1Department of Physiology and 2Oklahoma Medical Research Foundation, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73034
Submitted 6 November 2003 ; accepted in final form 7 February 2004
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ABSTRACT |
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sulphonylurea receptor; inflammation; dextran sulfate; real-time PCR; voltage clamp
In several different animal models of colonic inflammation, voltage-dependent Ca2+ currents of smooth muscle have been shown to be suppressed due to either decrease in membrane expression (19) or a possible alteration in the channel regulation (17). Previously, in a dextran sulfate sodium (DSS) model of experimental colitis in mice, we observed that in addition to decreased Ca2+ currents, colonic inflammation also results in selective changes in K+ currents of colonic smooth muscle (3). The activation of ATP-sensitive K+ channel currents (KATP) in response to lemakalim, a potassium channel opener, were markedly enhanced, whereas the biophysical properties of the transient outward current remained unaltered. KATP channels have been previously described in gastrointestinal smooth muscle (11, 18); however, their role in the pathophysiology of smooth muscle is not clear.
KATP is a heterooctomer comprised of two subunits, a sulfonylurea receptor (SUR) and the inwardly rectifying, pore-forming K+ channel, Kir 6.x. The two isoforms, Kir 6.1 and Kir 6.2, are inwardly rectifying channels encoded by two separate genes that associate with either of the two isoforms of the SUR receptor, SUR1 and SUR2. SUR2 is alternatively spliced producing SUR2A and SUR2B (5, 7, 27, 28). It is now well recognized that Kir 6.2/SUR1 and Kir 6.2/SUR2A form the major KATP complex in the pancreatic -cell and the heart, respectively (13, 14, 28); however, the subunits comprising those of gastrointestinal smooth muscle are unclear. Koh and colleagues (16, 18) have suggested that Kir 6.2/SUR2B underlie the KATP channel in the murine colonic smooth muscle on the basis of the presence of transcripts identified in single cells. However, the small single-channel conductance of 27 pS (18) appears to be more consistent with Kir 6.1. The characterization of the KATP complex is generally defined on the basis of the single-channel conductance that distinguishes the isoforms of the Kir subunit and pharmacologically for the SUR subunit (28). Heterologously expressed Kir 6.1 demonstrates a conductance of
35 pS, whereas Kir 6.2 has a conductance of
80 pS (13, 14, 26, 36). In some smooth muscle cells, it is likely that coexpression of Kir 6.1/Kir 6.2 leads to an intermediate conductance (9, 26, 33).
Recent studies (32) demonstrating the effects of various knockout of either of the genes encoding the KATP channel illustrate the pathophysiological role of Kir 6.2/SUR1 in relation to persistent hyperinsulinemic hypoglycemia of infancy, whereas Kir 6.2/ mice demonstrated depressed ST elevation during myocardial ischemia/reperfusion. Kir 6.1/ mice show a phenotype similar to Pirnzmetal angina in humans, which may be due to loss of KATP function in vascular smooth muscle (5, 20). In gastrointestinal smooth muscle, the physiological role of the KATP channel may be related to modulation of cell excitability (11, 18). However, whether changes in KATP channel function occur in pathophysiological conditions is not known. In the present study, we examined KATP channels in the mouse experimental colitis model and demonstrate that the major sarcolemmal isoform of the Kir channel in mouse colon is composed of Kir 6.1 and SUR2B and the channel demonstrates increased bursting activity in response to the KATP channel opener after inflammation. Inflammation also enhanced the gene expression of Kir 6.1.
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MATERIALS AND METHODS |
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Colonic inflammation was induced by administering 5% DSS in drinking water ad libitum for 7 days. All protocols were approved by the Institutional Animal Care Committee. The mice were killed, and the distal colon was excised and placed in Tyrode's solution. To assess the severity of colitis, the daily disease activity index (DAI) was calculated by determining the daily weight of each animal, the presence of blood occult (detected by hemoccult strips; Smith Kline Diagnostics, San Jose, CA) or gross blood in the feces and stool consistency. Each of the parameters was assigned a score based on previously described criteria (31), which was used to calculate an average DAI for each animal. Briefly, the scores ranged from 0 to 4, with 0 being no weight loss, normal stool consistency, and no rectal bleeding, whereas 4 was assigned when the weight loss exceeded 20% with diarrhea and gross bleeding. Histological assessment of colonic lesions was also determined. After the removal of the colon, at least two cross sections from the distal diseased area were immediately fixed in 10% formalin. The tissue was then embedded in paraffin and stained with hematoxylin and eosin.
Immunohistochemistry.
Mice colon samples were embedded in Tissue-Tek OCT and snap-frozen in liquid nitrogen-cooled isopentane. Cryosections (7-µm thick) were blocked with 3% bovine serum albumin in PBS (containing 0.01% saponin) for 1 h at room temperature. The slides were subsequently incubated with primary antibodies: goat anti-Kir 6.1 (antigen sc-11224, Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-Kir 6.2 (antigen sc-11225, Santa Cruz Biotechnology) (20 µg/ml) for 1 h at RT. After sections were thoroughly washed with PBS (0.01% saponin), they were incubated with secondary antibody, Cy3 conjugated rabbit anti-goat IgG (Zymed, 1:100), fixed with 4% paraformaldehyde for 5 min and mounted with Vectashield (Vector) containing 0.001% TO-PRO (Molecular Probes) for nuclear staining. Sections were analyzed by confocal laser scanning microscopy using a Nikon C1 system. Controls were carried out by preabsorption of the primary antibodies with the blocking peptide.
Cell isolation.
Colonic smooth muscle cells were isolated from 5- to 6-wk-old BALB/c mice as described previously (12). Mice were euthanized and the distal colon was quickly removed. The colon was opened along the mysenteric border, and muscle strips were dissected away from the mucosa and submucosa in low Ca2+ Tyrode's solution. Strips of colonic muscle, which include both circular and longitudinal layers, were transferred to 5 ml of Tyrode's solution containing 2 mg collagenase, 2 mg bovine serum albumin, and 1.5 mg trypsin. Incubation in the enzyme solution was carried out at 37°C for 1012 min. The partially digested tissues were then incubated in enzyme-free solutions containing serum albumin and were gently agitated with a wide-bore fire-polished pipette to release smooth muscle cells. Isolated cells were stored on ice and used within 6 h of dispersion. All electrophysiological recordings were performed at room temperature (2225°C).
Electrical recordings.
Standard whole cell, cell-attached, and inside-out patch configurations were used. The voltage-clamp amplifier was an Axopatch 200A (Axon Instruments). Data were low-pass filtered at 5 kHz and sampled at a rate of 10 to 100 µs per sample. The pipettes were prepared on a Flaming-Brown horizontal puller (model P-87; Sutter Instruments) and fire polished. Resistance of the pipettes was 5 M
for whole cell configuration and 810 M
for cell-attached or inside-out single-channel recordings when filled with pipette solution.
Solution.
Solutions used for electrophysiological recordings and cell isolation are listed in Table 1. The low-Ca2+ Tyrode's solution was equilibrated with 95% O2-5% CO2. All of the bathing solutions were equilibrated with 100% O2. The pH was adjusted to 7.4 with KOH. For inside-out patch recordings, the same external solution was used as that in cell-attached recordings with the addition of 1 mM ADP-Mg.
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Single-channel currents were analyzed by using Fetchan and Pstat subroutines in the pClamp 8.2 software (Axon Instruments) and Origin for Windows software (Microcal Software, Northampton, MA). For analysis of the single-channel kinetics, the amplitudes of current, distribution of open and closed times, and burst analysis were measured by constructing histograms after events-list analysis from raw records. Events were detected by setting a threshold at one-half of the amplitude of the open-channel current. The opened- and closed-time histograms were fitted with exponential components by the method of maximum likelihood. The number of exponential densities used for fitting the histograms was determined in pStat on the basis of examination of the F statistic (P > 0.99).
Bursts were defined as an opening or series of openings separated by closures shorter than a critical time. An optimal interburst interval was calculated from closed-time distribution using the algorithms in PClamp 8.2 software. Burst analysis were then performed to define the duration of openings within burst, duration of closings within burst, and total burst durations. Channel activity was calculated as channel open probabilities (NPo). NPo was measured by integrating idealized records of channel opening and closing transitions and dividing this by the time integral of single-channel current.
Intracellular microelectrode recordings.
Colonic muscle strips were dissected away from the mucosa and cut into small strips. Each strip was pinned mucosal side up and placed in the recording chamber containing Krebs solution (in mM): 120.9 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 11 glucose (pH 7.4) and oxygenated in 95% O2-5% CO2 solution. Strips were superfused at 3 ml/min and maintained at 37°C in the presence of nifedipine (1 µM) and atropine (1 µM). Microelectrodes have resistances of 3060 M when filled with 3 M KCl. The transmembrane potential was measured with an amplifier IE-210 (Warner Instruments, Holliston, MA), and the signals were digitized by using an analog-to-digital convertor and analyzed by AxoScope 8.2 (Axon Instruments).
Total RNA extraction, conventional and real-time RT-PCR.
Colonic muscle strips (devoid of mucosa) were frozen in liquid N2, and total RNA was extracted by using the Micro-to-Midi total RNA isolation kit (Invitrogen) according to the manufacturer's instructions. All RNA samples were quantified by spectrophotometer and run on denaturing RNA gels to check the quality and integrity of the RNA samples. First-strand cDNA was synthesized by using the Super Script first-strand synthesis system for RT-PCR (Invitrogen). Each cDNA sample was synthesized by using 50 units of Superscript II RT at 42°C for 50 min in the presence of 5 µg of total RNA, 0.5 µg of oligo(dT) primers, and 10 mM dNTPs in a total of 20 µl reaction. PCRs were performed on a Robocycler PCR machine (Stratagene, La Jolla, CA) using 3 µl of cDNA, 2.5 units of Taq DNA polymerase, 10 mM dNTPs, and 10 µM each forward and reverse primers in 50-µl volume. Samples with minus RT or water templates were used as controls. PCR was performed under the following conditions: 1 cycle at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 30 s, and extension at 72°C for 1 min. Thirty microliters of RT-PCR products were separated on 2% agarose/1x Tris-acetate-EDTA gels, and DNA bands were visualized by ethidium bromide staining.
Real-time PCR was performed on the SmartCycler (Cephid) using SYBRgreen as the intercalating dye. Four independent control and five inflamed total RNA samples were examined. For each mRNA sample, colon samples from three mice were used to get sufficient mRNA. For each sample, PCR was conducted in a 25-µl volume of reaction mixture containing SYBRgreen (1:125,000 dilution; Molecular Probes), Ready-to-Go-PCR beads (Amersham) that include dNTPs and Taq polymerase, 2.5 µl both forward and reverse primer, 2 µl MgCl2 (25 mM), and 2 µl template cDNA prepared from equal amounts of total RNA from control and inflamed samples. The PCR protocol consisted of three steps with an initial denaturation (95°C for 120 s), followed by 35 cycles of denaturation (95°C for 15 s), annealing (5760°C for 15 s), and extension (72°C for 15 s). A melt curve was derived at the end of the PCR by heating the PCR product from 60 to 95°C. A single peak in the melt curve is indicative of the specificity of the PCR product. The absence of peaks in water controls suggested lack of primer-dimer formation. This was further confirmed by gel electrophoresis of the RT-PCR products. The semiquantitative comparison between control and inflammation was calculated according to a previously published method (10). The cycle threshold value, (CT count), for each sample was normalized by subtracting it from the CT value of the housekeeping gene -actin to establish
CT (
CT = target gene CT
-actin CT). To determine the relative enhanced expression of the target gene by inflammation, inflamed sample
CT was compared with control sample
CT to obtain
CT (
CT = inflamed
CT control
CT). The fold change was measured as 2
CT.
Primer design.
Primers used are listed in Table 2. Primers for real-time PCR experiments were designed to amplify smaller-sized products. An additional set of primers for Kir 6.1 were designed to span an intron (primer set 3) to verify any possible genomic DNA contamination. The -actin primers were also designed to span an intron in addition to two exons (30).
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Diazoxide, glibenclamide, bovine serum albumin, and trypsin were purchased from Sigma (St. Louis, MO). Collagenase was obtained from Yakult (Tokyo, Japan). Levcromakalim was obtained from Tocris (Ellisville, MO). Antibodies for Kir 6.1 (antigen sc-11224) and Kir 6.2 (antigen sc11225) were purchased from Santa Cruz Biotechnology.
Statistical analysis.
Statistical difference was evaluated by using Student's paired or unpaired t-test. Differences with values of P < 0.05 between control and test group were considered to be significant. Values in the text are as given as means ± SE.
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RESULTS |
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To identify KATP in colonic smooth muscle cells, conventional whole cell recordings were made in isolated single cells dialyzed with low ATP (0.1 mM) and initially bathed in normal HEPES solution at a holding potential of 70 mV from control and DSS-treated mice colonic cells, henceforth referred to as inflamed cells.
The average capacitance was 62.4 ± 3.8 pF (n = 22) in control and 60.0 ± 3.4 pF (n = 23) in inflamed cells. Changing the external solution from 5 mM K+ to that containing 140 mM K+ resulted in a small, steady, inward current measuring 1.9 ± 1.08 pA/pF in control cells, and 4.8 ± 2.6 pA/pF in inflamed cells. Perfusion with levcromakalim (20 µM) in this symmetrical K+ gradient further increased the inward currents to 7.7 ± 1.1 pA/pF (n = 7) in control cells and 26.3 ± 8.6 pA/pF (n = 9) in inflamed cells (Fig. 2), representing an almost fourfold difference in the response to the potassium channel opener. Currents in both control and inflamed cells were reversed by either glibenclamide (10 µM) or by returning to 5 mM K+ bathing solution.
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Single-channel recordings.
The single-channel properties of the KATP channel were determined in cell-attached patches. To avoid activation of the large-conductance Ca2+-activated K+ channel, 200 nM charybdotoxin was included in the patch pipette, and currents were measured at negative potentials. Cells were bathed in high-K+ solution containing 10 µM levcromakalim. KATP channels demonstrated brief burst-like openings, separated by long, closed intervals. This bursting activity appeared to be increased in inflamed cells (Fig. 4A). The amplitudes at each potential, obtained from amplitude histograms, such as those shown in Fig. 4C, were plotted against voltage to obtain the single-channel conductance. Whereas single-channel activity was markedly increased in inflamed cells, there were no significant differences in the single-channel conductance from control and inflamed cells. The conductance obtained from the slope of the I-V curve was 43.3 ± 1.7 pS (n = 8) in control cells and 42.1 ± 2.5 pS (n = 9) in inflamed cells (Fig. 4, B and D). Single-channel currents were abolished in the presence of glibenclamide (10 µM). The single-channel conductance was similar in inside-out patches.
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Previously, Koh et al. (18) identified transcripts for Kir 6.2 and SUR2B in the mouse colon but not for Kir 6.1. We reexamined the presence of Kir 6.1 and 6.2 by conventional RT-PCR. Figure 9A shows the presence of transcripts for both Kir 6.1 and 6.2 in the mouse colon. To negate any possibility of genomic contamination, we also designed primers spanning an intron between exons 2 and 3 of Kir 6.1 (primer set 3, Table 2). Figure 9B shows that a single band of 342 bp was obtained corresponding to the predicted size of Kir 6.1 mRNA. This was confirmed in three different groups of cDNA from control and inflamed tissues.
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To define the protein localization of the Kir 6.1 and Kir 6.2 isoforms, cross sections of the mice colon were processed for immunostaining with anti-Kir 6.1 and anti-Kir 6.2 antibodies. As shown in Fig. 10, Kir 6.1 was localized to the plasma membrane of both longitudinal and circular smooth muscle as shown in the transverse sections of the colon. On the other hand, Kir 6.2 staining appeared diffuse and more intense around the nucleus. There was no particular evidence of membrane localization. Thus it appears that Kir 6.1 is the major isoform in the plasma membrane consistent with the small single-channel conductance.
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To determine the changes in the gene expression of Kir 6.1 and SUR2B, we examined mRNA levels by real-time PCR. In an initial analysis, we determined that neither of the primers for Kir 6.1 nor SUR2B produced primer dimers. This was measured both by gel electrophoresis and by the melting curves. cDNA was prepared from four groups of control mice and five groups of DSS-treated mice. In each group, two to three mice colons were pooled for RNA isolation. For each sample of mRNA, the CT for -actin was also determined and used to establish
CT as described in MATERIALS AND METHODS. The
CT count for controls was 10.7 ± 0.08 (n = 4) for Kir 6.1 and 9.8 ± 0.36 (n = 4) for SUR2B. When measured for mRNA from inflamed smooth muscle, the
CT count for Kir 6.1 was 6.28 ± 0.14 (n = 5) and SUR2B was 11.4 ± 0.56 (n = 5). An example of the fluorescence signal curves is shown in Fig. 11A for control and inflamed mRNA. Melt curves showed single peaks for control and inflamed samples (Fig. 11B). A quantitative comparison between control and inflamed cells was measured by normalizing the
CT counts (see MATERIALS AND METHODS). mRNA for Kir 6.1 was increased by almost 22-fold, whereas that for SUR2B was decreased by almost threefold (Fig. 12).
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DISCUSSION |
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Previously, Koh et al. (18) measured the single-channel conductance of 27 pS in the mouse colon and identified the transcript for Kir 6.2 and SUR2B, but not Kir 6.1 in smooth muscle cells. However, the small single-channel conductance of
42 pS and previous reports that KATP in gastrointestinal smooth muscle is inhibited by protein kinase C (11, 16, 33) are more consistent with the major isoform being Kir 6.1. We detected transcripts for both Kir 6.1 and Kir 6.2. By using primers spanning an intron, we confirmed that there was no genomic contamination. The protein expression of both transcripts was identified by immunohistochemical localization. Kir 6.1 is strongly expressed on the plasma membrane, consistent with the single-channel conductance, whereas Kir 6.2 appeared to be restricted to the cytosol, particularly around the nucleus. The significance of Kir 6.2 in the cytosol is not clear at present but could reflect defects in protein trafficking of the immature channel in the colon. Inflammation did not alter the conductance of the channel, indicating that increased current was not due to change in the expression of the Kir isoforms.
The finding that KATP channel activation leads to an increased hyperpolarization of inflamed tissues is consistent with an increase in whole cell currents and increased burst durations in cell-attached patches. The increase in currents would be expected with an increase in expression of the pore-forming Kir 6.1 with inflammation. An analysis of the bursting behavior indicated that neither the mean closed time nor the mean open time for a single-channel event within a burst was altered by inflammation. However, the total mean close durations within a burst was significantly reduced. This could reflect either an increase in the number of channels, or increase in the frequency of openings. The increase in mRNA for Kir 6.1, as determined by quantitative PCR, suggests that transcriptional regulation of the pore-forming Kir subunit may result in an increase in the bursting duration observed with inflammation.
Activation by levcromakalim was necessary to demonstrate the increased responses. Because the major binding site for the K+ channel opener lies within the SUR subunit, it is surprising that SUR2B was not upregulated by inflammation but rather was slightly downregulated. Similar observations have been made with regard to changes in Kir 6.1 and SUR2 in cardiac myocytes and kidney cells after ischemia. Akao et al. (2) noted an upregulation of Kir 6.1, but not SUR2, after myocardial ischemia/reperfusion. The mRNA for Kir 6.1 increased by 2.5-fold in both ischemic and nonischemic regions of the rat left ventricle after LAD ligation (2). In the heart, the functional activation of the KATP channel during myocardial ischemia results in the shortening of the action potential leading to a decrease in Ca2+ influx, thus protecting against cardiac damage (21). Similarly, Sgard et al. (29) also demonstrated an increase in the mRNA for Kir 6.1 but not SUR2 in rat kidney after ischemic injury. It is not clear how the altered transcriptional regulation of the two subunits affects the functional channel complex. One possibility may be that there is sufficient copy number of the SUR subunit present after inflammation, thus allowing for a functional heterooctomer channel complex. Alternatively, variants of the SUR may be expressed that were not detected by the primers used in the present study. The SUR subunit in smooth muscle is SUR2B, as demonstrated by RT-PCR and by sensitivity to diaoxide (18). Neither SUR1 nor SUR2A could be readily detected in our study, similar to that reported by Koh et al. (18) in the mice colon. Furthermore, inflammation did not appear to induce transcription of SUR1 or SUR2A. Although our studies show that SUR2B transcription was not increased, there are several tissue-specific variants due to alternative splicing of SUR2 receptors that may exist in a single cell. These isoforms may have different sensitivities to the agonist. One such variant that involves alternative splicing of exon 17 has differential sensitivity to ATP (6). It is therefore conceivable that inflammation may alter the expression of specific SUR variants.
In addition to the increased expression of Kir 6.1, responses to levcromakalim may also be shifted by a change in the metabolic state of the inflamed cells. Electrophysiological studies from inside-out patches have suggested that the effects of K+ channel openers may be potentiated in the presence of ADP (1). Thus either an increase in ADP or a change in ATP-to-ADP ratio in inflamed cells could enhance the response to levcromakalim, in conjunction with increased Kir 6.1. Further studies to test this possibility would require experiments using inside-out patches. It is noteworthy, however, that the increased response to levcromakalim was also observed in whole cell recordings in which the concentrations of ATP were identical between control and inflamed cells while these were dialyzed through the patch pipette.
In summary, the present results provide electrophysiological and molecular evidence of changes in K+ channels after inflammation of the colon. In concert with the reported downregulation of the Ca2+ influx through voltage-dependent Ca2+ channels (3, 17, 19), enhanced K+ is likely to result in decreased excitability contributing to the clinical expression of decreased motility of colonic smooth muscle after inflammation. There is also now increasing evidence of changes in transcriptional regulation of channels after insult and injury. This has been particularly well characterized for neuronal sodium channels after injury and inflammation and has been termed transcriptional channelopathy (35). Our present findings suggest that inflammation-induced changes in ATP-sensitive K+ channels likely belong to this class of disorders.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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