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. 2003 Feb 21;548(Pt 2):475–484. doi: 10.1113/jphysiol.2002.036806

Regulation of Cl− secretion by α2-adrenergic receptors in mouse colonic epithelium

Rebecca S Lam *, Ernst M App *, Drew Nahirney *, Artur J Szkotak *, Maria A Vieira-Coelho †, Malcolm King ‡, Marek Duszyk *
PMCID: PMC2342847  PMID: 12598592

Abstract

Previous studies have shown that α2 adrenoceptor (α2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of α2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that α2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that α2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 μg ml−1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+–K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 μm). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of α2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that α2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by α2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, α2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

The α2-adrenergic receptors (α2ARs) belong to the seven transmembrane domain superfamily of G protein-coupled receptors (GPCRs) and mediate many of the physiological effects of the native catecholamines, adrenaline and noradrenaline (Guimaraes & Moura, 2001). The primary signal transduction pathway of α2ARs is through pertussis toxin (PTX)-sensitive Gi/o proteins, which leads to inhibition of adenylyl cyclase and a reduction in intracellular cAMP (Cotecchia et al. 1990). However, under certain circumstances α2ARs can also couple to Gs proteins leading to activation of adenylyl cyclase (Eason et al. 1992). Other physiological signalling pathways mediated by α2ARs include stimulation of phospholipases (A2, C and D), and inhibition of voltage-gated Ca2+ channels (Graham et al. 1996). Three distinct α2AR subtypes have been described (α2A, α2B and α2CAR) based on molecular and pharmacological criteria (Guimaraes & Moura, 2001). These subtypes exhibit different cellular and tissue distributions, suggesting distinct physiological functions.

The presence of α2ARs in the intestinal mucosa has been demonstrated in earlier studies (Valet et al. 1993). These receptors were shown to be of the α2A subtype, and their distribution suggested preferential localization in the basolateral membranes of the proximal colon. The source of native α2AR agonists may be the noradrenergic fibres that extensively innervate the intestinal mucosa, endocrine cells within the epithelial layer, or circulating catecholamines. While the presence of α2ARs on enterocyte membranes implies a direct interaction between catecholamines and the epithelium, the mechanisms of α2AR-mediated effects and the nature of their molecular interaction with ion channels remain poorly defined.

Classically, regulation of epithelial transport processes occurs in response to agents that alter cyclic nucleotide or [Ca2+]i levels, affecting mainly apical anion channels and basolateral K+ channels. Although CFTR Cl− channels represent a major pathway for anion movement across the apical membrane, the contribution of outwardly rectifying Cl− channels, Ca2+-dependent Cl− channels and members of the ClC Cl− channel family may also be important (Tarran et al. 1998; Barrett & Keely, 2000). At least four biophysically and pharmacologically distinct types of K+ channel have been shown to contribute to the basolateral K+ conductance: a cAMP-activated K+ channel (KCNQ1), an intermediate conductance Ca2+-activated K+ channel (IK-1), a large conductance Ca2+-activated K+ channel (BK), and an ATP-dependent K+ channel (KATP) (Cuthbert et al. 1999b; McNamara et al. 1999; Schultz et al. 1999). These channels are thought to play a crucial role in the regulation of the overall process of chloride secretion.

α2AR agonists have been shown to inhibit electrolyte secretion in human colonic epithelial cell lines (Warhurst et al. 1993; Holliday et al. 1997), rabbit ileum (Fondacaro et al. 1988) and rat jejunum (Vieira-Coelho & Soares-da-Silva, 1998). Although this type of regulation may be of clinical and pharmacological relevance in diseases characterized by abnormal intestinal secretion, the molecular mechanisms involved in this process are not well understood. Therefore, our main objective was to identify ion channels and transporters affected by α2AR agonists. Our data indicate that the main targets of α2AR action are basolateral KATP channels. These channels are inhibited by a process that requires activation of Gi/o proteins but is independent of the cAMP- or Ca2+-mediated pathways.

METHODS

Epithelial cells

The colonic epithelia used in this study came from four different strains of mice: BALB/c, NMRI, C57BL/6J and cystic fibrosis (CF) mice. The breeding colony of CF mice (B6.129S6-Cftrtm1Kth, Jackson Laboratory, Bar Harbor, ME, USA) was housed in a specific pathogen-free environment (Health Sciences Laboratory Animal Services, University of Alberta, Canada). Breeder pairs were heterozygous for a CTT deletion mutation of the CFTR gene, which results in the loss of a phenylalanine residue in exon 10 and corresponds to the human position 508. Pups were weaned at 21 days of age and genotyped by PCR amplification of ear clip genomic DNA, according to established methods (Jackson Laboratory). Pups were either wild-type homozygous (+/+), heterozygous (+/ΔF), or homozygous for mutant CFTR (ΔF/ΔF) (Cftrtm1Kth, referred to as CF). No differences in the behaviour of intestinal epithelium from CFTR(+/+) and CFTR(+/ΔF) mice have been found (Zeiher et al. 1995), and no distinction between the two types is made in this study. All experiments described in this study were carried out with the approval of the Health Sciences Animal Policy and Welfare Committee, University of Alberta, Canada.

Mice were killed by exposure to a rising concentration of CO2 gas, and 6 cm-long pieces of colon were removed from ∼2 cm below the caecum and immediately placed in cold Krebs-Henseleit (KH) solution containing (mm): 116 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11.1 glucose, pH 7.4. The colons were opened up and the muscle layers dissected away. Usually, two pieces of 0.2 cm2 were taken from proximal and distal colon, and mounted in Ussing chambers. In experiments requiring Cl−-free KH solution, NaCl and KCl were replaced by equimolar sodium gluconate and potassium gluconate, respectively, and 2.5 mm CaCl2 was replaced by 5 mm calcium gluconate to compensate for the Ca2+-buffering capacity of gluconate. In experiments requiring HCO3−-free KH solution the composition was (mm): 141 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 11.1 glucose and 10 Hepes, pH 7.4.

Transepithelial measurements

Standard techniques were used in Ussing chamber studies. The tissues were bathed on apical and basolateral sides with 10 ml of KH solution which was warmed to 37 °C and continually circulated with a gas lift by bubbling with 95 % O2–5 % CO2. Chemicals were added from concentrated stock solutions and both chambers were continuously and separately perfused to ensure proper oxygenation and stirring of the solutions. The transepithelial potential difference was clamped to zero using a DVC 1000 amplifier (WPI, Sarasota, FL, USA) and the resulting short circuit current (Isc) was recorded through Ag-AgCl electrodes and 3 m KCl agarose bridges. The Isc was allowed to stabilize for 10–15 min before application of α2 agonists or other tested chemicals. Positive currents were defined as anion secretion or movement from the basolateral to the apical side. The transepithelial resistance was continuously monitored and calculated, using Ohm's law, by measuring current changes in response to 0.5 mV pulses. The data were collected and stored using a PowerLab 8SP series (ADInstruments, NSW 2154, Australia).

For basolateral membrane K+ current measurements, apical NaCl was replaced by equimolar potassium gluconate, while basolateral NaCl was substituted with sodium gluconate and the Ca2+ concentration was increased to 5 mm in both solutions. In addition, 100 μm ouabain was added to the basolateral compartment to inhibit the Na+–K+-ATPase. Subsequent permeabilization of the apical membrane with nystatin (90 μg ml−1) allowed measurement of K+ current as these ions move down their concentration gradient through basolateral K+ channels.

Unidirectional ion fluxes

To measure chloride flux from the basolateral to apical side, epithelia were short circuited as described above. The radioisotope 36Cl− (3 μCi; Amersham Pharmacia Biotech, UK) was added to the basolateral side, 30 min allowed for equilibration and then two 0.5 ml samples were taken from the apical side and replaced with fresh KH solution; this point was considered time zero. Samples were taken thereafter at 10 min intervals for the next 30 min, followed by the addition of UK 14,304 and further sampling for another 30 min. Basolateral samples taken just prior to UK 14,304 addition gave the specific activity for chloride. This information together with that from the apical samples allowed the calculation of the Cl− flux change caused by UK 14,304. This change was compared to the change in the Isc response, calculated from the integral of the Iscversus time record. 36Cl− fluxes in the apical to basolateral direction were measured in exactly the same fashion, except that the radioisotope was added to the apical bathing solution.

Measurement of intracellular Ca2+ concentration

Changes in [Ca2+]i were measured in isolated colonic crypts using fura-2 as the reporter molecule. A ∼6 cm-long piece of mouse colon was everted, tied at both ends and filled with low calcium (LC) solution as previously described (Duszyk et al. 2001). The LC solution contained the following (mm): 127 NaCl, 5 KCl, 1 MgCl2, 5 glucose, 10 Hepes, 5 EDTA, and in addition 1 % bovine serum albumin (BSA), pH 7.4. The distended and everted colon was submersed in LC solution at 37 °C for 5 min with gentle shaking. At the end of 5 min the colonic preparation was shaken vigorously for 2 min, the tissue discarded and the crypt suspension separated by gentle centrifugation. The crypts were resuspended in KH solution and loaded with 2.5 μm fura-2 AM at 37 °C for 15 min, and then washed with KH solution to remove untrapped dye before the beginning of each experiment. Groups of cells (typically 5) were illuminated with light delivered from a 100 W mercury lamp via a × 40, 1.3 NA, oil-immersion objective. Cell fluorescence was analysed with an imaging system (TILL Photonics, Eugene, OR, USA), which included a monochrometer capable of switching the excitation wavelength in < 1 ms and a cooled CCD camera synchronized to integrate the emitted light at each excitation wavelength. Ratiometric imaging of fura-2 fluorescence was performed at 0.2 Hz with sequential 340 nm/380 nm excitation. Each fluorescence image was collected at 640–480 pixels resolution via a long-pass 510 nm dichroic mirror/emission filter and integrated for 10 ms. After subtraction of background fluorescence at each wavelength of excitation, the fluorescence ratio (R) of fura-2 at 340 nm/380 nm excitation was displayed as a continuous record showing the time course of changes of R from an individual region of interest.

Conversion of R into [Ca2+]i was performed in separate calibration experiments in which individual cells were dialysed in whole-cell recording with intracellular solutions of known [Ca2+] and fura-2 (0.1 mm, potassium salt from Molecular Probes). The three solutions for this calibration were identical to those previously used for calibrating indo-1 (Tse & Tse, 2000) and had [Ca2+] of < 0.1 nm, 212 nm and 15 μm, respectively. R values were converted to [Ca2+]i using the following equation (Grynkiewicz et al. 1985):

graphic file with name tjp0548-0475-mu1.jpg

In this study, Rmin was 0.132, Rmax was 3.4. K is a constant that was determined empirically, and was 2. 724 μm.

Cyclic AMP radioimmunoassay

A radioimmunoassay procedure was used in which the samples were acetylated before analysis (Harper & Brooker, 1975). Colonic epithelia (20 mm2) prepared as for Isc recording were exposed either to 1 μm UK 14,304, or to 10 μm forskolin for 10 min in 50 μl of KH solution, either with or without 3-isobutyl-1-methylxanthine (IBMX) (100 μm) at 37 °C. Acetic anhydride, 50 μl (final concentration 5 mm) was added to stop the reaction. The whole tissue was then frozen and thawed three times, to disrupt the cells, using solid CO2. Aliquots were taken for protein estimation while the remainder was boiled for 5 min, centrifuged at 20 000 g and used for immunoassay. Each measurement was the average of triplicate determinations.

Tissue noradrenaline measurement

The noradrenaline assay was performed by HPLC with electrochemical detection as previously described (Vieira-Coelho & Soares-da-Silva, 1993). Briefly, 0.5 ml tissue samples in perchloric acid were placed in 5 ml vials with 50 mg alumina, and the pH was adjusted to 8.6 with Tris. The adsorbed catecholamines were eluted from the alumina with 200 μl of 0.2 m perchloric acid on Costar Spin-X microfilters, and 50 μl of the eluate was injected into HPLC (Gilson Medical Electronics, France) by an automatic sample injector (Gilson model 231). The detection was carried out electrochemically with a carbon electrode, a Ag-AgCl reference electrode and an amperometric detector (Gilson model 141) operated at 0.75 V. The lower limit of noradrenaline detection ranged from 0.35 to 0.50 pmol.

RT-PCR

Total RNA was isolated from mouse colonic epithelium using the Qiagen RNeasy kit (Qiagen, Mississauga, ON, Canada). First-strand cDNA was synthesized by reverse transcription of the RNA using Superscript II (Invitrogen, Burlington, ON, Canada) and random hexamer primers (200 ng). PCR was performed using the following sets of primers (5′ to 3′): Kir6.1 (GenBank accession number D88159) forward nucleotides 126–145, reverse 1041–1022; Kir6.2 (D50581) forward 243–262, reverse 1066–1047; SUR1 (L40624) forward 2091–2110, reverse 2622–2603; SUR2A/B (D86038) forward 4237–4256, reverse 5076–5057 for SUR2A and 4900–4881 for SUR2B. PCR was performed using the hot-start method. Ten percent of the reverse transcription reaction was combined with 1 μm of each primer, 0.2 mm of each dNTP, 0.75 mm MgCl2, 50 mm KCl, 20 mm Tris-HCl (pH 8.4), 2.5 U of Taq polymerase, and autoclaved double distilled H2O to a final volume of 20 μl. After 3 min at 94 °C, amplifications proceeded for 35 cycles (94 °C, 30 s; 58 °C, 60 s; 72 °C, 3 min) with a final elongation period at 72 °C for 7 min. PCR products were separated and visualized on an ethidium bromide-stained 1.5 % agarose gel by electrophoresis. The expected sizes (bp) of the PCR products were: 916 (Kir6.1), 824 (Kir6.2), 532 (SUR1), 840 and 664 (SUR2A and SUR2B, respectively).

Chemicals

Amiloride (10 mm), clonidine (1 mm), ouabain (10 mm), rauwolscine (1 mm) and tetrapentylammonium chloride (TPA, 100 mm) were dissolved in H2O. Furosemide (frusemide) and IBMX were prepared as a 100 mm stock solution in H2O with a drop of 1 m NaOH. Charybdotoxin was prepared as a 10 μm stock in KH solution containing 0.1 % BSA. Bumetanide, clotrimazole, forskolin, medetomidine, phentolamine, tolbutamide and UK 14,304 were made as at least 1000-fold stock solutions in ethanol. Diazoxide (300 mm) and prazosin (10 mm) were dissolved in dimethyl sulphoxide (DMSO). Nystatin was prepared as a 180 mg ml−1 stock solution in DMSO and sonicated for 30 s just before use. XE991 (a generous gift from Dr B. S. Brown, DuPont, Wilmington, DE, USA) was prepared as a 10 mm stock solution in 0.1 m HCl. Medetomidine and HMR 1098 were generous gifts from Professor K. Starke, University of Freiburg, Germany, and Dr P. E. Light, University of Alberta, Canada, respectively. All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Nystatin and diazoxide were prepared fresh before each experiment. Other chemicals were used from stock solutions stored at either 4 °C or −20 °C.

Data analysis

All data are expressed as means ±s.e.m. along with the number of preparations used (n). Statistical difference was determined by Student's t test. Values of P < 0.05 were considered statistically significant.

RESULTS

Noradrenaline levels in mouse intestine

The total amount of noradrenaline in the proximal colon (including mucosa, submucosa, and muscular layers) was 3.49 ± 0.75 nmol g−1 (n = 5), of which 1.08 ± 0.21 nmol g−1 (or 31.2 %) was from the epithelial layer. For comparison, mouse jejunum contained 1.70 ± 0.37 nmol g−1 (n = 5), of which 0.04 ± 0.02 nmol g−1 (or 2.0 %) was from the epithelial layer. The fact that noradrenaline levels in the proximal colonic epithelium are ∼20-fold higher than in the jejunal epithelium suggests that α2ARs could play a significant role in colonic epithelial cell function.

Effect of α2AR agonists and antagonists on Isc

Application of the specific α2AR agonist, UK 14,304, produced a concentration-dependent (1–10 000 nm, both sides) decrease in the baseline current, with IC50= 34.7 nm. The effect of UK 14,304 (1 μm) was similar in the presence and absence of 10 μm apical amiloride (−21.5 ± 4.1 and −23.6 ± 3.9 μA cm−2, respectively, n = 5, P > 0.05), indicating that α2AR activation had no effect on electrogenic Na+ absorption by epithelial Na+ channels (ENaCs). Similar experiments performed in the presence or absence of apical BaCl2 (5 mm) to inhibit K+ secretion have shown that UK 14,304 does not affect K+ secretion (−18.7 ± 3.6 and −20.1 ± 4.4 μA cm−2, n = 4, P > 0.05). Therefore, all subsequent experiments were performed in the presence of amiloride (10 μm) in the apical compartment. In addition, in Cl− current studies, BaCl2 (5 mm) was also present in the apical compartment.

α2AR agonists are more effective inhibitors of Isc in the proximal colon than the distal colon (Fig. 1). UK 14,304 (1 μm) decreased the Isc from 40.7 ± 4.4 to 19.6 ± 4.2 μA cm−2 (n = 14, P < 0.001, paired t test) in the proximal colon, and from 34.7 ± 6.3 to 23.9 ± 4.2 μA cm−2 (n = 13, P < 0.01, paired t test) in the distal colon. Thus, activation of α2ARs inhibits ∼52 % of the proximal and ∼31 % of the distal colon baseline Isc. Similar results were obtained with two other α2AR agonists, medetomidine (1 μm, both sides, n = 3) and clonidine (10 μm, both sides, n = 6; data not shown). The α2AR antagonists, rauwolscine (1 μm, n = 4) and phentolamine (1 μm, n = 11), as well as the α1 adrenoceptor antagonist, prazosin (1 μm, n = 4), did not affect the Isc in any segment of the colon. In addition, the presence of prazosin (1 μm, n = 4) in the bath solution had no effect on Isc inhibition by UK 14,304. Tissue conductances of the proximal and distal colon in KH solution were 31.1 ± 1.1 mS cm−2 (n = 57) and 20.5 ± 0.9 mS cm−2 (n = 49), respectively, and were not significantly changed by UK 14,304 treatment (data not shown).

Figure 1. Inhibition of Isc by the α2AR agonist UK 14,304 in the distal and proximal colon.

Figure 1

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Isc was more effectively inhibited by UK 14,304 (1 μm, both sides) in the proximal than in the distal colon. The data were obtained in Krebs-Henseleit (KH) solution and show means ±s.e.m. from 12 different preparations in each segment. Amiloride (10 μm) and BaCl2 (5 mm) were present in the apical compartment to inhibit Na+ absorption and K+ secretion, respectively.

α2ARs inhibit CFTR-mediated Cl− secretion

Figure 2 shows that UK 14,304 (1 μm) inhibited baseline Isc in control but not in CF colonic epithelia. The data are representative of four different experiments with CF tissues. For comparison, the subsequent responses to forskolin and furosemide in both epithelia are also shown. Sequential application of forskolin and furosemide is known to exert opposite effects on Isc in control compared to CF epithelia (Cuthbert et al. 1999a). The results shown in Fig. 2 suggest that activation of α2ARs does not affect forskolin or furosemide responses in either tissue.

Figure 2. Effect of UK 14,304 on Isc in control and CF colonic epithelia.

Figure 2

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UK 14,304 (1 μm, both sides) inhibited baseline Isc in control but not in ΔF508 CF colonic epithelia. For comparison, the responses to forskolin (10 μm, both sides) and furosemide (1 mm, basolateral) in control and CF tissues after UK 14,304 treatment are also shown. The data are representative of four different preparations from CF mice.

Further characterization of the effects of α2AR agonists on anion secretion has been performed in anion replacement studies. In the proximal colon, in Cl−-free solution, UK 14,304 inhibited Isc by 10.8 μA cm−2 (from 29.9 ± 3.9 to 19.1 ± 3.6 μA cm−2, n = 4), whereas in HCO3−-free solution it was inhibited by 41.6 μA cm−2 (from 81.3 ± 5.8 to 39.7 ± 4.0 μA cm−2, n = 13). Despite the marked differences in the baseline Isc in these solutions, the relative effect of UK 14,304 in KH and HCO3−-free solutions was similar (52 % vs. 51 %), but significantly reduced in Cl−-free solution (∼33 %). This suggests that UK 14,304 affects mainly Cl− secretion in colonic epithelia.

A direct way to show that UK 14,304 inhibits Cl− secretion is to measure chloride movement using 36Cl−. Chloride fluxes were measured in both the basolateral to apical and in the apical to basolateral directions. The change in chloride movement in each direction in response to UK 14,304 (1 μm, both sides) was calculated from the specific activity and expressed as a percentage of the Isc response to UK 14,304, obtained by integrating the area under the Isc-time record. The experiments were unpaired, that is each preparation was used to measure flux in only one direction. The results of these studies show that UK 14,304 decreased Cl− flux in the basolateral to apical direction, and significantly increased Cl− backflux (Fig. 3). In all experiments, amiloride (10 μm) and BaCl2 (5 mm) were present in the apical compartment to inhibit Na+ absorption and K+ secretion, respectively, as described earlier (Duszyk et al. 2001). Overall, the net flux decreased by 0.44 μequiv cm−2 h−1, which corresponded to 96 % of that predicted by the Isc.

Figure 3. Effect of UK 14,304 on Cl− fluxes.

Figure 3

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Unidirectional and net Cl− fluxes are shown before and after addition of UK 14,304 (1 μm, both sides). Under baseline conditions the basolateral-to-apical (JBA) and the apical-to-basolateral (JAB) fluxes were approximately equal, resulting in a lack of net Cl− flux (JNET). UK 14,304 decreased JBA and increased JAB (n = 6 in each direction). Thus inhibition of Cl− flux by UK 14,304 is mainly due to an increase in the Cl− backflux rather than a reduction in the forward flux. In all experiments, amiloride (10 μm) and BaCl2 (5 mm) were present in the apical compartment to inhibit Na+ absorption and K+ secretion, respectively. *P < 0.05, **P < 0.01, single-tailed Student's paired t test.

α2ARs control basolateral KATP channels

Chloride flux measurements and the fact that UK 14,304 has no effect on Isc in CF epithelia indicate that α2ARs regulate CFTR-mediated Cl− secretion. Primary control of transepithelial anion secretion occurs at the level of apical anion channels as well as basolateral Na+–K+-ATPases, Na+–K+–2Cl− cotransporters and K+ channels. Each of these components could be a target for transport modulation. The effects of α2AR activation on CFTR Cl− channel function were evaluated by measuring forskolin-activated Isc. Forskolin (10 μm, both sides) increased Isc from 39.2 ± 4.5 to 264.6 ± 2.7 μA cm−2, n = 6. Subsequent application of UK 14,304 reduced Isc by 24.3 ± 8.1 μA cm−2. Under baseline conditions UK 14,304 reduced the Isc from 36.8 ± 4.1 to 19.9 ± 2.4 μA cm−2, and subsequent application of forskolin increased Isc by 216.8 ± 7.8 μA cm−2, n = 6. Statistical analysis of these data indicated that UK 14,304 had similar effects on the baseline and forskolin-activated Isc (P > 0.05).

An inhibitor of the basolateral Na+–K+–2Cl− cotransporter, furosemide (1 mm), reduced forskolin-activated Isc by ∼65 %, but had no significant effect on the baseline current (ΔIsc=−3.8 ± 4.1 μA cm−2, n = 6, P > 0.05 Student's paired t test). In the presence of furosemide, UK 14,304 reduced the Isc by 19.8 ± 3.9 μA cm−2 (n = 4), which is not significantly different from experiments under control conditions. Similar results were obtained with another inhibitor of the basolateral Na+–K+–2Cl− cotransporter, bumetanide (10 μm, n = 6, data not shown). These results indicate that α2ARs do not affect Na+–K+–2Cl− cotransporter function in mouse colonic epithelia.

The effect of α2ARs on basolateral K+ channels was assessed in the presence of ouabain (100 μm, basolateral side) to inhibit the Na+–K+-ATPase, and with the use of the ionophore nystatin in order to bypass the apical membrane (Fig. 4A). In the presence of an apical-to-basolateral-directed K+ gradient, nystatin (90 μg ml−1, apical side) evoked a strong increase in Isc, which rose to a peak value of 325.6 ± 59.8 μA cm−2 (n = 4). When the tissue was pretreated with UK 14,304 (1 μm, both sides), the effect of nystatin was decreased by about half, i.e. the increase in Isc amounted to only 151.6 ± 26.3 μA cm−2 (n = 4). The effect of UK 14,304 on Na+–K+-ATPase activity was investigated in the absence of a K+ gradient, i.e. with standard KH solution on both sides of the tissue (Fig. 4B). Nystatin increased Isc by 108.6 ± 16.9 μA cm−2 and 99.5 ± 10.3 μA cm−2 in the absence and presence of UK 14,304 (1 μm, both sides), respectively (n = 4 in both sets, P > 0.05, Student's unpaired t test). The effect of nystatin was abolished in the presence of basolateral ouabain (100 μm), indicating that the nystatin-induced current in the absence of a K+ gradient is caused by the Na+–K+-ATPase. Thus, these experiments demonstrate that UK 14,304 inhibits basolateral K+ channels but not the Na+–K+-ATPase. Further experiments were designed to identify the K+ channels inhibited by α2ARs.

Figure 4. Effect of UK 14,304 on nystatin-stimulated Isc.

Figure 4

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A, epithelia, pre-treated with ouabain (100 μm, basolateral), were subjected to an apical-to-basolateral K+ gradient and nystatin (90 μg ml−1, apical) was added at the time indicated. B, in the absence of a K+ gradient, i.e. with standard KH solution on both sides, apical nystatin produced a similar increase in Isc in the absence and presence of UK 14,304 (1 μm, both sides). The effect of nystatin was abolished in the presence of ouabain (100 μm, basolateral), indicating that the nystatin-induced current in the absence of a K+ gradient is caused by the Na+–K+-ATPase. Furthermore, UK 14,304 does not affect Na+–K+-ATPase activity. The data are shown as means ±s.e.m. from 4–7 different experiments. The insets show the direction of K+ gradients. gluc, gluconate; ap, apical; bl, basolateral.

At least four biophysically and pharmacologically distinct types of K+ channels contribute to the basolateral K+ conductance. The KCNQ1 channel can be specifically blocked by the cognitive enhancer XE991 (Wang et al. 2000), the IK-1 channel-by an antifungal antibiotic, clotrimazole, the BK channel by charybdotoxin or TPA, and the KATP channel by tolbutamide (McNamara et al. 1999). We used the above-mentioned blockers to identify K+ channels inhibited by α2ARs. We found that XE991 (30 μm, n = 4), clotrimazole (50 μm, n = 6), TPA (100 μm, n = 5) and charybdotoxin (50 nm, n = 4) did not affect Isc inhibition by UK 14,304. However, in the presence of tolbutamide (100 μm), Isc inhibition by UK 14,304 was reduced by 46.2 ± 5.4 % (n = 8), suggesting the involvement of KATP channels. In another study we used the specific KATP opener diazoxide (300 μm) to study its effects on Isc inhibition by α2AR agonists. Diazoxide increased Isc by 15.2 ± 2.8 and by 82.5 ± 10.4 μA cm−2, in KH and HCO3−-free solutions, respectively (n = 4). The diazoxide-activated Isc could be completely inhibited by UK 14,304, confirming the critical role of KATP channels in inhibition of anion secretion by α2ARs (Fig. 5).

Figure 5. Activation and inhibition of KATP channels in colonic epithelia.

Figure 5

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Diazoxide (300 μm, basolateral) causes a strong activation in Isc, which is completely inhibited by UK 14,304 (1 μm, both sides). The data show average responses (mean ±s.e.m.) from four experiments in HCO3−-free solution.

KATP channels are heteromultimers composed of inwardly rectifying K+ channel subunits (Kir6.x) and sulfonylurea receptors (SURs) that associate in a 4:4 stoichiometry to form an octameric unit. Glibenclamide has been shown to inhibit KATP channels containing the SUR1 isoform at nanomolar concentrations, whereas inhibition of channels containing either SUR2A or SUR2B subunits required significantly higher concentrations (Russ et al. 2001). We found that glibenclamide (200 nm, basolateral) changed the baseline current from 32.7 ± 5.8 to 29.9 ± 4.0 μA cm−2 (n = 6, P > 0.05, Student's paired t test), indicating that the KATP channel in mouse colonic epithelium does not contain the SUR1 isoform. We have not used glibenclamide at higher concentrations, where this chemical is also known to inhibit CFTR. However, it has recently been shown that the sulphonylurea derivative HMR 1098 is a selective inhibitor of KATP channels containing the SUR2A subunit (Fox et al. 2002). We used this chemical to investigate the composition of KATP channels in mouse colonic epithelia. HMR 1098 (20 μm, basolateral) changed the baseline Isc from 35.2 ± 9.9 to 33.8 ± 7.8 μA cm−2 (n = 6, P > 0.05, Student's paired t test), indicating that epithelial KATP channels do not contain the SUR2A isoform. This result is consistent with Isc activation by diazoxide, which is known to activate KATP channels containing SUR1 or SUR2B but not SUR2A subunits (Koh et al. 1998; Aguilar-Bryan & Bryan, 1999).

Further identification of the epithelial KATP channel composition was performed using RT-PCR studies. Figure 6 shows the presence of Kir6.1, Kir6.2, SUR1 and SUR2B in mouse colonic epithelia. SUR2A mRNA was not detected.

Figure 6. Expression of KATP channel subunits in murine colonic epithelial cells.

Figure 6

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RT-PCR experiments show the expression of Kir6.1, Kir6.2, SUR1 and SUR2B but not SUR2A mRNAs. The marker lane contains a 100 bp ladder, with the characteristic thick band corresponding to 600 bp. The identity of PCR products was confirmed by sequencing.

α2ARs inhibit anion secretion by activating Gi/o proteins

In many cell types, α2ARs transduce their signal through pertussis toxin (PTX)-sensitive Gi/o proteins. In order to investigate their role in inhibition of Cl− secretion by α2ARs, epithelia were incubated with PTX (500 ng ml−1, 2 h) before application of UK 14,304. In two mouse colon preparations PTX completely prevented Isc inhibition by UK 14,304 (1 μm, both sides). However, in three other preparations less than 45 % of the UK 14,304 response was reduced by PTX. The reason(s) for this considerable variability between animals that have been maintained under the same conditions and are otherwise normal is not certain. Overall, UK 14,304 reduced the Isc by 5.2 ± 1.6 μA cm−2 (n = 14) after PTX treatment. This indicates that more than 75 % of the UK 14,304 response is sensitive to PTX.

Studies with cultured human colonic epithelial cells have shown that both cAMP and Ca2+ signalling pathways mediate inhibition of Cl− secretion by α2ARs (Warhurst et al. 1993; Holliday et al. 1997). Therefore, we used radioimmunoassay to measure cyclic AMP concentrations following exposure of colonic epithelia to either UK 14,304 or to forskolin in the presence or absence of IBMX. The cyclic AMP content of the epithelial tissue plus that released into the medium were measured. We found that UK 14,304 had no significant effect on the intracellular level of cAMP in mouse colonic epithelium (Fig. 7A). To measure if α2ARs affected [Ca2+]i, we used Fura-2 as a reporting molecule and challenged isolated colonic cells with UK 14,304 or ionomycin (Fig. 7B). UK 14,304 treatment changed [Ca2+]i from 242.8 ± 57.1 nm to 251.2 ± 53.2 nm (n = 6 mice, P > 0.05, Student's paired t test). Subsequent application of ionomycin increased [Ca2+]i to 754.8 ± 171.3 nm.

Figure 7. cAMP and intracellular Ca2+ are not involved in the signal transduction pathway activated by UK 14,304.

Figure 7

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A, the amount of cAMP refers to that present in the tissue and that lost into the medium under basal conditions, during UK 14,304 (1 μm) exposure, or during forskolin (10 μm) exposure, in the presence or absence of IBMX (100 μm). Forskolin was used as a positive control. Values are means of 4 triplicate measurements ±s.e.m.*P < 0.05, single-tailed Student's t test, compared with the basal values. B, UK 14,304 (1 μm) has no effect on [Ca2+]i. Ionomycin (10 μm) was used as a positive control. The data show a representative recording of six different preparations.

DISCUSSION

Earlier studies have shown that human intestinal mucosa possessed the highest α2AR density in the proximal colon, and that their number decreased gradually towards the distal section (Valet et al. 1993). Our study shows that α2AR agonists inhibited Cl− secretion with the greatest potency in the proximal section, suggesting a similar gradient of α2AR distribution in the mouse colon.

The α2AR family is comprised of three subtypes of receptors, α2A, α2B and α2C, encoded by distinct genes (Guimaraes & Moura, 2001). The rodent α2AAR differs pharmacologically from the human α2AAR, and is sometimes called α2DAR (Bylund et al. 1994). Studies of α2AR subtype tissue distribution have shown that intestinal epithelia express α2AAR but not α2BAR or α2CAR (Valet et al. 1993; Saunders & Limbird, 1999). These receptors are present in the basolateral membrane of colonic epithelial cells (Valet et al. 1993). The α2ARs have differential sensitivity to prazosin: α2AAR are insensitive (Ki > 300 nm), α2BAR are very sensitive (Ki= 5 nm), and α2CAR have an intermediate sensitivity (Ki= 15–36 nm) (Blaxall et al. 1991). The fact that in our studies prazosin did not affect Isc inhibition by UK 14,304 supports the conclusion that α2AARs mediate the effects of UK 14,304 in colonic epithelial cells.

Experiments with CF mice, Cl− flux measurements, and ion replacement studies indicated that in mouse colonic epithelium α2ARs inhibit transepithelial Cl− secretion. Other studies have shown that α2AR agonists inhibit cAMP-dependent Cl− secretion in human colon (Holliday et al. 1997) and rat jejunum (Vieira-Coelho & Soares-da-Silva, 1998), but activate K+ secretion in rat colon via Ca2+-dependent pathways (Horger et al. 1998; Schultheiss & Diener, 2000). This indicates that the effect of α2AR activation varies amongst species and tissues and may involve activation of different targets and signal transduction pathways.

Intestinal Cl− secretion depends on the coordinated activity of apical Cl− channels as well as basolateral Na+–K+-2Cl− cotransporters, Na+–K+-ATPases and K+ channels. Inhibition of one of these transport mechanisms leads to the inhibition of Cl− secretion. Our data show that in mouse colon α2ARs affect Cl− secretion by inhibiting basolateral KATP channels, in a process that probably requires activation of Gi/o proteins, but does not involve intracellular second messengers such as cAMP and Ca2+.

A number of K+ channels have been identified on the basolateral membrane of mammalian colon (Barrett & Keely, 2000). Interestingly, our studies show that, of these, only KATP channels are affected by α2ARs. These channels are constitutively active, since their blockers inhibit baseline Isc by more than 50 %. KATP channels are heteromultimers composed of inwardly rectifying K+ channel subunits (Kir6.x) and sulfonylurea receptors (SURs) that associate in a 4:4 stoichiometry to form an octameric KATP channel (Aguilar-Bryan & Bryan, 1999). Various combinations of these two subunits convey the heterogeneity in channel properties observed in native cells such as Kir6.2/SUR1 in pancreatic β-cells and neural tissue, Kir6.2/SUR2A in cardiac and skeletal muscles, and Kir6.1/SUR2B or Kir6.2/SUR2B in vascular smooth muscle (Aguilar-Bryan & Bryan, 1999). RT-PCR experiments and pharmacological studies have shown that colonic epithelial cells do not express the SUR2A subunit, and the lack of sensitivity to glibenclamide suggests that the epithelial KATP channel complex does not contain SUR1. Thus, KATP channels in colonic epithelia are likely to be similar to vascular smooth muscle KATP channels, composed of SUR2B and either Kir6.1 or Kir6.2 subunits.

α2ARs are members of the G protein-coupled receptor superfamily that interact primarily with Gi/o proteins (Cotecchia et al. 1990). Most (but not all) physiological signalling pathways linked to α2AR activation involve these PTX-sensitive G proteins. For example, α2AARs were shown to inhibit adenylyl cyclase upon stimulation by UK 14,304 in a PTX-resistant manner (Wong et al. 1992), and to have the potential to couple physically and functionally to other G proteins (Eason et al. 1992). In addition, during the past few years, several reports have described various physiological consequences of GPCR stimulation that were not mediated by G protein activation (for review see Hall et al. 1999). In our experiments, pretreatment of cells with PTX reduced Isc inhibition by α2AR agonists by ∼75 %. This suggests that interaction with Gi/o proteins is an integral part of α2AR function in colonic epithelium, but does not exclude the possibility that some effects of α2AR activation could be mediated via a Gi/o protein-independent pathway.

The regulation of KATP channels by Gi/o proteins has been demonstrated by several authors (Fosset et al. 1988; Kirsch et al. 1990; Ishizaka et al. 1999). These studies showed that PTX-sensitive Gi/o proteins activated KATP channels in pancreatic cells (Fosset et al. 1988), cardiac myocytes (Kirsch et al. 1990), and neurons (Galeotti et al. 1999). Interestingly, our data show that in murine colonic epithelium, activation of Gi/o proteins by α2ARs inhibits KATP channels. This result is consistent with studies showing that α2ARs inhibit Cl− secretion via activation of PTX-sensitive G proteins (Warhurst et al. 1993), and that blockers of KATP channels reduce anion secretion in human colonic epithelial cells (McNamara et al. 1999).

ATP inhibits KATP channels with IC50 values generally in the low micromolar range, 5–20 μm (Aguilar-Bryan & Bryan, 1999). Since the intracellular ATP concentration in most cells lies between 3 and 7 mm, it may be argued that KATP channels should never be open under ATP-rich, physiological conditions. Interestingly, our data show that KATP channels are tonically active in the colonic epithelium. This fact suggests that other regulatory mechanisms are involved in the control of KATP channel activity. One such mechanism may involve increased levels of Mg-ADP, which has been shown to activate K+ channels inhibited by ATP (Dunne & Petersen, 1986; Kakei et al. 1986). Another mechanism may involve phosphatidylinositol phosphates, which facilitate channel activity in the presence of ATP by antagonizing the ATP-induced channel inhibition (Baukrowitz et al. 1998; Shyng & Nichols, 1998). In addition, there is significant evidence that supports the existence of subcellular compartmentalization in different cells (Steinberg & Brunton, 2001). This suggests that not all ATP could gain access to KATP channels, limiting effective ATP concentration in the vicinity of the channel.

In conclusion, our results provide new insights into the mechanisms of α2AR regulation of anion secretion. We have shown that α2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers, such as cAMP or Ca2+. This type of regulation may be of clinical and pharmacological relevance in understanding the molecular mechanisms of diseases characterized by abnormal intestinal secretion.

Acknowledgments

Measurements of [Ca2+]i were performed in Dr Fred Tse's laboratory, Department of Pharmacology, University of Alberta, by Ms Fenglian Xu. Their help with these studies is greatly appreciated. We also thank Dr Anthony Ho, Department of Physiology, University of Alberta, for help with the cAMP assay, and Professor K. Starke, University of Freiburg, for helpful discussions at the initial stages of this project. This work was supported by grants from the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research.

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