Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone

A. W. Cuthbert, M. E. Hickman, P. Thorn, and L. J. MacVinish

Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Concentration-response curve for 1-ethyl-2-benzimidazolone (EBIO), determined by cumulative addition, in 4 separate colonic epithelia. Means ± SE are shown. Data were fitted to Hill equation with Kaleidagraph, which gave following values: maximal response, 156.7 ± 8.5 µA/cm2; EC50, 576.7 ± 15.4 µM; Hill slope, 6.4 ± 1.2. Delta SCC, change in short circuit current.

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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Fig. 2.   SCC records from 1 pair of colonic mucosae from 1 mouse. Each was exposed to 600 µM EBIO on both sides. Charybdotoxin (ChTX; 50 nM) and 293B (100 µM) were applied on basolateral side only, but order was reversed for the 2 tissues.

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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Fig. 3.   Cumulative data for experiments of type shown in Fig. 2. A: effects of EBIO on SCC in the 2 groups of tissues. B: effects of 50 nM ChTX alone and after 293B. C: effects of 293B alone and after ChTX. Means ± SE are shown, and P values were determined by paired t-test.

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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Fig. 4.   Partial concentration-response curves for 293B and ChTX for reduction in SCC (-Delta SCC) after 600 µM EBIO addition. For 293B (A) inhibition was determined in absence and presence of ChTX (50 nM). Similarly, for ChTX (B) inhibition curves were determined in absence and presence of 293B (100 µM). Also shown are effects of ChTX and 293B on responses to forskolin (10 µM). By each curve is shown extent of SCC responses to either EBIO or forskolin, before inhibitory responses were measured by cumulative addition. A paired t-test was used to determine P values indicated on figure.

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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Fig. 5.   Comparison of effects of 2 concentrations of EBIO (600 and 200 µM) on SCC (A) and effects of ChTX (50 nM) on SCC responses (B). C: percentages of inhibition by ChTX at the 2 concentrations of EBIO used. A t-test was used to test for significance; n = 16 for 600 µM EBIO, and n = 4 for 200 µM EBIO. NS, not significant.

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 Delta F508 mutants.


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Fig. 6.   Effects of EBIO on cystic fibrosis (CF) colonic mucosae. A, left: SCC record from a CF colon exposed sequentially to EBIO (600 µM), then to forskolin (10 µM) (both agents were applied to both sides), and finally to furosemide (1 mM) applied basolaterally. B, left: experiments similar to those in A, except that the EBIO concentration was increased to 4,200 µM and ChTX (50 nM; added basolaterally) and IBMX (1 µM) were present throughout. C, left: CF colons were exposed to IBMX (1 µM) and, after a steady state had been reached, to furosemide (1 mM; basolaterally), EBIO (4,200 µM), and finally to ChTX (50 nM) together with 293B (10 µM). For A, B, and C, cumulative data (means ± SE) are shown at right.

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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Fig. 7.   A: SCC responses to different concentrations of EBIO in apically permeabilized colon mucosae in presence of an inwardly directed K+ gradient. Also shown is current removed by ChTX (50 nM) and by 293B (100 µM). For all values n = 7, and P values were determined by nonparametric t-test (Mann-Whitney). B: similar data for permeabilized CF colons for a single concentration of EBIO (600 µM). P was determined by nonparametric paired t-test; n = 7. C: SCC responses of permeabilized colonic mucosae in presence of a K+ gradient to 8-bromo-cAMP (8-BrcAMP; 1 mM) + IBMX (100 µM) (cAMP) and current remaining after addition of 293B (10 µM). P was determined by nonparametric paired t-test; n = 6.

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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Fig. 8.   Examples of SCC traces made with apically permeabilized colonic epithelia showing ability of EBIO to generate a ChTX-sensitive current (A) and ability of cAMP to generate a 293B-sensitive current (B). Concentrations: EBIO, 600 µM; ChTX, 50 nM; 8-BrcAMP, 1 mM (+ 100 µM IBMX); and 293B, 10 µM. Nystatin concentration was 180 µg/ml and was given in divided doses in A.

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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Fig. 9.   Demonstration of changes in intracellular Ca2+ in isolated colonic crypts. Fura 2-loaded crypts were exposed first to 600 µM EBIO and then to 5 µM ionomycin. Fluorescence intensity at 340 and 380 nm, with excitation at 510 nm, was recorded (A), and ratio of intensity at 340 nm to that at 380 nm (340/380 ratio) was measured (B). EBIO had no effect on 340/380 ratio.

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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Table 1.   Effect of EBIO and forskolin on cAMP in isolated murine colonic crypts

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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Table 2.   Effect of forskolin and EBIO on SCC in apically permeabilized colonic epithelia subject to apical-to-basolateral K+ gradient


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 10.   Model for secretory effects of EBIO on intact and permeabilized colonic epithelial cells. AC, adenylate cyclase; CFTR, CF transmembrane conductance regulator.

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 Delta F508 CFTR to the apical membrane.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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