Selective inhibition of Cl
conductance in toad
skin by blockers of Cl
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 |
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
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INTRODUCTION |
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.
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METHODS |
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.
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RESULTS |
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.
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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; ), ERY (n = 9;
), or FURO (n = 11;
).
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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.
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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.
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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 ( ), and FFA
( ).
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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;
, control 2; , FURO; ,
DCDPC. Control 2 was done 20 min after washout of FURO (not shown).
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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.
 |
DISCUSSION |
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.
 |
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