Pfliigers Arch (1991) 418:479--490

EIl~ri]e"anJournal of Physiology

003167689100102R

9 Springer-Verlag 1991

Inhibition of epithelial chloride channels by cytosol* K. Kunzelmann, M. Tilmann, Ch.P. Hansen, and R. Greger Physiologisches Institut der Albert-Ludwigs-Universit~tt, Hermann-Herder-StraBe 7, W-7800 Freiburg, Federal Republic of Germany Received October 17, 1990/Received after revision March 6, 1991/Accepted March 11, 1991

Abstract. Chloride channels that have an intermediate conductance and are outwardly rectifying were studied by the patch-clamp technique in cell-excised membrane patches from respiratory epithelial cells in primary culture (REC) of normal and cystic fibrosis tissue, HTz9 and T84 human colon carcinoma cells and placenta trophoblast cells (PTC). Chloride channels were immediately activated by the exposure of the cytosolic side of the patch to a Ringer-type solution, which lacked cytosolic components normally inhibiting chloride channels in the "on" cell configuration. Tentatively, we labelled the cytosolic component (or components) responsible for this inhibition cytosolic inhibitor (CI). The presence of CI in cytosol derived from HT29 cells was shown by assaying crude cytosol extracts from these cells on C1- channels from HT29 cells (n = 2) and REC from normal subjects and cystic fibrosis patients (n = 4). In order to examine CI further, PTC were used as a source of cytosol. The cytosol of PTC inhibited HT29 C1- channels in a dosedependent manner with a half-maximal inhibition observed at a 1 : 6 dilution (n = 11) of the native cytosol. CI from PTC was heat-stable (10min at 100~ n = 8). When cytosol extract was partitioned into a chloroform phase, CI- channel inhibition was shown for the lipophilic extract (n = 12) as well as for the aqueous phase (n = 10). The inhibitory potency of the lipid extract was slightly larger than that of the aqueous phase. Several separation procedures were used to determine the molecular size of CI. When CI was filtered through 30-kDa filters at 6000 rpm for 45 min, inhibitory potency was observed in the filtrate and the retained fraction (n = 3). The same was observed with 10-kDa filters (n = 6). When CI was dialysed through a 12-kDa membrane, inhibitory capacity was recovered from the dialysate. Similarly, gel filtration indicated that CI was < 5 kDa (n = 13) and

* Preliminary accounts of this report have been given at the cystic fibrosis conferences in Sestri Levante (March 1990) and in Arlington (October 1990) Offprint requests to: K. Kunzelmann

probably < 1.5 kDa (n = l 1), but > 700 kDa (n = 9). CI was exposed to bead-coupled hydrolysing enzymes (trypsin, non-specific protease, lipase, a-amylase, nucleotidase), but none of the enzymes used destroyed the inhibitory potency of CI. These data indicate that CI is present in HT29 as well as in PTC. It inhibits reversibly intermediate-conductance outwardly rectifying C1- channels in REC, HT29, and PTC. CI is heat-stable and amphiphilic and has an apparent molecular mass of 0.7-1.5 kDa. Given this nature of CI, several putative ion-channel regulators were examined on C1- channels of HT29 cells. It was found that inositol triphosphate, GTP, GTP [7-S], ATP, cAMP, cGMP and dioleoylglycerol all had no effect from the cytosolic side. Non-saturated fatty acids (n = 23) inhibited the open probability o f these C1channels from the cytosolic side after some delay reversibly at concentrations of 5 ~tmol/1 for arachidonic acid and more than 1 mmol/1 for linoleic acid. Saturated fatty acids had no effect. The present data indicate that this type of C1- channel may be inhibited by some cytosolic inhibitor with the above properties. Excision of membrane patches containing this channel leads to instantaneous disinhibition ( = excision activation). It is possible that an increased concentration of CI or an increased sensitivity to CI may be responsible for the "tonic inhibition" of C1- channels observed in cystic fibrosis REC.

Key words: Cystic fibrosis - Chloride channel - Patch clamp - Chloride secretion - Respiratory epithelial cells - Placenta trophoblast cells

Introduction Previously [22] we have reported that excision o f apical cell membranes from cystic fibrosis respiratory epithelial cells (REC) led to an immediate activation of chloride channels at 37 ~ We have called this phenomenon "excision activation". Furthermore we have postulated that some cytosolic component keeps these chloride channels

480

in a closed state in the intact cell (cell-attached). In order to examine further the phenomenon of excision activation and the concept of cytosolic inhibition we have undertaken the present experiments. We wanted to address the following questions. (a) Is the excision activation of CI- channels unique and present only in cystic fibrosis cells or is it also observed in other (normal) ceils? From previous studies it has been concluded that the outwardly rectifying intermediate-conductance C1- channel was demonstrable in hormone (e.g. catecholamine)-treated normal REC, but was absent in cystic fibrosis ceils [4, 22, 30, 32]. On the other hand, this same type of C1- channel has only been noticed with sizeable incidence in excised but not in cellattached patches of other normal cells [8, 18, 25]. Taking together both sets of findings, one may suggest that this kind of C1- channel, even though it may be present in cell-attached patches of several ceils such as REC or coIonic carcinoma cells, may be down-regulated by some cytosolic factor. The balance between these inhibitory and activating mechanisms (i.e. phosphorylation, volume control, and Ca 2+) would then determine the number of C1- channels that are open at a given time. The excision of the patch would then result in a disinhibition, i.e. an increase in the channel open probability. In cystic fibrosis tissues the situation might be different in the sense that one of the activating mechanisms could be defective [16, 1% 23, 24, 28] or that the inhibition might be enhanced [22]. Both defects would lead to a more marked reduction of the channel open probability on the intact cell. This phenomenon has been called "tonic inhibition" in previous reports [22, 30]. The present report will demonstrate that immediate excision activation is, in fact, a more general phenomenon observed in colonic carcinoma cells (HT29 , T84), normal and cystic fibrosis REC, and placenta trophoblast ceils (PTC). These findings support the above view that the open probability of this type of C1channel is governed by inhibitory as well as activating mechanisms. (b) Given the marked difference of the channel open probability in cell-attached and excised patches, which we and others [4,6,22] have noticed in cystic fibrosis tissues and will show here also for other C1--transporting epithelial ceils, the next question has to address the mechanism by which this inhibition occurs. We have approached this question by preparing crude cytosolic extracts and by examining these extracts on excised C1channels. In a further series we have attempted to characterize the cytosolic component (or components) responsible for this inhibition. They will be called "cytosolic inhibitor" (CI). In an independent and parallel study by Krick et al. [20] it will be shown that a cytosolic inhibitor of electrogenic C1- transport and of epithelial C1- channels can also be isolated from renal cortex and other sources such as HT29 and CFPAC 1 cells. Materials and methods Culture methods. Primary cultures of respiratory epithelial cells (REC) from normal subjects and cystic fibrosis patients have been obtained as

described previously [21, 22]. The methods of culturing HT29 cells have also been published previously [15]. For the HT29 cells a glucose-free Dubecco's modified Eagle's medium (DMEM) was used, to which fetal calf serum (100 ml/1) and L-glutamine (2 retool/l) were added. T84 cells were grown in a I : 1 mixture of glucose-free DMEM and F 12, to which fetal calf serum (100 ml/1) and L-glutamine (1.5 mmol/1) were added. Placenta trophoblast ceils (PTC) were prepared as follows. After removal of amnion and chorion, trophoblast tissue (about 30 pieces of approx. 1 mm 3) was removed from the placenta. This tissue was rinsed in sterile Ringer solution to remove blood, transferred into DMEM with antibiotics [21] and was incubated at 37 ~ for 30 min. The rinsing and incubation procedure were repeated three or four times. Thereafter the material was transferred into a disaggregation solution [21] and was shaken in the refrigerator (4 ~ for 15-17 tl). After addition of 200 ml/1 fetal calf serum the material was centrifuged (600 rpm, 10 min) to separate individual cells and cell clusters. With the sediment this procedure was repeated. The sediment was then resuspended and cultured in DMEM to which the following components were added: 200 ml/l fetal calf serum, 200kU/1 penicillin, 100mg/1 streptomycin, 20mrnol/1 HEPES and 2 mmol/1 L-glutamine. Cultures were kept in an incubator at 37 ~ and an atmosphere of 95% air and 5% O z for 1 - 4 clays in the case of HT29and PTC and for up to 2 weeks in the case of T84 and REC.

Preparation ofcytosoL In the case of placenta cytosol, the fresh placenta was perfused through one of the umbilical arteries with Ringer solution to remove blood. Then the amnion, chorion, decidua and umbilical cord were removed. The remaining tissue was cut into small pieces of approximately 1 cm3. Larger blood vessels and connective tissue were removed mechanically by dissection. Thereafter the material was frozen in liquid nitrogen and there by mechanically disrupted. After thawing, the material was homogenized and ultracentrifuged at 100000 g for I h. For HT29 and REC cells this ultracentrifugation step was preceded by disruption in a Potter homogeniser (20 strokes). The supernatant (cytosol) was then analysed with respect to its ionic composition (C1and pH) and was adjusted to a C1- concentration of 150 mmol/1 and a pH of 7.2. This solution was then bioassayed on excised chloride channels (see below), or was used for further purification.

Purification of eytosol. (a) Filtration of the cytosol was carried out by centrifugation at 5000 g for 45 rain through molecular filters of defined sizes: 10 kDa and 30 kDa (Centricon-10 and Centricon-30, Amicon, Mass., USA). The retained fluid as well as the filtrate were tested on excised chloride channels. Cytosol was also subjected to gel filtration using gels for different molecular sizes 700, 1500 and 5000 Da (G 10, G 15, G25, respectively, Pharmacia Freiburg, FRG). The cytosol passing through the gel filter was then examined on excised chloride channels. In addition, cellulose dialysis membranes (pore size 12 kDa) were used to determine the molecular mass of the cytosolic inhibitor (dialysing sacks, D9402, Sigma GmbH, Deisenhofen, FRG). The dialysate was examined for its effect on excised C1- channels. (b) Separation of the cytosol into a lipophilic and a hydrophilic fraction was carried out by shaking the cytosol with the same volume of organic solvents: chloroform, chloroform/isoamyl alcohol 25: 1, or light petroleum 60/70. After phase separation the aqueous phase was used for testing on excised chloride channels. The organic solvents were removed from the lipid phase by evaporation. Then the solutes were dissolved in dimethylsulphoxide and diluted 1 : 1000 into the Ringer-type solution (see below) to yield a volume equal to that of the aqueous phase. These lipophilic extracts were then also examined on excised chloride channels. (c) In another series the cytosol was treated with different enzymes immobilized on Sephadex beads (Sigma Chemie, Deisenhofen, FRG). The cytosol was !ncubated with either one of the following enzymes: lipase (60000U/1, L2764), trypsin (20000 U/l, T1763), protease (4000U/1, P8790), a-amylase (10000U/1, A5386) and ribonuclease (4000 U/1, R1626) for 5 h at 37 ~ After incubation, the cytosol was centrifuged at 10000 rpm for 15 min to sediment the beads and the supernatant was examined on excised chloride channeis. (d) In a further series of experiments fatty acids as well as other known constituents of the cytosol were added to the Ringer-type solu-

481 tion (see below) and examined on excised chloride channels. These chemicals were all obtained from Sigma (Deisenhofen, FRG). The dissolved fatty acids were kept frozen and light protected under an argon atmosphere until use. All solutions were prepared fresh, immediately prior to testing.

Patch clamp studies - bioassay. Patch clamp studies were performed as customary in our laboratory [9, 11]. Cell-attached and excised recordings were made on subconfluent cultures of HT29 and T84 cells, respiratory epithelial ceils (REC), and placenta trophoblast cultures (PTC). The patch pipettes were gently pressed against the apical surface of these cells. Giga seal formation was observed in about 20% - 30% of all trials. The excised patches were mostly of inside-out configuration [14]. With the outwardly rectifying C1- channel present in the patch, the orientation of the membrane could be verified by the direction of current rectification. Crude cytosol or partially purified extracts were usually tested on the cytosolic side of excised inside-out patches. All experiments were carried out at 37 ~ The patch pipette and the bath solution contained a modified NaC1 or KC1 Ringer solution, which contained in mmol/l: NaC1 Ringer: NaC1 145, K2HPO4 1.6, KH2PO4 0.4, CaC12 1.3, MgC1z t.0, D-glucose 5; KC1 Ringer: KC1 145, Na2HPO4 1.6, NaH2PO4 0.4, CaC12 1.3, MgC121.0, D-glucose5. Regarding the properties of the C1channels studied in this report the main cation made no difference. All cytosolic preparations were diluted at least by a factor of 2-5, as judged from the C1- concentration of the crude cytosol. Before use the chloride concentration and the pH were adjusted to 150mmol/1 and 7.2, respectively. The excised C1- channel was examined first in the Ringer-type solution, then the test solution was perfused through the bath (volume approx. 200 gl) at approximately 1 ml/min. The experimental period lasted for some 1 -3 min, after which the bath solution was changed back to the control solution. Usually the experimental phases were bracketed by pre- and post-experimental controls. The data were sampled with a low-pass filter of 1 kHz and stored on analogue tape. For data analysis the analogue recordings were digitized with a sampling rate of 2000/s and further processed with a PDP 11 computer system [9, 11]. The definition of current and voltage polarities conforms to the usual standards, i.e. anions flowing from the outside to the cytosolic side are referred to as positive currents.

Results

The properties of outwardly rectifying intermediateconductance CI- channels The properties o f the outwardly rectifying intermediatec o n d u c t a n c e C1- channels o f HT29 and R E C have been described extensively in previous reports [10, 15, 22, 29, 33]. This C1- channel is independent o f Ca 2+ and pH, has a m e a n slope conductance, at 0mV, o f about 4 0 - 7 0 pS, and is reversibly blocked by low concentrations (0.1 - 10 ~mol/1) o f 5-nitro-2-(3-phenylpropylamino)-benzoate. C1- channels with identical properties were f o u n d in the present study in HT29 (n = 95) and T84 (n = 3) colon carcinoma cells, R E C (n = 38), and PTC (n = 2).

Excision activation of CI -~ channels I m m e d i a t e excision activation o f C1- channels was f o u n d in non-stimulated n o r m a l (n = 10) and cystic fibrosis (n = 17) REC, HT29 (n = 15) and T84 (n = 3) colon carcinoma cells and, in a single pilot experiment, in a PTC. A n example for each different cell type is shown in Fig. 1. The condensed traces reveal that no C I - channels were active in the cell-attached recordings, but that

C1- channels were activated immediately when the m e m branes were excised. Immediate excision activation was, however, observed only in the m i n o r i t y o f all attempts. We believe [22] that the m a j o r i t y o f patches that stayed silent after excision had f o r m e d vesicles. Alternatively, some o f these patches m a y have been, in fact, free o f C I - channels. Even t h o u g h immediate excision activation occurred at depolarized voltages, it should be m a d e clear that excision activation was also observed at hyperpolarized voltages (also cf. [221). Since C I - channels were activated simply by excision and exposure to an artificial bath solution, we concluded that a cytosolic factor inhibited C1- channels in the intact cell and that the absence o f this factor in our artificial b a t h solution was responsible for instantaneous excision activation. Tentatively, we label this factor cytosolic inhibitor (CI).

Cl- channel inhibition by the HT29 cytosol In a first series the cytosol prepared f r o m HT29 ceils was examined in C1- channels present in inside-out m e m brane patches f r o m REC. These C I - channels f r o m normal (n = 4) as well as cystic fibrosis (n = 4) R E C were reversibly blocked by HT29 cytosol with a significant reduction in the channel open probability (P0) f r o m 0.64+0.14 to 0.29+0.14 and from 0.38+0.1 to 0.13+0.06, respectively. I n several pilot experiments we also obtained inhibition o f HT29 C I - channels by HT29 cytosol (P0 reduction to a b o u t 1/3, n = 2) and by the cytosol o f n o r m a l respiratory cells (P0 reduction to 1/3, n = 1).

Effect of placenta cytosol In another pilot series o f experiments we examined the effect o f cytosol from PTC o n C1- channels o f PTC and n o r m a l REC. It was shown that placenta cytosol significantly reduced P0 to some 50% o f its control value (n = 1 and n = 3, respectively). In a larger series, where placenta cytosol was applied to HT29 C1- channels, the channel P0 was reduced f r o m 0.76+0.03 to 0.35+_0.03 (n = 34). In these experiments the cytosol was diluted on average by a factor o f a b o u t 4, as estimated f r o m the a m o u n t o f volume added during the preparation and electrolyte adjustment (cf. Materials and methods). A typical experiment is shown in Fig. 2. It is evident that the inhibition by cytosol was instantaneous, i.e. it occurred at the speed with which the bath solution was changed. The effect was also rapidly reversible. A close inspection o f Fig. 2 reveals that the block is o f a flicker type. This flicker-type block has been observed in m a n y but not in all experiments. A s u m m a r y o f all experiments o f this series is shown in Fig. 3 B. It is apparent that the reduction in P0 was reversible in all but one experiment. A dose/response curve for the cytosol has been obtained in 11 experiments. This was achieved by diluting the cytosol with the standard Ringer type o f solution (cf. above). The data are summarized in Fig. 3 A. This figure indicates that the inhibitory effect o f the cytosol was dose-dependent. A significant inhibitory effect was already seen for the 1 : 300 dilution. The highest concentration tested was the 1 : 3 dilution, which gave an approximately 60% inhibition. If

482

Nasal CQ]I, Vc=5OmV Excision

25

p*,[__ 230

m~

400

ms

800

ms

~

HT29 Ceil, Vc=2OmV Excision a

pA

T84 Ceil. Vc=4OmV Exclsion

7

p^

Placenta Trophoblast, Vc=8OmV Excision

26

pA

,,

800

one extrapolates this dose/response curve to undiluted cytosol, the approximate inhibition would be 80%. In two pilot experiments we have also tested whether cytosol had any effect on the extracellular side of excised outside-out-oriented C1- channels, and we found that it was entirely ineffective. Hence, the effect o f cytosol is confined to the cytosolic side o f the channel.

Attempts to characterize the cytosolic inhibitor All experiments o f the following series were done with the cytosol obtained from PTC. Excised inside-out patches of HTa9 cells containing C1- channels were used as the bioassay system. First, we examined whether the Ca a+ activity on the cytosolic side had any influence on the inhibitory potency of the cytosol. Therefore, 5 mmol/1 EGTA was added to the cytosol, reducing the Ca z+ activity to below 10 nmol/1 [11,21]. This had no effect on the inhibitory potency of the cytosol. Cytosol with a low Ca 2§ activity reduced P0 from 0.68+_0.19 to 0.35+0.06 (n = 3).

Next we examined whether CI was heat-stable. Cytosol was heated to 100~ for 10min. Then the

~s

Fig. 1. Excision activation of C1- channels of various cells. For each recording the clamp voltage (Vc) during the excision, marked by an arrow, is indicated. Note that excision activation occurred instantaneously. The apparent current fluctuations in the second recording (HT29) in the control period (cell-attached) represents digitized noise. In the lowest trace Vc was stepped back from +80mV to +20mV immediately after the channel was activated by excision

yolume, pH and C1- concentration were adjusted and the heat-treated cytosol was examined. It reduced P0 from 0.68_+0.07 to 0.35+0.06 (n -- 9). This is shown in Fig. 4 A and a summary of this series is given in Fig. 4B. Note that even the magnitude of inhibition was unchanged by this procedure. The factor responsible for cytosolic inhibition therefore appears to be heat-stable. Another series tested whether the inhibitory potency of cytosol could be abolished by exposure to enzymes. The enzymes were coupled to sephadex beads. This property of the enzymes enabled us to remove them by centrifugation after the cytosol had been exposed to the respective enzyme (cf. Materials and methods). Incubation of the cytosol with (a) lipase (n = 5), (b) a-amylase (n = 5), (c) trypsin (n = 5), (d) ribonuclease (n = 3) and (e) protease (n -- 3) did not destroy the inhibitory effect of the cytosol. Just as with the native cytosol, the channel-open probability was reduced from: (a) 0.61 +0.03 to 0.29_+0.05; (b) 0.55+0.16 to 0.28_+0.1; (c) 0.65_+0.02 to 0.21+_0.03; (d) 0.65+_0.02 to 0.25+_0.01; and (e) 0.77+_0.07 to 0.16+_0.04. These data suggest that the factor responsible for cytosolic inhibition withstands rather rigorous treatment with several hydrolytic enzymes.

483 Cyt.osol

KCI/NoCI

(4)-~ (8) " ~

1.0 0.8

(9) ~

0 0.6 C~

Po = O.86

A

O Q_ 0.4

(9)

0.2 C~

Po = O.24

0.0

~/~36oo

1:36o 1:s6

l:k

Dilution

C2

Po = O.g2 pk

[__ 400

m~

Fig. 2. Inhibition of a HT29 C]- channel by placenta cytosol. The patch was in the inside-out configuration, the holding voltage was +30 inV. The pipette was filled with KC1 Ringer and the bath contained NaC1-Ringer. The upper trace shows the instantaneous effect of 1 : 2-diluted cytosol added to the cytosolic side (bath). Note that the inhibitory effect is rapidly reversible when the cytosol is removed. C ~ = the zerocurrent level = baseline current. The lower three traces show the effect o f cytosol (third trace) at higher time resolution. Note that cytosol apparently reduces the mean open time of the channel (flicker-type block). Note also that in this, unlike in many other recordings (cf. Fig. 11), cytosol had no effect on the baseline current

In the next group of experiments the cytosol was partitioned into a hydrophilic and a lipophilic fraction (chloroform and other solvents, cf. Materials and methods). The inhibitory effect was recovered from both fractions: the hydrophilic fraction reduced P0 from 0.76+_0.12 to 0.43_+0.19 (n = 10) and the lipophilic fraction had a slightly stronger effect, reducing P0 from 0.63___0.07 to 0.34+-0.08 (n = 9). In these experiments the lipophilic fraction was taken up in a volume equal to that of the hydrophilic fraction. The results with the different organic solvents (cf. Materials and methods) were identical, and were therefore pooled. A typical experiment is shown in Fig. 5 A. This figure indicates that the inhibitory effects of the diluted native cytosol, the hydrophilic, and the lipophilic fractions were all comparable. Hence, no inhibition was lost in this experiment. Fig. 5B summarizes the experiments of this series. As a control series, Ringertype solution underwent the same partitioning and, as expected, neither fraction had any inhibitory effect on CIchannels (n = 6).

Attempts to define the molecular size of the cytosolic inhibitor

0.8 0.6 0.4 ] 0.2 o.o

C

CI

Fig. 3A, B. Effect of placenta cytosol on the open probability of HTa9 C1--channels. A Dose/response curve showing mean + SEM (n). The open probability for the experimental phase, expressed as a fraction of the pre- and post-experimental control (POe/POc) is plotted versus the dilution of the cytosol. The inhibitory effect was significant for dilutions ~ 1.5 k D a (C) and > 0.7 k D a (D). Note that a small inhibitory effect is seen with the filtrate containing molecules above 5 k D a and below 1 kDa. No effect is seen with molecules above 1.5 kDa. These two findings suggest that the inhibitory molecule has a molecular size less m a n , 1.5.kDa. The fact that complete inhibition is seen w k h molecules greater t h a n 0.7 kDa indicates that the size of the inhibitor should be 0 . 7 - 1 . 5 kDa.

-4

Fig. 9A, B. Effect of fatty acids on C1- channels. A Original trace of a typical experiment. Linoleic acid (LA) was examined on the cytosolic side of an inside-out oriented HT29 C1- channel. The pipette contained KCI Ringer and the bath NaC1 Ringer. C ~ = the zero-current level. The clamp voltage was + 2 0 mV. Note that linoleic acid induces a dose-dependent and reversible flicker-type block. B Effects of fatty acids on the relative open probability of HT29 C1- channels (Po experimental/Po control). The concentration on the cytosolic side is given on the x-axis and Poe/Po c is shown as the m e a n _+ SEM, n. A A Arachidonic acid; L N A , linolenic acid; LA, linoleic acid; OA, oleic acid; PA, palmitic acid. The inhibitory effects o f A A , LNA, a n d L A are statistically significant

Effects of fatty acids on Cl- channels In another experimental series several fatty acids were examined on the cytosolic side of excised C1- channels. Care was taken that the fatty acids were always freshly prepared. An example is shown in Fig. 9A. When linoleic acid was added to the Ringer-type solution at 5 0 - 500 gmol/1 an inhibitory effect was noted. Unlike results with the cytosol, this effect developed with a delay of 2 - 3 min. The block was of slow onset, of flicker type, and was only slowly reversible. Figure 9 B summarizes the effects of four non-saturated fatty acids: arachidonic acid (n = 4), linolenic acid (n = 5), linoleic acid (n = 6), and oleic acid (n = 3). It is evident that only the first three fatty acids had a significant inhibitory effect at the concentration of 10~mol/1 used. The saturated fatty acid, palmitic acid (n = 3), was without any significant effect even at 0.1 mmol/1. P0 was reduced to 37-+18% (of the control value) by arachidonic acid, to 75-+16070 by linoleic acid and to 71 _+15% by linoleic acid, but was unchanged (91_+6%) by oleic acid and (96_+13%) by palmitic acid. The effects of arachidonic acid, linolenic acid, and linoleic acid were examined more closely, and dose/response curves were obtained. The data are summarized in Fig. 1 0 A - C . The half-maximal inhibitory concentrations were around 5 ~tmol/1 for arachidonic acid, 0.4mmol/1 for linolenic acid, and larger than 1 mmol/1 for linoleic acid. Note that the dose/response

486

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B

C

(2)

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0.4 0,2 0.0 0

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curves for all fatty acids were rather flat, especially in the case of linoleic and linolenic acid.

Experiments with known cellular regulators Another experimental series examined whether cytosolic compounds, known to influence ionic channels, had any effect on excised C1- channels. Inositol triphosphate (10 ~tmol/1, n = 6), dioleoylglycerol (0.1 mmol/1, n = 4), ATP (1 mmol/1, n = 5), GTP (1 mmol/1, n = 4), GTP [7-S] (lmmol/1, n = 2 ) , cAMP (0.1mmol/1, n = 2 ) , cGMP (0.1 mmol/1, n = 1) and butyrate (1 mmol/1, n = 2) all had no detectable inhibitory effect on the C1channels in this study. Phosphatitic acid (0.1 mmol/1) reduced Po from 0.77+0.03 to 0.42+0.09 (n = 6).

Cytosol from other cells To test for the specificity of the cytosolic inhibition, reported here for REC, HT29 and placenta cytosol, we also examined whether cytosol from non-epithelial ceils (e.g. skeletal muscle, red blood cells, and fibroblasts) might have a comparable effect on C1- channels. The cytosols were prepared as described above for PTC. Cytosol from human skeletal muscle reduced Po only slightly and not significantly from 0.68+_0.19 to 0.55+_0.19 (n = 10). The cytosol from human fibroblasts (n = 2) and human red blood cells (n = 5) had no detectable effect. These data indicate that the cytosol inhibition of C1- channels is exhibited by some tissues but is not a general property of all cytosols.

Eytosol

-6 ocid

-5 -4 (Io9 r n o l / I )

-3

-2

Fig. 1 0 A - C . Effect of fatty acids on C1- channels. Dose/response curves. The relative open probability (Po experimental/Po control; Poe~Poe) as the mean _+ SEM (n) o f inside-out-oriented HT29 C I channels is plotted as a function of the fatty acid concentration on the cytosolic side. Note that the dose-response curves are rather flat for all three (A, B, C) fatty acids. Halfmaximal inhibition was obtained at about 5 ~mol/1 for arachidonic acid (A); 0.4 mmol/1 for linolenic acid (B); and > 1 mmol/1 for linoleic acid (C)

Other effects of the placenta cytosol In about 90% of all experiments with placenta cytosol we observed a small or sometimes marked shift in the baseline current when the cytosol was applied to the cytosolic side of excised inside-out oriented CI- channels. An example is shown in Fig. 11. As apparent from this condensed recording, there is a clear-cut reduction in the open-channel probability of the intermediate-conductance chloride channel, but, beyond this, there is also a shift of the baseline current by about 2 pA. This shift cannot be explained by liquid-junction potentials because the cytosol had an ionic composition very similar to that of the control NaCI solution. Indeed, current/voltage relationships for the baseline current, as well as for the single-channel current, were not altered with respect to the zero-current potential by cytosol (n = 7). It was, however, noted that the slope conductance of the baseline current was reduced markedly. This may indicate that cytosol, by some unknown mechanism, improves the interaction of the membrane and the glass surface of the patch pipette, or, more likely, that it has some additional inhibitory effect on the membrane conductance. This question was examined directly in yet another experimental series. CI was isolated from the lipophilic fraction and dissolved in NaC1 Ringer or choline chloride Ringer. It was found (n = 3) that CI inhibited the excision-activated intermediate-conductance outwardly rectifying C1- channel and induced the reduction of baseline current in NaC1 as well as in the choline chloride solution. These data suggest that the baseline current, sensitive towards CI, is in fact a C1- current.

KCI/NoCI

Discussion 10 s e c Fig. 11. Effect o f placenta cytosol on a HT29 C1- channel and on the baseline current. Typical experiment at low time resolution. Placenta cytosol was examined on the cytosolie side of an inside-out oriented HT29 C1- channel. The pipette and bath contained NaC1 Ringer. The clamp voltage was +30mV. Note that cytosol induces an immediate flicker-type block of the C1- channel and, simultaneously, a 2-pA downward shift of the baseline current. This downward shift in the baseline current corresponds to a threefold increase in the apparent seal resistance of this patch. Both effects are rapidly and completely reversible

Excision activation The present study shows that the outwardly rectifying C1- channel with intermediate conductance is activated upon excision in a variety of chloride transporting cells. This activation occurs immediately and is not voltage dependent. Delayed activation of this kind of channel has meanwhile been reported in a large number of studies on respiratory epithelial cells [16, 19, 2 3 - 2 5 , 28, 32], sweatgland secretory cells [7], salivary gland cells [18], cultured

487 PAC cells [20], epididymis cells [25], keratinocytes [8], lymphocytes [2,13], fibroblasts [1] etc. In all these studies the channel activation was delayed and highly variable in onset, probably because excision was carried out at room temperature. In contrast, when excision is carried out at 37 ~ channel activation is instantaneous. This immediate activation has been shown in our previous study [22], it has meanwhile been confirmed by others [33], and is again documented in the present report. Consequently it follows that in the intact cell this type of chloride channel appears to be silent or down-regulated, and this phenomenon has been called "tonic inhibition" in the specific case of cystic fibrosis cells [22, 30]. Even though in severel studies this type of excision activation of the intermediate-conductance outwardly rectifying chloride channel in cystic fibrosis has been claimed to be pathophysiologically relevant [16, 19, 23, 24, 28], present evidence with whole-cell current measurements [3] (own unpublished observations and Fr6mter et al. personal communication) would favour the view that this channel itself is probably not causally related to cystic fibrosis. At the end of the discussion we shall return to this point. At this stage several arguments favour the conclusion that this channel is silent (cystic fibrosis) or reduced in its open probability in cell-attached patches (other normal cells), not because it is not activated by phosphorylation [16, 19, 23, 24, 28], but rather because it is down-regulated by a cytosolic inhibitory factor: (a) excision activation occurs instantaneously; (b) this activation does not require agonists nor even a specific voltage polarity; (c) the activation can be reversed by the addition of cytosol, and this inhibition is instantaneous and easily reversible. The first point has been already discussed above. The second and third points will require some additional comments. The present data show that excision activation occurs when the cytosolic side of the channel is exposed to rather simple artificial solutions devoid of ATP, kinases and second messengers. Also, neither the Ca 2+ activity nor the pH (over a wide range) has any influence on the excision activation or the properties of these channels. In fact, we have been unable to see any effect of protein kinase A or C in our experiments, since the excised patches already contain maximally activated chloride channels. The addition of ATP or alkaline phosphatase to the cytosolic side was without effect in our studies (unpublished results). The present data further show that tonic inhibition of this channel can be restored simply by the addition of cytosol to the cytosolic side of the channel. It did not matter whether the cytosol was prepared from respiratory epithelial cells (REC), HTz9 colon carcinoma ceils or placenta trophoblast cells (PTC). Furthermore, the different cytosols were effective in chloride channels of HT29 cells, REC and in one pilot experiment also in PTC. These data indicate that the cytosolic factor, responsible for inhibition, is probably very similar (if not identical) in the various cells. The degree to which this type of channel would be active in cell-attached patches would then be determined by the sensitivity of the CI- channel towards the inhibitor or, more likely, by the concentration of this cytosolic inhibitor.

The mechanism of action of cytosolic inhibitor We are aware that the cytosol used in many of our experiments is a crude extract, and, hence, the observed inhibition of the outwardly rectifying intermediate-conductance C1- channel by cytosol could be caused by any difference between this poorly defined extract and the normal Ringer-type solution. First we have examined several points in which the two fluids obviously differ. Our experiments indicate that cytosolic inhibition is not caused by possible changes in ionic composition (pH, Ca 2§ , CI-, K+). Nor is it reproduced by cytosol derived from any other cell; e.g. non-epithelial cells. This conclusion is supported by our observation that no significant inhibition was obtained with the cytosols prepared from skeletal muscle, fibroblasts and red blood cells. A comparable observation to ours is reported in an accompanying report [20] (also cf. below). In this study, a cytosolic factor was extracted from bovine renal cortex and is shown to inhibit rheogenic C1- flux in renal brush-border membranes and outwardly rectifying REC CI- channels. It is also pertinent to this discussion that inhibition by cytosol was only observed from the cytosolic side of the C1channel. The dose-response curves would indicate that some relatively potent component of the cytosol is responsible for this inhibitory effect and that dilution of this component reduces the inhibitory effect correspondingly. All our attempts to purify the cytosol: removal of many proteins by boiling, dissolving the cytosol into organic solutes, separation of the cytosol by filtration, dialysis and gel filtration, did not remove the inhibitory effect or influence the type of block. This would make it unlikely that we are looking at a large variety of different inhibitory molecules. It is obvious, however, that we cannot be certain that we deal with one single inhibitory molecule. Nevertheless, we label these factors tentatively as cytosolic inhibitor (CI). The present data suggest that CI acts directly on the channel and does not require biochemical modification of the channel such as phosphorylation or dephosphorylation. This conclusion is based on the findings that the effect of cytosol is instantaneous and easily reversible. It should also be noted that CI (in many experiments, cf. Figs. 2, 6, 7, 11) induces channel flickering, suggesting that CI interferes with the channel itself or its immediate surroundings. Even though the present data clearly indicate that the intermediate conductance outwardly rectifying C1channel is inhibited by cytosol, it is important to note that in the majority of our experiments the inhibition of this channel was accompanied by a marked reduction in the baseline current, indicating an increase in "seal resistance". The fact that the seal resistance represents two parallel conductance pathways (one between membrane and glass, and the other through the membrane) explains why this phenomenon was variable. In other words, if this phenomenon was due to the inhibition of other ionic channels in the membrane with single channel conductances well below the resolution of the patch clamp method, it would only be noticed as a reduction in the baseline current if the seal between glass and membrane was suffi-

488 ciently tight. It is worth mentioning in this context that this shift in baseline current was also observed with purified fractions of cytosol (cf. below). Hence, this phenomenon seems to be related to CI and not simply due to other cytosolic components present in crude extracts. Our experiments with CI dissolved in choline chloride solution suggest that CI also inhibits another C1- channel with a conductance not resolved by our methods (subpicosiemens conductance).

The chemical nature of cytosolic inhibitor Cytosol has been processed further in order to determine the nature o f CI and its molecular size. The present data suggest that CI has an apparent molecular mass somewhere around 7 0 0 - 1 5 0 0 Da. We are aware that our data are not conclusive since, especially with small molecular sizes, the steric characteristics o f the molecule will also strongly depend on its polarity and on its charges. The present factor is heat-stable and amphiphilic. The data from the enzymatic hydrolysis assay suggest that this factor is not a simple oligopeptide or a nucleic acid. In such a case we would have expected a destruction by non-specific proteases or nucleotidases. The fact that the factor also withstood the exposure to other enzymes may indicate that its chemical bonds require other, and maybe more specific, hydrolases. It is very likely that the CI described here is similar or identical to that described in a parallel study by Krick et al. [20]. The similarity between some properties of CI and fatty acids, membrane lipids, and phospholipids has p r o m p t e d the examination of some of these compounds in excised C1- channels. The phorbol ester dioleoylglycerol and inositol trisphosphate, at rather high concentrations, were without any effect. Fatty acids, on the other hand, showed an inhibitory effect. Comparable data have meanwhile been published by two other laboratories [12, 34]. The inhibitory effect o f fatty acids appears to be restricted to some nonsaturated forms. It is not found with the saturated aliphatic acids, butyric acid and palmitic acid. Even with the effective compounds, rather high concentrations were required for inhibition. Also it was observed that the mechanism of fatty acid inhibition looked quite different from the inhibition by CI. Fatty acids needed some time to act (usually 2 - 3 min) and it also took several minutes to reverse the inhibition. This may be caused by an interaction of the fatty acids with the membrane lipid and is in contrast to CI where the inhibition occurred as rapidly as we changed solutions. Also, it is worth noting that the dose-response curves for fatty acids were rather flat (Fig. 10), whilst that of CI was steeper (Fig. 3). At this stage it is not possible to make a decisive statement about whether fatty acids may in fact be the CI. On the grounds of circumstantial evidence we conclude that this is unlikely; (a) fatty acids do not fit into the molecular mass range estimated for CI; (b) their mode of inhibition and the dose-response curves are different; (c) the required concentrations are rather high; (d) a partition coefficient of about 1, as determined for CI, does not agree with the coefficient of the fatty acids used in this study; (e) nonsaturated fatty acids are much more heat-labile than CI.

Relevance of cytosolic inhibitor for the pathophysiology of cystic fibrosis Current concepts of the pathophysiology of cystic fibrosis (CF) in respiratory epithelial cells favour the view that the cAMP-dependent C1- conductance is absent in the affected tissues. Along these lines two major findings have been reported by several laboratories. (a) Unlike the situation in normal respiratory cells, C1- channels are absent in cell-attached patches of CF cells even after exposure to agonists that increase cytosolic cAMP [4, 6, 22]. C1- channels are active, however, in excised patches [16, 19, 2 2 - 2 4 , 28]. While several laboratories have reported that protein kinase A and C activation was defective in these excised patches of CF cells but not in normal cells [16, 19, 23, 28], we have argued that excision in itself, without any addition of protein kinases, activated these channels in both normal and CF cells [22]. (b) More recent whole-cell patch-clamp studies indicate that the C1-- conductance, defective in CF, is not outwardly rectifying, but shows linear I / V curves and has an inhibitor sensitivity and ion selectivity different from that of the outwardly rectifying C1- channel [3] (cf. also accompanying report by Krick et al.). Provided that these wholecell data are representative and provided that the excision in itself does not alter the qualitative properties of the C1- channel, these latter data would suggest that the outwardly rectifying C1- channel is, if anything, only of minor importance in the pathophysiology of CF. On the other hand, such a conclusion would be in conflict with the above mentioned phosphorylation studies on excised patches [16, 19, 23, 28], which would also be in conflict with the observation that these outwardly rectifying C1channels are absent in cell-attached patches of CF cells treated with, e.g., catecholamines, but are present in normal cells [ 4 - 6, 3 0 - 32]. Provided that the outwardly rectifying C1- channel is relevant in the pathophysiology of CF, it appears pertinent to discuss the present findings on CI in the light of what has been called tonic inhibition of C1- channels in CF cells [22, 30]. Tonic inhibition has been claimed to be due to the fact that the phosphorylation mechanism distal to cAMP was defective in CF cells. The present data would suggest an alternative explanation, namely that the sensitivity of the C1- channel towards CI was increased in CF, or that the concentration of CI was increased in CF cells. Our preliminary data with the cytosol of HT29 cells examined in normal and CF C1- channels suggest a comparable inhibition. This, if confirmed in ongoing experiments, would render it unlikely that the C1- channel o f CF cells has an increased sensitivity towards CI. Additional experiments comparing the dose-response curves of cytosol from CF and normal cells in, e.g., HT29 C1channels, will be required to answer the question whether the difference between normal and CF cells lies in the concentration of CI present in the cytosol. It should be clear that the two concepts of tonic inhibition, as they are discussed above, are not mutually exclusive. In fact, a CF cell might have both a defective activation as well as an increased inhibition. Also the possibility is not excluded that the activation mechanism operates via the inhibitor,

489

i.e. phosphorylation modification could act on CI and reduce its inhibitory potency. Regarding the discussion of which C1- channel may be inhibited or prevented from activation in CF, it should also be noted that the present study indicates that CI has not only an inhibitory effect on the outwardly rectifying C1- channel but also on another C1- channel with a very small conductance, which has been noticed in this study as a baseline C1- current. Finally, the recent description of the CF gene and its gene product cystic fibrosis trans membrane conductance regulator (CFTR) [27] demands some comments on how CFTR defects might be correlated with the present findings. CFTR may not be the C1- channel itself [17, 26] but rather a membrane transport protein controlling some other factor in the cytosol, which then acts directly or indirectly on the C1- channel. Provided that CF was related to some cytosolic inhibitor (cf. above), a defect in CFTR might increase the cytosolic concentration of a cytosolic inhibitor and this increase in the concentration of inhibitor might then be responsible for the down-regulation of the C1- conductance. Further work is required and is proceeding to test this hypothesis. In conclusion, the present study indicates that excision activation of intermediate-conductance outwardly rectifying chloride channels is demonstrable in several C1--transporting epithelial cells. The reduction of the open probability of these C1- channels in cell-attached patches is probably caused by some cytosolic inhibitor. This inhibitor is amphiphilic, heat-stable, and has a molecular mass of 700-1500 Da. Acknowledgements. This study has been supported by DFG Gr 480/10 and BMFT 01 GA 8816. We are very grateful to Mr. H.Chr. Estler (Biochemisches Institut der Albert-Ludwigs-Universit/it, Freibnrg) for very helpful suggestions during the course of this study, to Prof. C.A.D. Boyd (University, Oxford), who stimulated us to test placenta cytosol, and to Dr. I. Novak and Prof. Z.I. Cabantchik for the critical evaluation of this manuscript. The technical assistance of Mrs. J. Siebert, Mrs. A. Hauser, Mrs. R.B. Nitschke and Mrs. I. Burhoff is gratefully acknowledged.

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