Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, United Kingdom
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ABSTRACT |
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1-Ethyl-2-benzimidazolone (EBIO) caused a sustained increase in
electrogenic Cl secretion
in isolated mouse colon mucosae, an effect reduced by blocking
basolateral K+ channels. The
Ca2+-sensitive
K+ channel blocker charybdotoxin
(ChTX) and the cAMP-sensitive K+
channel blocker 293B were more effective when the other had been added
first, suggesting that both types of
K+ channel were activated. EBIO
did not cause Cl
secretion
in cystic fibrosis (CF) colonic epithelia. In apically permeabilized
colonic mucosae, EBIO increased the
K+ current when a concentration
gradient was imposed, an effect that was completely sensitive to
ChTX. No current sensitive to trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethylchromane (293B) was found in this condition. However, the presence of
basolateral cAMP-sensitive K+
channels was demonstrated by the development of a 293B-sensitive K+ current after cAMP application
in permeabilized mucosae. In isolated colonic crypts EBIO increased
cAMP content but had no effect on intracellular
Ca2+. It is concluded
that EBIO stimulates Cl
secretion by activating
Ca2+-sensitive and cAMP-sensitive
K+ channels, thereby
hyperpolarizing the apical membrane, which increases the electrical
gradient for Cl
efflux
through the CF transmembrane conductance regulator (CFTR). CFTR is also
activated by the accumulation of cAMP as well as by direct activation.
colonic electrogenic chloride secretion; cystic fibrosis transmembrane conductance regulator; charybdotoxin; 293B
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INTRODUCTION |
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IT IS ALMOST AXIOMATIC THAT sustained electrogenic
Cl secretion in gut
epithelia can only result from effects exerted at both the apical and
basolateral poles of the epithelial cells. When movement of
Cl
across the basolateral
membrane into the cell is favorable, no secretion can occur if there
are no apical channels available for
Cl
exit. Similarly, in the
presence of Cl
channels
there will be no secretion unless the
Cl
concentration in the
cell can be increased to above its electrochemical equilibrium. This
study concerns electrogenic
Cl
secretion in the mouse
colon, where an increase in intracellular cAMP causes a maintained
increase in Cl
secretion
for at least 6 h (13). This agent activates both apically located
cystic fibrosis transmembrane conductance regulator (CFTR)
Cl
channels (14) and
transport components on the basolateral side, namely, the
Na+-K+-2Cl
cotransporter (12) and cAMP-activated
K+ channels (7). The
cotransporter, together with the
Na+-K+-ATPase,
is responsible for the accumulation of
Cl
in the cell, whereas the
activation of the K+ channel
hyperpolarizes the apical membrane to favor
Cl
exit. By contrast,
agents that increase intracellular
Ca2+
[Ca2+]i only give a transient
increase in Cl
secretion,
which rapidly wanes to a low-level plateau (3). In this situation
[Ca2+]i activates both the
cotransporter and Ca2+-sensitive
K+ channels in the basolateral
membrane but has no effect on apical CFTR
Cl
channels. Thus in the
cystic fibrosis (CF) mouse colon there is no
Cl
secretion because no
alternative Ca2+-sensitive
Cl
channels occur in the
colon (3). The independent actions of Ca2+- and cAMP-dependent agonists
lead to synergism between the effects of pairs of agents, such as
forskolin and carbachol (10). Some Ca2+-dependent agonists, such as
kinins, also cause liberation of prostaglandins, which in turn activate
adenylate cyclase, producing a mixed agonist response (10). A recent
study (11) on the effects of carbachol on the human colon epithelium
concluded that Cl
secretion
can only occur in the presence of cAMP, because the agonist effects are
strictly basolateral. A further example of unilateral action, this time
on the apical membrane, is found with the compound NS-004, an agent
that activates CFTR Cl
channels. This agent has very little effect on
Cl
secretion because of a
lack of effect on the basolateral faces of the cells (5).
The present study concerns the compound 1-ethyl-2-benzimidazolone
(EBIO), which increases electrogenic
Cl secretion in a variety
of epithelial model systems, such as T84 monolayers (5, 6). It was
shown that the large and sustained increase in short circuit current
(SCC) depended on two distinct actions of EBIO. First, EBIO activated
basolaterally located, charybdotoxin (ChTX)-sensitive,
Ca2+-sensitive
K+ channels (6). Second, EBIO
activated an apically located, cAMP-dependent
Cl
channel, namely, CFTR
(5). As discussed above, coordinated actions at both apical and
basolateral membranes are required to produce maintained
Cl
secretion. Thus EBIO and
forskolin are similar in that both activate an apical
Cl
conductance and a
basolateral K+ conductance. To
reach these conclusions, use has been made of specific blockers of
K+ channels, namely, ChTX for
Ca2+-sensitive
K+ channels (4) and the flavone
293B for cAMP-sensitive K+
channels (9). Devor et al. (6) showed that the EBIO current was
unaffected by 293B but was inhibited by ChTX (50 nM) by 67% in T84
monolayers. When T84 monolayers were activated with forskolin, 293B
(100 µM) caused 56% inhibition, whereas ChTX had no effect. One
experimental paradigm not investigated by Devor et al. (5, 6) was the
effects of 293B in EBIO-stimulated T84 monolayers after treatment with
ChTX. This was the starting point for our own investigation made with
murine colonic epithelia. It was found that after
Cl
secretion had been
stimulated with EBIO the effectiveness of ChTX and 293B to inhibit
secretion depended on the order in which the agents were added. The
data suggest not only that coordinated activity between apical and
basolateral membranes is needed to give maintained electrogenic
Cl
secretion but also that
EBIO can activate two different basolateral K+ channels.
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MATERIALS AND METHODS |
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All experiments were performed on the isolated colonic mucosae of mice.
Mice were killed by CO2 narcosis,
and the colons were immediately removed and placed in cold
Krebs-Henseleit solution (KHS). The composition, in mM, was 117 NaCl,
4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, and 11.1 glucose, pH
7.4. The colons were opened up, and the muscle layers were dissected
away. One or two pieces were taken from each distal colon, each of 20 mm2, and mounted in Ussing
chambers. The tissues were bathed on both sides with KHS, 12 ml, which
was warmed to 37°C and continually circulated with a gas lift by
being bubbled with 95% O2-5%
CO2. The epithelia were short
circuited exactly as described in a recent publication (20). In a few
experiments the colons from CF mice were used, either CF nulls
(Cftr tm1Cam) (16) or
F508 mutants
(Cftr tm2Cam) (1). The
colons of these two types of CF mice have the same phenotype (1, 16),
and no distinction between the two types is made in this study.
To examine the properties of the colon basolateral membranes, we used nystatin to permeabilize the apical membrane and applied a K+ gradient. This protocol has been used previously for both cultured (5, 6) and native epithelia (8, 17). The polyene antibiotic was applied, usually at 180 µg/ml and sometimes in divided doses, so that a steady increase in current, which eventually reached a semi-steady-state value, was obtained. The experimental measurements were made at this time. SCC was measured with an inward K+ gradient (apical solution, in mM: 120 potassium gluconate, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 4 CaCl2, and 10 glucose; the inside solution was the same except that potassium gluconate was replaced with sodium gluconate), and membrane conductance was measured by intermittently applying known voltage pulses across the epithelium and recording the resultant current change.
To examine the effects of EBIO on intracellular Ca2+ and cAMP contents, isolated colonic crypts were prepared by a modification of a method described elsewhere (18). Briefly, descending colons were everted, tied at both ends, and then washed and immersed in a solution of, in mM, 127 NaCl, 5 KCl, 1.25 CaCl2, 1 MgCl2, 5 glucose, 5 sodium pyruvate, and 10 HEPES, pH 7.4, along with 1% BSA. The colons were injected with 1 ml of low-Ca2+ medium and suspended in 5 ml of the same medium, which had the same composition as that of the above medium except that CaCl2 was omitted and 5 mM EDTA was added, and incubated at 37°C for 5 min. The colonic crypts were then dislodged by vigorous shaking, and after the colons had been removed the crypts were pelleted by centrifugation (2,000 rpm for 2 min). The crypts were then resuspended in the Ca2+-containing solution.
To measure the effect of EBIO on [Ca2+]i in colonic crypts, the method described elsewhere (15) was used. Briefly, colonic crypts were loaded with 8 µM fura 2-AM for 60 min at 37°C. Afterwards they were suspended in culture medium (DMEM) and placed on a rocking shaker for 1 h at 37°C. The crypts were recovered by gentle centrifugation and resuspended in medium of, in mM, 137 NaCl, 5.3 KCl, 1.0 CaCl2, 0.4 KH2PO4, 0.3 MgSO4, 10 HEPES (pH 7.4), and 11.0 glucose, along with 0.1% BSA. Fluorescence was measured in a spectrophotometer at 340 and 380 nm with irradiation at 510 nm, and the ratio of fluorescence intensity at 340 nm to that at 380 nm (340/380 ratio) was calculated. No attempt was made to calibrate the results for [Ca2+]i.
For measurement of cAMP an enzyme immunoassay system (Biotrak; Amersham Pharmacia Biotech UK, Buckinghamshire, England) was used. Briefly, aliquots of colonic crypts were exposed to either EBIO or forskolin for 10 min in KHS at 37°C, in the presence or absence of IBMX (100 µM). The crypts were then lysed, and the resulting solution was used for cAMP measurement. Aliquots of the lysate were transferred to a 96-well titer plate precoated with an antibody against a rabbit anti-cAMP antibody. The cAMP in the lysate competed with a coupled cAMP-peroxidase for the anti-cAMP antibody, which bound to the plate. After the addition of substrate (3,3',5,5'-tetramethylbenzidine-H2O2) and the release of color from the peroxidase reaction, the plate was read at 450 nm. Each value was the mean of triplicate determinations. Separate measurements for the colonic crypts from individual mice were made, and the results are given as femtomoles per milligram of protein. Protein estimations were made from aliquots of the original preparations. Thus the measurements represent cAMP generated and that released into the medium during the period of exposure to the drugs.
Drug sources were as follows: EBIO was from Aldrich; forskolin and ionomycin were from Calbiochem; ChTX, amiloride, and furosemide were from Sigma; and 293B was from Dr. R. Greger. Forskolin, ionomycin, and EBIO were made as 1,000× stock solutions in 95% alcohol. 293B was used as a 1,000× stock solution made in DMSO.
Statistical analysis was performed either by a standard
t-test or by nonparametric methods, as
indicated in RESULTS. Values of
P 0.05 were considered significant.
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RESULTS |
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Application of EBIO (600 µM) to both sides of murine colonic
epithelia produces an increase in SCC, which is maintained at a plateau
level of 141.4 ± 7.1 µA/cm2
(n = 31). In a further set
of measurements, 1 mM furosemide reduced the stable plateau current by
82.2% (600 µM EBIO applied after amiloride gave a stable plateau
current of 109.9 ± 20.4 µA/cm2,
n = 6, which was reduced to 19.9 ± 2.1 µA/cm2,
n = 6, after addition of furosemide).
The concentration-response relationship for EBIO is given in Fig.
1. This shows that the EC50 for EBIO is close to the
standard concentration of EBIO used throughout most of the present
study, i.e., 600 µM. The response curve was steep, with a Hill slope
of 6.4.
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The following data address the question of what kind of
K+ channels are involved by using
two specific K+ channel
blockers, ChTX (specific for
Ca2+-sensitive
K+ channels) and 293B (specific
for cAMP-sensitive K+ channels).
The starting point for the investigation is provided by the experiments
illustrated in Fig. 2. In these and in all others described in this report, amiloride (100 µM) was present in
the apical bathing solution throughout to eliminate electrogenic Na+ absorption, so that no
attention need be paid in the discussion to the possibility that the
movement of this ion contributed to the recorded
currents. 293B produced little or no inhibition of the
EBIO-induced current in mouse colon epithelia, but the subsequent addition of ChTX produced a substantial inhibition of current, more so
than when ChTX was added first. Furthermore, when 293B was added after
ChTX, the former produced a marked inhibition of the EBIO-induced
current. Figure 2B suggests that EBIO
predominantly activates
Ca2+-sensitive
K+ channels, whereas Fig.
2A suggests that the effect of EBIO is predominantly on cAMP-sensitive K+
channels.
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The result of the single paired experiment of Fig. 2 is amply confirmed
in the data given in Fig. 3. The response
to ChTX (50 nM) is doubled if 293B is given first, whereas the response to 293B (100 µM) is trebled if ChTX is given first. The differences cannot be attributed to differences in the extent of the responses to
EBIO because Fig. 2 shows that the responses in the two groups of
tissues used were virtually identical.
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To examine the concentration dependence of the potentiating effects of
one blocker on another, partial concentration-response curves were
obtained and are shown in Fig. 4. Two
observations stand out from the data. First, the potentiation of 293B
by prior administration of ChTX and the potentiation of ChTX by prior
addition of 293B are present whether the inhibitors are given by
cumulative addition or as a single concentration, as in Fig. 2. Second,
the potentiation seen with a single high concentration is also found at
low concentrations. Furthermore, after EBIO and ChTX addition, 293B
(100 µM) caused 54% inhibition of the current, virtually the same as
that found by Devor et al. (6) for the inhibition of forskolin currents
by 293B in T84 monolayers. Similarly, after EBIO and 293B addition,
ChTX (50 nM) caused 58% inhibition of the current, marginally less
than that found by Devor et al. (67%) (6) for the inhibition of EBIO
currents in T84 monolayers. Figure 4 also shows for comparison the
effects of ChTX and 293B on the responses to forskolin (10 µM). 293B
was as effective after forskolin addition as after the addition
of EBIO plus ChTX. By contrast, ChTX was virtually inactive at
inhibiting forskolin-sensitive SCC. Plateau values for the SCC after
EBIO and forskolin addition are also given in Fig. 4, leaving no
argument that the extents of the inhibitory responses were
limited by the sizes of the maintained responses after EBIO.
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The inability of either ChTX or 293B to substantially affect the
current alone after EBIO (600 µM) addition may arise if the basolateral membrane is already driven towards the equilibrium potential for K+
(EK) by
excessive opening of K+ channels,
so that the apical membrane limits the extent of secretion. Blockade of
one type of channel might still not affect
EK substantially; transport would then become more vulnerable to the second blocker. If
so, it might be predicted that a smaller concentration of EBIO, producing a less profound effect on
K+ channels, would increase the
apparent potencies of both ChTX or 293B to inhibit SCC. Data from
experiments using 200 µM EBIO are given in Fig.
5 and show that ChTX produces less,
although not significantly less, inhibition when the SCC response to
EBIO is reduced to one-third, rather than more as was predicted.
Therefore either the model is incorrect or the apical membrane remains
rate limiting even at low EBIO concentrations.
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Further consideration of a model in which the apical membrane dominated the level of transport led to three predictions that could be tested: 1) CF epithelia, where no CFTR is present, represent an extreme condition of apical membrane limitation and will not respond to EBIO; 2) if an agent to permeabilize the apical membrane is used in the presence of a K+ gradient, a current that is sensitive to ChTX or 293B without prior addition of the other should be revealed after EBIO addition; and 3) CF colonic epithelia should behave like wild-type tissues after permeabilization of the apical membrane in the presence of a K+ gradient.
The first of these predictions was tested by using colonic epithelia
from CF mice (1, 16) (Fig.
6A). A
small, slowly developing inhibitory current was seen after EBIO
addition in the example illustrated, a current which was increased by
forskolin and reversed by furosemide, characteristic of the
K+ secretory current in CF colons
when cAMP content is increased (3). Overall there was no effect of EBIO
on Cl secretion, in accord
with the first prediction, indicating that in some of the four colons
the SCC must have increased slightly after EBIO addition (Fig.
6A). It is to be noted that the SCC increase after furosemide addition is greater than the SCC decrease after forskolin addition, indicating that some
K+-secretion was occurring in the
absence of either forskolin or EBIO (2). To maximize the possibility of
showing a forskolin-like effect of EBIO, the following protocol was
used. ChTX (50 nM) was added to the basal side to block
Ca2+-sensitive
K+ channels, IBMX (1 µM) was
added to prevent cAMP breakdown, and the concentration of EBIO was
increased to 4,200 µM. Despite these changes EBIO gave a small SCC
increase, but one that was less than that caused when furosemide was
added subsequently (Fig. 6B). This
suggests that EBIO is causing a reduction of
K+ secretion, which is entirely
possible if the balance between apical hyperpolarization and
intracellular K+ concentration is
different than it is with forskolin. CF mouse colons show basal
K+ secretion (2), and the
elimination of this appears as an SCC increase with the convention
used. In a further investigation, furosemide, EBIO, and ChTX together
with 293B produced a progressive increase in SCC,
consistent with the elimination of
K+ secretion (Fig.
6C). The SCC increase obtained with
EBIO after furosemide addition makes it unlikely that the current
increase was due to Cl
secretion, even though some of the mice were
F508 mutants.
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To deal with the second and third predictions, the epithelia were
subjected to an apical-to-basolateral
K+ gradient, as detailed in
MATERIALS AND METHODS. Then, under
short circuit conditions, the apical surface was permeabilized by the application of nystatin, and the current and conductance were monitored
until a semi-steady-state condition was achieved (this phase took
10-30 min). At this time the conductance in 34 colonic mucosae had
increased from 2.42 ± 0.4 to 2.88 ± 0.4 mS/cm2
(P < 0.0001; paired
t-test). The technique is not a
straightforward one. If nystatin was left in contact with the tissue
for too long, the SCC fell rapidly as conductance increased, presumably
because nystatin was attacking the basolateral membrane. Thus it was a matter of judgment to decide when the nystatin effect had reached a
semi-steady-state condition, allowing the effects of other agents to be
investigated. EBIO applied after nystatin caused an increase in current
and conductance, which was sensitive to ChTX. Figure 7A shows
how virtually all the current increase caused by EBIO in the
permeabilized epithelium is removed by the addition of ChTX. By
contrast, 293B only produced a 15% inhibition of the K+ current, which did not reach
significance. Figure 7B shows that CF
colonic epithelia also respond to EBIO, once the apical face has been
permeabilized, the responses again being sensitive to ChTX.
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Thus all three predictions are shown to be true, with the exception
that the EBIO-induced responses in permeabilized epithelia are not
significantly sensitive to 293B. To further investigate the presence of cAMP-sensitive K+
channels in permeabilized epithelia, the epithelia were treated with
the membrane-permeable 8-bromo-cAMP (8-BrcAMP; 1 mM) plus IBMX (100 µM). Together these agents produced a rapid increase in SCC that was
sensitive to 293B (10 µM), giving 93% inhibition of the
K+ current. The SCC data from
these experiments are shown in Fig. 7C, and examples of SCC traces are
given in Fig. 8.
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To further clarify the effects of EBIO, an investigation of the effects
on second messenger systems was made. Because the Cl secretory current arises
from the colonic crypts, measurements were made with preparations of
isolated crypts. On the basis of fura 2 fluorescence, there was no
evidence that EBIO affected [Ca2+]i (Fig.
9) because EBIO had no effect on the
340/380 ratio. Two further experiments like the one illustrated were
performed. The average change in the 340/380 ratio was zero for 600 µM EBIO and 3.6 ± 1.6 (n = 3)
for 5 µM ionomycin.
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An enzyme immunoassay was used to measure the accumulation of cAMP
after exposure to EBIO. Colonic crypt preparations varied considerably
in their abilities to generate cAMP, presumably reflecting variable
quality. A paired t-test was therefore
used to compare the values with and without drugs. Accumulation after
the addition of either agent was increased by the presence of IBMX in
the reaction mixture (Table 1), suggesting
that EBIO affected adenylate cyclase directly, rather than inhibiting
phosphodiesterase and reducing cAMP metabolism. The more complete set
of data obtained in the presence of IBMX suggests that EBIO is less
active than forskolin at generating cAMP, even at the highest
concentrations used.
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Because it has now been shown that EBIO increases cAMP in isolated
colonic crypts, the reason for the failure of 293B to significantly reduce the K+ current generated by
EBIO was revisited. From the cAMP data of Table 1 it is clear that 10 µM forskolin generates cAMP as efficiently as (absence of IBMX) or
more efficiently than (with IBMX) the highest concentrations of EBIO
used in the assay. Furthermore, it has been shown that
the currents generated by forskolin in intact epithelia (Fig. 4) and by
cAMP in permeabilized epithelia (Fig. 7) are sensitive to 293B.
Consequently, experiments have been carried out with forskolin, rather
than EBIO, to activate adenylate cyclase in permeabilized epithelia. Of
six experiments, 10 µM forskolin failed to give any current increase
in three and produced only a short-lived transient rise in current in
the rest (Table 2). We were careful to show
that the solvent for forskolin (alcohol; final concentration 0.1%) was
without effect. The subsequent addition of EBIO after forskolin did not
prevent the usual effect, i.e., a maintained current
comparable in size to that seen in the absence of forskolin (Fig.
7A; Table 2). The transient effect of
forskolin was small compared with the response to 8-BrcAMP obtained in
permeabilized epithelia (Fig. 7C;
Table 2). The data for these experiments are given in Table 2, and the
conclusion must be that the adenylate cyclase system in
nystatin-permeabilized epithelia subjected to a
K+ gradient is impaired, a
conclusion that supports the view that EBIO does not act directly on
cAMP-sensitive K+
channels.
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DISCUSSION |
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It has been shown that EBIO, at a concentration close to the
EC50, produces a sustained
increase in SCC of ~150 µA/cm2
due to electrogenic Cl
secretion. The presence of amiloride in all experiments precludes the
possibility that electrogenic Na+
transport contributes to the response. The failure of furosemide to
completely inhibit the response (82.2% inhibition) is expected, because experiments were conducted in KHS containing bicarbonate. Furosemide essentially converts the protocol to a
Cl
-free situation, allowing
bicarbonate to be secreted via CFTR (2).
Evidence to indicate that in the mouse colonic epithelium EBIO activates more than one type of K+ channel, namely, the ChTX-sensitive, Ca2+-sensitive K+ channel and the 293B-sensitive, cAMP-sensitive K+ channel, has been derived. The presence of these two types of K+ channels in transporting epithelia has been described for other epithelia, such as T84 monolayers (12). The assumption that both ChTX and 293B are specific blockers of Ca2+- and cAMP-sensitive K+ channels, respectively, must be made. However, this assumption is reasonably sure because neither agent has much effect without the prior addition of the other (Fig. 3), which would not be the case if either agent affected both types of channel. Furthermore, the interaction between the two channel blockers is shown at low concentrations (Fig. 4).
To investigate the effect of EBIO directly on the basolateral membrane, we have used nystatin to permeabilize the apical membrane in the presence of an inwardly directed K+ gradient. Nystatin increased the SCC and increased membrane conductance under these conditions. We were able to show that EBIO caused an increased K+ current, which was virtually eliminated by 50 nM ChTX. This result, coupled with the lack of effect of EBIO on [Ca2+]i, argues for a direct effect on Ca2+-sensitive K+ channels, as shown by Devor et al. (5, 6). Similar data were obtained for CF colons, indicating the similarity with wild-type tissues once the apical permeability barrier is removed.
The failure of 293B to influence the EBIO current in apically
permeabilized epithelia was unexpected, considering its effectiveness in untreated epithelia after ChTX addition. Indeed, the failure provides a further example of the specificity of this
channel blocker, because from the above it is clear that
Ca2+-sensitive
K+ channels were activated under
these conditions. The conclusion is that EBIO fails to activate
cAMP-sensitive K+ channels in the
permeabilized preparations, eliminating the possibility that it has a
direct effect on these K+ channels
in the basolateral membrane. Clearly such channels are present in
permeabilized epithelia, because they can be activated by cAMP and
blocked by a low concentration (10 µM) of 293B. As pointed out by
others (7), 293B is equally effective in inhibiting colonic forskolin
responses whether applied apically or basolaterally. Because the drug
is lipid soluble, it can penetrate to either membrane from the
contralateral side. Furthermore, if there are cAMP-sensitive apical
K+ channels, these too, when
activated, would favor apical hyperpolarization and enhance
Cl secretion. We have shown
that forskolin, like EBIO, is unable to generate a 293B-sensitive
K+ current in permeabilized
epithelia even though both agents activate adenylate cyclase (Tables 1
and 2) and both increase Cl
secretion, which is sensitive to 293B. We conclude that
permeabilization or the application of a
K+ gradient or both disrupt the
adenylate cyclase system. We can only speculate why this is, but it has
been shown that nystatin strongly inhibits both adenylate cyclase and
cAMP phosphodiesterase (19). Forskolin, however, did not prevent EBIO,
added subsequently, from activating a sustained SCC increase of the
expected magnitude (Table 2). These results again argue for a direct
effect of EBIO on Ca2+-sensitive
K+ channels and an indirect effect
of EBIO and forskolin on basolateral cAMP-sensitive
K+ channels and possibly apical
cAMP-sensitive channels also in intact tissues (Fig.
10).
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In intact CF colons, EBIO produced either no effect on SCC (at 600 µM) or a small SCC increase (at 4,200 µM in the presence of IBMX and ChTX; Fig. 6). It is suggested that the increase in SCC in CF colons induced by EBIO is due to a reduction in K+ secretion. Whether K+ secretion or absorption is obtained depends on a balance between the amount of apical hyperpolarization (which will inhibit K+ secretion) and intracellular K+ concentration (which will be dependent on the activity of the cotransporter and will likely be more strongly activated with forskolin). There is no a priori reason why EBIO and forskolin will possess similar profiles with respect to these parameters, even when EBIO is used in the presence of ChTX. Further, the sensitivity of apical K+ channels (7) in the mouse colon to either EBIO or cAMP is unknown.
We have not specifically investigated whether CFTR channels are
directly activated by EBIO, but have no reason not to accept the data
of Devor et al. (5, 6) indicating that this channel is activated.
Indeed, we argued in the INTRODUCTION
that agents that produce a sustained increase in
Cl secretion must have
coordinated actions on both faces of the epithelial cells. Certainly it
is clear that CF epithelia show significant responses to EBIO only
after the apical membrane is removed. Thus CFTR is necessary for EBIO
to be effective and could well be activated directly (5, 6) as well as
by the accumulation of cAMP, as shown in this study. Whether CFTR is
more sensitive to cAMP than K+
channels or the cotransporter is unknown for the mouse colon.
Thus our data are in agreement with that of Devor and colleagues (5, 6)
for T84 monolayers. In the mouse colon, there is the extra dimension of
concurrent activation of cAMP-sensitive K+ channels, giving extra impetus
to Cl secretion via apical
hyperpolarization and CFTR activation. A model for the activation by
EBIO of the secretory process in intact and apically permeabilized
mouse colon epithelial cells is given in Fig. 10. Therapeutic compounds
with actions like those of EBIO may well have a place in the treatment
of CF, either to increase the effectiveness of gene therapy or to
combine with agents that increase the delivery of
F508 CFTR to the
apical membrane.
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ACKNOWLEDGEMENTS |
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We thank Dr. Rainer Greger for a generous gift of 293B. We are grateful to Drs. W. H. Colledge and M. J. Evans for the CF colons used in this work.
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FOOTNOTES |
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This work was supported by funding from the Medical Research Council, Wellcome Trust, and the Cystic Fibrosis Trust.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. W. Cuthbert, Dept. of Pharmacology, Univ. of Cambridge, Tennis Court Rd., Cambridge CB2 1QJ, UK (E-mail: awc1000{at}cam.ac.uk).
Received 26 January 1999; accepted in final form 27 April 1999.
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REFERENCES |
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1.
Colledge, W. H.,
B. S. Abella,
K. W. Southern,
R. Ratcliff,
C. Jiang,
S. H. Cheng,
L. J. MacVinish,
J. R. Anderson,
A. W. Cuthbert,
and
M. J. Evans.
Generation and characterisation of a F508 cystic fibrosis mouse model.
Nat. Genet.
10:
445-452,
1995[Medline].
2.
Cuthbert, A. W.,
M. E. Hickman,
and
L. J. MacVinish.
Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium.
Br. J. Pharmacol.
126:
358-364,
1999
3.
Cuthbert, A. W.,
L. J. MacVinish,
M. E. Hickman,
R. Ratcliff,
W. H. Colledge,
and
M. J. Evans.
Ion transporting activity in the murine colonic epithelium of normal and cystic fibrosis animals.
Pflügers Arch.
428:
508-515,
1994[Medline].
4.
Devor, D. C.,
and
R. A. Frizzell.
Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1271-C1280,
1993
5.
Devor, D. C.,
A. K. Singh,
R. J. Bridges,
and
R. A. Frizzell.
Modulation of Cl secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L785-L795,
1996
6.
Devor, D. C.,
A. K. Singh,
R. A. Frizzell,
and
R. J. Bridges.
Modulation of Cl secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L775-L784,
1996
7.
Diener, M.,
F. Hug,
D. Strabel,
and
E. Scharrer.
Cyclic AMP-dependent regulation of K+ transport in the rat distal colon.
Br. J. Pharmacol.
118:
1477-1487,
1996[Abstract].
8.
Halevy, J.,
E. L. Boulpaep,
H. J. Binder,
and
J. P. Hayslett.
Aldosterone increases maximal turnover rate of the sodium pump.
Pflügers Arch.
410:
476-480,
1987[Medline].
9.
Lohrmann, E.,
I. Burhoff,
R. B. Nitschke,
H. J. Lang,
D. Mania,
H. C. Englert,
M. Hropot,
R. Warth,
H. Rohm,
M. Bleich,
and
R. Greger.
A new class of inhibitors of cAMP-mediated Cl secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance.
Pflügers Arch.
429:
517-530,
1995[Medline].
10.
MacVinish, L. J.,
R. J. Pickles,
and
A. W. Cuthbert.
Cyclic AMP and Ca2+ interactions affecting epithelial chloride in human cultured colonic epithelia.
Br. J. Pharmacol.
108:
462-468,
1993[Abstract].
11.
Mall, M.,
M. Bleich,
M. Schurlein,
J. Kuhr,
H. H. Seydewitz,
M. Brandis,
R. Greger,
and
K. Kunzelmann.
Cholinergic ion secretion in human colon requires coactivation by cAMP.
Am. J. Physiol.
275 (Gastrointest. Liver Physiol. 38):
G1274-G1281,
1998
12.
Mandel, K. G.,
J. A. McRoberts,
G. Beuerlein,
E. S. Foster,
and
K. Dharmsathaphorn.
Ba2+ inhibition of VIP- and A23187-stimulated Cl secretion by T84 cell monolayers.
Am. J. Physiol.
250 (Cell Physiol. 19):
C486-C494,
1986
13.
Manson, A. L.,
A. E. O. Trezise,
L. J. MacVinish,
K. D. Kasschau,
N. Birchall,
V. Episkopou,
G. Vassaux,
M. J. Evans,
W. H. Colledge,
A. W. Cuthbert,
and
C. Huxley.
Complementation of null CF mice with a human CFTR Yac transgene.
EMBO J.
16:
4238-4249,
1997
14.
Morris, A. P.,
S. A. Cunningham,
A. Tousson,
D. J. Benos,
and
R. A. Frizzell.
Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl secretion in epithelial cells.
Am. J. Physiol.
266 (Cell Physiol. 35):
C254-C268,
1994
15.
Pickles, R. J.,
and
A. W. Cuthbert.
Failure of thapsigargin to alter ion transport in human sweat gland epithelia while intracellular Ca2+ concentration is raised.
J. Biol. Chem.
267:
14818-14825,
1992
16.
Ratcliff, R.,
M. J. Evans,
A. W. Cuthbert,
L. J. MacVinish,
D. Foster,
J. R. Anderson,
and
W. H. Colledge.
Production of a severe cystic fibrosis mutation in mice by gene targeting.
Nat. Genet.
4:
35-41,
1993[Medline].
17.
Sandle, G. I.,
N. Higgs,
P. Crowe,
M. N. Marsh,
S. Venkatesan,
and
T. J. Peters.
Cellular basis for defective electrolyte transport in inflamed human colon.
Gastroenterology
99:
97-105,
1990[Medline].
18.
Siemer, C.,
and
H. Gogelein.
Activation of nonselective cation channels in the basolateral membranes of rat distal colon crypt cells by prostaglandin E2.
Pflügers Arch.
420:
319-328,
1992[Medline].
19.
Surarit, R.,
and
M. G. Shepherd.
The effects of azole and polyene antifungals on the plasma membrane enzymes of Candida albicans.
J. Med. Vet. Mycol.
25:
403-413,
1987[Medline].
20.
Teather, S.,
and
A. W. Cuthbert.
Induction of bradykinin B1 receptors in rat colonic epithelium.
Br. J. Pharmacol.
121:
1005-1011,
1997[Abstract].