Cholinergic ion secretion in human colon requires coactivation by cAMP

M. Mall1,2, M. Bleich1, M. Schürlein2, J. Kühr2, H. H. Seydewitz2, M. Brandis2, R. Greger1, and K. Kunzelmann1

1 Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg; and 2 Kinderklinik der Albert-Ludwigs-Universität Freiburg, D-79106 Freiburg, Germany

    ABSTRACT
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Procedures
Results
Discussion
References

Cl- secretion in the colon can be activated by an increase of either intracellular Ca2+ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 µmol/l, basolateral), via an increase in cytosolic Ca2+ concentration ([Ca2+]i), activated a transient lumen-negative equivalent short-circuit current (Isc) [change (Delta ) in Isc = -79.4 ± 7.5 µA/cm2]. Previous studies indicated that intracellular Ca2+ directly acts on basolateral K+ channels, thus enhancing driving force for luminal Cl- exit. Increased intracellular cAMP (by basolateral addition of 100 µmol/l IBMX and 1 µmol/l forskolin) activated a sustained lumen-negative current (Delta Isc = -42.4 ± 7.2 µA/cm2) that was inhibited by basolateral trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 µmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (Delta Isc = 167.7 ± 32.7 µA/cm2), suggesting cooperativity of the Ca2+- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 µmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positive Isc was probably due to activation of apical K+ channels because it was blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 µmol/l, basolateral), an increase of cAMP activated a sustained negative Isc. Under these conditions, CCH induced a large further increase in lumen-negative Isc (Delta Isc = -100.0 ± 21.0 µA/cm2). We conclude that CCH acting via [Ca2+]i can induce Cl- secretion only in the presence of cAMP, i.e., when luminal Cl- channels are already activated. The activation of a luminal and basolateral K+ conductance by CCH may be essential for transepithelial KCl secretion in human colon.

cystic fibrosis transmembrane conductance regulator; epithelial transport; potassium channels; Ussing chamber; microelectrodes; transepithelial voltage; carbachol

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

SODIUM CHLORIDE AND WATER secretion across the human colon is generated mainly by epithelial cells lining the crypts but also and to a lesser degree by the surface epithelium (9, 19). Secretion is under the control of a variety of hormones and neurotransmitters and is affected in common diseases like secretory diarrhea and cystic fibrosis (CF). Therefore, detailed knowledge about the ion conductances involved is essential for the understanding of electrolyte secretion in human colon. Activators of electrolyte transport can be subdivided into those that act via the intracellular cAMP-, cGMP-dependent pathway and others that require an increase in intracellular Ca2+. During stimulation of electrolyte transport by either pathway, ion channels are activated in apical or basolateral membranes of colonic epithelial cells. Previous reports demonstrated that an apical Cl- conductance is activated when intracellular cAMP is enhanced to upregulate Cl- secretion (13). This apical Cl- conductance is formed by the CF transmembrane conductance regulator (CFTR), the protein that was demonstrated to be defective in CF (26). In addition to the opening of apical Cl- conductances, basolateral K+ channels are activated (34) in rat colonic epithelium. The basolateral K+ conductance is formed by very small-conductance K+ channels, corresponding to the recently cloned KvLQT1 channel. This novel type of K+ conductance can be inhibited specifically by a new class of chromanol compounds (4).

Whereas CFTR and KvLQT1 are regulated by intracellular cAMP, other classes of ion channels are activated by intracellular Ca2+. Accordingly, basolateral K+ channels with a larger single-channel conductance (~16 pS) are activated by agonists that increase intracellular Ca2+ such as carbachol (CCH) (5, 28). These channels are inhibited by the common K+ channel blockers Ba2+ and tetraethylammonium (TEA+) but not by chromanols. It has been shown for the rat colonic epithelium that Ca2+-dependent K+ channels are shut off when the small-conductance K+ channels are turned on during an increase of intracellular cAMP (34). It is, however, not clear whether or not an increase of intracellular Ca2+ also leads to the activation of apically localized Ca2+-regulated Cl- channels in colonic epithelial cells. Furthermore, CCH might further upregulate cAMP-dependent Cl- channels. Alternatively, CCH-induced Cl- secretion could be solely due to an activation of basolateral K+ channels, which hyperpolarizes these cells and thus enhances the driving force for luminal Cl- exit (6). These hypotheses have thus far not been examined in human colonic epithelium.

The aim of the present study was to gain a more detailed knowledge about the process of electrolyte secretion in the human colonic epithelium. To this end, it was essential to make use of freshly isolated human colonic epithelium rather than cultured cells. Using a novel type of miniature Ussing chamber allowing for the continuous exchange of luminal and basolateral bath solutions, we demonstrate the presence of a recently cloned new type of K+ channel in the basolateral membrane of human colonic epithelial cells. This channel could be an important pharmaceutical target for the treatment of secretory diarrhea (21). Moreover, the results uncover the relationship between Ca2+- and cAMP-activated electrolyte secretion in the human colon. Former studies demonstrated a relationship between cholinergic and cAMP-dependent colonic ion secretion and described defective function for both in CF (11, 15, 31-33). The conclusions from this study are essential for the understanding of previous results showing altered Ca2+ (i.e., CCH)-induced electrolyte secretion in CF (32, 33).

    EXPERIMENTAL PROCEDURES
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Patients. Colonic tissue preparations were obtained from 29 patients with a mean age of 55.9 ± 4.5 yr (ranging from 1 mo to 88 yr) who underwent routine surgical procedures at the University Hospital Freiburg. Two- to three-millimeter forceps biopsies were taken either from surgical resections or directly from the patients. There was no muscle layer left after superficial forceps biopsies. Intestinal segments examined in the present study comprised distal descendent colon, sigmoidal colon, and rectum. The responses in the presence of indomethacin were similar in the preparations derived from the various segments. The tissues used for Ussing chamber experiments were not affected by the primary disease that was the cause for surgical intervention. The study was approved by the ethics committee and the patients had given their written informed consent.

Ussing chamber experiments. Small pieces of the removed colon that were not affected by the primary disease were immediately put into an ice-cold buffer solution of the following composition (mmol/l): 127 NaCl, 5 KCl, 5 D-glucose, 1 MgCl2, 5 sodium pyruvate, 10 HEPES, 1.25 CaCl2, and 10 g/l albumin. Small samples (2-4 mm in diameter) were taken from the tissue and mounted into a modified Ussing chamber. To obtain stable measurements even with small pieces of tissue, we constructed a sandwich chamber with a circular aperture of 0.95 mm2. The luminal and basolateral sides of the epithelium were perfused continuously at a rate of 10-20 ml/min (chamber volume 1 ml), allowing for the paired examination of the effects of CCH in the presence or absence of cAMP. The bath solution, which was replaced continuously, had the following composition (mmol/l): 145 NaCl, 0.4 KH2PO4, 1.6 K2HPO4, 5 D-glucose, 1 MgCl2, and 1.3 calcium gluconate. pH was adjusted to 7.4. Bath solutions were heated by a water jacket to 37°C. Experiments were carried out under open-circuit conditions with values for transepithelial voltage (Vt) referring to the serosal side of the epithelium. We found open-circuit measurements more adequate because 1) they more accurately reflect the in vivo situation, 2) we were able to keep the tissue preparations functional and responding for a longer time period (up to 7 h), and 3) the resulting calculated short-circuit current (Isc) was larger and tissues responded better to stimulation with the agonists used in this study. Transepithelial resistance (Rt) was determined by applying short (1 s) current pulses [change (Delta ) in I = 0.5 µA]. Voltage deflections obtained under conditions without the mucosa present in the chamber were subtracted from those obtained in the presence of the tissues. Rt was calculated according to Ohm's law (Rt = Delta Vt /Delta I). Tissue preparations were only accepted if Rt exceeded that obtained for an empty chamber at least by a factor of 2. From each of patients 1-6, in most cases three biopsies were examined and recordings were usually stable for 3-4 h. Typically, after stabilization of basal Vt and Rt, amiloride (10 µmol/l) was added to the luminal side of the colonic mucosa. Under these conditions the effect of basolaterally added CCH (100 µmol/l) was examined. Subsequently, the effect of CCH was examined in the presence of activators of the intracellular cAMP pathway (IBMX, 100 µmol/l, and forskolin, 1 µmol/l, basolateral solution). In another series of experiments, recordings were performed in the presence of indomethacin (10 µmol/l, basolateral solution) to suppress synthesis of endogenous prostaglandins and intracellular cAMP.

Compounds and analysis. Amiloride, indomethacin, TEA+, Ba2+, and IBMX were all obtained from Sigma and Merck (Deisenhofen and Darmstadt, Germany). Forskolin and trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (293B) were obtained from Hoechst (Frankfurt, Germany). All used chemicals were of highest grade of purity available. Data are shown as individual recordings or as mean ± SE (n = number of observations). Paired Student's t-test was used for analysis of paired data (P < 0.05).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Basal properties of human rectal mucosa. Under control conditions, Isc of the tissue biopsies was -52.3 ± 5.3 µA/cm2 (n = 41). Basal Vt was -1.6 ± 0.3 mV and Rt was 27.8 ± 3.1 Omega  · cm2. Addition of amiloride (10 µmol/l) to the mucosal side of the epithelium reduced Vt and Isc slightly but significantly to -1.5 ± 0.2 mV and -48.1 ± 5.1 µA/cm2, respectively (n = 41). Rt under these conditions was 29.1 ± 3.0 Omega  · cm2.

Cooperativity of Ca2+- and cAMP-dependent Cl- secretion. In the presence of amiloride, Ca2+-dependent Cl- secretion was stimulated by adding CCH (100 µmol/l) to the basolateral side of the epithelium. CCH invariably increased the lumen-negative Isc from -41.3 ± 5.7 to -122.4 ± 22.7 µA/cm2 (n = 10). Vt was increased from -1.0 ± 0.4 to -2.4 ± 0.5 mV and Rt was slightly reduced from 24.6 ± 5.8 to 23.2 ± 5.2 Omega  · cm2 (n = 10) (Fig. 1, A and B). The effect of CCH was only transient, and Isc returned to control values within 2-4 min and was due to increase of intracellular Ca2+ without any change of intracellular cAMP (unpublished data from our laboratory).


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Fig. 1.   Effect of basolateral carbachol (CCH, 100 µmol/l) on transepithelial voltage (Vt) and resistance (Rt) of human colon epithelium. Rt was determined from the Vt downward deflections obtained by pulsed current injection. Impact of parallel activation of cAMP pathway is shown. A: CCH transiently enhanced lumen-negative Vt under control (Con) conditions. Activation of the cAMP pathway by stimulation with IBMX (100 µmol/l) and forskolin (Fors, 1 µmol/l) persistently enhanced the lumen-negative Vt and thus increased equivalent short-circuit current (Isc = Vt /Rt). In the presence of IBMX and forskolin, effect of CCH on Vt was more pronounced and thus activation of Isc by CCH was augmented by costimulation via the cAMP-dependent pathway. Time gap between both records was 5 min. B: summary of Isc obtained from experiments as shown in A. IBMX and forskolin induced a slight and nontransient increase in Isc. No. in parentheses indicates no. of experiments. § Effect of CCH was significantly enhanced in presence of parallel activation of the cAMP pathway. All experiments were performed in presence of 10 µmol/l amiloride. * Significantly different from control (P < 0.05).

Intracellular cAMP was enhanced by the inhibitor of phosphodiesterase IBMX (100 µmol/l) and the stimulator of the adenylate cyclase forskolin (1 µmol/l), both applied to the basolateral side of the epithelium. Intracellular Ca2+ was not affected by these agonists (unpublished data from our laboratory). This enhanced lumen-negative Isc from -40.5 ± 7.1 to -71.8 ± 9.8 µA/cm2 (Vt increased from -1.0 ± 0.4 to -2.0 ± 0.5 mV; Rt decreased from 34.2 ± 3.1 to 33.6 ± 2.8 Omega  · cm2, n = 28). After activation of the cAMP-dependent pathway, the effects of CCH on lumen-negative Isc were significantly enhanced: Isc was enhanced from -71.8 ± 9.8 to -248.3 ± 38.2 µA/cm2 (Vt was increased from -1.0 ± 0.4 to -5.2 ± 0.8 mV; Rt was decreased from 25.3 ± 3.8 to 22.7 ± 3.8 Omega  · cm2, n = 10) (Fig. 1, A and B). These paired experiments indicate that Ca2+ and cAMP increase Cl- secretion cooperatively.

Role of basolateral K+ channels for Cl- secretion. We further examined the impact of basolateral K+ channels, activated by either cAMP or Ca2+, on Cl- secretion in human colon epithelium. After stimulation of the tissues by IBMX and forskolin, the effects of BaCl2 (5 mmol/l) and a specific blocker of the cAMP-activated KvLQT1 K+ channel [chromanol 293B (21)] were added to the basolateral side. In this series, IBMX and forskolin enhanced Isc from -43.4 ± 3.5 to -72.9 ± 5.0 µA/cm2 (Vt was increased from -1.5 ± 0.2 to -2.4 ± 0.3 mV and Rt was decreased from 33.4 ± 3 to 32.1 ± 2.4 Delta cm2, n = 36). Addition of BaCl2 (5 mmol/l) to the basolateral side completely inhibited Isc activated by increase of intracellular cAMP and reduced total Isc to -17.3 ± 3.1 µA/cm2 (n = 6) (Fig. 2, A and C). This effect was mimicked by the chromanol 293B (10 µmol/l), which also led to complete inhibition of Isc activated by the increase of intracellular cAMP and reduced total Isc to -29.8 ± 3.0 µA/cm2 (n = 36) (Fig. 2, B and C). Figure 2D depicts the concentration-response curve for 293B. The approximate IC50 value was 5 µmol/l. Therefore, the activation of a basolateral K+ conductance is essential for cAMP-dependent stimulation of electrolyte secretion in the human colon, and the K+ channel involved is most likely the KvLQT1 channel (4).


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Fig. 2.   Increase of lumen-negative Vt and increase in Isc (Isc = Vt /Rt; Rt was determined from the Vt downward deflections obtained by pulsed current injection) by stimulation of the cAMP pathway require a basolateral K+ conductance. A and B: increase of intracellular cAMP by IBMX (100 µmol/l) and forskolin (1 µmol/l) enhanced lumen-negative Vt, which was completely blocked by basolateral Ba2+ (5 mmol/l; A). Effect of Ba2+ could be mimicked by the K+ channel blocker trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (293B; B), which was applied to the basolateral side. Time gaps between both records in A and B were 3 min. C: summary of Isc data calculated from experiments shown in A and B. IBMX- and forskolin-induced Isc were completely blocked by either Ba2+ or 293B. D: concentration-response curve for effects of 293B on cAMP-activated Isc. max, Maximum. All experiments were performed in the presence of 10 µmol/l amiloride. * Significantly different from control, §significantly different vs. IBMX (P < 0.05).

To examine the impact of the above-described K+ channel blockers on Ca2+-dependent Cl- secretion, the colonic tissue was first stimulated by IBMX and forskolin, and subsequently the effects of CCH were examined in the presence or absence of Ba2+ or 293B, respectively. CCH (100 µmol/l) enhanced Isc from -47.1 ± 6.6 to -151.4 ± 27.5 µA/cm2 (n = 27). The effect of CCH was completely abolished in the presence of Ba2+ (Delta Isc = 1.0 ± 3.3 µA/cm2, Delta Rt = 1.4 ± 0.2 Omega  · cm2, n = 6) (Fig. 3, B and C). In contrast, 293B inhibited sustained lumen-negative Isc activated by cAMP from -72.3 ± 5.2 µA/cm2 to -32.5 ± 3.5 µA/cm2 (Delta Rt = 0.7 ± 0.1 Omega  · cm2), but the CCH-induced transient changes of Isc were not attenuated by 293B (Delta Isc = -102.2 ± 14.4 µA/cm2, Rt = 4.1 ± 0.31, n = 26) (Fig. 3, A and C). We conclude from these data that different types of K+ channels are activated during stimulation of electrolyte secretion in the human colon by the two second messengers cAMP and Ca2+. One type of K+ conductance must be activated to maintain electrolyte transport. In this respect, the properties of the human colon as found in the present study are very similar to those found in rat colon (34).


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Fig. 3.   Effects of basolateral application of K+ channel blockers on CCH-induced changes in Vt. A: CCH (100 µmol/l) induced an increase in negative Vt in the presence of IBMX (100 µmol/l) and forskolin (1 µmol/l). Application of 293B (0.1-10 µmol/l) to the basolateral side of the epithelium inhibits lumen-negative Vt to a large degree. Subsequent basolateral application of CCH in the presence of both IBMX and forskolin and 293B induced an effect similar to that in the absence of 293B. B: CCH (100 µmol/l) induced an increase in negative Vt in the presence of IBMX and forskolin. Application of Ba2+ (5 mmol/l) to the basolateral side of the epithelium inhibits lumen-negative Vt to a large degree. In the presence of Ba2+ the effect of CCH was almost abolished. Time gaps between both records in A and B were 5 min. C: summary of the equivalent Isc (Isc = Vt /Rt; Rt was determined from the Vt downward deflections obtained by pulsed current injection) before and after stimulation with CCH. All experiments were performed in the presence of IBMX and forskolin and in the presence of 10 µmol/l amiloride. The effect of CCH on Isc was completely abolished by Ba2+ but was not altered by 293B. * Significantly different from control (P < 0.05; paired t-test).

Ca2+-dependent Cl- secretion requires activation of the cAMP-dependent pathway. To further investigate a possible cooperativity of Ca2+- and cAMP-activated Cl- secretion, we examined in paired experiments the effects of CCH under three different conditions: 1) under control conditions, 2) in the presence of indomethacin (10 µmol/l) to suppress endogenous production of prostaglandins and thus intracellular cAMP, and 3) after maximal activation of the cAMP-dependent pathway by IBMX and forskolin. Before treatment with indomethacin, basal Isc was -32.1 ± 3.7 µA/cm2 (Vt = -1.1 ± 0.3 mV, Rt = 33.2 ± 4.5 Omega  · cm2) and was further increased by CCH to -99.6 ± 13.7 µA/cm2 (n = 26). Subsequently, indomethacin was added to the basolateral side of the mucosa and the effect of CCH was examined repetitively in intervals of 10-20 min. After only ~1 h of perfusion with indomethacin, the basal Isc was inhibited almost completely to -8.8 ± 1.8 µA/cm2 (Vt = -0.4 ± 0.1 mV, Rt = 38.3 ± 3.6 Omega  · cm2, n = 26). In 18 of 26 experiments (70%), we observed positive deflections of Vt after the application of CCH (Fig. 4A) resulting in a transient increase of Isc to 14.3 ± 4.7 µA/cm2 (Delta Rt = 3.6 ± 0.5 Omega  · cm2) In 6 of these 18 experiments the response was monophasic and consisted only of a lumen-positive Isc. In 20 experiments, residual negative deflections of Vt were observed with Isc increasing to -14.3 ± 2.1 µA/cm2 (Delta Rt = 0.1 ± 0.2 Omega  · cm2). In eight experiments the response was monophasic negative. This variability was observed for all colonic segments and rectal tissues, respectively, and is most likely due to variable inhibition of endogenous cAMP synthesis in different tissue preparation because of variable incubation with indomethacin.


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Fig. 4.   Effect of indomethacin (10 µmol/l) on CCH (100 µmol/l)-induced changes in Vt. A: effect of basolaterally applied CCH in the absence of indomethacin. Application of indomethacin inhibited lumen-negative Vt. After 45 min of incubation with indomethacin, stimulation with CCH induced a lumen-positive Vt. After recovery from CCH, stimulation with IBMX (100 µmol/l) and forskolin (1 µmol/l) induced a nontransient lumen-negative Vt in the presence of indomethacin. The effect of CCH in the presence of both indomethacin and IBMX and forskolin was augmented. Time gaps between records were 40 min (first gap) and 20 min (second gap). B: summary of the equivalent Isc (Isc = Vt /Rt; Rt was determined from the Vt downward deflections obtained by pulsed current injection) under control conditions (solid bars), after inhibition with indomethacin, and after subsequent stimulation with forskolin and IBMX in the presence of indomethacin (n = 26; open bars). All experiments were performed in the presence of 10 µmol/l amiloride. All experimental Isc values were significantly different from the respective pre- and postexperimental controls [*Significantly different from control (P < 0.05; paired t-test)]. Of 26 experiments, 18 showed positive deflections and 20 showed residual negative deflections of Vt after the application of CCH. Twelve experiments showed a biphasic response.

After complete inhibition of the prostaglandin synthesis, cAMP production was again increased by basolateral addition of IBMX (100 µmol/l) and forskolin (1 µmol/l). This procedure enhanced lumen-negative Vt significantly (-0.4 ± 0.1 vs. -1.6 ± 0.2 mV). Rt fell (36.7 ± 3.0 vs. 32.2 ± 2.6 Omega  · cm2) and lumen-negative Isc was increased from -7.6 ± 1.7 µA/cm2 to -55.7 ± 8.0 µA/cm2 (n = 26). Now, the effect of CCH was examined in the presence of both indomethacin and IBMX and forskolin. A further increase of the lumen-negative Isc to -151.4 ± 27.5 µA/cm2 was observed (Vt = -3.5 ± 0.4 mV, Rt = 30.4 ± 2.7 Omega  · cm2, n = 26) (Fig. 4, A and B). As shown above for the absence of indomethacin, the CCH response was significantly enhanced under these conditions. These results suggest that Ca2+-dependent Cl- secretion in human colonic epithelium requires coactivation of the cAMP-dependent pathway and is only demonstrable when the endogenous cAMP pathway is activated, e.g., due to stimulation by the major autacoid prostaglandin. These results also suggest that the only relevant apical Cl- conductance in human colon epithelial cells is that by cAMP-dependent Cl- channels, corresponding to CFTR. Because, in CF, CFTR is mutated and cannot function as a Cl- channel, colonic Cl- secretion is defective, thus leading to the well-described intestinal manifestations of CF (12).

CCH activates luminal K+ secretion. The reversed lumen-positive response induced by CCH after inhibition of the cAMP pathway could be either due to activation of a basolateral Cl- conductance or, more likely, due to an unmasked parallel activation of a luminal K+ conductance. We addressed this question by comparing the effects of CCH on lumen-positive Vt in the presence or absence of luminal BaCl2 and TEA+. In nine paired experiments with indomethacin in the bath, CCH (100 µmol/l) induced an increase of lumen-positive Isc from -6.3 ± 3.1 to 20.9 ± 6.7 µA/cm2 (Delta Rt = 4.8 ± 0.5 Omega  · cm2) (n = 9). After addition of BaCl2 (5 mmol/l) and TEA+ (10 mmol/l) to the luminal side, the lumen-positive CCH-induced Isc was completely abolished and only a very small lumen-negative Isc of -4.6 ± 4.2 µA/cm2 remained. Furthermore, inhibition of the lumen-positive Isc by BaCl2 and TEA+ was completely reversible on removal (Delta Isc = 29.5 ± 5.7 µA/cm2, n = 9; Fig. 5, A and B). These experiments clearly indicate activation of a K+ conductance in the luminal membrane of human colonic epithelial cells by CCH, which is unmasked when cAMP-dependent apical Cl- channels are blocked by indomethacin.


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Fig. 5.   Inhibition of luminal K+ secretion by luminal Ba2+ (5 mmol/l) and tetraethylammonium (TEA+; 10 mmol/l). A: effect of basolaterally applied CCH (100 µmol/l) in the presence of indomethacin (Indo; 10 µmol/l). After 45 min of incubation with indomethacin, stimulation by CCH induced a lumen-positive Vt. This response was reversibly inhibited by Ba2+ and TEA+. Time gaps between the 3 records were 5 min each. B: summary of the equivalent Isc (Isc = Vt/Rt; Rt was determined from the Vt downward deflections obtained by pulsed current injection) in the presence of indomethacin (10 µmol/l). Mean values ± SE are shown. The CCH effect is inhibited reversibly by Ba2+ and TEA+. * Significantly different vs. without CCH (P < 0.05).

    DISCUSSION
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Procedures
Results
Discussion
References

cAMP and Ca2+ activate different types of K+ channels. The results of the present study indicate that at least two different types of K+ channels exist in the basolateral membrane of human colonic epithelial cells: one activated by Ca2+ and the other by cAMP. Intracellular Ca2+ was increased by CCH, which binds to M3- type receptors on the basolateral side of colonic epithelial cells (25). Although cholinergic stimulation may increase intracellular inositol trisphosphate and Ca2+ as well as diacylglycerol and thus may activate protein kinase C (PKC), Ca2+ probably is the primary mediator because basolateral K+ channels in the colon are directly activated by an increase in intracellular Ca2+ (5). Both types of K+ channels can be distinguished on the basis of their sensitivity toward the recently designed K+ channel blocker 293B (21); the cAMP-activated K+ channel is inhibited by 293B, whereas the Ca2+-activated K+ channel is not (5, 34). Thus electrophysiological properties of the human colon resemble those of the rat. Although the molecular nature of the Ca2+-activated K+ channel is not definitively clarified at this stage (5, 28), the present data strongly suggest that the cAMP-activated K+ channels in the basolateral membranes of human colonic epithelial cells are most likely identical to the KvLQT1 channels that were recently cloned from human heart (2, 29). In addition, overexpression of KvLQT1 in COS-7 cells and Xenopus oocytes identified KvLQT1 as the target for 293B (4, 7, 23). Activation of KvLQT1 channels by cAMP in the basolateral membrane of human colon epithelial cells is essential for cAMP-dependent electrolyte secretion. In this respect, the compound 293B may add a new therapeutic tool for the treatment of secretory diarrhea (21).

Activation of luminal K+ conductance by CCH. Inhibition of the endogenous production of prostaglandins and hence a fall in cytosolic cAMP unmasked activation of apical K+ conductance by an increase in intracellular Ca2+. This occurs in parallel to the activation of basolateral K+ channels. Apical K+ channels were identified in the rat colon in previous reports (8, 30). Normally, the positive Isc due to activation of apical K+ conductance is masked by the parallel activation of luminal Cl- channels. As a net result, CCH enhances lumen-negative Isc. Because of difficulties in performing patch-clamp recordings from luminal membrane of colonic epithelial cells (unpublished observations from our laboratory), it is not currently clear which class of K+ channels accounts for the apical K+ conductance. According to a previous report, expression of luminal K+ channels is modified by dietary K+ and by aldosterone (10, 22, 27). Thus colonic KCl secretion apparently is activated by an increase in intracellular Ca 2+ and depends on dietary K+ uptake and aldosterone.

Cooperativity of Ca2+- and cAMP-activated membrane conductances. Previous patch-clamp studies identified Ca2+-activated Cl- channels in nonpolarized cultured colonic epithelial cells, whereas other studies failed to demonstrate Ca2+-activated Cl- channels in colonic epithelial cells (6, 20). The data of the present study on native human colonic tissue demonstrate that Ca2+-induced Cl- secretion requires activation of apical CFTR Cl- channels and therefore confirm results of previous studies (3, 11, 14, 15, 24, 31). Moreover, inhibitors of Ca2+-activated Cl- channels such as DIDS failed to show inhibitory effects on CCH-induced Cl- secretion when applied to the luminal side of the epithelium (data not shown). Hence, a separate Ca2+-regulated Cl- conductance could not be demonstrated in the luminal membrane of human colonic crypts. The absence of a Ca2+-activated Cl- conductance in the present experiments could also be caused if this specific conductance would require the coactivation of CFTR by cAMP. In fact, in previous experiments with HT-29 colonic carcinoma cells, the amplitude and time course of the Ca2+-activated Cl- currents depended on the expression of CFTR and its prestimulation with cAMP (1). At any rate, although contribution of other cAMP-dependent Cl- conductances cannot be ruled out completely by this and previous studies (17, 18), CFTR seems to be the predominant Cl- conductance in the luminal membrane of colonic crypt cells.

CCH and thus an increase in intracellular Ca2+ seem to activate basolateral and apical K+ conductances in human colon epithelial cells. In addition, the increase of intracellular Ca2+ during stimulation by CCH apparently enhances the activity of CFTR Cl- channels through the activated PKC pathway (16). When CFTR is mutated and thus the apical Cl- conductance is impaired as in CF and in CFTR (-/-) knockout mice, colonic Cl- secretion is abolished (3, 11, 14, 15, 31, 33). From these reports and the present study it becomes obvious that CFTR is essential not only for cAMP-dependent but also for Ca2+-dependent Cl- secretion in the colon.

CCH responses in CF colon and implications for the measurement of CFTR activity. As demonstrated in the present report, characteristic changes as they occur in CF can be mimicked by treatment of the tissue with inhibitors of the prostaglandin synthesis. In this respect the results of another report (12) have to be reconsidered. For that report, residual activation of Isc on CCH-dependent stimulation of colonic mucosa biopsies derived from CF patients were taken as a measure for the severity of the disease. To that end, the tissues were treated with indomethacin for only 10 min, which is, according to our data, too short for complete inhibition of prostaglandin synthesis. Therefore, data obtained under these conditions are difficult to interpret. We suggest that quantification of residual CFTR function by measuring CCH-induced Isc in colonic biopsies of CF patients with different CF phenotype should be obtained only in paired experiments. To that end, endogenous prostaglandin synthesis should be completely inhibited by indomethacin and the effect of CCH should be examined in both the absence or presence of cAMP in paired fashion.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of H. Schauer and G. Kummer.

    FOOTNOTES

K. Kunzelmann is supported by a Heisenberg fellowship. This work was supported by Deutsche Forschungsgemeinshaft Grant Ku756/2-2, AFLM, Zentrum Klinische Forschung 1, and Fritz Thyssen Stiftung.

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: K. Kunzelmann, Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strabeta e 7, 79104 Freiburg, Germany.

Received 15 May 1998; accepted in final form 24 August 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(6):G1274-G1281
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