Selective inhibition of Clminus conductance in toad skin by blockers of Clminus channels and transporters

Wolfram Nagel1, Petra Somieski1, and Uri Katz2

1 Department of Physiology, University of Munich, 80336 Munich, Germany; and 2 Department of Biology, Technion, Haifa, Israel


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We compared the effects exerted by two classes of Cl- transport inhibitors on a Cl--selective, passive anion transport route across the skin of Bufo viridis, the conductance (GCl) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated GCl with IC50 of 6 ± 1, 54 ± 12, and 607 ± 125 µM, respectively; they had no effect on the cAMP-induced GCl. The voltage for half-maximal activation of GCl (V50) increased compared with controls, but effects on the maximal GCl at more positive clamp potentials were small. Cl- channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated GCl with IC50 of 8.3 ± 1.2, 10.5 ± 0.6, 16.5 ± 3.4, and 36.5 ± 11.4 µM, respectively, and also inhibited the cAMP-induced GCl, albeit with slightly larger IC50. V50 was not significantly changed compared with controls; the maximal GCl was strongly reduced. We conclude that the pathway for Cl- is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.

diphenylamine-2-carboxylic acid; fenamates; eosin; furosemide; voltage-activated chloride conductance


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOVEMENT of Cl- across the isolated toad skin is mostly passive. The conductance (GCl) of the associated transport pathway can be activated by imposing serosa-positive clamp potentials (termed "voltage-activated GCl") and may increase from baseline conductance below 0.5 mS/cm2 to values in excess of 5-6 mS/cm2 (for references, see Refs. 11 and 16). Comparably high stimulation of the Cl- pathway can be achieved under short-circuit conditions by the application of membrane-permeable, nonmetabolized analogs of cAMP (termed "cAMP-induced GCl") (12, 35). Evidence has been presented and discussed previously (11, 16, 22, 35) showing that both voltage-activated and cAMP-induced GCl share a common route, but the morphological site(s), molecular pathways, and principles of regulation responsible for the transepithelial Cl- translocation have not been resolved. One of the reasons for this is the lack of appropriate means to isolate and investigate the structures involved. Unequivocal evidence indicates, however, that the major cellular compartment of the epithelium, the principal cell, which is preferentially involved in active Na+ uptake, does not provide a route for transepithelial Cl- movement (for references, see Ref. 22). The epithelial glands can also be excluded from a role in voltage-activated or cAMP-induced GCl, because split epithelia devoid of glands show a magnitude and response pattern identical to those of intact tissues (12, 24). This implies that the route involves the mitochondria-rich (MR) cells, a minor fraction only of the epithelial cells, or the paracellular path. Techniques for the study of these structures, particularly in a native tissue such as the amphibian skin, are sparse. Information on putative ion channels for Cl- might be obtained from the examination of effects exerted by stimulators and/or inhibitors of the activated GCl. In the present study, we analyze the response of GCl to application of different Cl- channel blockers from the group of arylaminobenzoates and particular inhibitors of cotransporters or antiporters involved in transmembranal Cl- movement. As inhibitors of Cl--dependent cotransport, the loop diuretics furosemide and bumetanide (31) were selected, along with two derivatives of fluorescein, eosin and erythrosine, which inhibit the band 3 anion exchanger in red blood cells (13). The inclusion of the latter compounds was prompted by our observation that eosin-5-maleimide reversibly blocks the voltage-activated GCl in toad skin (23). In the group of arylaminobenzoates, comprising derivatives of diphenylamino-2-carboxylic acid (DPC), which includes the nonsteroidal anti-inflammatory fenamates, several potent blockers of Cl- channels in many tissues have been identified (33). Both groups inhibit the voltage-activated GCl in toad skin, but they differ in their influence on the voltage dependence of GCl and the effect on cAMP-induced GCl. This could give insight into the mechanism of Cl- movement across the limiting border of the pathway and contribute to the understanding of the mode of inhibition by Cl- channel blockers.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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The experiments were carried out on isolated abdominal skin from Bufo viridis using recently described techniques (23). In brief, skin pieces were mounted in an Ussing-type chamber with a 0.5-cm2 aperture and were continuously perfused with Ringer solution at 2-5 ml/min on both sides. For the analysis of serosal effects and some experiments in the presence of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP), split epithelia devoid of the connective tissue layer and glands were prepared by the collagenase method as described recently (24). The collagenase solution was supplemented with 0.1 mg/ml trypsin inhibitor to avoid interference from traces of trypsin (25). Ringer solution contained (in mM) 110 Na+, 2.5 K+, 1 Ca2+, 1 Mg2+, 114 Cl-, and 3.5 HEPES; pH was 7.5. The apical perfusion solution contained generally 20 µM amiloride to block transepithelial Na+ transport. Transepithelial voltage (Vt) and clamping current (It) were measured or delivered via Ag-AgCl electrodes and KCl-agar bridges, respectively. The tissues were kept short-circuited or voltage-clamped to -30 or +80 mV (with reference to the apical side) with the use of an automatic clamping instrument. An integrated sample-and-hold circuit served for the continuous on-line determination of the tissue conductance (Gt) from voltage perturbation (10 mV, 250-ms duration every 2.5 s). It was displayed on a multichannel line recorder (BD110; Kipp and Zonen, Delft, The Netherlands) and served to guide the timing of the experiments, which was necessary because the time course of conductance activation and action of inhibitors was rather variable. The approach of steady-state values was generally allowed to within 10% by inspection of the line records before continuation of the experiment. This protocol leads to slightly higher values for IC50 and the voltage for half-maximal activation of GCl (V50), estimated from concentration-response and conductance-voltage (G/V) relationships, respectively, but the errors are within the standard deviation of these estimates. In addition, data were stored to a computer file with a 14-bit analog-to-digital converter. From these files, line graphs were generated using graphics software (Origin, Microcal). Concentration-response relationships were derived from the data using a sigmoidal fitting procedure. Voltage-activated GCl is considered to be the difference between the respective steady-state values of Gt at the hold potential minus the baseline Gt at inactivating clamp potentials (-30 mV). G/V relationships were determined by imposing sequentially increasing hold potentials and awaiting the approach of steady states at each value of Vt, which took 3-5 min for each level of clamp. The derived values of Gt at the respective clamp potentials were plotted and fitted to a sigmoidal regression function (Origin, Microcal). Determinations under the influence of inhibitors were always preceded by measurements under control conditions for individual comparison, and significance of differences was calculated by paired two-values t-test.

Laboratory chemicals were purchased from Sigma. The structures of the inhibitors applied are shown in Fig. 1: eosin, erythrosine, CPT-cAMP, DPC, dichloro-DPC (DCDPC; kindly made available by R. Greger), flufenamic acid (FFA), niflumic acid (NA), furosemide, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Except for the first four, which are water soluble, substances were first dissolved in DMSO at concentrations of 2-5 mg/100 µl DMSO and added at the final concentration to Ringer solution. The concentration of DMSO in the Ringer solution did not exceed 0.2%. At this concentration, DMSO does not affect baseline conductance or voltage-activated or cAMP-induced GCl. Furosemide was dissolved in an equimolar amount of 0.1 M NaOH.


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Fig. 1.   Chemical structure of Cl- transport inhibitors examined in the present study DPC, diphenylamino-2-carboxylic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Inhibitors of cotransporters or antiporters. Figure 2 shows the typical response of the voltage-activated GCl to apical addition of furosemide at concentrations of 100-3,000 µM. GCl decreased promptly and dose dependently on application of furosemide. Inhibited levels of GCl were approached within 4 min at all concentrations of furosemide. GCl was completely inhibited at 3 mM furosemide. As in all other experiments, the reversibility of inhibition was very prompt, even at the highest concentration of furosemide, which permitted the subsequent test of the inhibitory action of erythrosine on the same tissue. Figure 2, middle, shows that the voltage-activated GCl had decreased already at the concentration of 3 µM erythrosine; complete blockage was achieved at ~30 µM. Return of the voltage-activated GCl after washout of erythrosine was not awaited because pilot experiments had indicated that reversibility is very slow and imperfect at concentrations >10 µM. Nevertheless, the elevation of GCl by CPT-cAMP, which was studied at short circuit due to its voltage insensitivity (12), was not prevented by the persisting attenuation of the voltage-activated GCl after erythrosine (Fig. 2, right). The magnitude of gain in conductance and the time course of increase induced by CPT-cAMP were not different from those typically observed in untreated toad skin. The cAMP-induced GCl was only slightly reduced by furosemide at concentrations of 1 and 3 mM and was practically unaffected by erythrosine at 300 µM. The same low inhibitory effects of furosemide and erythrosine were also observed in the majority of experiments in which tissues were studied under the influence of CPT-cAMP without preceding treatment with other inhibitors (n = 5). Similar experiments with eosin differed from erythrosine only in the sensitivity, i.e., concentrations one order of magnitude higher were needed to inhibit voltage-activated GCl, and the cAMP-induced GCl was completely insensitive to eosin.


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Fig. 2.   Influence of furosemide (FURO) or erythrosine (ERY) in the apical perfusion solution on the voltage-activated Cl- conductance (GCl) of toad skin. Reversibility was quick and complete after washout of FURO but poor after ERY. FURO (1 and 3 mM) and ERY (300 µM) were again applied from the apical side after stimulation with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) from the serosal side. Gt, tissue conductance.

The results of all experiments using inhibitors of cotransporters or antiporters are summarized in Fig. 3. The most powerful inhibitor is erythrosine with an IC50 of 5.8 ± 1.3 µM, followed by eosin, for which the IC50 is about 10 times larger than for erythrosine (54 ± 12 µM). Much less potent is furosemide, an with IC50 of 607 ± 125 µM. Nevertheless, all agents completely blocked the voltage-activated GCl at higher concentrations. In contrast, the cAMP-induced GCl was only minimally changed (<10% inhibition in 7 experiments using erythrosine up to 300 µM and 8 experiments with furosemide up to 3 mM). Additional tests revealed that both fluorescein (n = 5), the Cl--substituted congener of eosin and erythrosine, and the furosemide-related compound bumetanide (n = 3) affected neither the voltage-activated nor the cAMP-induced GCl at concentrations up to 1 mM (data not shown).


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Fig. 3.   Concentration-response relationships for the inhibition of voltage-activated GCl by eosin (EOS; n = 7; open circle ), ERY (n = 9; black-triangle), or FURO (n = 11; ).

In contrast to the inhibitory action at the mucosal side, the inhibitors were far less effective at the serosal side of isolated epithelia. The more hydrophilic agents eosin and erythrosine were virtually ineffective, whereas the slightly lipophilic compound furosemide retained the inhibitory effect on the voltage-activated GCl, albeit with an increase of the IC50 to >2,000 µM (n = 5). The time course of the onset of inhibition was similar to that after mucosal application (data not shown).

Channel blockers. The response of GCl to apical addition of FFA at different concentrations is shown in Fig. 4. The inhibition occurred within 3-4 min and was reversible after washout on a similar time scale. At the higher concentration, which eliminated GCl almost completely, reversibility was not complete during continuous voltage perturbation but returned after intermittent inactivation (at Vt = -30 mV) during subsequent voltage activation. FFA retained its inhibitory activity on GCl after application of CPT-cAMP, as shown in Fig. 4, right, but it appears that higher concentrations were required than for the inhibition of the voltage-activated GCl. In other experiments, NA at similar concentrations as FFA elicited responses of the voltage-activated and the cAMP-induced GCl comparable in magnitude to those with FFA. Slightly more inhibition was exerted by DCDPC, which was effective already at 1 µM and blocked the voltage-activated GCl completely at ~30 µM; slightly higher concentrations were required for the inhibition of the cAMP-induced GCl. Reversibility of the inhibition by higher concentrations of DCDPC (>30 µM) was poor over the time scale of the present experiments; at lower concentrations, partial return of GCl was observed. Similar experiments showed that NPPB, a powerful inhibitor of Cl- channels in several tissues (33), has notably lower affinity than the other derivatives of DPC. Nevertheless, the voltage-activated and the cAMP-induced GCl were also completely inhibited at concentrations of >300 µM NPPB. Reversibility was almost complete at all concentrations probed (up to 300 µM).


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Fig. 4.   Response of GCl of toad skin on application of flufenamic acid (FFA) from the apical side. FFA was first applied during voltage activation at 80 mV (serosa positive). After washout of FFA and return of GCl to control levels, the tissue was short-circuited, and CPT-cAMP was added to the serosal side. Thereafter, the response of the cAMP-induced GCl to FFA was tested.

Concentration-response relationships for the derivatives of DPC are summarized in Fig. 5. The sigmoidal fits reveal that half-maximal inhibition of the voltage-activated GCl was achieved at 5-40 µM. The values of IC50 for DCDPC, NA, FFA, and NPPB were 8.3 ± 1.2, 10.5 ± 0.6, 16.5 ± 3.4, and 36.5 ± 11.4 µM, respectively. A much lower degree of inhibition was found with another DPC derivative of the fenamate type, mefenamic acid, which reduced the voltage-activated GCl by ~45% only at 200 µM (n = 4). In view of this low affinity compared with the other fenamates, the concentration-response relationship was not further assessed. Also not specifically addressed was the concentration-response relationship for DPC, which inhibited the voltage-activated as well as the cAMP-induced GCl, albeit at more than threefold higher concentrations than required with DCDPC.


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Fig. 5.   Concentration-response relationship for the effect of 4 derivatives of DPC [FFA, niflumic acid (NA), dichloro-DPC (DCDPC), and NPPB] on the voltage-activated GCl observed in 4-8 individual experiments on toad skin for each of the compounds.

The sidedness of the inhibition of voltage-activated GCl by derivatives of DPC was tested on isolated epithelia. All of these inhibitors, which are notably lipophilic, exerted specific action also from the serosal side, but at slightly higher concentrations than required at the mucosal side. Half-maximal inhibition was observed at 25-40 µM for DCDPC, NPPB, and FFA. The approach of the inhibited state was also slightly delayed after serosal application compared with the response after mucosal addition.

The concentration-response relationships of the inhibition of the CPT-cAMP-induced GCl by DCDPC, NPPB, and FFA are summarized in Fig. 6. The data show that the sensitivity of the cAMP-induced GCl to the derivatives of DPC is lower than that of the voltage-activated GCl. Half-maximal inhibition was obtained at 26 ± 10, 55 ± 26, and 109 ± 11 µM for DCDPC, NPPB, and FFA, respectively. It should be noted that four experiments of this series (2 for DCDPC, 1 each for NPPB and FFA) were done on split epithelia devoid of glands, which confirms that the inhibition occurs at the epithelial cell layer.


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Fig. 6.   Concentration-response relationship for the inhibition of the cAMP-induced GCl in toad skin by DCDPC (), NPPB (triangle ), and FFA ().

Influence of the inhibitors on the G/V relationship. G/V relationships were analyzed to investigate the influence of the different inhibitors on the degree of voltage sensitivity of GCl. Figure 7 depicts the protocol for the determination of the G/V relationships and a typical result. Under control conditions, GCl reached the maximal level at a Vt of ~100 mV. With 600 µM furosemide, which inhibited ~60% of the activated GCl at 80 mV, a clamp potential higher than 140 mV was required for the approach of the maximal value of GCl. It should be noted that the final value of GCl at the higher clamp potential was only slightly lower than the maximal GCl under control conditions. After washout of furosemide, the G/V relationship of the tissue (not shown) returned close to the control level and permitted the subsequent testing of DCDPC. The latter agent did not shift the voltage sensitivity of GCl, notably in contrast to furosemide. Figure 8 depicts the G/V relationships of the experiment. Half-maximal activation occurred at a Vt of ~60 mV before and after washout of furosemide, and the same value was observed in the presence of DCDPC, which merely reduced the maximal level of GCl. Furosemide, in contrast, increased the value of half-maximal activation to >90 mV but had little influence on the final level of GCl.


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Fig. 7.   Original record showing the protocol for the analysis of conductance-voltage (G/V) relationships. After stabilization of voltage-activated GCl by repeated alternation of the transepithelial voltage (Vt) between -30 and +80 mV, the response of GCl on consecutive elevation of Vt by steps of 20 mV was tested (Gt). The same protocol was followed after application of 600 µM FURO, washout of FURO, and addition of 10 µM DCDPC to the mucosal perfusion solution.



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Fig. 8.   G/V relationships derived from the experiment shown in Fig. 7: , control 1; black-triangle, control 2; , FURO; triangle , DCDPC. Control 2 was done 20 min after washout of FURO (not shown).

Similar experiments were performed with erythrosine and FFA. Table 1 shows the mean values of voltage required for V50 and the ratio of the maximal GCl to the value of GCl at 80 mV (the potential for activation according to our protocol). It is evident that the inhibitors differ in their influence on the G/V relationship, because those affecting cotransporters or antiporters (furosemide and erythrosine) shift the G/V relationship to significantly higher clamp potentials, whereas the blockers of Cl- channels (FFA and DCDPC) have no influence on the V50. This shift of the G/V relationship is the reason for the much larger difference between the maximal value of GCl and the conductance at standard clamp potential of our protocol (80 mV). This difference is <30% for control conditions and in the presence of the channel blockers (derivatives of DPC) but is more than doubled with the cotransport or antiport inhibitors furosemide and erythrosine.

                              
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Table 1.   Effect of Cl- transport inhibitors on the voltage dependency of the voltage-activated GCl in toad skin


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The compounds investigated in the present study with regard to the influence on GCl in toad skin can be grouped into two general classes according to their established action on Cl- transport in other tissues: 1) the inhibitors of Cl--related cotransporters or antiporters, furosemide (6) and erythrosine/eosin (13); and 2) the blockers of Cl- channels, DPC derivatives and NPPB (4, 33). Both types of substances inhibit the voltage-activated GCl completely, but the potency differs considerably among agents of either group. Erythrosine and the DPC derivatives (DCDPC, FFA, and NA) are among the most powerful inhibitors of the voltage-activated GCl in toad skin. Comparable inhibitory potency has hitherto only been reported for aminoperimidine (27) and cyanide (26). Less powerful compounds are eosin, furosemide, DPC, and NPPB, which nevertheless are still more effective than another previously reported inhibitor, MK-196 (5).

The response of voltage-activated GCl to erythrosine, eosin, and furosemide shows that these agents have the potential to inhibit a conductive Cl- pathway. This kind of interference is surprising for agents that are supposed to be inhibitors of cotransporters or antiporters for Cl- movement across membranes. Erythrosine and eosin inhibit the anion exchanger (band 3) in red blood cell ghosts (13), and furosemide is well known as a selective blocker of the Na-K-2Cl symporter (6). Carrier-mediated transport affected by erythrosine, eosin, and furosemide is electroneutral, whereas GCl represents the conductive translocation of Cl- across channels or channel-like structures. From the data, we may speculate that similar epitopes, which are determinative for Cl- translocation, exist in the exchanger protein and the Cl- pathway and are occupied by the inhibitors. Half-maximal inhibition of the anion exchanger in red blood cell ghosts is achieved at 56.3 and 3.12 µM for eosin and erythrosine, respectively (13), and the reported values for the IC50 of furosemide range between ~10 and >500 µM in different tissues (for references, see Refs. 6 and 30). These values are comparable to those observed in the present study for toad skin GCl. Particularly intriguing is the concentration-response relationship among the three anthracene compounds, which corroborates the view of similar epitopes for binding of these inhibitors. Whereas the Cl--substituted congener fluorescein has negligible effects on GCl (this study) and is a poor inhibitor of anion transport in red blood cells (29), the replacement of Cl- by Br- or I- leads to inhibitory compounds. Particularly effective is the substitution by I- on the anthracene ring. For the interference with the band 3 anion exchanger, it has been suggested that the larger lipophilicity of the I- moiety in erythrosine compared with that of Br- in eosin could facilitate the access to a binding site within hydrophobic environments (13). It should be noted that a band 3-like protein has been localized immunologically in toad skin MR cells (10). This protein might be involved in anion transport, but it remains to be shown that the transport is electrogenic, i.e., that it reflects voltage-activated GCl at this site. Other specific inhibitors of the band 3 anion exchanger, the stilbene derivatives SITS and DIDS, have very little effect on transepithelial GCl (Katz and Nagel, unpublished results).

For furosemide, inhibition of Ca2+-activated Cl- channels has been reported (2, 8, 9), and the inhibitory effect of this compound on GCl in amphibian skin by furosemide has been documented previously (3, 14, 19, 36). The concentrations of furosemide for effective inhibition are very similar to those of the present study. Interestingly, our data show that the inhibition of GCl by bumetanide is negligible, even at concentrations up to 1 mM. In contrast, bumetanide is a considerably more effective blocker of the Na-K-2Cl symporter than furosemide, with an IC50 <2 µM (6). This indicates that no simple parallelism can be drawn between the responses of the cotransporter and the conductive Cl- pathway.

The inhibition of GCl by the derivatives of DPC is perhaps more readily comparable with the interference reported for different types of Cl- channels, including the cystic fibrosis transmembrane conductance regulator (CFTR) (20, 33, 34, 37, 38). For this inhibition, a direct action on the Cl- channel has been inferred generally. The inhibition of CFTR-type Cl- channels requires comparatively high concentrations (>100 µM) of DPC or its derivatives, but other Cl- channels show higher affinity for analogs of DPC. Slightly higher inhibitory power on Ca2+-activated Cl- channels in Xenopus oocytes has been reported for NA compared with FFA (IC50: 17 vs. 28 µM, respectively) (34). The present study shows a similar relationship for GCl in toad skin. Substitution on the tolyl ring with Cl- leads to agents of even larger inhibitory power. DCDPC is one of most potent inhibitors in the loop of Henle among the DPC derivatives, with an IC50 of 8 µM (33). Similar values have been observed for the inhibition of the spontaneous GCl in frog skin under short-circuit conditions (21). NPPB, which appeared to be the most potent inhibitor of Cl- channels in many tissues, was considerably less potent than DCDPC. These patterns point to the variability in Cl- channel behavior.

The most pertinent difference between the two groups of inhibitors is their influence on GCl under conditions when cAMP in supraphysiological concentrations has been used to shift the Cl- pathway into the voltage-insensitive open state (12). Under these conditions, GCl is only marginally influenced by the inhibitors of cotransporters or antiporters but readily inhibited by the channel blockers, albeit at a notably lower affinity. Similar decrease in the sensitivity of the cAMP-induced GCl has been observed previously with other inhibitors of the Cl- pathway such as aminoperimidine (27), N-ethylmaleimide (23), and CN- (26), which hints at the possibility that high concentrations of cAMP alter the internal structure of the Cl- pathway. Under these conditions, the sensitivity of toad skin GCl to the derivatives of DPC is shifted toward the range observed for CFTR (20, 34), although the cAMP-induced GCl of toad skin is still notably more sensitive. Results from patch-clamp analyses of apical membranes of isolated MR cells (32) and immunolocalization of antibody binding against human CFTR to apical membranes of MR cells (1) have been considered evidence for the involvement of CFTR Cl- channels in Cl- movement across toad skin. It is questionable, however, whether the voltage-activated or the cAMP-induced Cl- pathway in toad skin can be associated with CFTR, since the detected quantity of the putative channel is far less than would be required to account for the stunning magnitude of GCl. Moreover, it was noted that the functional properties of Cl- channels identified by patch-clamp analyses do not show the characteristic patterns of transepithelial Cl- movement (17, 22, 32).

The distinction made above between the two groups of inhibitors is further extended by the G/V relationship analysis. These experiments showed that the channel blockers do not change the activation of GCl by voltage but effectively reduce the Cl- current across the open pathway. Similar response behavior can be derived from published data for another blocker of GCl in amphibian skin, MK-196 (see Fig. 5 in Ref. 5). The inhibitors of the cotransporters or antiporters, on the other hand, reduced the voltage sensitivity considerably and shifted the curve to the right, i.e., to higher voltages. This might be explained on the basis of a model in which the anion conductance pathway in amphibian skin is composed of two components, i.e., the conductance path proper and its voltage-sensitive regulator (23). Whereas the conductive path exhibits poor anion selectivity (7), activation of the pathway, i.e., the transition to the open state, requires necessarily the presence of Cl- on the apical face of the tissue (11, 15, 18). The cotransport or antiport inhibitors interfere with the activation process rather than with the permeation. It may be interesting to note in this context that the inhibition of the band 3 anion exchanger by eosin or erythrosine occurs by interference with a site separate from the transport site for anions (13). The resulting shift of the G/V relationship is the first example of such an inactivation and would be opposite to the effect exerted by theophylline (12), IBMX, and other xanthine derivatives lacking inhibition of the phosphodiesterase (Nagel, unpublished observations). These agents shift the G/V relationship to lower clamp potentials, which is presumably not due to an increase of intracellular cAMP (22). It should be pointed out that the inhibition by the blockers of cotransporters or antiporters does not prevent the opening of the pathway by supramaximal doses of cAMP, which conforms with the observation that the cAMP-induced GCl has lost the dependence on voltage (12), i.e., cannot be influenced by the voltage sensor.

Intraepithelial localization of the anion conductance pathway is not resolved, given that electrical measurements could not localize >20% of the total activated GCl to MR cell sites (28). It might be concluded from the present data that the pathway contains several parallel or consecutive binding sites for the different inhibitors that are differently modified by cAMP. Another possibility would be that some of the compounds act on the signaling chain, which leads to the opening of the pathway by voltage. Both of these mechanisms could explain the slow onset of action compared with the blockage of Na+ uptake by amiloride, which suggests that no simple binding to apically accessible sites leads to the inhibition of GCl. The present data cannot distinguish between these alternatives.


    ACKNOWLEDGEMENTS

We thank Dr. John Davis for careful reading of the manuscript and improvement of the language and Inge Kirmeyer for valuable technical assistance.


    FOOTNOTES

The work was supported by Deutsche Forschungsgemeinschaft Grant Na27/16-5 (to W. Nagel) and by the Fund for Promotion of Sponsored Research at the Technion (to U. Katz).

Address for reprint requests and other correspondence: W. Nagel, Dept. of Physiology, Univ. of Munich, Pettenkoferstr. 12, 80336 Munich, Germany (E-mail: W.Nagel{at}lrz.uni-muenchen.de).

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

Received 14 December 2000; accepted in final form 12 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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