Production of a severe cystic fibrosis mutation in mice by gene targeting.

We have used gene targeting in embryonic stem cells to introduce an HPRT mini-gene into the coding sequence of the murine cystic fibrosis gene (cftr). This insertion introduces a termination codon in frame with the cftr coding sequence to terminate prematurely the CFTR protein within the first nucleotide binding domain. Animals homozygous for the cftr disruption fail to thrive and display a range of symptoms including meconium ileus, distal intestinal obstructions, gastrointestinal mucus accumulation and blockage of pancreatic ducts. The animals also show lacrimal gland pathology. Tracheal and caecal transepithelial current measurements demonstrate the lack of a cAMP activatable Cl- channel. These animals will prove useful for the evaluation of new therapeutic drugs and gene therapy strategies.

Generation and characterization of a delta F508 cystic fibrosis mouse model.

We have generated mice carrying the most common mutation in cystic fibrosis (CF), delta F508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. delta F508-/- mice die from peritonitis and show deficiencies in cAMP-activated electrogenic Cl- transport. These mice produce delta F508 transcripts and show the temperature-dependent trafficking defect first described for the human delta F508 CFTR protein. A functional CFTR Cl- channel not demonstrated by null CF mice or present at 37 degrees C was detected following incubation of epithelial cells at 27 degrees C. Thus, these mice are an accurate delta F508 model and will be valuable for testing drugs aimed at overcoming the delta F508 trafficking defect.

Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice.

Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero-injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (Cftr(tmlCam)) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr(-/-) mice treated with the CFTR-adenovirus and those treated with the control vector.

Chloride transporting capability of Calu-3 epithelia following persistent knockdown of the cystic fibrosis transmembrane conductance regulator, CFTR.

BACKGROUND AND PURPOSE: Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available. EXPERIMENTAL APPROACH: A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability. KEY RESULTS: All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent. CONCLUSIONS AND IMPLICATIONS: It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity.

Mechanisms of anion secretion in Calu-3 human airway epithelial cells by 7,8-benzoquinoline.

(1) Cultured epithelial monolayers of Calu-3 human airway cells were used to measure anion secretion in response to a number of phenanthrolines and benzoquinolines, using short-circuit current measurements. Calu-3 cells are derived from serous cells of submucosal glands of the airways and are a target for conditions in which muco-ciliary clearance is compromised. (2) Compounds studied were 5,6-benzoquinoline, 5-chloro-1,10-phenanthroline, 7,8-benzoquinoline, 5-nitro-1,10-phenanthroline, benzo[c]cinnoline and 1,10-phenanthroline, which gave EC50 values of 34, 48, 123, 235, 192 and 217 microm, respectively. Of these, 7,8-benzoquinoline was chosen for further detailed study. Concentration-response relationships for all the compounds had Hill slopes greater than 1. (3) Permeabilisation of the apical surface of epithelia with nystatin in the presence of an apical to basolateral potassium ion gradient reduced the EC50 for 7,8-benzoquinoline to 31 microm and altered the Hill slope to close to 1. (4) Using apically permeabilised epithelia it was shown that 7,8-benzoquinoline activates an intermediate-conductance calcium-sensitive potassium channel (KCNN4) and a cAMP-sensitive potassium channel (KCNQ1/KCNE3) in the basolateral epithelial membranes. (5) 7,8-Benzoquinoline was shown to increase chloride conductance of apical epithelial membranes, presumed to be by activation of the cystic fibrosis transmembrane conductance regulator. (6) 7,8-Benzoquinoline had a minor effect on cAMP accumulation in Calu-3 cells, probably by inhibition of phosphodiesterase, which may contribute to its effect on CFTR- and cAMP-sensitive potassium channels. (7) The usefulness of these novel actions in promoting secretion in airway submucosal glands is discussed.

Kinin-induced chloride permeability changes in colony 29 epithelia estimated from 125I- efflux and MEQ fluorescence.

1. The changes in apical Cl- permeability of Colony 29 human colonic epithelial monolayers were estimated from the rate constant of 125I- efflux from tissues loaded with the isotope. 2. Forskolin was used to increase intracellular concentrations of adenosine 3:5' cyclic-monophosphate (cyclic AMP), and A23187 to increase intracellular free Ca2+ (Cai). Both treatments increased the rate constant for 125I- efflux, indicating an increase in apical Cl- permeability. 3. Lysylbradykinin (LBK) also increased the rate constant for 125I- efflux, sometimes biphasically. Chelation of intracellular Ca2+ with BAPTA or prevention of prostaglandin formation with piroxicam, attenuated but did not eliminate the effect of LBK. It is concluded that LBK affects 125I- efflux through the agency of both cyclic AMP and Ca2+. 4. Ba2+ attenuated the effect of LBK and A23187 on 125I- efflux, but had no effect on the action of forskolin. It is concluded that Ca2+ has a major effect on K+ channels, the resulting hyperpolarization increasing the driving force for 125I- efflux. A secondary effect on Ca(2+)-sensitive Cl- channels is possible. By contrast, cyclic AMP exerts it major effect on apical Cl- channels. 5. Using a Cl- sensitive fluorescent dye, MEQ, the intracellular chloride concentration, Cli was estimated to be around 30 mM, which was increased to around 50 mM by forskolin, suggesting cyclic AMP could activate the Na-K-2Cl co-transporter. 6. MEQ fluorescence was used to estimate Cl- influx and efflux rates of epithelial cells. These were increased three fold by forskolin and dibutyryl cyclic AMP and two fold by LBK and histamine. 7. It is concluded that LBK increases electrogenic chloride secretion in Colony 29 epithelia through the generation of second messengers cyclic AMP and Ca2+, each of which may act on both apical and basolateral membranes.

Antagonism of kinin effects on epithelial by Hoe 140: apparently competitive and non-competitive interactions.

1. Hoe-140, a potent kinin receptor antagonist, was investigated for its ability to inhibit the effects of lysylbradykinin (kallidin) on a cultured colonic epithelium, HCA-7 Colony 29, derived from a human adenocarcinoma. 2. Measurements of electrogenic chloride secretion (as short circuit current), and of intracellular Ca2+ (from Fura-2 fluorescence) were used to assess the action of lysylbradykinin in the absence and presence of Hoe 140. 3. From short circuit current data, Hoe 140 appeared to be a competitive antagonist with a Ki value of 5 nM. However, with measurements of intracellular Ca2+ Hoe 140 was apparently a non-competitive antagonist with a Ki of between 4-6 nM. 4. Because of the unexpected finding of non-competitive antagonism, measurements were made with a second antagonist pair, histamine and mepyramine. Mepyramine behaved as a competitive antagonist against responses to histamine with a Ki value of approximately 5 nM when short circuit current measurements were evaluated. However, when intracellular Ca2+ concentration was used as a measure mepyramine, 30 nM, produced a near parallel shift in the response curve, but at 100 nM the maximal response was depressed. 5. The reasons why the apparent type of antagonism depends upon the method of measurement is discussed, bearing in mind that the increase in intracellular Ca2+ is a signal which precedes the increase in short circuit current.

Type 1 hypersensitivity reactions in reconstructed tissues using syngeneic cell types.

1. Type 1 hypersensitivity reactions in response to antigen challenge have been measured as short circuit current (SCC) responses in reconstructed tissues consisting of syngeneic cell types. 2. In all instances reconstructed tissues consisted of an epithelial monolayer grown on collagen-coated Millipore filters and a pad of peritoneal cells. Monolayers were formed of either HCA-7 or HCA-7-Col 1 cells derived from a human adenocarcinoma. Peritoneal cells were derived from rats or guinea-pigs sensitized to either ovalbumin or beta-lactoglobulin. 3. The SCC responses of the monolayers were dependent upon the 'concentration' of peritoneal cells in the reconstructed tissue. The threshold concentration was 0.4 X 10(6) cells when rat peritoneal cells are combined with an epithelial monolayer of 0.2 cm2. 4. The SCC responses in response to antigen challenge were selectively inhibited by the H1-receptor antagonist, mepyramine. Similarly the effects of exogenously applied histamine were antagonised by mepyramine. 5. The responses to antigen challenge were not inhibited by tetrodotoxin in reconstructed tissues. This result is in contrast to that with isolated intestinal epithelia from sensitized animals where tetrodotoxin inhibits the SCC responses to external field stimulation and to challenge with antigens. The consequences of these results for understanding the mechanisms of epithelial Type 1 hypersensitivity reactions are discussed. Suggestions are made to illustrate how the methods developed here may be employed to ask questions about the nature of mediators released and the types of cell responsible in human disease conditions.

Cyclic AMP and Ca2+ interactions affecting epithelial chloride secretion in human cultured colonic epithelia.

1. Chloride secretion in three types of cultured epithelial monolayers derived from a single human colonic adenocarcinoma was measured in terms of short circuit current. The three cell types were designated HCA-7, Colony 3 and Colony 29. 2. Responses of HCA-7 monolayers to basolaterally applied lysylbradykinin (LBK) (10-1000 nM) or carbachol (1-100 microM) were potentiated by pre-exposure to forskolin (10 microM) for 5 min. Forskolin itself increased short circuit current (SCC), so that the total response to forskolin and LBK or carbachol were non-additive. 3. Colony 3 cells did not respond to LBK on either face but did to carbachol on the basolateral side, while Colony 29 epithelia responded to LBK on both sides and to carbachol and histamine basolaterally. Unlike HCA-7 epithelia, responses in Colony 3 and Colony 29 epithelia were not potentiated by forskolin, but were attenuated by piroxicam. 4. In the presence of piroxicam, both forskolin and prostaglandin E2 were able to potentiate the action of both LBK and carbachol in Colony 29 epithelia. 5. LBK receptor activation in Colony 29 epithelia is transduced into an increase in intracellular Ca2+ and cyclic AMP, while in HCA-7 epithelia there is only an increase in intracellular Ca2+ (Cai). These conclusions are considered to apply to both apical and basolateral kinin receptors. 6. It is shown that forskolin has no effect on the elevation of Ca2+ by LBK in either HCA-7 or Colony 29 cells. 7. It is concluded that potentiation of agonist responses occurs when cyclic AMP is raised at the time that intracellular Ca2+ increases. No potentiation of LBK or carbachol by forskolin occurs in Colony 29 monolayers as these agonists increase cyclic AMP via eicosanoid production.

Kinin effects on ion transport in monolayers of HCA-7 cells, a line from a human colonic adenocarcinoma.

Using epithelial monolayers of HCA-7 cells, derived from a primary human colonic adenocarcinoma and grown on pervious supports, it is shown that responses to lysylbradykinin can be elicited from either side. It is proposed that kinin receptors are inserted into both apical and basolateral membrane domains.

Formal analysis of electrogenic sodium, potassium, chloride and bicarbonate transport in mouse colon epithelium.

1. The mammalian colonic epithelium carries out a number of different transporting activities simultaneously, of which more than one is increased following activation with a single agonist. These separate activities can be quantified by solving a set of equations describing these activities, provided some of the dependent variables can be eliminated. Using variations in the experimental conditions, blocking drugs and comparing wild type tissues with those from transgenic animals this has been achieved for electrogenic ion transporting activity of the mouse colon. 2. Basal activity and that following activation with forskolin was measured by short circuit current in isolated mouse colonic epithelia from normal and cystic fibrosis (CF) mice. 3. Using amiloride it is shown that CF colons show increased electrogenic sodium absorption compared to wild type tissues. CF mice had elevated plasma aldosterone, which may be responsible for part or all of the increased sodium absorbtion in CF colons. 4. The derived values for electrogenic chloride secretion and for electrogenic potassium secretion were increased by 13 and 3 fold respectively by forskolin, compared to basal state values for these processes. 5. The loop diuretic, frusemide, completely inhibited electrogenic potassium secretion, but apparently only partially inhibited electrogenic chloride secretion. However, use of bicarbonate-free solutions and acetazolamide reduced the frusemide-resistant current, suggesting that electrogenic bicarbonate secretion accounts for the frusemide-resistant current. 6. It is argued that the use of tissues from transgenic animals is an important adjunct to pharmacological analysis, especially where effects in tissues result in the activation of more than one sort of response.

4-Chloro-benzo[F]isoquinoline (CBIQ) activates CFTR chloride channels and KCNN4 potassium channels in Calu-3 human airway epithelial cells.

1 Calu-3 cells have been used to investigate the actions of 4-chloro-benzo[F]isoquinoline (CBIQ) on short-circuit current (SCC) in monolayers, whole-cell recording from single cells and by patch clamping. 2 CBIQ caused a sustained, reversible and repeatable increase in SCC in Calu-3 monolayers with an EC50 of 4.0 microm. Simultaneous measurements of SCC and isotopic fluxes of 36Cl- showed that CBIQ caused electrogenic chloride secretion. 3 Apical membrane permeabilisation to allow recording of basolateral membrane conductance in the presence of a K+ gradient suggested that CBIQ activated the intermediate-conductance calcium-sensitive K(+)-channel (KCNN4). Permeabilisation of the basolateral membranes of epithelial monolayers in the presence of a Cl- gradient suggested that CBIQ activated the Cl(-)-channel CFTR in the apical membrane. 4 Whole-cell recording in the absence of ATP/GTP of Calu-3 cells showed that CBIQ generated an inwardly rectifying current sensitive to clotrimazole. In the presence of the nucleotides, a more complex I/V relation was found that was partially sensitive to glibenclamide. The data are consistent with the presence of both KCNN4 and CFTR in Calu-3. 5 Isolated inside-out patches from Calu-3 cells revealed clotrimazole-sensitive channels with a conductance of 12 pS at positive potentials after activation with CBIQ and demonstrating inwardly rectifying properties, consistent with the known properties of KCNN4. Cell-attached patches showed single channel events with a conductance of 7 pS and a linear I/V relation that were further activated by CBIQ by an increase in open state probability, consistent with known properties of CFTR. It is concluded that CBIQ activates CFTR and KCNN4 ion channels in Calu-3 cells.

Chloride channels and anion fluxes in a human colonic epithelium (HCA-7).

1. Colonic epithelial cells, derived from a human adenocarcinoma (HCA-7), were examined by the patch clamp technique. 2. Outwardly rectifying anion (Cl-) channels were identified in the apical membrane. The conductance was g(in) approximately 26 pS, g(out) approximately 40 pS. The open state probability of the channels increased with depolarization and the selectivity for Cl- over K+ (PCl/PK) was approximately 7.5. 3. The channels were sensitive to intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP, 0.1 mM), but not to Ca2+ (at concentrations up to 1 mM). At depolarized potentials the channels were blocked by pirentanide (1-5 microM) applied intracellularly. 4. HCA-7 monolayers loaded with 125I- (as a marker for Cl-) were used to measure I- efflux and converted to instantaneous rate constants. 5. The rate constant for I- efflux was increased by forskolin and lysylbradykinin (LBK). The effects of forskolin were not effected by BAPTA (an intracellular calcium chelator). The effects of LBK were inhibited by BAPTA and by Ba2+, indicating that LBK raised intracellular Ca2+ (Cai) which activates Ca(2+)-sensitive K-channels, the latter being blocked by Ba2+. 6. Although it cannot be conclusively proved that the outwardly rectifying chloride channels described here are solely or partially responsible for the increased anion efflux or transepithelial chloride secretion, the channels are likely to be more relevant for cyclic AMP-requiring rather than Ca(2+)-requiring secretagogues.

Calcium- and cyclic AMP-dependent chloride secretion in human colonic epithelia.

Three stable epithelial cell lines (HCA-7, HCA-7-Col 1 and HCA-7-Col 3) all derived from the same human adenocarcinoma have been cultured on collagen-coated Millipore filters. These epithelial monolayers have been used to record short circuit current (SCC) in response to of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. Lysylbradykinin, applied to either side of the monolayers, increased SCC in HCA-7 cells but had little effect on the other two lines. The responses showed rapid desensitization, which could be prevented by cooling to 4 degrees C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo-oxygenase. Other secretagogues, vasoactive intestinal polypeptide (VIP) and carbachol also increased SCC in monolayers. The responses to VIP were greatest in HCA-7-Col 1 monolayers while responses were virtually absent in HCA-7-Col 3. A similar profile was seen with carbachol except that responses of HCA-7 and HCA-7-Col 1 monolayers were more equal. With one exception the responses to VIP and carbachol showed sidedness, acting only from the basolateral side. The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. Treatment with forskolin increased SCC in HCA-7 and HCA-7-Col 1 monolayers with little effect in HCA-7-Col 3. Nevertheless cyclic AMP levels were elevated most in HCA-7-Col 3 and least in HCA-7-Col 1 monolayers, in reciprocal relationship to the functional response. A23187 increased SCC when applied to HCA-7 and HCA-7-Col 3 monolayers with little effect on HCA-7-Col 1. The differential responses of the three human cell lines provide unique opportunities to discover the functional responsibilities of entities involved in the chloride secretory process. HCA-7-Col 3 cells which generate high levels of cyclic AMP in response to forskolin but which fail to show a substantial chloride secretory response may be a useful model of some disease conditions.

Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.

1. Guanylin, a 15 amino acid endogenous gut peptide, increased the short circuit current (SCC) in the epithelium of the mouse colon, but only when applied to the apical and not the basolateral surface. 2. By use of selective blockers of epithelial ion transport and modification of the bathing solution, it was concluded that guanylin increased electrogenic chloride secretion but also had a minor effect on electrogenic sodium absorption. In addition there were small residual currents which remained unresolved. 3. The threshold concentration of guanylin causing a SCC increase was less than 50 nM, but at concentrations 40 times greater no indication of a maximally effective concentration was found. 4. Two guanylin isomers with the same amino acid sequence but with the disulphide bridges joined in an alternate fashion showed no activity. Thus only guanylin with the greatest structural homology to heat stable enterotoxin (STa) showed biological activity. 5. The action of guanylin was virtually eliminated in colonic epithelia from transgenic cystic fibrosis (CF) mice. As these animals lack the chloride channel coded by the CF gene sequence, it is likely that the final effector process in murine colonic epithelia involves the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. 6. Opportunistic infections of the gut generating STa lead to diarrhoeal conditions via an action of the toxin on apical guanylin receptors. Thus, as discussed, the CF heterozygote may have a genetic advantage in this circumstance.

Kinin-stimulated chloride secretion in mouse colon requires the participation of CFTR chloride channels.

The effect of lysylbradykinin on electrogenic chloride secretion in the epithelium of the mouse colon has been investigated. The peptide was active only when applied to the basolateral surface and its effects were inhibited by the B2 receptor antagonist, Hoe 140, also applied to the same surface. The chloride channel blocker, niflumic acid, also inhibited the response to kinin when added apically. Cyclo-oxygenase inhibition with piroxicam attenuated the responses to kinin, indicating involvement of prostaglandins in the responses. It is concluded that lysylbradykinin increases chloride secretion by acting via B2 receptors and, as with other tissues, brings about secretion through the agency of multiple messengers. In colonic epithelia from cystic fibrosis (CF) mice lysylbradykinin was without effect, suggesting that the final effector process involves apically located cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels.

Bicarbonate-dependent chloride secretion in Calu-3 epithelia in response to 7,8-benzoquinoline.

Stimulation of Calu-3 epithelia with 7,8-benzoquinoline, under short circuit current conditions, produced a current increase that was completely accounted for by the net flux of chloride, measured simultaneously with 36Cl-. Nevertheless the current stimulated by 7,8-benzoquinoline was sensitive to acetazolamide, which caused up to 50 % inhibition of the stimulated current, the remainder being sensitive to the Na+-K+-2Cl- cotransport inhibitor bumetanide. The effects of acetazolamide could be mimicked by either amiloride or by the di-sodium salt of 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) added to the basolateral side of the epithelium, but their actions were not additive. Amiloride was needed in sufficient concentration to inhibit the sodium-proton exchanger NHE1. DNDS blocks both the chloride-bicarbonate exchanger AE2 and the sodium-bicarbonate transporter NBC1. However, since 7,8-benzoquinoline activates basolateral K+ channels, causing hyperpolarisation, it is unlikely NBC1 is active after addition of 7,8-benzoquinoline. The effect of DNDS is, therefore, mainly on AE2. It is concluded that chloride enters the basolateral aspect of the cells using the Na+-K+-2Cl- cotransporter and a parallel arrangement of NHE1 with AE2, these latter two being sensitive to acetazolamide because of their association with the cytoplasmic form of carbonic anhydrase CAII. The effects of acetazolamide could be mimicked by removal of HCO3-/CO2 from the bathing medium, and furthermore showed that the NHE1-AE2 mechanism is particularly important when the transport rate is high. Thus part of the current stimulated by 7,8-benzoquinoline and inhibited by acetazolamide or HCO3-/CO2 removal can be said to represent bicarbonate-dependent chloride secretion.

EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes.

The effect of 1-ethyl-2-benzimidazolone (EBIO) on electrogenic chloride secretion in murine colonic and nasal epithelium was investigated by the short-circuit technique. In the colon, EBIO produces a sustained current increase in the presence of amiloride, which is sensitive to furosemide. In nasal epithelium EBIO causes only a small, transient current increase. Sustained increases in current were obtained in response to forskolin in both epithelia. To examine the mechanisms by which EBIO increases chloride secretion, the effects on intracellular mediators were measured in colonic crypts. There was no effect on [Ca(2+)]i but cAMP content was increased, more so in the presence of IBMX, indicating a direct effect on adenylate cyclase. In colonic epithelia in which the apical surface was permeabilized by nystatin, and the tissue subjected to an apical to basolateral K(+) gradient, EBIO caused a current increase that was entirely sensitive to charybdotoxin (ChTX). In similarly permeabilized colons Br-cAMP caused a current increase that was entirely sensitive to 293B. Thus EBIO increases chloride secretion in the colon by coordinated actions at both the apical and basolateral faces of the cells. These include direct and indirect actions on Ca(2+)-sensitive and cAMP-sensitive K(+) channels respectively, and indirect actions on the basolateral cotransporter and apical CFTR chloride channels via cAMP. In CF colonic epithelia EBIO did not evoke chloride secretion. It is not clear why the nasal epithelium responds poorly to EBIO whereas it gives a sustained response to the related compound chlorzoxazone.