shows traces recorded from an excised outside-out patch in which the pipette (intracellular) solution was NaCl rich and the bath (extracellular) solutions had a similar composition (see Table 1). Traces are shown at 80 and 0 mV, respectively. At 80 mV, outward currents were seen; the unitary current was 5.3 pA. At 0 mV, no currents were seen. In Fig. 4B the bath solution had been changed to 500 mM NMG glutamate. Again outward currents were seen at 80 mV, but this time the unitary current was only 2 pA (note change in scale from Fig. 4A). At 0 mV, small inward currents were seen.

Current-voltage relations obtained from the experiment illustrated are shown in Fig. 4C. When Cl- was the main bath anion, Erev was ~0 mV. When glutamate was the main bath anion, Erev shifted to 28 mV. Application of the GHK equation with the Erev obtained for glutamate gave a PGlu/PCl of 0.09; similar results were obtained in three separate experiments. The corresponding permeability ratio found by Banderali and Roy (3) in MDCK cells was 0.18.

Effects of pharmacological agents on the enterocyte ORCC. Many Cl- channel blockers act primarily on the extracellular surface of the membrane. Because of this, the effects of blockers at the single-channel level were studied on excised outside-out patches.

Whole cell recordings from isolated guinea pig villus enterocytes have shown that the blocker NPPB at a concentration of 10 µM strongly inhibits the outwardly rectifying Cl- conductance. Figure 5A shows traces recorded from an outside-out patch held at 50 mV. In the top trace of Fig. 5A, current fluctuations in a patch containing two ORCCs are shown. The bottom trace of Fig. 5A was recorded after the addition of 50 µM NPPB to the bath solution. Under control conditions the two channels were seen to be open with high Po. The addition of NPPB resulted in a flickery-type blockade that reduced the current going through the patch by 82%; similar results were obtained in two separate experiments. These results are consistent with those previously obtained in the whole cell configuration (43).


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Fig. 5.   Effects of inhibitors on guinea pig enterocyte ORCCs. A: traces recorded from an excised outside-out patch held at 50 mV. Pipette solution was NaCl rich. Top trace: bath solution was normal saline; bottom trace: 50 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) had been added to the bath solution. Arrows indicate closed level. B and C: traces recorded as in A but from different patches and with blockers indicated.

It has been shown that the Ca2+ channel blocker verapamil inhibits an outwardly rectifying Cl- conductance modulated by changes in cellular volume (12, 53). Verapamil also inhibits similar Cl- channels in colonic carcinoma cells (6). The effect of this compound on the guinea pig enterocyte Cl- channel was explored. Figure 5B shows traces recorded from an outside-out patch before and after verapamil addition. The compound caused a strong flickery-type blockade. The inhibition of the Cl- channel was reversible (results not shown). 1,9-Dideoxyforskolin (DDFSK), like verapamil, has recently been shown to inhibit volume-activated outwardly rectifying Cl- conductances (12, 53). Figure 5C shows traces recorded from an excised outside-out patch with pipette and bath solutions rich in NaCl. As with verapamil, addition of DDFSK to the bath solution caused a flickery-type blockade. The channel inhibition by DDFSK was reversible (results not shown).

Anti-estrogen interaction with guinea pig enterocyte ORCCs. The anti-estrogen tamoxifen was used to explore further the relationship between the ORCCs in guinea pig villus enterocytes and volume-activated ORCCs. Tamoxifen has been shown to block completely the Cl- conductance modulated by cell volume increase at concentrations <= 10 µM (54, 60). When 10 µM tamoxifen was added at the extracellular surface of excised outside-out patches from isolated guinea pig villus enterocytes, the patches rapidly deteriorated and broke. Thus it was not possible to look at the effect of tamoxifen on the Cl- channel at this concentration. Figure 6A shows traces recorded from an excised outside-out patch in which the pipette solution was NaCl rich and the bath solution was normal saline. In Fig. 6A, the top trace shows channel activity under control conditions, whereas the bottom trace shows channel activity after the addition of 5 µM tamoxifen to the bath solution. There was no effect on either the Po or current amplitude of the channel. In two separate experiments with 5 µM tamoxifen, three experiments with 2 µM tamoxifen, and three experiments with 1 µM tamoxifen, no effect was seen on the Cl- channel. Although tamoxifen failed to block the guinea pig enterocyte Cl- channel, the derivative 4-hydroxytamoxifen (4-OH-tamoxifen) was tested to see if it would have any effect. This compound has been shown to inhibit volume-activated outwardly rectifying Cl- conductance and, like tamoxifen, does so at concentrations in the low micromolar range. As shown in Fig. 6B the same is true for the Cl- channel from guinea pig enterocytes. The top trace in Fig. 6B shows the control condition, in which no 4-OH-tamoxifen had been added to the bath solution. There were two active channels in the patch, both of which had very high Po values. The middle and bottom traces of Fig. 6B show the effect of adding 2 and 10 µM 4-OH-tamoxifen to the bath solution. It can be seen that this resulted in an increasingly marked flickery-type blockade of the Cl- channel. There was also a slight decrease in the current amplitude in the presence of 4-OH-tamoxifen. The current amplitude under control conditions was 3.0 pA, and it decreased to 2.4 pA at 10 µM tamoxifen. A dose-response curve for the inhibition of the Cl- channel by 4-OH-tamoxifen is shown in Fig. 7. Even at 10 µM 4-OH-tamoxifen the channel was only inhibited by ~38%. Using higher concentrations of 4-OH-tamoxifen caused damage to excised outside-out patches. The line is a fit of a rectangular hyperbola to the data. From this fit the maximal inhibition was 50%; for 4-OH-tamoxifen, the concentration required for half-maximal inhibition of the channel (IC50) was 3.2 µM.


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Fig. 6.   Effects of inhibitors on guinea pig enterocyte ORCCs. A: traces recorded from an excised outside-out patch held at 50 mV. Pipette solution was NaCl rich. Top trace: bath solution was normal saline; bottom trace: 5 µM tamoxifen had been added to bath solution. Arrows indicate closed level. B: traces recorded as in A, but from a different patch and with 4-hydroxytamoxifen (4-OH-tamoxifen) at indicated concentrations.


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Fig. 7.   Dose-response curves for guinea pig enterocyte ORCC inhibition. Percent inhibition of the Cl- current at indicated concentrations of 4-OH-tamoxifen (open circle ), 1,9-dideoxyforskolin (DDFSK; down-triangle), and verapamil (triangle ). Effect of NPPB (square ) at a single concentration is also shown. Values are means ± SE.

Figure 7 also shows the concentration dependence for the effects of verapamil and DDFSK. The continuous lines are fits of rectangular hyperbolas to the data. For verapamil inhibition, maximal blockade of the channel deduced from the fit was 78%. The IC50 for Cl- channel inhibition by verapamil was 12 µM. The dose-response curve for the inhibition of the Cl- channel by DDFSK gave an IC50 of 52 µM.

Blockade of guinea pig enterocyte ORCCs by trinitrophenyl-ATP. ATP and some derivatives can block ORCCs. Trinitrophenyladenosine 5'-triphosphate (TNP-ATP), reported to be a powerful blocker (55), has been used here. Figure 8A shows traces recorded from an outside-out patch maintained at 50 mV throughout the experiment. The patch, seen in the control condition in the top trace of Fig. 8A, contained three active ORCCs. The middle and bottom traces of Fig. 8A were recorded after addition of 10 and then 50 nM TNP-ATP to the bath solution, respectively. The addition of TNP-ATP resulted in a strong inhibition, which greatly reduced Po without any effect on the single-channel current. In Fig. 8B a rectangular hyperbola has been fitted to the concentration dependence of TNP-ATP inhibition, yielding an IC50 value of 18 nM.


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Fig. 8.   Effects of trinitrophenyladenosine 5'-triphosphate (TNP-ATP) on guinea pig enterocyte ORCCs. A: traces recorded from an excised outside-out patch held at 50 mV. Pipette solution was NaCl rich. Top trace: bath solution was normal saline; bottom traces: recordings were from the same patch after perfusion with TNP-ATP. Arrows indicate closed level. B: dose-response curve for TNP-ATP effect. Results are means ± SE from 4 experiments.

The high potency of blockade of TNP-ATP would make it an excellent tool to investigate the role of the ORCC in enterocyte physiology. As a way to link the whole cell currents previously described in villus enterocytes (43) and the single-channel activity studied here, the effect TNP-ATP was studied. Figure 9A shows whole cell recordings measured with voltage protocols clamping the cell between -120 and 120 mV in 40-mV steps from a holding potential of 0 mV, with Cl--rich intra- and extracellular solutions (see Table 1 for composition). As described previously, an outwardly rectifying current was obtained with little evidence for voltage dependence except for the most hyperpolarizing pulse. Surprisingly, addition of 100 nM ATP to the medium did not affect the currents markedly. This is seen in Fig. 9B, in which the corresponding current-voltage relations are shown. An experiment, also shown in Fig. 9B, in low Cl- concentration (gluconate replacement, see Table 1) confirmed the anionic nature of the charge carrier in these experiments, as there was a marked decrease in outward current and a displacement of the Erev to the right. If the channels described above were nevertheless responsible for the macroscopic current, a possible explanation of the lack of effect could be a marked voltage dependence of TNP-ATP inhibition coupled to a slow on-rate. This was tested by increasing the length of depolarizing pulses. Figure 9C shows that by the end of an 80-mV pulse a marked inhibition by 100 nM TNP-ATP could be observed. With 1 µM TNP-ATP complete inhibition could be observed after 2 s. When the same current-voltage protocols as in Fig. 9A were applied from a holding potential of 80 mV, inhibition was observed at all potentials. This is shown in Fig. 9D, in which it can be seen that inhibition by 100 nM TNP-ATP was voltage dependent (i.e., it was more marked as the cell was more depolarized). The voltage dependency of TNP-ATP blockade was also evident in experiments (not shown) in which a conditioning pulse was given before voltage was returned to 80 mV and the tail currents were measured. Little inhibition was seen with a -120-mV prepulse, but blockade became progressively more marked as the conditioning prepulses became more positive. The voltage dependence of blockade by 100 nM TNP-ATP could be described reasonably well by a Boltzman distribution with effective valence of -0.8 and 50% inhibitory voltage of 14 mV.


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Fig. 9.   Effect of TNP-ATP on whole cell Cl- currents. A: whole cell Cl- currents obtained under the indicated conditions. Cell was held at 0 mV and pulsed to potentials from -120 to 120 mV in 40-mV steps. Bath and pipette solutions were NMGCl rich (see Table 1). B: current-voltage relations, where result of partial replacement of extracellular Cl- by gluconate is also shown. C: effect of long pulses to 80 mV under control conditions or in the presence of 2 concentrations of TNP-ATP. D: current-voltage relations for whole cell currents measured with same protocol and solutions as in A, but at a holding potential of 80 mV.

That similar voltage dependence was present at the single-channel level could be observed in outside-out patches as shown in Fig. 10. ORCC activity was studied by pulsing for 5 s to -80 mV and then to 80 mV for 20 s under control conditions (Fig. 10A) or after perfusion with 100 nM (Fig. 10B) and then 1 µM TNP-ATP (Fig. 10C). A similar degree of activity was observed at -80 mV regardless of the presence of the nucleotide. On application of the depolarizing step, progressive blockade was apparent, which resulted in complete inhibition by the end of the pulse. In Fig. 10D averaged traces are shown, which show no differences between control and in the presence of the inhibitor at -80 mV. On switching to 80 mV in the presence of TNP-ATP, rapid blockade occurred that was virtually complete after 5 s at 80 mV. Similar results were obtained in five separate patches. These observations link the single ORCC activity and the macroscopic Cl- current in enterocytes, although they might correspond to different channels coincidentally affected by TNP-ATP in a similar fashion.


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Fig. 10.   Time course of the effect of TNP-ATP on guinea pig enterocyte ORCCs. A-C: traces were recorded from an excised outside-out patch held at 0 mV and pulsed to -80 mV for 5 s and then switched to 80 mV for a further 20 s. Pipette and bath solutions were NaCl rich. Control traces and traces in the presence of TNP-ATP are from same patch. D: averaged traces obtained from 22 sweeps (control) and 15 sweeps each in the presence of 100 nM or 1 µM TNP-ATP.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Cell-attached patches on villus enterocytes isolated from guinea pig small intestine contain a spontaneously active Cl- channel. Outward currents through this channel were seen by depolarizing the membrane patch. However, in the cell-attached configuration, inward currents could not be resolved. A spontaneously active Cl- channel of intermediate conductance (35-40 pS) that did not rectify has been observed in cell-attached patches from isolated rat colonic enterocytes (10). ORCC activity in cell-attached patches has been reported, but only after some form of stimulation. ORCC activity has been seen in cell-attached patches of HT-29 cells after strong depolarizing voltage pulses (14) were applied, in MDCK cells after exposure to hypotonicity (2), and in colonic T84 cells under the action of cAMP-mediated secretagogues (21). Most reports describe activation of ORCCs after excision of previously silent cell-attached patches, often requiring strong depolarization (20, 21, 33, 57). Kunzelmann et al. (31) found that the ORCC activated instantaneously on excision of cell-attached patches at 37°C, irrespective of the clamp potential. It was later shown that the cytosol of some cells (including HT-29 cells, T84 cells, and CFPAC-1 cells) can inhibit the ORCC, and this might explain "excision activation" of ORCCs (28, 32). Although it appears that the channel studied here does not require any form of activation to be recorded, some unintentional stimulus might have arisen during the isolation procedure. If the enterocyte ORCC were sensitive to cell swelling or direct or indirect mechanical stimulation, it is conceivable that this type of activation might have taken place during the isolation procedure. It is not possible to say whether the volume of the cells is at a normal level after isolation. Similarly isolated cells, however, can respond to anisotonicity or changes in volume consequent to active transport with regulatory volume adjustments (34, 35) and have near-physiological ion gradients (9).

Thus the spontaneously active Cl- channel reported here in cell-attached patches is unusual in two ways: it required no apparent stimulation in order to activate, and only outward currents could be seen for this channel in the cell-attached mode. It is conceivable that inward currents (Cl- leaving the cell) are not detectable, as Goldman-Hodgkin-Katz rectification would further decrease the already small inward currents at negative potentials. Another explanation would be that some intracellular component blocks Cl- exit (inward current). This may be related to the proposed cytoplasmic inhibitor of this type of channel (28, 32). A possible physiological role for this channel might be to allow the efflux of Cl- from the cell during the Na+-coupled absorption of nutrients (34, 35).

When cell-attached patches containing outward Cl- currents were excised, both outward and inward currents were always seen. There were no exceptions to this observation, suggesting that the outward and inward currents were through the same channel and not through two different channel types. In symmetrical 145 mM Cl- solutions the chord conductance for outward currents (at 80 mV) was ~62 pS and for inward currents (at -80 mV) was ~19 pS. These values are typical for intermediate-conductance ORCCs (51). Interestingly, a recent report (26) has demonstrated that ORCC activity probably accounts for volume-regulated anion currents as previously proposed (46, 58).

The anion permeability sequence for the Cl- channel, derived from shifts in the Erev in response to bathing excised inside-out patches in different anion-containing solutions, was very similar to selectivity sequences reported for other epithelial Cl- channels (15, 17, 19). Earlier whole cell experiments in guinea pig villus enterocytes had shown that the outwardly rectifying Cl- conductance had Panion/PCl values of 2.57, 0.75, and 0.27 for SCN-, F-, and gluconate, respectively (43). The permeability sequence obtained was SCN- > I- > Br- > Cl- > F- > (gluconate, glutamate, SO<SUP>2−</SUP><SUB>4</SUB>). This sequence corresponds to Eisenman's sequence I (59), suggesting that the channel pore contains weak binding sites.

The strong outwardly rectifying nature of the Cl- channel described here, together with its high selectivity for anions over cations and its anionic permeability sequence, suggests that this channel may be the channel responsible for the whole cell Cl- currents reported previously in isolated guinea pig small intestinal villus cells (43).

Roy and Sauvé (40) have reported that after exposure to hypotonic medium, MDCK cells undergo a regulatory volume decrease (RVD) that involves the loss of K+, Cl-, and amino acids from the cells. It was later found that the pathway for amino acid loss during RVD was selective for neutral and anionic amino acids over cationic amino acids (39). In MDCK cells (2) the loss of K+ during RVD occurs by activation of a highly selective K+ channel; loss of Cl- was found to be through the activation of an ORCC that was not highly selective among anions. Single-channel patch-clamp recordings from MDCK cells have now shown that the ORCC will allow the permeation of the amino acids glutamate, taurine, and aspartic acid (3), and it is proposed that neutral and anionic amino acid (and some sugar) losses observed during RVD occur via the ORCC (see Ref. 48 for a recent comprehensive review on the subject).

It has been demonstrated in this report that the ORCC in guinea pig villus enterocytes will allow the large anion gluconate and the anionic amino acid glutamate to permeate. The permeability of glutamate relative to that of Cl- was 0.09 and that of gluconate relative to Cl- was 0.23. These values suggest that the permeation of organic molecules through the ORCC could be of physiological importance.

Although it is well known that the role of villus enterocytes is to absorb nutrients from the gut lumen, relatively little has been reported on how these nutrients leave the cell at the basolateral membrane. Sugars and amino acids exit across the basolateral membrane of absorptive cells by what appears to be either simple diffusion or facilitated diffusion (7, 47). As these molecules do not readily cross the lipid bilayer, one could speculate that the enterocyte ORCC would be a possible route for the exit of nutrients (both amino acids and sugars) after Na+-dependent uptake at the brush border membrane. Banderali and Roy (3) postulate that for MDCK cells a sudden increase in amino acid uptake at the apical membrane would lead to an important rise in cell amino acid content and cell volume increase that would trigger the opening of the basolaterally located ORCC, thus permitting a rapid exodus of amino acids (45). Thus it may be that cells that have a physiological capability to absorb nutrient across the apical membrane require the ORCC in the basolateral membrane as an exit route for the absorbed nutrients.

In conclusion, cell-attached and excised patches from guinea pig villus enterocytes have been used to demonstrate the presence of a Cl- channel, whose characteristics suggest that it is responsible for the whole cell Cl- currents reported previously (43). This channel has many of the characteristics of ORCCs present in other epithelial cells. The guinea pig villus enterocyte ORCC is novel in that it is spontaneously active in cell-attached patches.

Blockade by NPPB of the ORCC in HT-29 colonic cells has been studied extensively (6, 13, 14, 24, 51). NPPB is a potent blocker of the HT-29 ORCC, and it acts by binding to the extracellular aspect of the channel. Swelling-activated outwardly rectifying Cl- currents and ORCC single-channel activity are also blocked by NPPB, with IC50 values of 2-25 µM (11, 12, 17, 30). Whole cell recordings from guinea pig small intestinal villus enterocytes have previously shown that the outwardly rectifying Cl- currents present were strongly and reversibly inhibited by 10 µM NPPB (43). When applied to the extracellular surface of an enterocyte excised outside-out patch, 50 µM NPPB caused a >80% blockade of the ORCC, suggesting that IC50 lies in the low micromolar range.

The Ca2+ channel blocker verapamil caused a potent, dose-dependent, fully reversible inhibition of the ORCC of guinea pig small intestinal villus enterocytes. DDFSK, an analog of forskolin that has no effect on cAMP levels, fully blocks the enterocyte ORCC with an IC50 of 50 µM. The ORCC of HT-29 (D4) cells was inhibited with an IC50 of 100 µM by verapamil added to the extracellular surface of excised outside-out patches (6). Verapamil and DDFSK also inhibit the volume-activated outwardly rectifying Cl- currents (12, 41, 53) with IC50 values <100 µM. Thus the spontaneously active ORCC of guinea pig small intestinal villus enterocytes is blocked to a similar extent by verapamil and DDFSK as volume-activated outwardly rectifying Cl- currents are blocked.

The anti-estrogen tamoxifen is a potent blocker of volume-activated outwardly rectifying Cl- currents, which are inhibited with an IC50 of 0.3 µM when tamoxifen is added extracellularly (60). Tamoxifen has no effect on either the cAMP-activated or Ca2+-activated Cl- currents of T84 cells (54). The fact that both the guinea pig small intestinal villus enterocyte ORCC and volume-activated Cl- currents are blocked by verapamil and DDFSK suggested that these currents might be related. One might expect that tamoxifen would be a potent blocker of the guinea pig small intestinal villus enterocyte ORCC, but it was without effect. 4-OH-tamoxifen, which also blocks volume-activated Cl- currents (60), caused a dose-dependent blockade of the guinea pig small intestinal villus ORCC with IC50 of 3.2 µM. 4-OH-tamoxifen caused a flickering blockade and also slightly decreased the current amplitude. It is surprising that 4-OH-tamoxifen can block the ORCC, whereas tamoxifen, of similar chemical structure, does not. It has been shown, however, that 4-OH-tamoxifen has a 100-fold greater affinity for the estrogen receptor than tamoxifen (27), showing that a small change in the chemical structure can lead to dramatic changes in properties. The finding that 4-OH-tamoxifen but not tamoxifen blocks the guinea pig villus enterocyte ORCC suggests that this family of potential blockers may also be used to distinguish between different types of Cl- channel.

Voltage-dependent blockade of various Cl- channels by ATP has been reported (1, 25, 36), and an ORCC from rat colonic crypts was blocked with high affinity by TNP-ATP (55). An even more potent blockade than previously reported was observed for the small intestinal enterocyte ORCC by TNP-ATP in the present work, with an IC50 of 18 nM compared with 270 nM in the rat colonic channel. Inhibition was voltage dependent, making TNP-ATP a poor inhibitor at physiological voltages and therefore decreasing the value of its use in functional intact cell work. The compound probably exerts its effect crossing part of the membrane field and should be valuable as a probe for the channel structure, although it is not known whether it inhibits other channel types. The similarity in TNP-ATP effects on whole cell current and ORCC, coupled with the similarity in permeability sequence, outward rectification, and sensitivity to NPPB, make the ORCC a good candidate to mediate the macroscopic currents described before.

In summary, an ORCC of spontaneous activity has been described in enterocytes from small intestinal villus of the guinea pig. It is proposed that it underlies the Cl- currents previously described in these cells and could serve to provide an exit pathway for Cl- taken up across the apical membrane (43). In addition, it is speculated that it might be the pathway responsible for Cl-, and perhaps nutrient, efflux from enterocytes swollen osmotically or by nutrient uptake.

    ACKNOWLEDGEMENTS

This work was supported by Fondecyt (Chile) Grant 1961208 and by a grant from the Volkswagen Stiftung (Germany). Institutional support to Centro de Estudios Científicos de Santiago from a group of Chilean private companies (Compañía Manufacturera de Papeles y Cartones S.A., Compañía General de Electricidad Industrial, Corporación Nacional del Cobre de Chile, Compañía de Petróleos de Chile S.A., Minera Escondida Limitada, Nova Gas International, Business Design Associates, and Xerox de Chile S.A.) is also acknowledged. A. S. Monaghan was supported by a Research Studentship from the Agricultural and Food Research Council of the UK (AFRC), and G. M. Mintenig was supported by AFRC Grant LRG-111. F. V. Sepúlveda is in receipt of an International Fellowship of the Howard Hughes Medical Institute and a Cátedra Presidencial en Ciencias.

    FOOTNOTES

Address for reprint requests: F. V. Sepúlveda, Departamento de Medicina Experimental, Facultad de Medicina, Universidad de Chile, Casilla 70058, Santiago-7, Chile.

Received 13 January 1997; accepted in final form 8 August 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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