| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand
Submitted 20 April 2005 ; accepted in final form 23 August 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89
In particular, CF affects various epithelia of the body by reducing Cl– secretion. The CF gene codes for the CF transmembrane conductance regulator (CFTR), which functions as a cAMP-dependent Cl– channel (53, 54). Mutations in the CF gene lead to defects in CFTR, resulting in reduced Cl– and fluid secretion manifested as dehydration of the respiratory and intestinal epithelia. Intestinal complications of CF include meconium ileus (impacted ileum), malnutrition, and DIOS. Indeed, 10–15% of infants with CF suffer from meconium ileus (48) and have a higher risk of malnutrition (37) or DIOS (51) in later life. In addition, children with CF have severely reduced Cl– transport, as determined by jejunal biopsies (45, 64). Pharmacological modulation of ion transport has been suggested as a possible therapeutic treatment of intestinal complications of CF (36, 55).
The benzimidazolones are a class of compounds that were first characterized as modulators of K+ channels. Indeed, Olesen and colleagues (46, 47) first described the effects of 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazol-2-one (NS004) and 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-1,3-dihydro-2H-benzimidazol-2-one (NS1619) as activators of large-conductance K+ channels of bovine smooth muscle cells and mouse cerebellar granule cells. However, Gribkoff et al. (24) demonstrated that NS004 activated both wild-type CFTR and 
F508-CFTR expressed in Xenopus oocytes. Al-Nakkash et al. (1) likewise reported that NS004 activated phosphorylated F508-CFTR expressed in NIH3T3 mouse fibroblast cells. In addition, Becq and colleagues (12) have reported that NS004 activates phosphorylated (via forskolin) wild-type CFTR and G551D-CFTR expressed in Chinese hamster ovary cells. Recently, a benzimidazolone has been implicated in the modulation of the Na+-K+-2Cl– cotransporter (63). Therefore, benzimidazolones can modulate several transport proteins involved in the Cl– secretory response.
1-Ethyl-2-benzimidazolinone (1-EBIO), another benzimidazolone, has been demonstrated to stimulate Cl– secretion in several epithelia, including the T84 colonic cell line (14), mouse jejunum (28) and colon (11, 38), rat colon (14, 69), and Calu-3 human airway cells (16), for example. 1-EBIO is a known activator of K+ channels (34), in particular the intermediate-conductance Ca2+-dependent K+ channel (IKCa, KCNN4) (14, 28, 33, 69). 1-EBIO has been reported to stimulate intracellular levels of cAMP in native tissue (11, 39); however, others do not concur (67). 1-EBIO increases the activity of CFTR in cultured cells (13, 16) and native epithelia (10, 38).
Recently, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a derivative of 1-EBIO, has been demonstrated to be an extremely potent activator of Cl– secretion in T84 colonic cells (61). However, the effects of DCEBIO are yet to be determined in native epithelia. The aims of this study were twofold: first, to examine the effect of DCEBIO on the short-circuit current (Isc) response of a native epithelium, the mouse jejunum; and second, to determine the mechanism of action of DCEBIO on the Cl– secretory response of the mouse jejunum.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
Electrophysiological measurements–Ussing chamber experiments. The Ussing chamber short-circuit current experiments were performed as previously described (3, 5, 28). Once the tissues were isolated and pinned, the tissues were glued (Loctite 454; Henkel Loctite Adhesives, Welwyn Garden City, UK) to plastic annuli (0.7 cm2) and mounted in modified Ussing chambers. In all experiments, tissues were bathed on both mucosal and serosal sides with 10 ml of the NaCl Ringer solution. Solutions were aerated (100% O2) and maintained at 37°C by water-jacketed solution reservoirs.
Tissues were voltage clamped to 0 mV (Biodesign, South Campus Electronics, University of Otago), and Isc was continuously recorded with a MacLab data-acquisition system (ADInstruments, Castle Hill, Australia) (3, 5, 28). Initially, tissues were rinsed three times with NaCl Ringer solution. After that, all tissues were pretreated with indomethacin (1 µM, mucosal and serosal) to reduce prostaglandin production (20) and tetrodotoxin (0.6 µM, serosal) to reduce enteric nerve activity (31) for 1 h (total time) to reduce transepithelial transport to basal conditions. Data analysis was performed with MacLab Chart (version 3.6.3; ADInstruments). The basal Isc was 87 ± 3 µA/cm2 (n = 175 tissues, N = 75 animals). The change in Isc (Isc) induced by a treatment was expressed as the difference from the former baseline to the steady state. In the case of a biphasic response, two Isc values were measured: 1) from the former baseline to the peak response and 2) from the former baseline to the steady-state response. Concentration-dependent response curves were fit with the Hill equation. The presence of the circular and longitudinal muscle layers surrounding the jejunum did not impede access of chemicals to the epithelium, as exhibited by very rapid responses of Isc with the addition of forskolin or bumetanide (see Figs. 1, 2, 4–6).
|
|
|
|
Statistical analysis.
Statistical significance was assessed by Student’s t-test (paired and unpaired). MacCurveFit (Kevin Raner Software, Victoria, Australia) was used for curve fitting and determining the EC50, Vmax, and Hill coefficients values of the concentration-dependent response experiments. Data are presented as means ± SE. In some figures, the SE bar is within the symbol. A P value of 
0.05 was considered significant. The number of tissues for a given protocol and the number of animals used in a series of experiments are provided in the text. Sometimes more than one tissue from an animal was used for a particular protocol within a given series of experiments.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
Isc of 74 ± 8 µA/cm2, n = 8, N = 6) (Fig. 1, A and D) compared with control tissues (Isc of –5 ± 10 µA/cm2, n = 5) (Fig. 1, C and D). DCEBIO was also effective when added from the mucosal side of the tissue. Mucosal addition of DCEBIO (100 µM) (Fig. 1B) activated Isc by 41 ± 1 µA/cm2 (n = 3, N = 3) (Fig. 1E), which was greater (P < 0.01) than paired control tissues (–5 ± 5 µA/cm2, n = 3). DCEBIO (1–125 µM, serosal) stimulated a concentration-dependent increase in Isc as shown in Fig. 2A. This relationship exhibited a Vmax of 102 ± 2 µA/cm2, an EC50 of 41 ± 1 µM, and Hill coefficient of 1.9 ± 0.1 (Fig. 2, A and C, circles) (n = 6–8 per data point, N = 4). However, 1-EBIO, the parent molecule of DCEBIO (61), was less potent than DCEBIO. 1-EBIO, too, increased Isc in a concentration-dependent manner with a Vmax of 86 ± 8 µA/cm2, an EC50 of 862 ± 150 µM and Hill coefficient of 1.6 ± 0.3 (n = 8–11 per data point, N = 7, data not shown) as has been demonstrated by us and others (14, 28).
We used bumetanide, a known blocker of the basolateral Na+-K+-2Cl– cotransporter the entry step for Cl– in the Cl– secretory response, to establish that the DCEBIO-stimulated Isc was, indeed, Cl– secretion. Bumetanide (20 µM, serosal) caused a rapid reduction of the DCEBIO (100 µM, serosal)-stimulated Isc (Isc of –59 ± 5 µA/cm2, n = 8, N = 6) (Fig. 1, A and D) compared with paired control tissues (Isc of –5 ± 4 µA/cm2, n = 7) (Fig. 1D), indicative of Cl– secretion. Bumetanide (20 µM) inhibited 82 ± 5% of the DCEBIO-stimulated Isc. Bumetanide (0.01–50 µM, serosal) exhibited a concentration-dependent inhibition of the DCEBIO (100 µM, serosal)-activated Isc with a Ki = 0.9 ± 0.1 µM (n = 5–10 per data point, N = 8, data not shown). A maximum reduction of DCEBIO-activated Isc was achieved at 10 µM bumetanide. Bumetanide (20 µM, serosal) reduced the Isc activated by mucosally applied DCEBIO (100 µM) by 85 ± 16% (n = 3, N = 3) (Fig. 1, B and E).
These data suggest that DCEBIO stimulates a concentration-dependent Cl– secretory response of the mouse jejunum. In addition, DCEBIO is a more effective activator of Cl– secretion than its parent compound, 1-EBIO.
Do CFTR and IKCa participate in the DCEBIO-stimulated Isc response? As shown above, DCEBIO activated a sustained increase in Isc, suggesting that more than one transport protein is involved in the Cl– secretory response. At least an apical Cl– channel and a basolateral IKCa must participate in the DCEBIO-stimulated Isc (52) along with the Na+-K+-2Cl– cotransporter to maintain a sustained Cl– secretory response.
CFTR is a key Cl– channel involved in Cl– secretory response of many epithelial tissues (19, 52) and is present in the mouse jejunum (2). Therefore, we tested for the participation of CFTR in the DCEBIO-stimulated Isc response with glibenclamide and NPPB, known blockers of CFTR (10, 58, 60, 73). We (3) recently demonstrated that mucosally applied glibenclamide reduced the forskolin-activated Isc of the mouse jejunum by 60%. Similarly, we (27) reported that mucosal rather than serosal applied glibenclamide reduced methoxsalen-stimulated Cl– secretion of the mouse jejunum, suggesting that glibenclamide does not have access to the opposite side of the epithelium. Seven animals were used in this series of experiments. For each animal, two tissues were pretreated (30 min) with glibenclamide (100 µM, mucosal), whereas a third tissue was treated with DMSO only. After that, DCEBIO (100 µM, serosal) was added to one of the glibenclamide-treated tissues and to the DMSO tissue. In control tissues, DCEBIO activated Isc by 63 ± 11 µA/cm2 (Fig. 3, A and D) (n = 6); however, DCEBIO increased Isc by only 12 ± 8 µA/cm2 in the presence of glibenclamide (Fig. 3, B and D) (n = 7) in paired experimental tissues. Therefore, pretreatment of tissues with glibenclamide reduced the action of DCEBIO by 81%. In addition, glibenclamide (100 µM, mucosal) added in the presence of DCEBIO (100 µM, serosal) reduced the DCEBIO-stimulated Isc by 77 ± 10% compared with paired DCEBIO control tissues (n = 5, N = 5, data not shown).
|
These results demonstrate that CFTR participates in the DCEBIO-stimulatory Cl– secretory response of the mouse jejunum.
One of the predominant K+ channels present in the basolateral membrane of mouse crypt cells appears to be KCNN4, the IKCa channel (6, 28). As far as we are aware, the molecular identity of IKCa in the mouse jejunum has yet to be reported (D. Vandorpe, unpublished observation). However, IKCa has been demonstrated in mouse stomach, proximal colon, and distal colon (66). We (6, 28) have functionally (patch clamp) demonstrated that 1-EBIO activates IKCa in the mouse jejunum, and others (11, 14) have reported similar findings in native epithelia and cultured cells. On the basis of excised patch experiments, Bridges and co-workers (61) demonstrated that DCEBIO directly activates IKCa expressed in human embryonic kidney (HEK)-293 cells. We (62) have confirmed those findings. So, it was of great interest to determine whether IKCa participated in the DCEBIO-stimulated Isc. Clotrimazole has been demonstrated to inhibit IKCa in both heterologous systems and native epithelia (15, 56, 57, 72). Therefore, the effect of clotrimazole (3 µM, serosal) on the action of DCEBIO (100 µM, serosal) was assessed with seven animals. Experimental tissues were pretreated (30 min) with clotrimazole, whereas control tissues were pretreated with DMSO. Pretreatment with clotrimazole significantly reduced the activation of Isc by DCEBIO (Isc of 13 ± 9 µA/cm2, n = 6) (Fig. 4, B and D) compared with paired DCEBIO control tissues (Isc of 55 ± 6 µA/cm2, n = 8) (Fig. 4, A and D). Clotrimazole reduced the DCEBIO-stimulated Isc by 67 ± 15%. These data suggest that the Ca2+-activated K+ channel participates in the DCEBIO-activated Isc response.
Do DCEBIO and forskolin share a common pathway in activating Cl– secretion?
Cuthbert and colleagues (11, 39) have demonstrated that 1-EBIO (
600 µM) elevates intracellular cAMP levels of isolated mouse colonic crypts. However, Wallace et al. (67) did not demonstrate an effect of 1-EBIO (600 µM) on intracellular cAMP levels of T84 colonic cells. We have demonstrated that DCEBIO-stimulated Cl– secretion is reduced by glibenclamide (Fig. 3) and NPPB, indicative of the participation of CFTR. Bridges and colleagues (61) reported that DCEBIO (60 µM) activates Cl– currents (via CFTR) in basolaterally nystatin permeablized T84 monolayers, which suggests that CFTR is activated by DCEBIO. Because CFTR participated in the DCEBIO response in the present study, then in the presence of a maximal concentration of forskolin, an activator of CFTR via adenylyl cyclase, and thus cAMP/PKA, we predict that the DCEBIO-stimulated Isc should be reduced. We used six animals to test this hypothesis. Tissues were either pretreated with maximal forskolin (10 µM, mucosal and serosal; unpublished data), followed by the addition of DCEBIO (100 µM, serosal) or treated with DMSO before DCEBIO. As noted above, DCEBIO stimulated a sustained increase in Isc (61 ± 6 µA/cm2, n = 6) in the absence of forskolin as shown in Fig. 5, B and D. Pretreatment of tissues with forskolin increased Isc by 82 ± 13 µA/cm2 (Fig. 5C) (n = 6). However, in the presence of a maximal forskolin-stimulated Isc response, the DCEBIO-activated Isc response was biphasic and reduced, reaching a peak response of Isc of 43 ± 5 µA/cm2 and then falling to a sustained response of 17 ± 10 µA/cm2 above the Isc stimulated by forskolin (Fig. 5, C and D) (n = 6). These experiments are summarized in Fig. 5D. These data suggest that DCEBIO and forskolin may share a common pathway (CFTR).
|
Isc of 31 ± 7 µA/cm2, n = 5) (Fig. 6, B and C) compared (P < 0.01) with paired forskolin control tissues (Isc by 84 ± 11 µA/cm2, n = 5) (Fig. 6, A and C). DCEBIO stimulated Isc by 45 ± 9 µA/cm2 (n = 5) in control tissues. Again, these results demonstrate that DCEBIO and forskolin increase the Isc of the mouse jejunum via a similar pathway and provide further evidence that CFTR participates in the DCEBIO-stimulated increase in Isc.
It was surprising that DCEBIO and forskolin did not have an additive effect on the stimulated Isc of the mouse jejunum. However, forskolin (10 µM) has been shown to increase intracellular Ca2+ levels in epithelial tissues such as mouse tracheal and nasal epithelia (26, 38). However, Greger and colleagues (4, 23) demonstrated in the rat colon that forskolin (5 µM) led to a reduction in intracellular Ca2+ and a suspected decreased activity of IKCa. In addition, Cuthbert and colleagues (40) demonstrated little effect of forskolin (10 µM) on intracellular Ca2+ of cultured human colonic cells (colony 29). Nonetheless, it is possible that forskolin modulates IKCa, as well as CFTR (via PKA), and thus Isc. To test this hypothesis, we examined the effects of clotrimazole on forskolin-stimulated Isc. We selected a higher concentration of clotrimazole (20 µM, serosal) in these experiments because Devor et al. (15) reported that a higher concentration of clotrimazole was required for cAMP-dependent compared with Ca2+-dependent agonists of Cl– secretion. Five animals were used in this series of experiments. Two tissues were pretreated with clotrimazole (for 30 min), followed by the addition of forskolin (10 µM, mucosal and serosal), whereas a control tissue was pretreated with DMSO followed by forskolin. As shown in Fig. 7, even in the presence of 20 µM clotrimazole, forskolin still activated Isc by 48 ± 5 µA/cm2 (n = 5) (Fig. 7, B and C), which was reduced compared (P < 0.01) with forskolin control tissues (Isc of 83 ± 9 µA/cm2, n = 10) (Fig. 7, A and C). Clotrimazole reduced the forskolin-activated Isc by 48 ± 5%. These data suggest that IKCa is modulated by forskolin in the forskolin-stimulated Isc response.
|
Isc by 18 ± 7 µA/cm2 (n = 6), after which DCEBIO (1–125 µM, serosal) increased Isc in a concentration-dependent manner (n = 6 per data point) (Fig. 2, B and C, filled triangles). However, as predicted, the EC50 for the DCEBIO concentration-dependent response in the presence of 0.25 µM forskolin, was shifted from 41 ± 1 µM (Fig. 2C, filled circles) (DCEBIO alone) to 53 ± 5 µM (Fig. 2C, filled triangles, DCEBIO with forskolin), whereas Vmax was reduced from 102 ± 2 µA/cm2 (Fig. 2C, filled circles) to 58 ± 4 µA/cm2 (Fig. 2C, filled triangles) (n = 6, N = 6) (Fig. 2C).
These data suggest that prephosphorylating CFTR with a submaximal concentration of forskolin reduces the effect of DCEBIO on Isc. Again, one could interpret these data to suggest that forskolin, via increasing intracellular Ca2+, has increased the activity of IKCa before the addition of DCEBIO, thus reducing the potential action of DCEBIO. However, we believe this is not the case for the DCEBIO concentration-dependent experiments that were conducted with a very low concentration of forskolin. Nonetheless, to further examine the hypothesis that the action of DCEBIO involved cAMP, as well as activating Isc via IKCa, we examined the effects of DCEBIO on Isc in the presence and absence of 8-BrcAMP, thus passing the level of adenylyl cyclase. 8-BrcAMP (1–300 µM, mucosal and serosal) stimulated a concentration-dependent increase in Isc with a Vmax of 78 ± 2 µA/cm2 and a EC50 of 58 ± 2 µM (n = 4–7 per data point, N = 4, data not shown). The experimental protocols were similar to those used in the DCEBIO and forskolin experiments described above (Figs. 5 and 6). On the basis of the forskolin-DCEBIO experiments (see Fig. 5), we predicted that pretreatment of tissues with a maximum concentration of 8-BrcAMP (300 µM, mucosal and serosal) would reduce the peak and steady-state Isc response of DCEBIO. In this series of experiments, three animals were used to test this hypothesis. One tissue was pretreated with 8-BrcAMP for 30 min, followed by the addition of DCEBIO (100 µM, serosal), whereas a paired tissue was treated with DMSO before DCEBIO. DCEBIO stimulated a sustained increase in Isc (55 ± 17 µA/cm2; n = 3) in the absence of 8-BrcAMP as shown in Fig. 8, A and C. However, in the presence of a maximal 8-BrcAMP-stimulated Isc response (Isc of 74 ± 3 µA/cm2, n = 3), the DCEBIO response was once again biphasic with a peak response of Isc of 44 ± 2 µA/cm2 and then falling to a sustained response of 8 ± 6 µA/cm2 above the Isc stimulated by 8-BrcAMP (Figs. 8, B and C) (n = 3).
|
Isc of 16 ± 6 µA/cm2, n = 4) (Fig. 9, B and C) compared (P < 0.05) with paired 8-BrcAMP control tissues (Isc of 68 ± 17 µA/cm2, n = 4) (Fig. 9, A and C).
|
Isc of 37 ± 3 µA/cm2, n = 5) (Fig. 10, B and C) compared with (P < 0.01) paired DCEBIO control tissues (Isc of 58 ± 4 µA/cm2, n = 5) (Fig. 10, A and C). In a similar fashion, the presence of H89 reduced the forskolin-stimulated Isc (Isc of 36 ± 10 µA/cm2, n = 5) (Fig. 10, E and F) compared (P < 0.01) with paired forskolin control tissues (Isc of 102 ± 16 µA/cm2, n = 5) (Fig. 10, D and F).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
The bumetanide-insensitive, DCEBIO-stimulated Isc may well be HCO3– secretion (25, 59); however, the present experiments were conducted in HEPES-buffered Ringer solution. Thus, if HCO3– secretion occurred, the CO2 source could have been from metabolically generated CO2.
DCEBIO: modulation of components of Cl– secretion. The sustained Cl– secretory response exhibited by DCEBIO suggests that multiple transport proteins of the Cl– secretory pathway are involved in the DCEBIO response. Indeed, Bridges and colleagues (61) reported that DCEBIO activated CFTR in nystatin-permeablized basolateral membranes of T84 monolayers, similarly to NS004 (13). They also reported that DCEBIO stimulated the activity of IKCa expressed in HEK-293 cells (61), which we have confirmed (62).
Several lines of evidence suggest that CFTR and IKCa participate in the DCEBIO response of the mouse jejunum. First, CFTR is expressed in the mouse intestine and in the jejunum in particular (2). Second, a maximum phosphorylation of CFTR via adenylyl cyclase (with forskolin) greatly reduced the action of DCEBIO (Fig. 5). In fact, DCEBIO in the presence of forskolin resulted in a biphasic increase of Isc, which had a reduced peak response compared with DCEBIO alone, and a steady-state Isc that remained above the increased Isc level activated by forskolin. The hypothesis for this experiment was twofold: 1) if DCEBIO alters cAMP levels, as some have described for 1-EBIO (11, 39), then maximal stimulation of Isc by DCEBIO, in the presence of forskolin, should be reduced compared with DCEBIO alone; and 2) in the presence of a maximal cAMP signal (via forskolin), DCEBIO could further increase the Isc by the stimulation of IKCa. This appears to be the case; however, we were surprised by the fall in DCEBIO-stimulated Isc to a lower steady-state level (Fig. 5C) in the presence of forskolin. We did not observe this response during similar experiments with 1-EBIO (unpublished data). At present, we are uncertain as to the complex transient activation of Isc stimulated by DCEBIO in the presence of forskolin. Undoubtedly, the transient activation of Isc could result from a further hyperpolarization of the membrane potential (i.e., activation of IKCa). However, the subsequent decline of Isc to a new lower steady state would depend on the following: 1) a reduced hyperpolarization (i.e., slight decrease in K+ conductance), 2) an increase in activity of CFTR or an as yet undescribed component of the Cl– conductance without activation of K+ conductance, or 3) possibly an altered function of the Na+-K+-ATPase. However, Koegel et al. (35) reported that 1-EBIO downregulated IKCa of keratinocytes over an exposure time of hours, whereas they noted a stimulation of IKCa over a time frame of minutes. This would not explain our current results, because we noted a reduction of DCEBIO-stimulated Isc in <5 min after the peak Isc response (Fig. 5C). Third, pretreatment of the mouse jejunum with a maximum concentration of DCEBIO greatly reduced the effect of forskolin (Fig. 6), suggesting that DCEBIO and forskolin might use a common pathway for activating Cl– secretion. Fourth, mucosally applied glibenclamide, a known inhibitor of CFTR (58, 60), reduced the DCEBIO-stimulated Isc by >80% (Fig. 3). We (27) have previously demonstrated that glibenclamide applied from the serosal solution did not affect Cl– secretion of the mouse jejunum, suggesting that the effect of glibenclamide is on a transport protein (e.g., CFTR) that resides in the apical membrane. Finally, mucosally applied NPPB also reduced the DCEBIO-stimulated Isc.
Although IKCa has yet to be verified in the mouse jejunum by molecular techniques, IKCa is present within the mouse stomach and proximal and distal colon (66). IKCa is the predominant K+ channel of the basolateral membrane of the mouse jejunal crypt cell (6, 28). Clotrimazole, a known inhibitor of IKCa (15, 56, 57), was used to demonstrate the participation of IKCa in the DCEBIO-stimulated Isc response. Clotrimazole greatly reduced the secretory response (Isc) of the mouse jejunum by DCEBIO (Fig. 4). We have not examined the effects of DCEBIO and clotrimazole at the single-channel level of IKCa of jejunal crypts. However, we have demonstrated, using excised patch-clamp experiments, that clotrimazole inhibits DCEBIO-stimulated IKCa channel activity with a Ki of 65 nM for IKCa expressed in HEK-293 cells (62). Recently, Fioretti et al. (21) reported a DCEBIO- and clotrimazole-sensitive IKCa channel in a murine skeletal myoblast cell line.
We have not examined the effects of DCEBIO on single- channel recordings of IKCa of isolated jejunal crypts; however, we suspect that DCEBIO directly activates IKCa rather than altering intracellular Ca2+ levels resulting in the activation of IKCa. First, we (62) and others (61) have reported, on the basis of inside-out patch experiments, that DCEBIO directly activates IKCa heterologously expressed in HEK-293 cells or Xenopus oocytes. Second, D. C. Devor (unpublished observation) demonstrated that DCEBIO (using fura-2 measurements) did not alter intracellular Ca2+ concentrations of either T84 colonic cells (containing native IKCa channels) or HEK-293 cells expressing IKCa. Third, Cuthbert et al. (11) reported that 1-EBIO did not alter intracellular Ca2+ levels of isolated mouse colonic crypts. Similarly, D. C. Devor (unpublished observation) determined on the basis of fura-2 experiments that 1-EBIO failed to enhance intracellular Ca2+ levels of T84 colonic cells. Finally, we have reported that 1-EBIO activates IKCa directly using inside-out patch experiments (28).
These data strongly suggest that DCEBIO stimulates a Cl– secretory response of the mouse jejunum. It would appear that in the mouse jejunum, the effect of DCEBIO involves both the activation of IKCa as well as the modulation of CFTR.
DCEBIO stimulates Cl– secretion via cAMP?
Our understanding of the mechanism of action of DCEBIO in the Cl– secretory response is just now emerging. The mechanism of action of 1-EBIO is still debatable; nonetheless, there is little doubt that 1-EBIO activates IKCa directly (14, 28, 33, 69) and stimulates CFTR in nystatin-permeablized preparations (13, 16). Cuthbert and colleagues (11, 39) have demonstrated that 1-EBIO (600 µM) increases cAMP via activation of adenylyl cyclase; however, Wallace et al. (67), using the same concentration of 1-EBIO, did not observe an effect of 1-EBIO on cAMP in T84 cells.
In the present study, we have several lines of evidence that suggest that in addition to activating IKCa, DCEBIO appears to increase Cl– secretion in a cAMP-dependent manner. First, pretreating tissues with 0.25 µM forskolin, an activator of adenylyl cyclase, reduced and shifted the DCEBIO concentration-dependence curve (Fig. 2) with a reduction in Vmax and a rightward shift of EC50. It could be interpreted that the modest amount of forskolin used in those experiments may have increased intracellular Ca2+; however, we think that this is not the case. Greger and colleagues (4, 23) demonstrated that forskolin (10 µM) reduced Ca2+ levels in isolated rat colonic crypts, whereas Cuthbert and colleagues (40) reported little effect of forskolin (10 µM) on intracellular Ca2+ of cultured human colonic cells (colony 29). Considering that we used a concentration of forskolin (0.25 µM) that was five times lower, forskolin may not alter intracellular Ca2+ but slightly raise cAMP and thus phosphorylate CFTR. Therefore, we think that in the presence of low forskolin, DCEBIO can further increase intracellular cAMP, thereby increasing the activity of CFTR while activating IKCa, resulting in a sustained Cl– secretory response.
Second, we demonstrated that pretreatment of tissues with 300 µM 8-BrcAMP (a maximal concentration for activating Isc in the mouse jejunum) reduced the DCEBIO-stimulated Isc (Fig. 8). Likewise, pretreatment of tissues with DCEBIO (100 µM) reduced the 8-BrcAMP-activated Isc (Fig. 9). It could be argued that pretreatment of 300 µM 8-BrcAMP may have increased either intracellular Ca2+ or activated IKCa before the subsequent addition of DCEBIO. However, it has been reported that 1 mM 8-BrcAMP altered neither intracellular Ca2+ levels nor the activity of IKCa in Calu-3 cells (32). Holz et al. (30) reported that 1 mM 8-BrcAMP resulted in a fast transient increase in intracellular Ca2+ in primary cultures of rat pancreatic 
-cells. In the present study, we have used a low concentration (300 µM) of 8-BrcAMP compared with these other studies, and therefore we suggest that DCEBIO increases cAMP, which is then augmented by the addition of 8-BrcAMP (Fig. 9). The small increase in Isc with 8-BrcAMP in the presence of DCEBIO was not surprising, because Cuthbert et al. (11) reported that high concentrations of 1-EBIO was not as effective as forskolin in stimulating cAMP levels of the mouse colon.
Finally, if DCEBIO activates Cl– secretion via a cAMP-dependent manner, as well as by activating IKCa, then a PKA inhibitor should reduce the effect of DCEBIO on Isc. Indeed, H89 significantly reduced the DCEBIO-stimulated Isc by 34 ± 6% (Fig. 10, A–C). Similarly in paired tissues, H89 reduced the forskolin-stimulated Isc by 66 ± 8% (Fig. 10, D–F). In addition, Turner et al. (65) demonstrated that H89 (10 µM) reduced forskolin- and epinephrine-stimulated Isc of rabbit conjunctival epithelium. Similarly, Erlenkamp et al. (18) reported that PKA was stimulated by forskolin and inhibited by H89 (50 µM) in ventricular myocytes of the guinea pig.
Potency of DCEBIO compared with 1-EBIO.
The only difference between DCEBIO and its parent molecule 1-EBIO is the presence of chloro groups at positions 5 and 6 on the phenyl ring of the benzimidazolone structure (61). The chloro groups at those positions fulfill an optimal size and electronic character not achieved by bromo or methyl groups (61). Indeed, this slight change in the structure of the benzimidazolone significantly enhances the potency of DCEBIO over 1-EBIO. Bridges and colleagues (61) reported that DCEBIO exhibited an EC50 of 45 µM, whereas 1-EBIO had an EC50 of 1,200 µM in Isc experiments of T84 colonic monolayers. Similarly, with a native epithelium, the mouse jejunum, we report herein the EC50 of DCEBIO and 1-EBIO on Isc were 41 µM and 862 µM, respectively (Fig. 2). We and others have reported that 1-EBIO exhibits an EC50 on Isc for Cl– secretion of between 0.5 and 1 mM for Cl– secretion in cultured cells and native epithelia (11, 14, 16, 28, 61). Similarly, with patch-clamp experiments, the EC50 of DCEBIO on the activation of IKCa channels expressed in Xenopus oocytes is 840 nM (61) and 4 µM for IKCa expressed in HEK-293 cells (62), However, the EC50 for activation of IKCa by 1-EBIO for native IKCa channels of T84 colonic cells (61) and IKCa expressed in HEK-293 cells (33) is 
80 µM. The lower EC50 in patch-clamp experiments of IKCa is not surprising, considering the accessibility of the drug to the channel. Without a doubt, DCEBIO is a more potent activator of Cl– secretion and IKCa than 1-EBIO.
In summary, we have used the Ussing chamber short-circuit current technique to demonstrate for the first time in a native epithelium that DCEBIO stimulates a Cl– secretory response of the mouse jejunum. Both CFTR and IKCa participate in the DCEBIO-activated Cl– secretory response. The action of DCEBIO, as well as activation of IKCa, appears to occur in a cAMP-dependent manner.
|
NOTE ADDED IN PROOF |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFGRANTSREFERENCES |
|---|
2. Ameen N, Alexis J, and Salas P. Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract. Histochem Cell Biol 114: 69–75, 2000.[ISI][Medline]
2a. Ayabe T, Wulff H, Darmoul D, Cahalan MD, Chandy KG, and Ouellette AJ. Modulation of mouse paneth cell 
-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel. J Biol Chem 277: 3793–3800, 2002.
3. Baker MJ and Hamilton KL. Genistein stimulates electrogenic Cl– secretion in the mouse jejunum. Am J Physiol Cell Physiol 287: C1636–C1645, 2004.
4. Bleich M, Riedemann N, Warth R, Kerstan D, Leipziger J, Hor M, VanDriessche W, and Greger R. Ca2+ regulated K+ and nonselective cation channels in the basolateral membrane of rat colonic crypt base cells. Pflügers Arch 432: 1011–1022, 1996.[CrossRef][ISI][Medline]
5. Butt AG, Bowler JM, and McLaughlin C. Villus and crypt cell composition in the secreting mouse jejunum measured with x-ray microanalysis. J Membr Biol 162: 17–29, 1998.[CrossRef][ISI][Medline]
6. Butt AG and Hamilton KL. Ion channels in isolated mouse jejunal crypts. Pflügers Arch 435: 528–538, 1998.[CrossRef][ISI][Medline]
7. Chang EB and Rao MC. Intestinal water and electrolyte transport–mechanisms of physiological and adaptive responses. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994, vols. 1 and 2, p. 2027–2081.
8. Clarke LL, Grubb BR, Gabriel SE, Smithies O, Koller BH, and Boucher RC. Defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis. Science 257: 1125–1128, 1992.
9. Clarke LL, Grubb BR, Yankaskas JR, Cotton CU, McKenzie A, and Boucher RC. Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in CFTR–/– mice. Proc Natl Acad Sci USA 91: 479–483, 1994.
10. Cuthbert AW. Assessment of CFTR chloride channel openers in intact normal and cystic fibrosis murine epithelia. Br J Pharmacol 132: 659–668, 2001.[CrossRef][ISI]
11. Cuthbert AW, Hickman ME, Thorn P, and MacVinish LJ. Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone. Am J Physiol Cell Physiol 277: C111–C120, 1999.
12. Derand R, Bulteau-Pignoux L, and Becq F. Comparative pharmacology of the activity of wild-type and G551D mutated CFTR chloride channel: Effect of the benzimidazolone derivative NS004. J Membr Biol 194: 109–117, 2003.[CrossRef][ISI][Medline]
13. Devor DC, Singh AK, Bridges RJ, and Frizzell RA. Modulation of Cl– secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK. Am J Physiol Lung Cell Mol Physiol 271: L785–L795, 1996.
14. Devor DC, Singh AK, Frizzell RA, and Bridges RJ. Modulation of Cl– secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel. Am J Physiol Lung Cell Mol Physiol 271: L775–L784, 1996.
15. Devor DC, Singh AK, Gerlach AC, Frizzell RA, and Bridges RJ. Inhibition of intestinal Cl– secretion by clotrimazole: direct effect on basolateral membrane K+ channels. Am J Physiol Cell Physiol 273: C531–C540, 1997.
16. Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 743–760, 1999.
17. Dray X, Bienvenu T, Desmazes-Dufeu N, Dusser D, Marteau P, and Hubert D. Distal intestinal obstruction syndrome in adults with cystic fibrosis. Clin Gastroenterol Hepatol 2: 498–503, 2004.[CrossRef][Medline]
18. Erlenkamp S, Glitsch KG, and Kockskamper J. Dual regulation of cardiac Na+-K+ pumps and CFTR Cl– channels by protein kinases A and C. Pflügers Arch 444: 251–262, 2002.[CrossRef][ISI][Medline]
19. Field M. Intestinal ion transport and the pathophysiology of diarrhea. J Clin Invest 111: 931–943, 2003.[CrossRef][ISI][Medline]
20. Field M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3',5'-AMP. Am J Physiol 221: 992–997, 1971.
21. Fioretti B, Pietrangelo T, Catacuzzeno L, and Franciolini F. The intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis. Am J Physiol Cell Physiol 289: C89–C96, 2005.
22. Fryklund J, Mattsson JP, Berglund ML, Helander HF, and Larsson H. Effects of chloride transport inhibitors on intestinal fluid and ion transport in vivo and in vitro. Acta Physiol Scand 149: 365–376, 1993.[ISI][Medline]
23. Greger R, Bleich M, Leipziger J, Ecke D, Mall M, and Kunzelmann K. Regulation of ion transport in colonic crypts. News Physiol Sci 12: 62–66, 1997.
24. Gribkoff VK, Champigny G, Barbry P, Dworetzky SI, Meanwell NA, and Lazdunski M. The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel. J Biol Chem 269: 10983–10986, 1994.
25. Grubb BR. Ion transport across the jejunum in normal and cystic fibrosis mice. Am J Physiol Gastrointest Liver Physiol 268: G505–G513, 1995.
26. Grubb BR, Paradiso AM, and Boucher RC. Anomalies in ion transport in CF mouse tracheal epithelium. Am J Physiol Cell Physiol 267: C293–C300, 1994.
27. Hamilton KL, Butt AG, Cheng S, and Carter DJ. Methoxsalen stimulates electrogenic Cl– secretion in the mouse jejunum. Exp Physiol 87: 437–445, 2002.[Abstract]
28. Hamilton KL, Meads L, and Butt AG. 1-EBIO stimulates Cl– secretion by activating a basolateral K+ channel in the mouse jejunum. Pflügers Arch 439: 158–166, 1999.[CrossRef][ISI][Medline]
29. Hidaka H and Kobayashi R. Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol 32: 377–397, 1992.[CrossRef][Medline]
30. Holz GG, Leech CA, Heller RS, Castonguay M, and Habener JF. cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic B-cells. J Biol Chem 274: 14147–14156, 1999.
31. Hubel KA. The effects of electrical field stimulation and tetrodotoxin on ion transport by the isolated rabbit ileum. J Clin Invest 62: 1039–1047, 1978.[ISI][Medline]
32. Ito Y, Son M, Sato S, Ohashi T, Kondo M, Shimokata K, and Kume H. Effects of fluoranthene, a polycyclic aromatic hydrocarbon, on cAMP-dependent anion secretion in human airway epithelia. J Pharmacol Exp Ther 308: 651–657, 2004.
33. Jensen BS, Strobaek D, Christophersen P, Jorgensen TD, Hansen C, Silahtaroglu A, Olesen SP, and Ahring PK. Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275: C848–C856, 1998.
34. Jensen BS, Strobaek D, Olesen SP, and Christophersen P. The Ca2+-activated K+ channel of intermediate conductance: a molecular
