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Am J Physiol Gastrointest Liver Physiol 290: G1219-G1227, 2006. First published August 25, 2005; doi:10.1152/ajpgi.00032.2005
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NEUROREGULATION AND MOTILITY

Subtractive hybridization unravels a role for the ion cotransporter NKCC1 in the murine intestinal pacemaker

Mira Wouters,1,2 Ann De Laet,3,4 Luc Ver Donck,2 Eric Delpire,5 Pierre-Paul van Bogaert,4 Jean-Pierre Timmermans,3 Alban de Kerchove d'Exaerde,1 Karine Smans,2 and Jean-Marie Vanderwinden1

1Laboratoire de Neurophysiology, Faculté de Médecine, Université Libre de Bruxelles, Brussels, Belgium; 2Department of Gastrointestinal Pharmacology, Johnson and Johnson, Pharmaceutical Research and Development, a Division of Janssen Pharmaceutica, Beerse, Belgium; 3Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium; 4Laboratory of Electrobiology, University of Antwerp, Antwerp, Belgium; and 5Department of Anesthesiology, Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee

Submitted 26 January 2005 ; accepted in final form 12 August 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na+-K+-2Cl– cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.

interstitial cells of Cajal; bumetanide; slow waves

IN THE GASTROINTESTINAL (GI) tract, the spatiotemporal organization of contractile activity is essential for efficient propulsion of luminal content. Besides the enteric nervous system, the role of the interstitial cells of Cajal (ICC) in the coordination of contractile activity has been established in recent years (18, 41, 43). Depending on their location, ICC are associated with several functions. In the small intestine, ICC at the level of the deep muscular plexus (ICC-DMP) mediate neurotransmission (3, 49, 62), whereas ICC surrounding the myenteric plexus (ICC-MP) generate slow waves (SW). These SW are transferred to the adjacent smooth muscle cells and can be recorded as spontaneous, rhythmic, electrical oscillations of the resting membrane potential (41, 49, 64).

ICC express the c-Kit gene (locus W), which encodes the tyrosine kinase receptor KIT. The KIT ligand is the cytokine stem cell factor (SCF), encoded at the steel locus (Sl). The SCF-KIT signaling pathway is vital for the development and maintenance of various cell types, including the ICC in the GI tract (64). Mice homozygous for a null mutant at either W or Sl loci are not viable, whereas a number of partial loss-of-function mutations for both the W locus (Wv) and Sl locus (Sld) has been reported (6, 27, 39, 63). In viable mice carrying subtotal loss-of-function mutations in the KIT gene (W/Wv) or its ligand (Sl/Sld), ICC-DMP are preserved, whereas ICC-MP are lacking, resulting in the loss of pacemaker activity and disturbed intestinal transit (20, 22, 49, 50). Although the lack of ICC is restricted to defined subpopulations of ICC, these two models, W/Wv and Sl/Sld, will further be referred to as "ICC-deficient" mice.

In humans, disturbed ICC networks have been reported in several disorders of the GI transit, e.g., chronic intestinal pseudoobstruction (5, 11, 25), infantile pyloric stenosis (57), Hirschsprung's disease (59), slow transit constipation (16, 36, 48, 66), and diabetic gastroparesis (17). Functional alterations of ICC have to be considered in other conditions where ICC distribution appears unaffected, and targeting the SW might be an innovative way to new therapeutic developments in GI motility disorders (46).

Whereas the electrophysiology of SW has been extensively investigated in tissues and dissociated ICC, the molecular mechanism underlying the pacemaking currents in ICC remains poorly understood. By using suppression subtractive hybridization (SSH), the present study aimed to identify transcripts expressed in the muscularis propria of the jejunum in wild type (WT) but lacking in ICC-deficient mice and to unravel their function. Here, we report that Na+-K+-2Cl– cotransporter NKCC1 is expressed in ICC-MP and is involved in the SW mechanism in the jejunal musculature.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animals. Mice were maintained and experiments performed in accordance with the local ethics committees for animal well-being of the Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium, the University of Antwerp, Antwerp, Belgium and of Johnson and Johnson, Pharmaceutical Research and Development, a subdivision of Janssen Pharmaceutica, Beerse, Belgium.

Heterozygous Sl/+ and Sld/+ mice were obtained from Jackson Laboratory (Bar Harbor, ME). Wv/+ and WLacZ/+ mice were a generous gift from J. J. Panthier (Ecole Nationale Vétérinaire, Maisons-Alfort, France). The product of the WLacZ transgene lacks kinase activity and is functionally equivalent to the spontaneous null allele W (4). Heterozygous mice were bred to obtain viable Sl/Sld, WLacZ/Wv, and WT littermates.

Disruption of the mouse Slc12a2/NKCC1 gene has been previously reported. Heterozygous couples were bred and litters were genotyped as described. Homozygous NKCC1 knockout (KO) animals were readily distinguished by their small size and shaker/waltzer behavior (8). WT littermates served as controls.

One-month-old mice were killed by cervical dislocation. The jejunum was quickly dissected out, flushed with ice-cold Krebs solution, and pinned onto a Petri dish cooled at 4°C. The mesentery was carefully removed under a binocular.

PCR suppression SSH. For poly A+ and total RNA isolation, the muscle layers were peeled off the mucosa and submucosa by blunt dissection with fine tweezers, and the mucosa was discarded. Muscle strips were immediately transferred into cryotubes, frozen in liquid nitrogen, and stored at –80°C until use. Total RNA was extracted using the Rneasy fibrous tissue mini kit (Westbur, Leusden, the Netherlands) and treated with DNAse I (On Column Dnase, Westburg) according to the manufacturer's instructions to avoid genomic DNA contamination.

Poly A+ RNA was isolated from total RNA using Oligotex mRNA spin columns (Westburg). The mRNA was precipitated (1/10 V 0.3 M NaAc, 2.5 V 100% EtOH) overnight at –80°C, spun at 4°C during 30 min, washed with 70% EtOH, and diluted to 1 µg/µl in Rnase-free water.

PCR select cDNA subtraction was performed starting from 2 µg poly A+ RNA according to the manufacturer's instructions (Clontech, Palo Alto, CA). Two populations of mRNA isolated from jejunum muscle layers of WT vs. ICC-deficient mice were compared by SSH to enrich for differentially expressed transcripts. The subtraction was carried out in two ways: 1) the forward subtraction, WT minus ICC deficient, contained cDNA enriched for ICC-related genes, whereas 2) the reverse subtraction, ICC deficient minus WT, served as negative control. The subtraction procedure was performed on both models of ICC-deficient mice, Sl/Sld and WLacZ/Wv. To minimize the influence of the different genetic background of Sl/Sld and WLacZ/Wv animals, only clones differentially expressed in both models were further considered.

The forward subtracted cDNA library was cloned in pGem-t-easy (Promega, Madison, WI) and chemically transformed in one-shot cells (N.V. Invitrogen, Merelbeke, Belgium). Four thousand bacterial colonies were randomly selected and amplified. Amplification of the PCR product was confirmed on a 1% agarose gel. After the DNA was sequenced, it was spotted on hybridization filters (Amersham Pharmacia Biotech, Buckinghamshire, UK). Clones downregulated in ICC-deficient vs. WT mice were identified by probing these filters with 10 counts·min–1·ml–1 of [32P]CTP forward and reverse subtracted probe (Megaprime DNA labeling system, Amersham) from both subtractions (Sl/Sld and WLacZ/Wv). Southern blots were analyzed by STORM gel and blot imaging system (Amersham Pharmacia Biotech) and quantified with Imagene 4.2 software (BioDiscovery, El Segundo, CA).

Real-time quantitative PCR. Expression levels of genes having a difference in hybridization signal greater than three with the forward vs. reverse probe from both ICC-deficient models were examined by SybrGreen quantitative PCR (qPCR; Applied Biosystems, Warrington, UK). Real-time quantitative PCR (RT-qPCR) comparisons were performed exclusively between WT and ICC-deficient animals from the same litter. Total RNA isolation from jejunum muscle layers was performed as described in the previous paragraph. Reverse transcription was performed with 25 pmol/µl oligo(dT) 15 primer using Powerscript reverse transcriptase (BD Biosciences, Erembodegem, Belgium) according to the manufacturer's instructions.

Amplification reactions were performed in triplicates with 1 x SybrGreen PCR master mix (Applied Biosystems), 200 nM of gene specific primers and 25 ng sample DNA in a 50-µl final volume. The primers were designed with Primer Express 1.5 according to the manufacturer's instructions (Table 1), and qPCR was performed on an ABI Prism 1700 Sequence detector. Identical thermal profile conditions (95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min) were used for all primer sets. Emitted fluorescence was measured during the annealing/extension phase, and amplification plots were generated using the sequence detection system (Applied Biosystems). To differentiate specific amplicons from nonspecific products, a DNA dissociation curve was generated after each reaction.


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  		Table 1. Sequence of primers used for RT-OPCR and for SMART-RACE

 
All mRNAs were quantified relative to GAPDH mRNA using the comparative threshold cycle number (Ct) method. The Ct difference ({delta}Ct = Ctgene – CtGAPDH) was taken as a relative quantity of the transcript. To ascertain the validity of the {delta}{delta}Ct calculation ({delta}{delta}Ct = {delta}CtICC deficient – {delta}CtWT), the amplification efficiency was checked and found to be identical for all the genes measured. Statistical analysis was performed with the unpaired t-test, and a P value <0.05 was considered to represent a statistically significant difference.

Rapid amplification of cDNA ends. The NKCC1 full-length clone was identified by the SMART-Rapid amplification of cDNA ends (RACE) cDNA amplification kit (Clontech) according to the manufacturer's specifications (primer in Table 1). DNA sequencing was performed using the ABI PRISM BigDye Terminators v3.0 Cycling Sequencing Kit according to the instructions of the supplier (Applied Biosystems, Foster City, CA) except for the amount of dyes used, which was reduced to 1 µl for a 20-µl reaction. The software used for sequence assembly was Sequencher version 4.0.5 from Gene Codes (Ann Arbor, MI).

Immunohistochemistry. For morphological experiments, small pieces of jejunum were harvested, fixed overnight at 4°C in fresh 4% paraformaldehyde solution in 0.1 M PBS, cryopreserved in graded solutions of sucrose (10, 20, and 30%; overnight each), oriented transversally, embedded in Tissue-Tek optimum cutting temperature compound (Miles, Elkhart, IN), snap-frozen in 2-methyl butane that had been cooled on dry ice and stored at –80°C until sectioning. Twelve-micrometer sections were cut on a cryostat and mounted on slides coated with 0.1% poly-L-lysine (Sigma, St. Louis, MO), air dried for 20 min, and stored at –20°C until use.

Besides mouse jejunum, two specimens of normal human jejunum from our tissue collection (58) were used. The use of human tissues was approved by the Medical Institutional Ethics Committees of the Hôpital Universitaire des Enfants Reine Fabiola and Faculté de Médecine, Université Libre de Bruxelles, Brussels, Belgium.

Sections were rinsed three times in 10 mM Tris in 0.15 M sodium chloride, pH 7.4 [Tris-buffered saline (TBS)], containing 0.1% (vol/vol) Triton X-100 (TBS-TX), incubated for 1 h in 10% normal horse serum (NHS; Hormonologie Laboratoire, Marloie, Belgium) in TBS-TX to reduce background staining, and incubated overnight with the primary antisera diluted in TBS-TX containing 2% NHS. The primary antibodies used were a goat anti-mouse polyclonal IgG, raised against KIT(M14) (1/900; Santa Cruz Biotechnology, Santa Cruz, CA), and a rabbit anti-rat NKCC1 polyclonal antibody (1/500; Gentaur, Belgium). The slides were rinsed in TBS, incubated in the dark for 2 h at room temperature in TBS containing the secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) coupled respectively to FITC and Cy3, rinsed in TBS, and mounted with Slow-fade Light Anti-fade (Molecular Probes, Eugene, OR). The optimal working dilution had been determined empirically for each antibody. Omission of one of the primary or of one of the secondary antibodies resulted in the absence of immunoreactivity. The protocol used for double immunofluorescence staining did not modify the distribution or the intensity of each individual labeling observed in corresponding single procedures.

Preparations were observed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) at excitation wavelengths 488 and 543 nm. The images were analyzed with Zeiss LSM 510 Image Examiner.

Extracellular recording of SW activity in vitro. SW were recorded from a 4 cm-long segment of proximal jejunum of WT and NKCC1 KO mice. The segment was opened and placed with the serosal surface facing upward in a 150-ml recording chamber continuously perfused at 50 ml/min with prewarmed (37°C) oxygenated (95% O2-5% CO2) Tyrode's solution of the following composition (in mM): 2.70 KCl, 1.8 CaCl2·2H2O, 1.04 MgCl2·6H2O, 11.9 NaHCO3, 0.42 NaH2PO4·H2O, 136.9 NaCl, and 5.55 glucose, pH 7.4. Tissue was allowed to equilibrate for 10 min. The extracellular electrical activity was recorded using a 16 Teflon-coated silver electrodes array designed by W. Lammers [Al Ain, United Arab Emirates (31–33) (http://www.smoothmap.org)] gently positioned on the serosal surface using a micromanipulator. Electrical signals were recorded using an NI-SCX-1000 data-acquisition system (National Instruments, Austin, TX) for 1 min before the addition of bumetanide (Burinex, LEO Pharmaceutical Products, Ballerup, Denmark), 10 min after addition of bumetanide, and after 10 min of washout. The signals were sampled at 200 Hz and digitally stored on disc using Labview 7.0 software (National Instruments). Signals were filtered using a 50-Hz notch filter and a five-point smoothing for display and analysis purposes. Data are expressed as means ± SE. Differences were evaluated using a paired t-test.

Intracellular recording of SW activity in vitro. SW were recorded from the smooth muscle cells of proximal jejunum of WT Swiss mice and NKCC1 KO mice with a standard microelectrode technique. A segment of intestine was opened along the mesenteric border, and mucosal and submucosal layers were removed under stereomicroscopic visualization. The muscle strip (15 x 6 mm) was then pinned, serosal side down, to the Sylgard (Dow-Corning Europe, Belgium) floor of a recording chamber placed on the stage of an inverted microscope (Diaphot, Nikon, Tokyo, Japan). The tissue was continuously superfused (10 ml/min; temperature 36.5–37°C) with oxygenated Krebs-Ringer solution of the following composition (in mM): 118 NaCl, 4.75 KCl, 2.54 CaCl2·2H2O, 1.2 MgSO4·7H2O, 1 NaH2PO4·2H2O, 25 NaHCO3, and 11.1 glucose. All experiments were performed in the presence of the L-type Ca2+ blocker nicardipine (3 µM, Sigma) to reduce mechanical activity. Smooth muscle cells were impaled with a borosilicate glass microelectrode (1-mm outer diameter; Clarck Electromedical Instruments, Reading, UK) pulled on a P-97 Brown-Flaming micropipette puller (Sutter Instruments, Novato, CA). The electrodes were back filled with 1 M KCl (resistance 50–70 M{Omega}). Potentials were recorded with an electrometer amplifier (Axoclamp 2A; Axon Instruments, Foster City, CA). After amplification and low-pass filtering (500 Hz), the signal was digitized at a sample rate of 1 kHz using a Labmaster TL-1 DMA Interface (Axon Instruments) and displayed and stored on a personal computer. Data were obtained from the same cell before and after addition of bumetanide (Sigma) to the superfusion solution. Data analysis was performed using pClamp 6.0.2 (Axon Instruments) and InStat 3.05 (GraphPad Software, San Diego, CA) software. Data were expressed as means ± SE. Differences were evaluated using a paired t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
NKCC1 is downregulated in the jejunal muscularis propria of ICC-deficient mice. Four thousand transcripts from the forward subtracted library were screened, from which 148 and 139 clones hybridized at least three times more to the ICC-enriched probes from Sl/Sld and WLacZ/Wv, respectively. After SSH, 56 of these clones appeared downregulated in both ICC-deficient models (Fig. 1). These 56 candidate genes were then assessed by RT-qPCR. A threefold higher expression in WT than in at least one of the ICC-deficient mouse models was arbitrarily set as cut off level. KIT (Y00864 [GenBank] ) and NKCC1 (U13174 [GenBank] ) emerged as significantly downregulated in ICC-deficient compared with WT mice. KIT expression was 5.3 ± 0.3 (n = 6, P = 0.001) and 4.2 ± 0.3 (n = 6, P = 0.001) times higher in WT than in ICC-deficient Sl/Sld and WLacZ/Wv, respectively. NKCC1 expression was 3.5 ± 0.2 (n = 6, P = 0.013) and 1.9 ± 0.3 (n = 6, P = 0.005)-fold higher in WT than in ICC-deficient Sl/Sld and WLacZ/Wv, respectively.


Figure 1
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  		Fig. 1. Identification of downregulated genes using Southern blot analysis. Four thousand clones of the forward subtraction, wild-type (WT) minus Sl/Sld (A) and WT minus WLacZ/Wv (B), were spotted in 96-well configurations on hybridization filters. They were hybridized with 32P-labeled forward and reverse subtracted, Sl/Sld minus WT (A) and WLacZ/Wv minus WT (B), probes. Fifty-six clones that were at least 3 times less expressed with the reverse than with the forward subtracted probe in the 2 models (arrowhead) were selected for further analysis with real-time quantitative PCR (RT-qPCR).

 
Because SSH identified only fragments of transcripts of 300–500 bp, RACE was performed to obtain the full-length clone of NKCC1. The isolated clone fully matched the published sequence of the Slc12a2 gene product (68). Exon 21, which encompass the unique PKA consensus site primarily lacking in the brain NKCC1 isoform (44) was present, and no splice variant was detected (35).

NKCC1 is expressed by the pacemaking ICC-MP in the mouse jejunum. In the jejunum of WT mice, NKCC1 immunoreactivity (ir) was observed in the region between the circular and longitudinal muscle layers. Kit-ir ICC-MP surrounding the myenteric ganglia exhibited strong NKCC1-ir, whereas myenteric neurons exhibited a fainter NKCC1-ir. No NKCC1-ir was observed in smooth muscle cells of both layers nor in KIT-ir ICC-DMP (Fig. 2, \. A-C). In the jejunum of ICC-deficient Sl/Sld mice, which lack KIT-ir ICC-MP, NKCC1-ir was present only in the myenteric ganglia (Fig. 2, D-F). Furthermore, in the jejunum of both WT and ICC-deficient animals, strong NKCC1-ir was present in the epithelial cells lining the mucosal crypts.


Figure 2
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  		Fig. 2. Immunofluorescent staining for Na+-K+-2Cl– cotransporter (NKCC1) and c-Kit in the jejunum. A-C: WT mice NKCC1-immunoreactivity (ir) was observed in Kit-ir interstitial cells of Cajal surrounding the myenteric plexus (ICC-MP), whereas myenteric neurons exhibited a fainter NKCC1-ir. NKCC1-ir was absent in KIT-ir ICC at the level of the deep muscular plexus (ICC-DMP; arrowheads). D-F: ICC-deficient (Sl/Sld) mice. In the muscularis propria of ICC-deficient animals, which lack KIT-ir ICC-MP, NKCC1-ir was only present in the myenteric ganglia (arrow). KIT-ir ICC-DMP (arrowheads) lack NKCC1-ir. In both WT and ICC-deficient animals, strong NKCC1-ir was also present in the epithelial cells lining the mucosal crypts (top portion in the figures). G-I: human jejunum. The pattern of NKCC1-ir staining was similar to the mouse jejunum, with NKCC1-ir labeling ICC-MP (arrowheads) and myenteric ganglia (*). Kit-ir mast cells occasionally encountered in the circular muscle layer (arrow) were NKCC1 negative. Out of the field of view, on top, the epithelium was also NKCC1-ir positive, whereas the Kit-ir ICC-DMP did not exhibit NKCC1-ir (data not shown). Scale bars: 25 µm.

 
The NKCC1-ir staining pattern was similar in the human jejunum (Fig. 2, G-I). KIT-ir mast cells occasionally found in the circular muscle layer were NKCC1 negative (Fig. 2, G-I).

NKCC1 is involved in the SW activity of the mouse jejunum. Extracellular recordings showed spontaneous SW in WT small intestine with amplitudes of 400–600 µV and a frequency of 36.1 ± 2.1 SW/min (n = 8; Fig. 3). SW remained stable during the entire observation period (data not shown). In WT, application of 0.4 µM bumetanide had no effect on the SW (n = 5, Fig. 3A). However, 10 min after the addition of 4 µM bumetanide, the shape of the SW became irregular, and the amplitude decreased to 100–150 µV (n = 4, P < 0.0001; Fig. 3B), whereas the change in frequency was not statistically significant (P = 0.36). After the addition of 40 µM bumetanide (n = 8, Fig. 3C), the shape of SW became more irregular, the amplitude decreased to 50–100 µV (P < 0.0001), and the frequency decreased from 36.1 ± 2.1 to 27.4 ± 1.3 (P < 0.0001). After washout, SW recovered completely after 4 µM and partially after 40 µM bumetanide treatment.


Figure 3
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  		Fig. 3. Effect of bumetanide on slow waves in the WT and NKCC1-knockout (KO) murine jejunum recorded with an extracellular electrode. In WT, spontaneous slow waves (SW) with amplitudes of 400–600 µV and a frequency of 36.1 ± 2.1 SW/min remained stable under control conditions. SW were measured before (t = 0), 10 min (t = 10) after the addition of 0.4 (n = 5; A), 4 (n = 4; B) and 40 µM (n = 8; C) bumetanide, and 10 min after washout (WO). In A and B, SW recovered completely after WO. In NKCC1-KO animals (n = 4), the amplitude (50–100 µV) and frequency (26.0 ± 4.7) of the SW were very low, and no effect was detected after addition of 40 µM bumetanide (D). Scale bars: x-axis, 1 s; y-axis, 0.2 mV.

 
The small intestine of NKCC1 KO animals displayed slow-wave activity with amplitudes of 50–100 µV and a frequency of 26.0 ± 4.7 SW/min (n = 4). Addition of 4 and 40 µM (Fig. 3D) bumetanide (n = 4) had no effect on the amplitude or frequency of the SW.

When recording SW in control conditions using microelectrodes, SW were characterized by a steep depolarization followed by a plateau phase and a slower repolarization (Fig. 4, A-C, top). In WT animals, the addition of 0.4 µM bumetanide in the superfusion solution had no significant effect on the SW amplitude and shape (n = 5), whereas there was a small, but significant, decrease in SW frequency (Fig. 4A and Table 2). The addition of 4 µM (n = 10; Fig. 4B and Table 2) and 40 µM bumetanide (n = 9; Fig. 4C and Table 2) further significantly decreased the frequency of the SW and altered the shape of the SW within a few minutes. The amplitude decreased very significantly (P ≤ 0.0005, paired t-test) and, as can be seen in the figures (Fig. 3, B and C), there was a slower depolarization and a marked slower repolarization compared with the control situation. Furthermore, cells depolarized with both concentrations of bumetanide. In three of nine cases, the SW disappeared completely after the addition of 40 µM bumetanide. After washout, SW recovered, albeit slowly and not always completely during the recording period (Fig. 4C).


Figure 4
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  		Fig. 4. Effect of bumetanide on SW in WT animals recorded with an intracellular microelectrode. In WT, spontaneous SW remained stable under control conditions. Addition of 0.4 µM bumetanide (A) resulted in a small but significant decrease in SW frequency. Addition of 4 (B) and 40 µM bumetanide (C) decreased the SW frequency and resulted in a slower rate of rise, a slower repolarization, and a decrease in amplitude of the SW. After the addition of 40 µM bumetanide (C), in 3 of 9 cases, SW disappeared completely within a few minutes. After WO, SW recovered slowly. Scale bars: x-axis, 1 s; y-axis, 10 mV.

 

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  		Table 2. Effect of bumetanide on the smooth muscle cells of wild-type animals

 
Also, in the jejunum of NKCC1 KO animals, SW had a lower amplitude of 14 ± 2 mV and a frequency of 26.6 ± 1.7 SW/min (n = 14). The smooth muscle cells had a resting membrane potential of –46 ± 2 mV (n = 14). The addition of 4 µM (n = 7; Fig. 5A and Table 3) and 40 µM bumetanide (n = 7; Fig. 5B and Table 3) had no further effect on the shape and frequency of the SW nor on the resting membrane potential.


Figure 5
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  		Fig. 5. Effect of bumetanide on SW in NKCC1-KO animals recorded with an intracellular microelectrode. In NKCC1-KO animals, the addition of 4 µM (A) and 40 µM bumetanide (B) had no effect on the amplitude, shape, and frequency of the SW measured in the smooth muscle cells. Also the resting membrane potential of the cells remained stable after the addition of bumetanide. Scale bars: x-axis, 1 s; y-axis, 10 mV.

 

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  		Table 3. Effect of bumetanide on the smooth muscle cells of NKCC1 knockout mice

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Identification of genes expressed in the GI muscle layers of WT animals but lacking in ICC-deficient animals may lead to a better understanding of the mechanisms generating pacemaker currents in the GI tract. Various methods have been developed for analyzing differences in gene expression, including mRNA differential display (34), serial analyses of gene expression (SAGE) (60), cDNA microarray (7), and SSH (10). mRNA differential display is a fairly complex and labor-intensive procedure, whereas both SAGE and cDNA microarray require genomic information and are thus limited to already identified sequences. The SSH technique combines equalization of abundantly expressed cDNAs and over a 1,000-fold enrichment of rare sequences (10). However, a high false-positive rate has been reported, resulting in the identification of <20% of putative clones and confirmed differential expression of merely 2% of the candidates (45). In our hands, using SSH, 56 candidates of 4,000 clones analyzed (1.4%) were identified. Of these 56, 2 genes, namely KIT and NKCC1, were confirmed by RT-qPCR to be significantly downregulated two- to fivefold in ICC-deficient models. The diversity of cell types in the GI musculature and the low abundance of ICC, combined with the complex genetic background of the models used may explain this fairly low success rate.

The protein tyrosine kinase receptor KIT has been extensively used to reveal the networks of ICC in the GI tract by immunohistochemistry (50, 65). Although KIT is apparently not directly involved in the generation of pacemaker currents (13), the KIT-SCF signaling pathway is vital for the differentiation and maintenance of several populations of ICC, including the pacemaking ICC in the jejunum, which are lacking in the WLacZ/Wv and Sl/Sld mouse models (4, 50, 65, 67). The identification of KIT as a gene downregulated in WT vs. ICC-deficient mice established the specificity of the SSH method.

The other gene product identified here by SSH, NKCC1, is a bumetanide-sensitive sodium-potassium-chloride cotransporter encoded by the Slc12a2 gene. It belongs to the family of Na-K-Cl cotransporters, which encompass NKCC1, a widely expressed basolateral secretory cotransporter, and NKCC2, an apical absorptive cotransporter specific to the vertebrate kidney (42). NKCC cotransporters are characterized by their specific and reversible inhibition by bumetanide and other loop diuretics (47).

NKCC1 is expressed by sensory neurons, where it maintains the intracellular chloride concentration above electrochemical equilibrium, affecting the postsynaptic responses to presynaptic stimuli (2, 24, 38, 44, 51, 52, 55, 61). The cotransporter also regulates the vascular smooth muscle tone (1, 26, 40). In salivary and airway epithelia, NKCC1 is involved in chloride secretion, whereas in the gastric epithelium, basolateral NKCC1 raises the intracellular chloride concentration of the acid secretory cells above electrochemical equilibrium, providing the driving force for Cl– to exit the cell on the apical side (47, 53). In situ hybridization had revealed strong NKCC1 expression in mucosal crypts, but was not previously reported in the muscularis propria of the GI tract (14). We have shown here that, besides a very strong NKCC1 expression in mucosal crypts, NKCC1-ir was expressed in myenteric neurons and in KIT-ir ICC-MP, the ICC population that generates the pacemaking activity. Conversely, ICC-DMP, the other population of KIT-ir ICC in the muscularis propria of the jejunum, and smooth muscle cells were NKCC1 negative. The selective expression of NKCC1 in the pacemaking ICC-MP, and its downregulation in ICC-deficient models identified by SSH, raised the hypothesis that NKCC1 may be involved in the electrical SW activity, which is independent of neuronal activity (65).

In WT small intestine, SW are characterized by a steep depolarization followed by a plateau phase and a slower repolarization (29). The SW frequencies recorded in this study (37.3 ± 1.3 and 36.1 ± 2.1 SW/min, intracellular and extracellular recordings, respectively) were in line with published data (22, 65). Using both intracellular and extracellular methods, marked alterations of the SW amplitude, shape, and frequency were observed on pharmacological blockade of NKCC1 with 4 µM bumetanide, in line with the reported Ki value for bumetanide in the micromolar range (47). Bumetanide (40 µM) resulted in an even greater reduction of the SW amplitude and frequency. Reduction of the SW frequency by bumetanide has previously been reported in the guinea pig gastric antrum (56). The effect of bumetanide was reversible after washout, although recovery was slow and incomplete during the recording period with the 40 µM dose.

Bumetanide, even at the 40 µM dose, had no effect in NKCC1-KO animals, establishing the specificity of bumetanide for NKCC1 in the range of concentrations used. The constitutive lack of NKCC1 in KO animals resulted in a depolarization of the smooth muscle membrane potential and a decrease of the frequency and amplitude of SW in line with the effects of acute inhibition of NKCC1 by bumetanide.

Disturbed GI motility has been reported in mice deficient in pacemaking ICC (9, 21, 37, 65), and lack of ICC has been reported in a broad range of human GI motility disorders (46). Noteworthy, in NKCC1 KO mice, morbidity related to various severe GI dysfunctions, including hemorrhage, intussusception, and fecal impaction, has been reported around the weaning period (12). Another strain of NKCC1 KO appeared, however, essentially unaffected (14). The NKCC1 KO mice used in this study tend to be short lived, although no necropsy was performed. In NKCC1 KO mice, the intestinal ion and fluid flux are markedly altered but to a much lesser extent than in CFTR-KO mice, a model for cystic fibrosis in which severe impairment of the epithelial transport is blamed for the severe disturbance of GI transit. An unspecified "defect in the circulatory system" has been blamed for the GI morbidity observed in NKCC1 KO mice (12). Our observations on the role of NKCC1 in the pacemaker mechanism raises the possibility that the GI disturbances observed in NKCC1 KO mice may also be due, at least in part, to alterations of the electrical activity in the muscularis propria. Additional factors may be responsible for the variable phenotype observed (12, 14).

NKCC cotransporters are electroneutral, with a transport stoichiometry of 1Na+:1K+:2Cl–. The driving force for net transport is determined solely by the chemical gradients of these three ions. Under normal physiological conditions, NKCC cotransporters usually mediate net inward ion movement. The major effect of NKCC1 inhibition appears to be the disturbance of the intracellular Cl– equilibrium (47). Inhibition of Cl– currents leads to a decrease of SW amplitude and plateau component (19), in line with our results (23). NKCC1 inhibition also affects Na+ and K+ homeostasis and hence may influence inward Na+ and K+ currents. Noteworthy, a decrease of SW amplitude and an immediate hyperpolarization have been reported after removal of extracellular Na+ (54), whereas others reported a decrease of the SW amplitude and an accompanying depolarization (30). Furthermore, K+ currents also influence the resting membrane potential and excitability of ICC (15, 19, 28). The specific contribution of NKCC1 to the homeostasis of the different ions involved in the pacemaker mechanism needs to be further examined.

Our results suggest that NKCC1, which is expressed only in KIT-ir ICC-MP and myenteric neurons but not in ICC-DMP, participates to the complex mechanisms underlying the membrane potential of the smooth muscle cells and the amplitude and frequency of SW. Although NKCC1 is clearly not solely involved, it nevertheless appears to be one critical component, because adaptive mechanisms, if they exist, are unable to counterbalance the lack of NKCC1 in KO animals.

In conclusion, expression of the cotransporter NKCC1 is downregulated in the muscle layers of the jejunum in ICC-deficient mice, and NKCC1 is selectively expressed in the pacemaking ICC-MP in the mouse and human jejunum. Pharmacological inhibition in vitro and gene knockout of NKCC1 markedly altered the SW properties, indicating that NKCC1 is one of the components of the complex pacemaking mechanism in the jejunum. Further insight into the molecular mechanism of the GI pacemaker and its role in intestinal motor function may lead to new therapeutic approaches in GI motility disorders.


   GRANTS
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 MATERIALS AND METHODS
 RESULTS
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This work was supported by a research fellowship of the Born Bunge foundation to A. De Laet, grants from the National Fund for Scientific Research (Belgium), Fondation Médicale Reine Elisabeth, and Fondation Universitaire David et Alice Van Buuren (Belgium) to J. M. Vanderwinden.


   DISCLOSURES
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 MATERIALS AND METHODS
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This work was supported by a PhD fellowship from Johnson & Johnson Pharmaceutical Research & Development, a Division of Janssen Pharmaceutica (to M. Wouters).


   ACKNOWLEDGMENTS
 
We are indebted to V. Mussen, D. Kruijsen, K. Gillard, P. Hagué, and P. Janssen for the technical assistance and to J. M. Neefs for the bioinformatic analysis. We thank W. Lammers for providing the recording electrodes array and analysis software and for stimulating discussions.

J. M. Vanderwinden and A. de Kerchove d'Exaerde are Senior Research Associate and Research Associate of the National Fund for Scientific Research (Belgium), respectively. J. P. Timmermans and J. M. Vanderwinden are Partner and Associated Partner in IUAP/PAI5/20 from the Belgian Federal Science Policy Office, respectively.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-M. Vanderwinden, Université Libre de Bruxelles, Campus Erasme, CP 601, 808 route de Lennik, B-1070 Brussels, Belgium (e-mail: jmvdwin{at}ulb.ac.be)

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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