Pfl/igers Arch 0992) 420:46- 53

Journal of Physiology 9 Springer-Verlag1992

Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin F. Becq 1,z, M. Fanjul 1, I. Mahieu t, Z. Berger 3, M. Gola 2, and E. Hollande 1 Laboratoire de BiologicCellulaire,Universit6Paul Sabatier, 38 rue des Trente Six Ponts, F-31400 ToulouseCedex, France 2 Laboratoirede Neurobiologie, CNRS, LNB 4, 31 CheminJoseph Aiguier, F-13402 Marseille, Cedex09, France 3 Laboratoirede Pathologic Digestive,INSERM U. 315, 46 Boulevardde la Gaye, F-13009 Marseille, France Received April 4, 1991/Receivedafter revisionAugust 29, 1991/Accepted September 4, 1991

Abstract. Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi C1- channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying C1- channels were clustered in discrete loci, They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for C1- over monovalent cations (Na + and K +) and gluconate. They were blocked by 2 0 - 200 ~tM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and were insensitive to changes in the Ca 2 + concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion. Key words: Human pancreas - Adenocarcinoma - Pancreatic duct cells - Chloride channels - Patch clamp Cell culture - Disulphonic stilbene derivatives

Offprint requests to: M. Gola

Introduction The ability of pancreatic duct cells to secrete a bicarbonate-rich isotonic fluid plays an important part in digestion [1], although little is known about the underlying mechanisms. On the basis of recent patch-clamp studies on duct cells isolated from rat [5] and human fetal pancreas [6, 7], as well as studies on isolated perfused rat pancreatic ducts, it has been suggested that apical C1- channels and C1-/HCO~ exchanges mediate the movements of chloride in pancreatic duct cells. Two types of CI- channels have been detected in isolated duct cells from rat and human fetal pancreas: an apical cAMP-activated lowconductance (4 pS) non-rectifying C1- channel regulated by secretin [5], and two outwardly rectifying C1- channels [6, 7]. One of the rectifying channels ( 4 - 7 pS) resembles the non-rectifying C1- channel described in the rat, and appears to be involved in CI- recycling at the apical membrane of pancreatic duct cells in both rats and humans. The properties of the second rectifying C1- channel ( 2 0 - 50 pS) [6] are similar to those of the C1- channels described in various chloride-secreting epithelia and in several pancreatic (PANC-1 [22], CFPAC-I [20] and colonic cancer cell lines (HT-29 [10] and T84 [8]). There is scant information available on the distribution and properties of anionic channels in normal or pathological human pancreas. This information can be obtained by studying pancreatic cancer cell lines of ductal origin that have kept the ability to transfer chloride ions. In a previous study, we showed [17] that the human pancreatic adenocarcinoma cell line Capan-I of ductal origin forms domes, which are thought to reflect the transepithelial transport of water and electrolytes [18]. Histologically, the pancreatic tumour line Capan-1 has been described as a well-differentiated mucin-producing adenocarcinoma [16] resembling the tumour of origin. Chromosomal analysis showed a human karyotype with chromosomes numbering between 79 and 82. Since Capan-I cells are derived from duct cells [16], they may have conserved their ability to secrete ions, especially bicarbonate and chloride. These cells possess vasoactive

47 intestinal peptide receptors associated with adenylate cyclase [19], which m a y be involved in the secretion o f ions, as has been f o u n d to occur in the n o r m a l pancreas [1]. F u r t h e r m o r e , the high basal level o f c A M P in C a p a n 1 cells [16] m a y a c c o u n t for the existence o f ion t r a n s p o r t in the absence o f h o r m o n e stimulation. Capan-1 cells should therefore possess anion-selective channels on their luminal m e m b r a n e . In the present study using conventional p a t c h - c l a m p methods, we identified three types o f apical chlorideselective channels that m a y be involved in transepithelial solute transport.

Materials and methods

Cell culture. The Capan-I cell line, isolated in 1974 at the SloanKettering Institute of Cancer Research (New York, USA) from a liver metastasis of a pancreatic adenocarcinoma from a 40-year-old Caucasian man, was received at the laboratory of Cellular Biology in Toulouse at the 14th passage from the American Type Culture Collection (ATCC, Md., USA). Cells were maintained in RPMI-1640 medium (Gibco, N.Y., USA) containing penicillin (100 U/ml), streptomycin (100 gg/ml), amphotericin B (0.25 gg/ml) (all from Gibco) and 15% fetal calf serum (Intermed, FRG). The ionic composition of the RPMI medium was (in mM): 102.5 NaC1, 5.4 KC1, 0.4 MgSO4, 5.6 Na2HPO4, 28 NaHCO3, 0.4 Ca(NO3)2. Cells were grown in monolayers and were maintained through successive passages by treatment with 0.05% trypsin/0.02% EDTA. They were seeded at 2.5 x 105 cells/ml into 25-cm z plastic flasks (Coming Glass, Coming, N.Y.), and grown at 37~ in a humidified air atmosphere containing 5% CO2. The doubling time was 20 h. Cultures were regularly checked for mycoplasmic contamination. Electron-transmission microscopy. The cell cultures were fixed in situ in cold glutaraldehyde (2.5%)/paraformaldehyde (1%) in 0.1 M phosphate buffer (pH = 7.4), post-fixed in osmium tetroxide (1%) and embedded in epon/araldite. Semi-thin sections were examined after staining with toluidine blue in order to select areas of interest for more detailed examination. After contrasting with uranyl acetate and lead citrate, ultrathin sections were examined with a Hitachi HU 11C electron microscope. Patch-clamp study. Patch-clamp experiments were performed on confluent cells. The culture flask in which cells were grown was placed on the movable stage of an Olympus inverted microscope equipped with phase-contrast optics. An overall magnification of x 350 was employed. For cell-attached recordings, the RPMI-1640 culture medium was left in the chamber, Inside-out patches were formed by rapidly lifting the pipette to the solution/air interface. The pipette tip was then placed at the exit of a l-ram capillary tube perfused with the experimental solution. Experiments were performed at room temperature (20-22 ~C). Patch pipettes were formed by three-step pulling with soft glass tubes (Clark Electromedical Instrument, ref. GC 150F10) on a vertical puller (David Kopf Instrument, Tujunga, Calif., USA). They were heatpolished with a glass-coated platinum wire to reduce the tip diameter to 1 -1.5 gin. The pipette resistances ranged from 6 MQ to 12 MO. Currents were recorded with a List EPC-7 amplifier (Darmstadt, FRG) (filter setting 3 kHz), and low-pass filtered at 2 - 5 kHz using a six-pole Bessel filter. They were continuously recorded on a video cassette recorder after 16-bit digitization at 44 kHz with a pulse code modulator (PCM Sony) adapted to patch-clamp data storage by Biologic (Meylan, France). Stored data were further digitized at 1 - 5 kHz and transferred to an Olivetti M28 PC computer. In all illustrations, outward currents (flowing from the cell to the patch electrode) are displayed in the upward direction. Potentials

are expressed as the bath potential minus that in the patch electrode; in cell-attached recordings, they are the change in potential from the cell resting level. Junction potentials were routinely evaluated from the patch potential that gave a null-current baseline when channels were in the closed state. Reversal potentials and conductance data were obtained from current/voltage (I/V) relationships by least-square regression analysis, except for non-linear I/V relationships, which were drawn by hand. In all the figures, dashed lines are zero-current baselines with channels in the closed state.

Solutions. The standard solution used for filling the patch electrodes contained (in mM): 150 KC1, 2.5 CaC12, 1 MgC12, 5 TRIS (pH 7.8). Inside-out patches were perfused on the cytoplasmic side with the above KCl-rich saline in which the Ca 2§ content was varied from nominally 0 mM Ca 2 + (by adding 1 mM EGTA to the Ca-free saline) to 1 mM Ca 2 +. To test the ionic selectivity of the channels, KC1 was replaced by equimolar amounts of either NaC1 (NaCl-rich saline) or potassium gluconate. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) was used at 20 gM and 200 gM.

Results D u r i n g the exponential g r o w t h phase, Capan-1 cells adhered to the b o t t o m o f the flask. The cells had a large cytoplasm and a central nucleus with p r o m i n e n t nucleoli. A t this stage, they f o r m e d an epithelial monolayer. After 4 days in culture, the cells reached confluency and the m o n o l a y e r pattern was preserved. S p o n t a n e o u s loss o f adhesion o f the m o n o l a y e r occurred in discrete zones between days 5 a n d 7 (Fig. 1 A). These areas f o r m e d d o m e structures with an average diameter o f 7 0 - 1 6 0 g m (Fig. 1 B). A t the end o f the exponential g r o w t h phase, the cells were polarized with the apical pole pointing towards the culture medium. T h e y were elongated with p o o r l y developed microvilli, and were joined together by apical junctional complexes comprising tight and intermediate junctions (Fig. 1 C).

Chloride channels in the apical membrane of confluent cultures P a t c h - c l a m p experiments were p e r f o r m e d on 4- to 8-dayold cultures. The seal o f the pipette to the m e m b r a n e , even potentiated with suction, occurred in less than 20% o f the trials. Once established, seals h a d resistances ranging f r o m 2 G~2 to 10 Gf2. T h e y were sufficiently mechanically resistant for the p a t c h o f m e m b r a n e to be excised. A t t e m p t s to excise channels in the outside-out configuration were mostly unsuccesful, however; the rupture o f the m e m b r a n e in the patch pipette required a consistent suction, which altered the quality o f the seal. In seven cases, the resulting whole-cell configuration showed that the cell resting potential ranged between - 35 m V a n d - 4 3 inV. F o r the a b o v e - m e n t i o n e d reason, these values m i g h t have been below the actual m e m b r a n e potential (see below). I n 40% o f the successful seals (n = 140), channels were detected in the patch. Three different chloride channels were identified: two non-rectifying chloride channels (n = 35) and one o u t w a r d l y rectifying chloride channel (n = 12). ,An inward rectifying potassium channel o f 30 pS at negative potentials was present

48

Fig. 1A - C. General appearance of living Capan-1 ceils. A Appearance of a 7-day-old culture at passage 56. Cells were maintained in RPMI- 1640 medium supplemented with 15% fetal calf serum. The micrograph was focused on the cells adhering to the bottom of the flask. Areas with fuzzy appearance are domes. Phase contrast. Bar: 100 pm. B Semi-thin section performed at the level of a dome.

Domes result from a local loss of confluent monolayer adhesion. They are bounded by peripheral cells adhering to the flask (outlined by the dashed line). Bar: 5 gin. C Ultrastructure of confluent CapanI cells. Cells are polarized. The apical pole facing the culture medium exhibited microvillosities. Arrows indicate apical junctional complexes. Bar: ~ gm

in ten cell-attached patches. With a KCl-rich saline in the pipette, the reversal potential of the current through the potassium channel was + 52 mV (n = 6). If we assume that the potassium equilibrium potential under these conditions was around 0 mV, the cell resting potential would be around - 50 mV.

Most of the cell-attached patches were silent. In some of these patches, positive or negative voltage pulses of 2 0 - 40 mV amplitude revealed the presence of high-conductance ionic channels. In a few cases, the channels became active in response to large ( > 80mV) hyperpolarizing pulses; once induced, these channels stayed active for the duration of the experiment. This large-conductance channel had a linear current/voltage relationship and a reversal potential roughly equal to the cell resting potential. Its unitary conductance varied greatly from cell to cell ( 2 8 0 - 4 0 0 pS, mean 350 pS over 35 patches). Hereafter this channel will be referred to as g350. Superimposed on the large current fluctuations, we consistently observed fluctuations reflecting the presence of a medium-sized channel. In cell-attached patches, this

Chloride channels active in celI-attached patehes

The most prevalent channel types seen in the cell-attached mode turned out to be non-rectifying chloride-selective channels (25% of the successful patches). There was no significant difference in the number of channels per patch between adhering cells and cells located in domes.

49

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Fig. 2A-C. Cell-attached recordings of g50 and g350 chloride channels. Experimentperformed on a 6-day-oldcell culture. Patch electrode filled with the KCl-rich saline. A, B Sample recordings and correspondingcurrent/voltagerelationships from a patch that contained both channels. Note the subconductance levels of the g350 channel. Both g50 and g350currents reverseddirectionaround 0 mV. C Voltage-induced g350 channel inactivation. This patch contained three channels. The channel inactivationwas inducedby either positive(+ 70 mV) or negative( - 50 mV) voltagejumps from the cell resting potential

channel had a linear current/voltage relationship with a slope conductance of 3 5 - 7 0 pS (mean 50 pS over 35 patches). It will hereafter be denoted g50. The direction of the current through the g50 was reversed at around 0 inV. The g50 channel was characterized by bursts of openings with fast transitions between the closed and open states. Its opening probability was not obviously voltage-dependent. The large chloride channel was always observed in association with the g50 channel. It should be noted that opening of the g350 channel was always preceded by that of the g50 channel. Figure 2 illustrates the unitary currents from these two chloride channels. A prominent feature of the g350 channel was the presence of several subconductance levels (Fig. 2B). The g350 channel had a voltage-dependent gating mechanism, which tended to block the channel when the potential moved 4 0 - 50 mV from the resting level (Fig. 2 C). The presence of frequent subconductance levels further complicated the analysis of the channel kinetics. No rigorous quantification of the voltage dependence of the channel opening was therefore attempted.

Activity of chloride channels in excised inside-out patches Inside-out patches were readily obtained by pulling the patch electrode and exposing it for a short period to air.

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Fig. 3A, B. Ionicselectivityof the g50 and g350channels.A Channel excised in the inside-out configuration.Patch electrode filledwith the KCl-rich saline; bath medium: RPMI-1640, producing large positive and negativegradients for Na + and K + ions, respectively. 1, Unitary currents recorded at various patch potentials; small and large current fluctuationsreversed direction at about - 8 inV. The g350 channel had multiple conductance states. 2, Corresponding current/voltage relationships. A, g350 channel; O, g50 channel. B Current/voltage relationships of excised g350 (1) and g50 (2) channels. Pipette: KCl-richsaline. Bath: 0, II, KCl-richsaline; A, [~, NaCl-rich saline

The g50 and g350 channels remained active during these operations. In 5 experiment, the g350 channel was silent in cell-attached patches and was detected only after excision. Current fluctuations from an inside-out patch that contained both channels already active in the cell-attached mode are shown in Fig. 3AI. The excised g350 channel still appeared to be inactivated by positive or negative step voltage changes. As in the cell-attached mode, it was reversibly blocked by the application of 4- 40/50 mV patch potentials. The unitary current of both g50 and g350 channels was reversed at around - 8 mV (Fig. 3 A2); this level corresponds to the chloride equilibrium potential when the patch was bathed in the RPMI medium ( - 10.2 mV). The unitary conductance and reversal potential of the g50 and g350 channels showed no change when the bath was perfused with either the culture medium (Fig. 3 A2), the KCl-rich saline (black symbols in Fig. 3 B) or the NaCl-rich saline (open symbols in Fig. 3 B). These results indicated that these channels were either impermeant to monovalent cations, or that they did not discriminate between monovalent anions and cations. The selectivity of the g50 and g350 channels was firmly established by replacing chloride with gluconate in either the KCl-rich or the NaCl-rich salines. Shifting from the KCl-rich saline to the gluconate saline (Fig. 4Al) abolished the inward current (i. e. the outward movement of anions) and shifted the null-current voltage towards negative values (Fig. 4A2). Therefore, both channels were selectively permeable to chloride ions and not to gluconate or monovalent cations. We then tried to block the channel with DIDS. In 3 experiments, 20 laM DIDS

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Fig. 4A, B. Selectivity of the g350 channel for C1- over gluconate. Inside-out patch under symmetrical (KCl-rich) conditions. A 1, This patch contained two g350 channels. Effect of replacement of the bath KC1 saline by the 150 mM gluconate saline. Patch potential: -40 mV. 2, Corresponding current/voltage relationships. Gluconate abolished inward currents and shifted the null-current potential towards negative values. *, gluconatc; 0, KC1. B Reversible blockade of the g350 channel by DIDS (20 gM) added to the bath KC1rich saline

fully and reversibly suppressed the large current fluctuations from the g350 channel (Fig. 4B). We were unable to measure any change in the g50 channel induced by DIDS, owing to the background noise of the current records in the presence of DIDS. Around 60% of the cell-attached patches were silent. These patches were systematically excised in the insideout configuration. In 12 experiments, immediately after excision we observed the presence of channels that were active at both negative and positive potentials (Fig. 5 AI). These channels were not evenly distributed over the apical membrane, and they were found to be clustered in groups containing up to ten channels. Amplitude histograms (Fig. 5 A2) showed that the clustered channels had similar unitary currents and reversal potentials. The good fit obtained with a binomial distribution (dotted curves superimposed on the histograms in Fig. 5A2) led us to assume that these clusters were composed of identical independent channels. In inside-out patches bathed in the culture medium, the unitary current in these channels displayed a pronounced non-linear voltage dependence (Fig. 5 B). The channel had a larger conductance (70 pS) in the case of outward currents, i.e. it favoured the entry of anions into the cell. Under these asymmetrical conditions, the null-current potential was between - 10 mV and 0 inV. The opening probability was slightly voltagedependent. The amplitude histograms attached to the recordings in Fig. 5 were fitted to Gaussian curves (predicted by the binomial law) in the case of two identical, independent channels. The corresponding opening probability was 0.4 at + 50 mV and 0.58 at - 9 0 inV. The unitary conductance and reversal potential of the rectifying chloride channel remained unaltered when the culture medium was replaced by either the KCl-rich saline (symmetrical conditions, Fig. 6A1) or the NaCl-rich

Fig. 5A, B. Outward rectifying chloride channels in an inside-out patch. The patch contained two channels. A 1, Unitary currents at + 50 mV, -12 mV and -90 mV patch potentials. 2, Histograms show the amplitude distribution of the current fluctuations at + 50 mV and -90 inV. The superimposed dashed curves are the expected distributions in the case of two independent channels with an individual opening probability (Po) of 0.40 and 0.58 at + 50 mV and -90 mV respectively. B Unitary- current/voltage relationship from A. The current reversed direction at about -10 mV. Patch electrode filled with the KCl-rich saline. Bath saline: RPMI-1640 medium

saline. In both cases, the current/voltage relationship had a reversal potential of approximately 0 mV (Fig. 6A2). The fact that the rectification persisted under symmetrical ionic conditions showed that it was an intrinsic property of the channel. The ionic selectivity of this channel was then assessed by replacing chloride with gluconate in the bathing saline. Gluconate abolished or greatly reduced the inward current at negative potentials (Fig. 6 B) but had no effect on the outward current. Taken as a whole, these data indicated that the rectifying channel was selective for CI- over gluconate and monovalent cations. DIDS applied to the cytoplasmic side (n -- 3) induced a progressive blockade o f the clustered rectifying channels (Fig. 7). Tetraethylammonium ( I 0 - 2 0 mM) or EGTA (1 m M in Ca z § saline) applied to the cytoplasmic side of the patch had no effect on the opening probability or unitary conductance o f any of the three chloride channels. Figure 8 shows that all three channels remained active in the absence of calcium (Ca = 0), both at physiological calcium concentration (Ca = 0.1 laM) and in the presence of an excess of this ion (Ca = 1 mM).

Discussion The main result of the patch-clamp experiments was the relatively high density of chloride-selective channels in the apical membrane of Capan-1 cells. Out of 57 experiments in which channels were unambiguously identified on the basis of their ionic selectivity, 83% were found to be permeant to CI- ions. This is in line with the latest

51 Non-rectifying C I - channels

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Fig. 6A, B. Ionic selectivity of the outwardly rectifying chloride channel. A Inside-out patch containing four channels bathed in the KCl-rich saline. Exemplar recordings (1) and corresponding unitary current/voltage relationship (2): the null-current voltage was close to 0 inV. Bath saline: O, KCl-rich saline; A, NaCl-rich saline. The slope conductance and reversal potential were not altered by changing K + for Na + in the bath saline. B Inside-out patch perfused with the 150 mM gluconate saline. Note the reduction in the inward current at negative potentials (1). 2, corresponding unitary-current/ voltage relationship

DIDS

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Fig. 7. Outwardly rectifying chloride channel inhibited by DIDS. Inside-out patch containing at least ten channels. Pipette filled with the KCl-rich saline. Arrow: patch perfused with the same saline supplemented with 200 gM DIDS. Pipette potential: + 35 mV. Upper trace: continuous recording of channel activity on a slow time scale. Lower traces: details, on an expanded time scale, of channel activity before (left-hand recording) and during (right-hand recording) partial inhibition by DIDS

findings on the role of chloride-selective channels both in fluid secretion by epithelial tissues and in transepithelial solute transport [1]. We identified three different types of C1--selective channels in the Capan-1 cells: two non-rectifying channels and an outward rectifying channel.

The non-rectifying g50 and g350 Cl-channels were always found to be associated in groups containing a few channels of each type. We never observed any patches containing both rectifying and non-rectifying channels. The parallel distribution of the g50 and g350 channels strongly suggests that they are functionally linked. Medium-sized and maxi C1- channels have been reported to coexist in epithelial cells [9]. Krouse et al. [13] suggested that the maxi C1- channel in pulmonary epithelial cells may be a co-channel formed by the association of seven channels with a unitary conductance of 6 0 - 7 0 pS. The g50 C1- channel in Capan-1 cells could be an elementary constituent of the maxi g50 co-channel, although this will need to be substantiated by further studies on the properties of the g350 channel, especially as regards the distribution of its multiple substates. The non-rectifying g50 C1- channel resembled the medium-sized C1- channels detected in various tissues [3]. In Capan-I cells, the g50 channel was observed to be active in cell-attached recordings immediately after formation of the seal. This channel was not well-characterized at the unitary level, however, since it was always found to be associated with the maxi g350 C1- channel. It nevertheless emerged that the g50 channel, like the g350 channel, was selective for C1- ions over cations, and was not dependent on the intracellular calcium concentration. The g350 channel in Capan-1 cells has similar properties to those of the maxi chloride channels existing in a variety of cells [21, 23] including cells from several epithelia [2, 9, 13]. The salient characteristics of these channels are (a) unmasking of dormant channels (although spontaneously active g350 channels were present in Capan-1 cells) by prolonged voltage changes (voltage-induced activation); (b) complex gating properties with the appearance of multiple subconductance states; (c) steep voltage dependence with channels maximally opened at 0 mV applied patch potential. The biological significance of the maxi chloride channel is still obscure. The channel does not appear to be specific to any particular cell type as mentioned above. The maxi chloride channel has been found to be active in cell-attached patches in the M D C K cell line [12] and human sweat gland cells [14]. However, in most studies, the channel was either scarcely [21] or never [2, 23] observed in the cell-attached configuration. It becomes active only several minutes after the patch has been excised and after large positive or negative voltage steps [9, 23]. In Capan-/ cells, we observed that the maxi chloride channel was active in cell-attached patches either spontaneously or after voltage jumps. The channel was reversibly inactivated by applied patch potentials of _%+40 inV. A similar voltage-dependent inactivation was observed in inside-out patches in spite of the 40 to 50 mV shift in the transmembrane potential that resulted from excision. These findings suggest that the inactivation may not be directly related to the absolute level of polarization that the channel undergoes, but rather to the intensity of the current that flows through it. The overall

52

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g350 C l - c h a n n e l

Outwardly rectifying Cl-channel

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gating properties of the maxi C1- channel is still puzzling; it is likely that a common mechanism may underlie the voltage-induced activation of dormant channels and the voltage-induced reversible inactivation. Further understanding of this mechanism should provide new clues to the biological role of these channels.

Outwardly rectifying Cl- channel On the basis of unitary conductance and ionic selectivity, the outwardly rectifying chloride channel in Capan-1 cells appeared to be identical to that observed in various normal cell types such as airway [15], sweat gland [14], nasal [11], intestinal [4], choroid plexus [2] and pancreatic ducts [6] as well as in various cancer cell lines such as T-84 [8], HT-29 [10], CF PAC-1 [20], PANC-1 [22]. Another common feature of the rectifying C1- channels is their segregation in discrete loci containing several channels [4, 5, 10, 15, 20]. Under our conditions, this channel was not observed in cell-attached patches even when large voltage pulses were applied in an attempt to unblock quiescent channels. Little is known about the activity of such channels in cell-attached patches in epithelial cells. In a few reports in which this channel was well-characterized in excised patches, occasional openings were detected before excision [4, 11, 14, 15]. The only clear-cut evidence that cell-attached active channels may exist in non-stimulated cells has been recently obtained in pancreatic duct cells [6]. After excision, the rectifying chloride channel immediately became active. This property of "excision activation" therefore seemed to be independent of the patch voltage. This is consistent with the results obtained on CF (cystic fibrosis) airway cells [15], but it differs from the data on several epithelia [4, 6, 14, 20], where activation of the excised channel required large depolarizing voltages. The opening probabilty of the Capan-I rectifying chloride channel appeared to be almost independent of

Fig. 8. Effects of internal calcium upon the g50 (A), g350 (B) and the outwardly rectifying (C) chloride channels. Unitary currents from different inside-out patches under symmetrical ionic conditions (KCl-rich saline). Internal calciumwas changed from 0 mM (by adding I mM EGTA) to 0.1 gM and 1 mM. Patch potential: A - 80 mV; B - 60 mV; C +60mV

the voltage, at least in the physiological range, and cytosolic calcium concentration. Similar results have been reported for other rectifying chloride channels [2, 4, 6] (however, see [24]). Recent reports on electrolyte transport anomalies in cystic fibrosis demonstrate the existence of a failure in the cAMP pathway that regulates the chloride transport and suggest that the rectifying chloride channel is involved in fluid secretion by epithelial (for review, see [24]). The secretion of a bicarbonate-rich fluid by the pancreatic duct epithelium, however, has a basal level in the absence of hormonal stimulation. Under these conditions, the rectifying chloride channel has been found to be either quiescent or to have a very low probability of being active [6, 20, 22] (and this report). These data are not in agreement with the idea that the rectifying chloride channel plays a major role in anion secretion (C1- or HCO;-) and in chloride recycling. Data are rapidly being accumulated on the role of chloride-selective channels in fluid-secreting epithelial cells. There is no doubt that these channels are, to varying degrees, involved in recycling CI- ions in cells secreting either chloride-rich or bicarbonate-rich salines. In spite of the abundant recent literature, the circumstances of opening and hence the precise function of these channels are still unclear. Our data suggest that the non-rectifying g50 and g350 channels are candidates for C1- secretion at the apical membrane in Capan-1 cells. It nevertheless still remains to be demonstrated that these channels are actually involved in bicarbonate secretion, the main function of pancreatic duct cells.

Acknowledgements.The authors want to thank Mrs. M. Andre, Mrs. H. Chagneux, Mr. G. Jacquet, Mr. R. Fayolle for their technical assistance. We also want to acknowledgeProfessor J. W. Hanrahan (McGill University of Montreal), Professor R. Laugier (Marseille) and Dr. J. C. Dagorn (Marseille) for helpful discussions. This investigation was supported in part by the Institut National de la Sant6 et de la Recherche M6dicale (INSERM contrat 89007) and by the Centre National de la Recherche Scientifique(CNRS).

53

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