Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts

Geoffrey I. Sandle1,2, Geoffrey Warhurst2, Ian Butterfield2, Norman B. Higgs2, and Richard B. Lomax2

1 Molecular Medicine Unit, St. James's University Hospital, University of Leeds, Leeds LS9 7TF; and 2 Section of Gastroenterology, Department of Medicine, Hope Hospital, University of Manchester, Salford M6 8HD, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Somatostatin is a powerful inhibitor of intestinal Cl- secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K+ channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl- secretory process. Somatostatin (2 µM) elicited a >80% decrease in "spontaneous" K+ channel activity in cell-attached patches in nonstimulated crypts (50% inhibition =~8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100-200 µM dibutyryl cAMP, 2 µM somatostatin and its synthetic analog octreotide (2 µM) both produced similar degrees of K+ channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl- (5 mM) conditions. In addition, 2 µM somatostatin abolished the increase in K+ channel activity stimulated by 2 µM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca2+. These results indicate that somatostatin peptides inhibit 23-pS basolateral K+ channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca2+ sensitivity.

chloride secretion; G proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHLORIDE SECRETORY DIARRHEA, whether infective, inflammatory, or neurohumoral in origin, remains a common and costly clinical problem that often responds poorly to conventional antidiarrheal drugs. The dominant transport process, electrogenic Cl- secretion through apical Cl- channels, occurs primarily in small intestinal and colonic crypts and is triggered by soluble mediators acting through Ca2+-dependent and/or cAMP-dependent intracellular signaling pathways (5, 18). In the currently proposed model of electrogenic Cl- secretion in intestinal epithelia, in which an increase in apical Cl- conductance is the initial step in the secretory process, a subsequent increase in basolateral K+ conductance promotes cell hyperpolarization and recycling of K+ taken up via basolateral Na+-K+-2Cl- cotransport and Na+-K+-ATPase. This maintains the electrochemical gradient required for sustained apical Cl- exit (1). As a result, maneuvers leading to a decrease in basolateral K+ conductance have a profound inhibitory effect on electrogenic Cl- secretion. Serosal application of Ba2+ to polarized monolayers of the T84 human colonic epithelial cell line markedly inhibits basolateral 86Rb+ (a proxy for K+) efflux and cAMP-stimulated Cl- secretion by blocking a basolateral K+ conductance (27, 29). Inhibition of basolateral K+ conductance has also been implicated in the mechanism of action of other pharmacological agents with antisecretory properties in vitro (25, 34). Patch-clamp studies have confirmed that Ba2+-sensitive 23-pS K+ channels are present in abundance in the basolateral membrane of native human colonic crypt cells (35).

Somatostatin is a tetradecapeptide normally present in intestinal mucosa and has long been recognized as a potent antisecretory peptide capable of inhibiting all forms of Ca2+- and cyclic nucleotide-mediated Cl- secretion (10, 22, 41). This peptide exerts its antisecretory effects via G protein-coupled receptors on the basolateral membrane of the epithelium (39, 41), which in turn interact in a complex way with the Cl- secretory process. Although somatostatin lowers intracellular cAMP concentrations in epithelial cells via G protein-dependent inhibition of adenylate cyclase (38), it has additional antisecretory effects distal to the intracellular second messenger production/activation cascades (41). Somatostatin and octreotide, its more widely used synthetic analog, may therefore act at a fundamental regulatory site in the Cl- secretory process. The aim of this study was to investigate whether basolateral K+ channels are a possible target for the antisecretory effects of these peptides.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of Colonic Crypts

With informed consent (studies approved by the Salford and Trafford Health Authority Ethics Committee), four to five endoscopic biopsies of macroscopically normal sigmoid colonic mucosa were obtained from patients with functional abdominal pain who were not receiving medication at the time of colonoscopy. Colonoscopies were performed after bowel preparation with Klean-Prep (Norgine). Routine histology confirmed the absence of mucosal disease. Biopsies were placed immediately in ice-cold 0.9% NaCl solution, and intact crypts were isolated using a Ca2+ chelation technique (35). Crypts were used for patch-clamp studies on the day of isolation or after overnight storage at 4°C, when more than 85% of crypt cells remained viable on the basis of trypan blue exclusion.

Measurement of K+ Channel Activity

Patch-clamp recordings were made from basolateral membranes of cells in the middle third of intact crypts using cell-attached and excised inside out configurations (19, 35). The bath solution routinely contained (in mM) 140 Na+, 4.5 K+, 1.2 Ca2+, 1.2 Mg2+, 149 Cl-, 10 glucose, and 10 HEPES titrated to pH 7.4 with NaOH. For experiments performed under low-Cl- conditions, all but 5 mM Cl- was replaced with 72 mM SO2-4 and 72 mM mannitol. Studies with monolayers of the cultured human colonic epithelial cell line HT-29-cl.19A showed that this degree of Cl- replacement largely abolished the Cl- secretory response ordinarily stimulated by dibutyryl cAMP, a poorly hydrolyzable membrane permeant analog of cAMP.1 For experiments in which crypt cells were completely depolarized, the bath solution contained (in mM) 145 K+, 1.2 Ca2+, 1.2 Mg2+, 149 Cl-, 10 glucose, and 10 HEPES titrated to pH 7.4 with KOH. Patch electrodes were filled with this high-K+ solution throughout. Experiments were done at 20-22°C because isolated human colonic crypts deteriorated rapidly at 37°C, a phenomenon previously shown to reflect apoptosis (37). Single-channel currents were recorded with a patch-clamp amplifier (model EPC-7, List Electronics, Darmstadt, Germany) at a command voltage of -40 mV referenced to the pipette interior. Currents were stored on videotape after pulse code modulation (model PCM 201ES, Sony). Stored currents were low-pass filtered (750 Hz) and loaded into computer memory (model PC-450, Elonex) via a Labmaster TL1 interface and TM40 A/D converter (Axon Instruments, Foster City, CA) using a sampling frequency of 2.5 kHz. Single-channel open probability (Po) was calculated using an analysis program (gift of Dr. M. Hunter, Department of Physiology, University of Leeds) written in QuickBasic 4.0 (Microsoft). Transitions between fully closed and fully open current levels occurred when the currents crossed a threshold set midway between these two states. Po was calculated as Po = (Sigma ntn)/N where N is the number of channels seen to be open simultaneously while recording (for 30 s) under a specific set of experimental conditions, n represents the state of the channel (0, closed; 1, one channel open; and so forth), and tn is the time spent in state n.

Because of the difficulty experienced in maintaining high-resistance membrane seals throughout the long experimental protocols (up to 40 min), some experiments on individual crypts from each patient had to be aborted. This invariably resulted in only one complete experimental protocol being completed in crypts from a single patient. The number of patches used for each protocol therefore is equal to the number of patients from whom crypts were obtained.

Effect of Somatostatin Peptides on Intracellular cAMP

Crypts were resuspended in Krebs bicarbonate buffer solution containing (in mM) 146 Na+, 4.2 K+, 1.2 Ca2+, 1.2 Mg2+, 126 Cl-, 26.6 HCO-3, 1.2 HPO2-4, 0.2 H2PO-4, 10 glucose, and 1 isobutylmethylxanthine, gassed with 5% CO2-95% O2 to maintain pH 7.4, to which was added either 2 µM somatostatin, 2 µM octreotide, or an equal volume of vehicle (0.001% acetic acid), and incubated at 37°C for 10 min.2 At the end of this period, either 2 µM forskolin or an equal volume of vehicle was added and the incubation was continued for a further 30 min. The reaction was stopped, and intracellular cAMP was extracted by placing the crypts in a boiling water bath for 5 min and then cooling on ice and centrifuging at 12,000 g at 4°C for 10 min. The supernatant was assayed for cAMP using a specific protein binding assay as previously described (41). The pellet was resuspended in 1 M NaOH, and the protein content was measured by the Lowry method. The cAMP levels were expressed as picomoles per milligram of cell protein.

Measurement of Intracellular Ca2+ Concentration

Intact human colonic crypts were isolated as previously described and prepared for fluorescence imaging by incubating in NaCl Ringer solution containing 200 µM dibutyryl cAMP and 5 µM fura 2-AM for 15 min at room temperature, followed by repeated washing (3 times) with fresh NaCl Ringer solution. The 200 µM dibutyryl cAMP was present during all subsequent manipulations of the crypts to maintain similar conditions to those used in the patch-clamp studies. Two or three drops of crypt suspension were placed on a polyethyleneimine-coated glass coverslip mounted in a small chamber on the stage of a Nikon Diaphot inverted microscope, which facilitated continuous perfusion (2 ml/min) of the fura 2-loaded crypts. To reduce possible loss of dye from the crypts, measurements of intracellular Ca2+ concentration ([Ca2+]i) were started immediately after a 5- to 10-min equilibration period, during which time the cells were perfused continuously with NaCl Ringer solution. Cells were epi-illuminated alternately at 340 nm and 380 nm, and the emitted light above 520 nm was captured through a ×40 1.3 numerical aperture objective by an extended ISIS-M video camera (Photonic Science, Robertsbridge, UK) and digitized using a PT50 frame grabber (Perceptics, Knoxville, TN). Consecutive frames obtained during 340 nm and 380 nm excitation were analyzed pixel by pixel using Ionvision software (Improvision, Coventry, UK) to give ratio images every 5 s. [Ca2+]i was calculated with reference to a calibration curve relating nanomolar concentrations of Ca2+ to the 340/380 ratio using sigmoid curve-fitting software. The calibration curve was constructed using commercially available Ca2+-EGTA buffer solutions yielding known free Ca2+ concentrations in the range 0-39.8 µM (Molecular Probes, Eugene, OR) and containing 50 µM fura 2.

Statistical Analyses

Data are expressed as means ± SE. Differences between two means were compared using paired or unpaired Student's t-test as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Patch-Clamp Studies

Studies in the absence of dibutyryl cAMP. To evaluate the effect of somatostatin on basolateral K+ channel activity in the absence of a Cl- secretory agonist, a series of experiments was performed using human colonic crypts not previously exposed to dibutyryl cAMP. Under these conditions, "spontaneous" low-conductance K+ channel activity was identified in 10 out of 34 cell-attached basolateral membrane patches in the middle third of crypts studied on the day of isolation. In excised inside out patches, current recordings over a range of holding voltages (Fig. 1A) provided a current-voltage relationship (Fig. 1B) that confirmed the high K+-to-Na+ permeability ratio (49 ± 6:1) and low unitary conductance (24 ± 3 pS; n = 8 patches) of the channel. Addition of 2 µM somatostatin markedly decreased the Po of spontaneously active K+ channels in six out of six cell-attached patches (Fig. 2A). Figure 2B shows the full time course of three of these experiments (in which recordings were continued during a washout period) in which 2 µM somatostatin produced reversible inhibition of K+ channel activity, Po decreasing from 0.41 ± 0.04 to 0.07 ± 0.06 (P < 0.01). A 50% inhibition occurred after ~8 min, with maximal inhibition at ~20 min after the addition of somatostatin.


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Fig. 1.   Characteristics of basolateral K+ channels. A: recordings from an inside out membrane patch of a human colonic crypt cell at different command voltages (Vcom) referenced to pipette interior (140 mM Na+ in bath, 145 mM K+ in pipette). Broken lines denote closed-channel current levels, downward deflections represent K+ flow from pipette to bath, and upward deflections represent K+ flow from bath to pipette. Unitary conductance at -40 mV was 24 ± 3 pS (n = 8 patches). B: current-voltage relationship of recordings in A. Data fit and reversal potential were obtained using Goldman-Hodgkin-Katz current and voltage equations (16, 20).



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Fig. 2.   Reversible inhibition of basolateral K+ channels by somatostatin (SOM). A: recordings from a cell-attached basolateral membrane patch on a human colonic crypt cell (command voltage of -40 mV, 140 mM Na+ in bath, 145 mM K+ in pipette). Broken lines denote closed-channel current levels, downward deflections represent K+ flow from pipette to cell, and upward deflections represent K+ flow from cell to pipette. Channel activity shown in basal state (top trace), 20 min after addition of 2 µM somatostatin (middle trace), and 5 min after washout of somatostatin (bottom trace). B: time course of reversible inhibition of K+ channel activity [single-channel open probability (Po)] by 2 µM somatostatin. Data points are means ± SE from 3 patches.

Because previous studies have shown that the 23-pS K+ channel is moderately voltage dependent (Po decreasing with progressive membrane depolarization) (35), the inhibitory effect of somatostatin on the K+ channel may simply reflect changes in membrane potential (resulting in a decrease in Po) rather than a specific action on the channel. To address this possibility, the effect of somatostatin was studied in crypts (not pretreated with dibutyryl cAMP) maintained in a high-K+ bath solution (145 mM K+ and 0 mM Na+), which completely depolarized the cells and allowed cell-attached membrane patches to be clamped at predetermined voltages. As shown in Fig. 3, A and C, under symmetrical K+ conditions, basal 23-pS K+ channel activity was voltage dependent, being most marked at hyperpolarizing voltages, and the channel exhibited weak inward rectification (Fig. 3B). The addition of somatostatin decreased 23-pS K+ channel activity over the entire range of command voltages, although the voltage dependency of the channel was retained (Fig. 3, A and C). Inhibition of the 23-pS K+ channel was not associated with a significant change in unitary current (Fig. 3, A and B). Furthermore, as shown in Fig. 3A, some basolateral membrane patches also contained the less-abundant 138-pS K+ channel (26), whose activity was unchanged by somatostatin, which indicates that the inhibitory effect of somatostatin is specific to the 23-pS K+ channel.


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Fig. 3.   Voltage-independent inhibition of basolateral K+ channels by somatostatin. A: recordings from a cell-attached basolateral membrane patch in human colonic crypt cells (145 mM K+ in bath and pipette) showing coexisting 23-pS and 138-pS K+ channels in basal state and 17 min after addition of 2 µM somatostatin to bath. Broken lines denote closed-channel current levels, downward deflections represent K+ flow from pipette to cell, and upward deflections represent K+ flow from cell to pipette. B: current-voltage relationship of 23-pS K+ channels showing weak inward rectification. Data points are mean values (error bars omitted for clarity) obtained before (open circle ) and 20 min after () addition of somatostatin (n = 5 patches). Data points pre- and post-somatostatin overlap at -40 mV and 40 mV. Unitary currents were unchanged by somatostatin. C: summary of corresponding K+ channel activity at hyperpolarizing and depolarizing voltages before (open circle ) and after () addition of somatostatin.

Studies in the presence of dibutyryl cAMP. We then went on to study the relation between intracellular cAMP, electrogenic Cl- secretion,1 and the ability of somatostatin to inhibit the 23-pS basolateral K+ channel. Somatostatin inhibits adenylate cyclase (see Effect of Somatostatin Peptides on Intracellular cAMP) and reduces intracellular cAMP concentrations in many cell types, including intestinal epithelial cells (23, 41). We therefore performed a further series of experiments to evaluate the inhibitory effect of somatostatin on 23-pS K+ channel activity using human colonic crypts pretreated for 30 min with 100 µM dibutyryl cAMP to maintain high intracellular cAMP levels. Somatostatin inhibited K+ channel activity in dibutyryl cAMP-pretreated crypts to a similar degree (Po decreasing from 0.45 ± 0.03 to 0.19 ± 0.05, n = 9 patches, P < 0.01) and over the same time course as that seen in crypts not pretreated with dibutyryl cAMP. Pretreatment with a higher concentration (200 µM) of dibutyryl cAMP also failed to attenuate the inhibitory effect of somatostatin on K+ channel activity (data not shown). Octreotide (2 µM), a long-acting synthetic somatostatin analog used in the treatment of secretory diarrhea, also markedly reduced K+ channel activity in crypts pretreated with dibutyryl cAMP (Po decreasing from 0.47 ± 0.03 to 0.18 ± 0.04, n = 5 patches, P < 0.05).

In common with most of the cellular actions of somatostatin peptides, the antisecretory effects of somatostatin in intestinal epithelial cells are mediated by activation of G protein-coupled receptors and can be blocked by pertussis toxin (41). With the use of crypts stored overnight at 4°C, we investigated whether the somatostatin-induced inhibition of the 23-pS basolateral K+ channel was mediated via a similar mechanism by pretreating crypts with pertussis toxin (200 ng/ml) for 18 h. Figure 4 shows that, in the presence of 100 µM dibutyryl cAMP, somatostatin had no significant inhibitory effect on K+ channel activity in crypts pretreated with pertussis toxin. In contrast, K+ channels in crypts stored overnight and subsequently exposed to 100 µM dibutyryl cAMP but not pretreated with pertussis toxin exhibited the characteristic inhibition by somatostatin. These data are consistent with somatostatin inhibiting this population of K+ channels via a G protein-dependent process.


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Fig. 4.   Effect of pertussis toxin on somatostatin inhibition of basolateral K+ channels. Channel activity (Po) was measured in cell-attached basolateral membrane patches on human colonic crypt cells (command voltage -40 mV, 140 mM Na+ in bath, 145 mM K+ in pipette) before and at 5-min intervals after addition of 2 µM somatostatin to bath (arrow). Crypts were pretreated with 200 ng/ml pertussis toxin (PTX, , n = 4) or solvent control (open circle , n = 4) for 18 h and with 100 µM dibutyryl cAMP for 30 min. Data points are means ± SE. * P < 0.05 compared with value at time (t) = 0.

In addition, we considered whether the decrease in basolateral K+ channel activity elicited by somatostatin peptides might result indirectly from their action at another site in the Cl- secretory process (e.g., inhibition of Cl- channel activation). We therefore studied K+ channel activity independently of electrogenic Cl- secretion by incubating dibutyryl cAMP-pretreated crypts for 1-2 h in a high-Na+, low-Cl- (5 mM) bath solution, a maneuver that virtually abolished dibutyryl cAMP-stimulated Cl- secretion in monolayers of the HT- 29-cl.19A human colonic epithelial cell line.1 Under these conditions, the inhibitory effect of somatostatin on K+ channel activity persisted, with 2 µM somatostatin decreasing Po from 0.41 ± 0.09 to 0.03 ± 0.02 (n = 5 patches, P < 0.01). This decrease in Po (Delta Po 0.38 ± 0.09) did not differ significantly (P > 0.2) from that produced by somatostatin under high-Cl- conditions (Delta Po 0.26 ± 0.05, see above). Together, these observations show that somatostatin peptides exert a specific inhibitory effect on basolateral K+ channels, which is independent of both the intracellular level of cAMP and the prevailing rate of electrogenic Cl- secretion.

Studies in the presence of thapsigargin. In addition to the evidence that somatostatin inhibits intestinal Cl- secretion independently of changes in intracellular cAMP concentration, this peptide has been shown to prevent Cl- secretion stimulated by Ca2+-dependent secretagogues in a human colonocyte line, apparently by acting at a site distal to the process(es) involved in raising [Ca2+]i (41). Because the 23-pS basolateral K+ channels in human colonic crypts are Ca2+ sensitive (26, 35), we explored the possibility that somatostatin might alter this channel characteristic. As shown in Fig. 5, addition of 2 µM thapsigargin in the absence of somatostatin produced a twofold increase in K+ channel activity after 10 min in cell-attached patches (n = 3 patches, P < 0.01). In contrast, when crypts were pretreated with somatostatin for 20 min, subsequent addition of thapsigargin had no effect on K+ channel activity (n = 4 patches). The ability of somatostatin to block Ca2+ activation of this channel appeared to be independent of intracellular [Ca2+]i per se, because thapsigargin elicited similar sustained increases in [Ca2+]i in both the absence (from 207 ± 24 to 321 ± 31 nM, n = 6 crypts) and in the presence (from 201 ± 12 to 304 ± 25 nM, n = 6 crypts) of somatostatin (Fig. 6). These data suggest that the antisecretory effect of somatostatin may reflect, at least in part, a crucial loss of the K+ channel's ability to respond to [Ca2+]i.


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Fig. 5.   Effect of somatostatin on thapsigargin-induced basolateral K+ channel activity. Channel activity (Po) was measured in cell-attached basolateral membrane patches on human colonic crypt cells pretreated with 100 µM dibutyryl cAMP for 30 min (command voltage -40 mV, 140 mM Na+ in bath, 145 mM K+ in pipette). Somatostatin (2 µM) was added to bath (A) in the somatostatin-treated group (+SOM, , n = 4), and thapsigargin (2 µM) was added to bath (B) in somatostatin-treated group and somatostatin-untreated group (-SOM, open circle , n = 3). Data points are means ± SE. * P < 0.01 and ** P < 0.05 compared with Po value at t = 30 min in somatostatin-untreated group. # P < 0.002 and ## P < 0.01 compared with equivalent time points in somatostatin-untreated group.



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Fig. 6.   Effect of somatostatin on thapsigargin (THAPS)-induced rise in intracellular Ca2+. Intracellular Ca2+ levels were measured at midpoint of crypts pretreated with 100 µM dibutyryl cAMP. Somatostatin (2 µM) was added to one group of crypts (+SOM, n = 6) 15 min before (during which time it had no effect on intracellular Ca2+ time zero levels), whereas somatostatin-untreated group (-SOM, n = 6) acted as controls. Thapsigargin (2 µM) was added to both groups at time shown. Data are mean responses for both groups (error bars omitted for clarity).

Effect of Somatostatin Peptides on Intracellular cAMP

With the use of crypt preparations from four patients, the intracellular cAMP level was 15.1 pM/mg cell protein under basal conditions, rising to 332 pM/mg cell protein after the addition of 2 µM forskolin to stimulate adenylate cyclase. Whereas the prior addition of 2 µM somatostatin or 2 µM octreotide had no effect on the basal intracellular cAMP level, both somatostatin peptides produced modest decreases in intracellular cAMP [to 88.7 ± 2.8% (P < 0.005) and 83.7 ± 2.7% (P < 0.005), respectively, of the maximal level] in the forskolin-treated crypts. These data indicate that the ability of somatostatin and octreotide to decrease spontaneous basolateral K+ channel activity does not reflect inhibiton of adenylate cyclase. In addition, although we used exogenous dibutyryl cAMP rather than forskolin to activate electrogenic Cl- secretion during the patch-clamp studies, it seems likely that even in the presence of somatostatin peptides, forskolin-stimulated intracellular cAMP remains at levels that would normally (that is, in the absence of somatostatin and octreotide) sustain the Cl- secretory process.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the accepted model of intestinal Cl- secretion, activation of basolateral K+ channels plays a key role in maintaining the driving force for Cl- secretion by promoting cell hyperpolarization and the recycling of K+ taken up across the basolateral membrane via the Na+-K+-2Cl- cotransporter and Na+-K+-ATPase (1). Studies based on transepithelial electrical measurements suggest the presence of two pharmacologically distinct basolateral K+ conductances in cultured monolayers of T84 human colonic adenocarcinoma cells (4, 9). One is activated during cAMP-stimulated electrogenic Cl- secretion and is profoundly inhibited by serosal Ba2+, whereas the other is stimulated by Ca2+-mediated Cl- secretory agonists and is Ba2+ insensitive. With the use of patch-clamp recording techniques, we have also identified two distinct types of basolateral K+ channel in human colonic crypt cells. One is the 23-pS K+ channel we describe here, which is activated by exogenous cAMP and carbachol (a Ca2+-mediated secretory agonist) and is readily inhibited by Ba2+ (35). The other is a 138-pS K+ channel, which is also Ba2+ sensitive but is unaffected by cAMP and carbachol (26). It is obviously difficult to make a direct comparison between single-channel data obtained from native human colonic crypt cells and transepithelial data obtained from a malignantly transformed colonic epithelial cell line. It therefore remains unclear whether the inwardly rectifying 23-pS basolateral K+ channel we have identified in human colonic crypts equates with either of the basolateral K+ conductances described in T84 cells. However, the 23-pS K+ channel may correspond to the Ca2+-activated inwardly rectifying K+ channel of intermediate conductance identified in single (nonpolarized) T84 cells (7). The apparent responsiveness of 23-pS K+ channels to dibutyryl cAMP in human colonic crypt cells (35) may therefore reflect basal [Ca2+]i being higher in these cells (207 ± 24 nM; see Fig. 6) than in T84 cells (117 ± 7 nM; see Ref. 9), leading to a complex interplay between cAMP- and Ca2+-mediated signaling pathways and secondary activation of basolateral K+ channels during cAMP-stimulated Cl- secretion. Indeed, a rise in basal [Ca2+]i during the crypt isolation procedure may account for the significant levels of spontaneous 23-pS K+ channel activity that we saw in some crypts even in the absence of dibutyryl cAMP.

Although we have shown that 23-pS K+ channels predominate in the basolateral membrane of native human colonic crypt cells (26), they differ widely from basolateral K+ channels in intestinal epithelia in other species. Rat small intestinal enterocytes possess Ca2+- and voltage-dependent 250-pS basolateral K+ channels (30), but their physiological role is unclear. The basolateral membrane of cells at the base of rat distal colonic crypts is rich in Ca2+- and voltage-dependent 12-pS K+ channels that may help to maintain cell membrane voltage (3), has prostaglandin E2-stimulated 27- to 39-pS nonselective cation channels that may be involved in active K+ secretion (36), and contains an infrequent 187-pS K+ channel with no known function (3). The 12-pS K+ channel in rat colon may correspond to the Ca2+-sensitive inwardly rectifying 10- to 20-pS basolateral K+ channel that is activated during carbachol-induced (that is, Ca2+-mediated) Cl- secretion and inactivated during cAMP-induced Cl- secretion (2, 17, 32). Thus despite the evidence that basolateral K+ channels are an important component of the generally proposed model of intestinal Cl- secretion (1), the precise nature of these channels and their mode(s) of regulation are likely to show appreciable species-to-species variability.

Studies over many years have shown that somatostatin and its related peptides are extremely effective inhibitors of intestinal Cl- secretion stimulated by all types of secretagogue, including those acting through cAMP- and Ca2+-dependent processes (12, 22, 41). This has been the rationale for the clinical use of long-acting somatostatin analogs (such as octreotide) in the treatment of persistent Cl- secretory diarrhea (15). Octreotide therapy is now widely used with varying degrees of success to treat refractory diarrheas, such as those associated with carcinoid syndrome as well as high-output ileostomies (6, 42). Although previous studies in several species have alluded to an inhibitory interaction between somatostatin peptides and enterocyte K+ channels, the cellular mechanisms by which they exert such a comprehensive inhibition of the Cl- secretory process remain unclear. Inhibition of adenylate cyclase, as observed in the HT-29-cl.19A colonic cell line (41) and forskolin-treated human colonic crypts in the present study, suggests that direct interference with the production of intracellular second messengers is likely to be one component of somatostatin's antisecretory action. However, these peptides are also effective against agents that activate secretion distal to second messenger production. In the same study using HT-29-cl.19A cells, Cl- secretion activated by the Ca2+-mediated agonist carbachol was markedly inhibited by somatostatin via a pertussis toxin-dependent process without affecting the agonist-stimulated rise in [Ca2+]i (41). Similarly, somatostatin peptides inhibited dibutyryl cAMP-activated Cl- secretion in HT-29-c1.29A cells and rat colon (39, 41). In rat colonic crypts, somatostatin decreased carbachol-activated whole-cell K+ currents, although the type of K+ channel involved and its membrane location were not defined (11). Furthermore, studies in guinea pig enterocytes showed that activation of G proteins by the intracellular application of GTPgamma S inhibited whole cell K+ currents (14), but no attempt was made to identify the physiological regulator of this process. Despite the implication from such studies that somatostatin receptors couple to a fundamental regulatory site in the Cl- secretory process, which lies downstream to the production of intracellular second messengers, the nature and location of this site have been unclear.

In the present study, we show that somatostatin and octreotide reversibly inhibit spontaneous basolateral K+ channel activity via a mechanism that involves pertussis toxin-sensitive inhibitory G protein(s). Moreover, their inhibitory effect is independent of raised intracellular cAMP levels and Cl- secretion per se. Indeed, based on the studies with thapsigargin (Figs. 5 and 6), one possible interpretation of our data is that somatostatin- and octreotide-induced inhibition of the 23-pS K+ channel reflects loss of the channel's inherent Ca2+ sensitivity. An alternative interpretation, that somatostatin peptides cause a loss of the channel's sensitivity to cAMP, is possible but is not supported by our finding that somatostatin and octreotide both inhibited spontaneous K+ channel activity in crypts not pretreated with dibutyryl cAMP. In any event, the inhibitory effect appears to be restricted to the 23-pS K+ channel because somatostatin had no effect on the 138-pS K+ channel, which is also located in the basolateral membrane of human crypt cells (Fig. 3A and Ref. 26).

The precise mechanism through which somatostatin peptides inhibit 23-pS basolateral K+ channels in human colonic crypts is unknown and will be an important goal of future studies. One possibility to be considered is that the proposed loss of K+ channel sensitivity to Ca2+ may reflect a change in the phosphorylation state of the channel protein, which is an important determinant of the Ca2+ sensitivity of intestinal K+ channels (21). Furthermore, in rat pituitary tumor cells, somatostatin induces dephosphorylation of Ca2+-activated K+ channels via activation of arachidonic acid release and metabolism (13). In this respect it is interesting that arachidonic acid has recently been shown to directly inhibit Ca2+-dependent K+ channels in the T84 human colonic cell line (8).

The selective blockade of basolateral K+ channels as a means of controlling epithelial Cl- secretion has been explored by several groups. At the simplest level, the nonspecific K+ channel blocker Ba2+ inhibits 23-pS basolateral K+ channels in human colonic crypts (35) and causes almost complete cessation of Cl- secretion when added to the basolateral surface of colonic cell monolayers (27, 29). Recently, novel K+ channel blockers have been shown to inhibit cAMP-activated K+ conductances and Cl- secretion in rabbit colon (25). The antifungal antibiotic clotrimazole has also been shown to prevent fluid and electrolyte secretion in rabbit and mouse intestine triggered by Ca2+- and cAMP-mediated agonists via specific inhibition of basolateral K+ conductances (34). Furthermore, levamisole (and other phenylimidazothiazoles) inhibits Ca2+- and cAMP-activated Cl- secretion in T84 cell monolayers and isolated human distal colon, apparently by blocking basolateral K+ channels (31). Our finding that somatostatin, one of the most potent and comprehensive inhibitors of intestinal secretion, has a marked inhibitory effect on 23-pS basolateral K+ channels in human colonic crypts highlights this component of the intestinal Cl- secretory process as the starting point for new antidiarrheal strategies. To this end, we should emphasize that somatostatin peptides elicit their diverse cellular actions by activating a family of G protein-coupled somatostatin receptors (SSTR) (33), several of which are expressed in colonic epithelia (24, 39, 40). Studies using receptor-selective agonists in rat colon indicate that the antisecretory actions of somatostatin in this epithelium are mediated by a specific receptor, SSTR2 (28, 39). Identification of the SSTR subtypes linked to basolateral K+ channels in human small intestinal and colonic epithelia and a better understanding of the signaling processes involved may provide a basis for the development of new antisecretory drugs with greater potency and specificity.


    ACKNOWLEDGEMENTS

This study was supported by grants from the Sir Jules Thorn Charitable Trust, Medical Research Council, Wellcome Trust, Royal Society, and the North West Regional Health Authority.


    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. §1734 solely to indicate this fact.

1 Dibutyryl cAMP-stimulated Cl- secretion was studied in confluent monolayers of the human colonic HT-29-cl.19A cell line mounted between Ussing chambers containing Krebs bicarbonate solution (38). Increases in short-circuit current (Isc) reflected dibutyryl cAMP-stimulated electrogenic Cl- secretion (41). For experiments under low-Cl- (5 mM) conditions, monolayers were bathed with a solution in which 121 mM Cl- was replaced with equimolar gluconate. Isc was monitored at 5-min intervals before and after the basolateral addition of 200 µM dibutyryl cAMP. Under high-Cl- conditions, dibutyryl cAMP elicited a peak rise in Isc of 20.3 ± 2.2 µA/cm2 after 30-35 min (n = 5). In contrast, under low-Cl- conditions dibutyryl cAMP stimulated only a small rise in Isc of 2.4 ± 0.5 µA/cm2 (n = 5). These data confirm that dibutyryl cAMP-stimulated electrogenic Cl- secretion by human colonocytes is substantially reduced (by 88%, P < 0.001) under low-Cl- conditions.

2 The aim of this study was to establish the principle that somatostatin peptides influence basolateral K+ channels in Cl- secretory epithelia. We therefore used both somatostatin and octreotide at a concentration of 2 µM, because the addition of 5 µM somatostatin to the basolateral bathing solution has been shown to inhibit prostaglandin E2-induced electrogenic Cl- secretion by 90% in HT-29-cl.19A cell monolayers (41).

Address for reprint requests and other correspondence: G. I. Sandle, Molecular Medicine Unit, St. James's Univ. Hospital, Beckett St., Leeds LS9 7TF, UK (E-mail: g.i.sandle{at}leeds.ac.uk).

Received 16 June 1998; accepted in final form 2 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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