SEARCH

SEARCH BY CITATION

Keywords:

  • gastrointestinal motility;
  • myenteric plexus;
  • Na+/Ca2+ exchanger

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References

Background  The Na+/Ca2+ exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca2+ concentrations. NCX is critical for Ca2+ regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. However, little is known about the physiological role of NCX in the myenteric neurons and smooth muscles of the gastrointestinal tract.

Methods  To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in NCX1- and NCX2-heterozygote knockout mice.

Key Results  We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in NCX2 heterozygous mice (HET) than in wild-type mice (WT). Under the nonadrenergic, noncholinergic (NANC) condition, EFS-induced relaxation in NCX2 HET was similar in amplitude to that of WT. In addition, an NCX inhibitor, YM-244769 enhanced EFS-induced relaxation but did not affect EFS-induced relaxation under the NANC condition, as in NCX2 HET. Unlike NCX2 HET, NCX1 HET displayed no marked changes in colonic motility. These results indicate that cholinergic function in the colon is altered in NCX2 HET. The magnitude of acetylcholine (ACh)-induced contraction in NCX2 HET was similar to that in WT. In contrast, EFS-induced ACh release was reduced in NCX2 HET compared with that in WT.

Conclusions & Inferences  In this study, we demonstrate that NCX2 regulates colonic motility by altering ACh release onto the myenteric neurons of the distal colon.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References

The movements of the contents of the gastrointestinal tract rely on the coordinated contractions and relaxations of the smooth muscles that surround each specialized region. Excitation–contraction coupling in smooth muscle cells controls the process from electrical excitation of smooth muscle cells to peristaltic movement in the gastrointestinal tract. Ca2+ entry signals are crucial to the regulation of smooth muscle contraction and result in the activation of myosin light chain kinase (MLCK), which induces contraction.1,2 Ca2+ influx triggers Ca2+ release from the sarcoplasmic reticulum (SR). The combination of Ca2+ influx and release raises the free intracellular Ca2+ concentration allowing Ca2+ to bind to a calcium-binding protein, which then activates MLCK. The Ca2+/calmodulin-dependent signaling pathway and, notably, Ca2+-independent kinase are also able to activate MLCK.3,4

For relaxation to occur, the free intracellular Ca2+ concentration must decline. This Ca2+ clearance requires Ca2+ transport out of the cytosol by pathways involving plasma membrane Ca2+-ATPase, the Na+/Ca2+ exchanger (NCX), the sarco/endoplasmic reticulum Ca2+-ATPase, and mitochondria.5,6 Desensitization of the contractile proteins to intracellular Ca2+, in addition to a decline in the intracellular Ca2+ concentration, is crucial for relaxation in gastrointestinal smooth muscles.6 Furthermore, smooth muscle relaxation is also induced by MLC phosphatase, which results in dephosphorylation of the MLC.7,8

In the present article, we focused on the effect of Ca2+ movement through NCX on gastrointestinal tract motility because Ca2+ homeostasis is central to the regulation of smooth muscle function. NCX is a plasma membrane transporter involved in regulating intracellular Ca2+ concentrations in tissues such as the brain, kidney, and smooth muscle. NCX electrogenically exchanges Na+ and Ca2+ across the plasma membrane depending on the membrane potential and transmembrane gradients. The mammalian NCX family comprises three isoforms: NCX1,9 NCX2,10 and NCX3.11 Several splice variants were identified for NCX1 and NCX3, whereas no alternate splicing variants were detected for NCX2.12 NCX1 is expressed at high levels in the heart but is also present in many other tissues in varying amounts.13,14 NCX1 plays an important role in arterial smooth muscle cells, regulating arterial tone and blood pressure.15,16 NCX2 and NCX3 are expressed primarily in the brain and skeletal muscle.10,11 The physiological roles by which NCX influences gastrointestinal tract motility are incompletely understood and vary by tissue, although its role in cardiac muscle and brain neurons is well understood. We initially found that NCX1 and NCX2 are expressed in the smooth muscles and the myenteric neurons of gastrointestinal tissues. Coordinated responses of gastrointestinal smooth muscle depend on the influences of the myenteric neurons, intrinsic pacemaker cells, and endocrine/paracrine mediators. Excitatory or inhibitory action to smooth muscle is controlled by the conventional neurotransmitters such as acetylcholine and noradrenaline but also nonadrenergic noncholinergic (NANC) transmitters. Ca2+ entering on the myenteric neuron are necessary for release of the transmitters. Therefore, the aim of the present study was to investigate the physiological roles of NCX1 and NCX2 on gastrointestinal tract motility. To specifically assess the physiological role of NCX, we used NCX1-17 and NCX2-heterozygote knockout mice. To improve the understanding of NCX action on gastrointestinal tract motility, we used an organ tissue bath system to characterize motor function of smooth muscle segments from the distal colon in NCX1- and NCX2-heterozygote knockout mice. Furthermore, we sought to identify the mechanism underlying altered motility of smooth muscle segments in NCX1- and NCX2-heterozygote knockout mice.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References

Drugs

Acetylcholine (ACh), atropine, guanethidine, and N-nitro-l-arginine (l-NNA) were purchased from Wako Pure Chemical (Osaka, Japan). An Alexa Fluor 488-labeled goat anti-rabbit IgG and an Alexa Fluor 568-labeled goat anti-mouse IgG were purchased from Molecular Probes Inc. (Eugene, OR, USA). A goat polyclonal antibody against choline acetyltransferase (ChAT; AB144P) was purchased from Chemicon International (Temecula, CA, USA). A mouse polyclonal antibody against PGP9.5 was purchased from UltraClone Limited (Isle of Wight, UK). NOR-1 [(±)-(E)-Methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexaneamide] was purchased from Dojin (Kumamoto, Japan). Rabbit polyclonal antibodies against NCX1 and NCX2 were produced as described previously.18 As reported earlier,19 YM-244769 [N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide] was synthesized in our laboratory.

NCX knockout mice

NCX1 knockout mice were produced as reported previously.17 NCX2 knockout mice were generated as follows. Murine NCX2 gene sequences were isolated from a 129/SV mouse genomic library. The targeting vector was constructed by replacing the 4.8 kb SphI-BamHI fragment containing exon 1 of the NCX2 gene with a Neo cassette. The diphtheria toxin-A fragment gene was ligated to the 3′ position of the targeting vector for negative selection. The targeted ES clones were confirmed by Southern blot analysis and used in the generation of germline chimeras. Chimeric male mice were crossed with female C57BL/6 mice to confirm germline transmission. Both NCX1 heterozygous mice (HET) and NCX2 HET on the C57BL/6 background appeared healthy and were comparable in all analyses to age-matched wild-type mice (WT). All procedures used in this study complied with the institutional policies of the Osaka Prefecture University Animal Care and Use Committee.

Immunofluorescent staining

For tissue sections, WT were fixed by transcardiac perfusion with 4% paraformaldehyde, and the distal colon was removed. Frozen sections (5 μm thick) were cut and prepared for immunofluorescent staining.20

For whole-mount preparations, the distal colon obtained from WT was opened longitudinally, washed in Tyrode’s solution, and pinned on a dish of Sylgard silicon rubber filled with Tyrode’s solution. The mucosal layers (the mucosa and the submucosa) were peeled away from the muscular layers (circular and longitudinal muscle layers) by tweezers. The myenteric plexus attached to the muscle layers was then further cut into 2 mm segments and placed in 96-well culture plates. The segments were fixed with 4% paraformaldehyde for 2 h at 4 °C. All micro-dissections were carried out under a dissection microscope, and the fixed segments were used for immunofluorescent staining.21

The expression of NCX1 and NCX2 was detected using rabbit anti-NCX1 and anti-NCX2 antibodies. The immunoreactivity of NCX1 and NCX2 was detected using an Alexa Fluor 488-labeled goat anti-rabbit IgG antibody. To detect neurons, a mouse antibody against PGP9.5 was used, and the immunoreactivity of PGP9.5 was detected using an Alexa Fluor 568-labeled goat anti-mouse IgG antibody.22 Non-specific immunoreactivity was blocked with 10% normal goat serum, 1% BSA, and 0.3% Triton X-100 in PBS. Confocal images were obtained under a laser-scanning microscope (C1si; Nikon Corporation, Tokyo, Japan).

Recording of responses to EFS of longitudinal smooth muscles of the distal colon

A recording of the responses to EFS was carried out using previously described methods.23,24 Briefly, the distal colon was removed from mice (10–15 weeks old). Whole-wall strips were prepared in the orientation of the longitudinal muscle layer. These strips were mounted vertically in organ baths and held at a resting tension of 0.5 g. Baths were filled with Tyrode’s solution, which was maintained at 37 °C and bubbled with 95% O2 and 5% CO2. Responses to EFS were detected using isotonic force transducers (TD-112A; Nihonkohden, Tokyo, Japan). Tissue strips were allowed to equilibrate for at least 30 min. Following this equilibration period, the strips were exposed to EFS with trains of 100 pulses of 0.5 ms, 30 V, and 10 Hz for 10 s to evoke smooth muscle contraction and relaxation. When NANC responses were recorded, atropine (1 μmol L−1) and guanethidine (5 μmol L−1) were used to block the cholinergic responses and exclude the adrenergic responses, respectively.25,26l-NNA (30 μmol L−1) and YM-244769 (1 μmol L−1) tested were directly added to the bath at least 10 min prior to EFS. Relaxations, relative to the baseline tone, were analyzed by measuring the extent of the maximal relaxations in response to Ca2+-free EGTA solution. Contractions relative to the baseline tone were analyzed by measuring the extent of the maximal contraction in response to high K+ solution (60 mmol L−1). Basal tones were expressed as percentages of the strip length, which accounts for the addition of the maximal relaxations in response to Ca2+-free EGTA solution and the maximal contractions in response to high K+ solution.

Acetylcholine release induced by EFS

Acetylcholine release was assayed using a previously described method.27 Briefly, the distal colon was opened longitudinally. The mucosa layers were then peeled away from the muscular layers by tweezers. Baths were filled with 1 mL of Tyrode’s solution containing physostigmine (5 μmol L−1) and choline (1 μmol L−1), maintained at 37 °C and bubbled with 95% O2 and 5% CO2. Tissue strips were allowed to equilibrate for at least 30 min. The strips were exposed to EFS with trains of 100 pulses of 0.5 ms, 30 V, and 10 Hz for 20 s. Acetylcholine release and content were assayed by HPLC.

Quantitative real-time PCR

Quantitative real-time PCR for NCX1 and NCX2 mRNA was performed using a previously described method.28 Briefly, the distal colon was removed from the mice. The mucosal layers were peeled away from the muscular layers. Total RNA was extracted from the muscular layers. Amplification of HPRT mRNA was used for each experimental sample as an endogenous control to account for differences in the amount and quality of total RNA added to each reaction.

Statistical analysis

The results were expressed as means ± SE. Statistical significance was determined by a one-way anova for non-repeated measures to detect differences among WT, NCX1 HET, and NCX2 HET. The differences between groups were determined using the Tukey–Kramer test. The statistical significance was determined using the two-tailed Student’s t-test (paired) for comparisons with the control group. A P value of less than 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References

Localization of NCX1 and NCX2

We initially investigated the expression and localization of NCX1 and NCX2 in the distal colon of WT using double immunofluorescent staining. As shown in Fig. 1A, strong immunoreactivity of NCX1 and NCX2 was observed within the myenteric plexus layers (left panels). Expression of NCX1 and NCX2 was observed in the longitudinal and circular muscle layers as well as in the myenteric plexus layers. Myenteric neurons, which were recognized with the PGP9.5 antibody, expressed NCX1 and NCX2 in the myenteric plexus layers (right panels). In accordance with these results, myenteric neurons express NCX1 and NCX2 within the ganglion of the myenteric plexus in whole-mount preparations (Fig. 1B). In contrast, neither NCX1 nor NCX2 is expressed in the glial cells, which were stained using the S-100 antibody (data not shown).

image

Figure 1.  NCX expression in the distal colon of wild-type mice (WT). Immunohistochemical staining of tissue sections in the distal colon of WT (A) and whole-mount preparations of myenteric plexus layers in the distal colon of WT (B). Tissue sections and whole-mount preparations were stained for NCX1 or NCX2 (green). Neurons were stained with PGP9.5 (red). Double staining with NCX1/2 and PGP9.5 appears yellow in the merged images. Images shown are representative of four experiments. CM, circular muscle layer; MP, myenteric plexus layer; LM, longitudinal muscle layer. Scale bar, 100 μm. (C) The expression levels of NCX1 and NCX2 in WT (= 10), NCX1 HET (= 5), and NCX2 HET (= 6) were examined by quantitative real-time PCR. The mRNA level of protein in the distal colon is shown as a fold increase relative to the level of HPRT mRNA in the distal colon. *< 0.05.

Download figure to PowerPoint

As shown in Fig. 1C, the expression level of NCX1 is significantly lower in NCX1 HET. NCX2 HET show significantly lower levels of NCX2.

Electric field stimulation-induced responses

We investigated the responses to EFS in the longitudinal muscle obtained from the distal colon in WT, NCX1 HET, and NCX2 HET. Fig. 2A shows representative recording traces of responses to EFS. EFS induced a relaxation that persisted during the stimulus, and a rebound contraction (off-contraction) was recorded after the end of the stimulus. There was no notable difference in the basal tone when NCX1 HET or NCX2 HET was compared with WT (Fig. 2B). Importantly, the magnitudes of EFS-induced relaxation were greater in NCX2 HET, but not NCX1 HET, compared with WT (Fig. 2C). In contrast, NCX1 HET and NCX2 HET showed off-contractions in a manner similar to WT (Fig. 2D). As shown in Fig. 2C, EFS-induced relaxation in NCX2 HET accounts for approximately 280% of the relaxations in WT. We also investigated frequency–response EFS curve to find whether enhanced relaxation in NCX2 HET is present at any stimulation. Like 10 Hz, relaxations were greater in NCX2 HET at 1 and 3 Hz. EFS-induced relaxation in NCX2 HET accounts for approximately 180% at 1 Hz and 230% at 3 Hz of the relaxations in respective WT (data not shown).

image

Figure 2.  Enhanced relaxation during electric field stimulation (EFS) in NCX2 heterozygous mice (HET). EFS-induced responses in longitudinal muscle strips isolated from the distal colon in wild-type mice (WT) (= 9), NCX1 HET (= 11), and NCX2 HET (= 12). (A) Representative recording traces of EFS-induced responses are shown. Bars indicate the duration (10 s) of EFS. After basal tones were recorded, the chart speed was increased to make the EFS-induced responses clear. (B) Quantitative data on the basal tone. The basal tone was expressed as a percentage of the length of the strips. (C) Quantitative data on the EFS-induced relaxations. EFS-induced relaxations were expressed as percentages of Ca2+-free EGTA induced relaxation. **< 0.01 for WT vs. NCX2 HET. (D) Quantitative data on the off-contractions. Off-contractions were expressed as percentages of KCl-induced contraction.

Download figure to PowerPoint

Relaxation of the gastrointestinal smooth muscle induced by the release of NANC transmitters from enteric nerves occurs in several physiological digestive reflexes.24 Therefore, we determined whether NCX1 and NCX2 deficiency affects the EFS-induced responses under the NANC condition and/or during l-NNA administration. As illustrated in Fig. 3A, EFS under NANC conditions elicited an increased relaxation. Neither NCX1 HET nor NCX2 HET changed the basal tone (Fig. 3B). Unlike the response observed in normal conditions (Fig. 2C), the magnitudes of EFS-induced relaxation under NANC conditions in NCX2 HET and NCX1 HET were similar to those observed in WT (Fig. 3C). Off-contractions were similar among WT, NCX1 HET, and NCX2 HET (Fig. 3D). To characterize the neurotransmission process, EFS was carried out after tissues were incubated with l-NNA. EFS induced a contraction in the presence of l-NNA (Fig. 4A, left panel). The magnitudes of the EFS-induced contraction were smaller in NCX2 HET, but not NCX1 HET, compared with that in WT (Fig. 4A, middle panel). In contrast, NCX1 HET and NCX2 HET showed off-contractions in a manner similar to WT (Fig. 4A, right panel). In the experiments in which l-NNA was added under NANC conditions following the EFS, the magnitudes of EFS-induced relaxation were smaller in NCX1 HET and NCX2 HET than those in WT (Fig. 4B). These results indicate that NCX2 HET causes a decrease in the concentration of an excitatory mediator such as ACh and/or an increase in the concentration of an inhibitory NANC mediator during EFS.

image

Figure 3.  Electric field stimulation (EFS)-induced responses under NANC. EFS-induced responses under NANC conditions in longitudinal muscle strips isolated from the distal colon in wild-type mice (WT) (= 8), NCX1 heterozygous mice (HET) (= 10), and NCX2 HET (= 8). (A) Representative recording traces of EFS-induced responses are shown. Bars indicate the duration (10 s) of EFS. After the basal tone was recorded, the chart speed was increased to make the EFS-induced responses clear. (B) Quantitative data on the basal tone. The basal tone was expressed as a percentage of the length of the strips. (C) Quantitative data on the EFS-induced relaxations. EFS-induced relaxations were expressed as percentages of Ca2+-free EGTA-induced relaxation. (D) Quantitative data on the off-contractions. Off-contractions were expressed as percentages of KCl-induced contraction.

Download figure to PowerPoint

image

Figure 4.  Differences in electric field stimulation (EFS)-induced responses in NCX1 heterozygous mice (HET) and NCX2 HET. (A) EFS in the presence of l-NNA in longitudinal muscle strips isolated from the distal colon in WT (= 3), NCX1 HET (= 3), and NCX2 HET (= 4). Representative recording traces of EFS-induced responses are shown. Bars indicate the duration (10 s) of EFS. Quantitative data on the EFS-induced contractions (middle panel). EFS-induced contractions were expressed as percentages of KCl-induced contraction. **< 0.01 for WT vs NCX2 HET. Quantitative data on the off-contractions (right panel). Off-contractions were expressed as percentages of KCl-induced contraction. (B) EFS in the presence of l-NNA under NANC conditions in the longitudinal muscle strips isolated from the distal colon in WT (= 3), NCX1 HET (= 3), and NCX2 HET (= 3). Representative recording traces of EFS-induced responses are shown. Bars indicate the duration (10 s) of EFS. After the basal tone was recorded, the chart speed was increased to make the EFS-induced responses clear. Quantitative data on the EFS-induced relaxations (right panel). EFS-induced relaxations were expressed as percentages of relaxation under NANC conditions alone. **< 0.01 vs WT.

Download figure to PowerPoint

Responses of smooth muscle cells

Because there is expression of NCX1 and NCX2 in longitudinal smooth muscles as well as in neurons of the myenteric plexus layers (Fig. 1A), we determined whether NCX deficiency affects contraction in response to ACh and relaxation in response to nitric oxide (NO) in smooth muscle cells. Acetylcholine induced contraction in a dose-dependent manner. NCX1 HET and NCX2 HET exhibited ACh-induced contractions with magnitudes similar to those of WT (Fig. 5A). NOR-1, which generates NO, induces relaxation. NCX1 HET and NCX2 HET demonstrated magnitudes of NOR-1-induced relaxation similar to those of WT (Fig. 5B). ATP in addition to NO play major roles as NANC neurotransmitters in the longitudinal muscle of the mouse distal colon.29,30 The binding of ATP to its receptor, P2Y receptor, activates the phospholipase C and inositol 1,4,5-trisphosphate (IP3) receptors. ATP induced an increase in Ca2+ levels near the plasma membrane (Ca2+ puffs), which induced relaxation by activating small conductance Ca2+-dependent K+ channels (SK channels), which leads to hyperpolarization. We also determined whether NCX deficiency affects relaxation in response to ATP in smooth muscle cells. However, NCX1 HET and NCX2 HET demonstrated magnitudes of ATP-induced relaxation similar to those of WT (data not shown). Therefore, we can exclude the possibility that the NCX deficiency of smooth muscle cells enhances their sensitivity to NO and ATP, and attenuates their sensitivity to ACh.

image

Figure 5.  Acetylcholine (Ach)-induced contraction and NOR-1-induced relaxation. (A) ACh-induced contractions in longitudinal muscle strips isolated from the distal colon in wild-type mice (WT) (= 4), NCX1 heterozygous mice (HET) (= 3), and NCX2 HET (= 4). Representative recording traces of ACh-induced contractions are shown. ACh-induced contractions are expressed as percentages of KCl-induced contractions. (B) NOR-1-induced relaxation in the longitudinal muscle strips isolated from the distal colon in WT (= 5), NCX1 HET (= 5), and NCX2 HET (= 7). NOR-1-induced relaxations are expressed as percentages of Ca2+-free EGTA-induced relaxation.

Download figure to PowerPoint

Acetylcholine release

In the distal colon of WT, the amount of ACh released spontaneously was 764 pmol g−1 tissue/20 s. EFS increased ACh release to 2389 pmol g−1 tissue/20 s (Fig. 6A). There was no significant difference in the values of spontaneous ACh released in NCX1 HET and NCX2 HET compared with WT. EFS-induced ACh release was notably smaller in NCX2 HET, but not NCX1 HET, compared to WT (Fig. 6A). We investigated total ACh contents in the distal colon from WT, NCX1 HET, and NCX2 HET. NCX1 HET and NCX2 HET showed a total ACh content similar to that of WT (Fig. 6B). These results suggest that NCX2 HET has decreased ACh release in myenteric neurons, as detected by anti-NCX2 antibody staining. We investigated the important possibility that NCX2 and ChAT co-localize in the myenteric plexus using double immunofluorescent staining. As shown in Fig. 6C, there is co-localization of NCX2 and ChAT within the myenteric plexus in whole-mount preparations.

image

Figure 6.  Repressed release of acetylcholine (Ach) during electric field stimulation (EFS) in NCX2 heterozygous mice (HET). (A) Spontaneous and EFS-induced release of ACh in longitudinal muscle strips isolated from the distal colon in WT (= 8), NCX1 HET (= 3), and NCX2 HET (= 5). *< 0.05 for WT vs NCX2 HET. (B) ACh content in the longitudinal muscle strips isolated from the distal colon in WT (= 3), NCX1 HET (= 3), and NCX2 HET (= 3). (C) Immunohistochemical staining of tissue sections in whole-mount preparations of myenteric plexus layers in the distal colon of WT. Whole-mount preparations were stained for NCX2 (green). ChAT staining is also shown in red. Double staining with NCX2 and ChAT appears yellow in the merged images. The images shown are representative of five experiments. Scale bar, 10 μm.

Download figure to PowerPoint

Effect of NCX inhibitors on EFS-induced responses

To further evaluate the physiological role of NCX in gastrointestinal motility, we investigated the effect of YM-244769, a specific NCX inhibitor,31 on EFS-induced responses. Fig. 7A shows representative recording traces of responses to EFS with or without YM-244769. When administered onto smooth muscles, YM-244769 (1 μmol L−1) had no effect on the basal tone of the tissue strips, even after a 30-min exposure period (Fig. 7B). As shown in Fig. 7C, YM-244769 enhanced the magnitudes of EFS-induced relaxation, whereas it attenuated the magnitudes of off-contraction after EFS (Fig. 7D). We determined whether it affected the EFS-induced responses under the NANC condition and during l-NNA administration. As illustrated in Fig. 8A, YM-244679 had no systematic influence on EFS-induced relaxation under NANC conditions. In the experiments where l-NNA was added under NANC conditions following EFS, YM-244679 did not affect the magnitudes of EFS-induced relaxation (Fig. 8B).

image

Figure 7.  Effects of the NCX inhibitor on electric field stimulation (EFS)-induced responses. Effects of YM-244769 (= 4) on the EFS-induced responses in longitudinal muscle strips isolated from the distal colon in wild-type mice (WT) (control; = 9). (A) Representative recording traces of EFS-induced responses are shown. Bars indicate the duration (10 s) of EFS. After the basal tone was recorded, the chart speed was increased to make the EFS-induced responses clear. (B) Quantitative data on the basal tone. The basal tone was expressed as a percentage of the length of the strips. (C) Quantitative data on the EFS-induced relaxations. EFS-induced relaxations are expressed as percentages of Ca2+-free EGTA induced relaxation. **< 0.01 for control. (D) Quantitative data on the off-contractions. Off-contractions are expressed as percentages of KCl-induced contraction. **< 0.01 for control.

Download figure to PowerPoint

image

Figure 8.  Effects of NCX inhibitor on electric field stimulation (EFS)-induced responses under NANC conditions in the absence or presence of l-NNA. (A) EFS under NANC conditions. Effects of YM-244769 (= 4) on the EFS-induced responses in longitudinal muscle strips isolated from the distal colon in wild-type mice (WT) (control; = 16). Quantitative data on the EFS-induced relaxations. EFS-induced relaxations are expressed as percentages of Ca2+-free EGTA induced relaxation. (B) EFS under NANC conditions in the presence of l-NNA. Effects of YM-244769 (= 3) on the EFS-induced responses in the longitudinal muscle strips isolated from the distal colon in WT (control; = 3). Quantitative data on the EFS-induced relaxations. EFS-induced relaxations are expressed as percentages of relaxation under NANC conditions. (C, D) Absence of effect of NCX inhibitor on acetylcholine (Ach)-induced contraction and NOR-1-induced relaxation. (C) Effects of YM-244769 (= 3) on ACh-induced contraction in longitudinal muscle strips isolated from the distal colon in WT (control; = 4). ACh-induced contractions are expressed as percentages of KCl-induced contractions. (D) Effects of YM-244769 (= 3) on the NOR-1-induced relaxations in longitudinal muscle strips isolated from the distal colon in WT (control; = 5–7). NOR-1-induced relaxations are expressed as percentages of Ca2+-free EGTA-induced relaxation.

Download figure to PowerPoint

We determined whether the NCX inhibitor affects contraction in response to ACh and relaxation in response to NO in smooth muscle cells. YM-244769 had no effect on the magnitudes of ACh-induced contraction (Fig. 8C) or on the magnitudes of NOR-1-induced relaxation (Fig. 8D).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References

The first aim of this study was to investigate the physiological role of NCX on gastrointestinal tract motility. Little is known about the role of NCX in regulating gastrointestinal tract motility. Our immunohistochemical results provide evidence indicating the presence of NCX-containing neurons in the myenteric plexus of the gastrointestinal tract of mice. Our findings indicate a role for NCX in regulating murine distal colon motility and highlight the importance of NCX for this regulation. We demonstrated that NCX2 HET show enhanced amplitudes of EFS-induced relaxation. This enhanced relaxation is not pronounced in NCX1 HET, suggesting that NCX1 and NCX2 have different responses in distal colon motility. It appears that the overall functional properties of NCX1 and NCX2 are not fundamentally different, although protein kinase A and C are found to activate NCX1, but not NCX2.32 Given that little is known about the differences between the functional properties of NCX1 and NCX2, we would like to note that the different phenotypes of NCX1 HET and NCX2 HET in the current study provide important information useful for elucidating NCX2-specific properties.

There are two possibilities for enhanced relaxation as observed in NCX2 HET. One possibility may be a decrease in the concentration of an excitatory component. Another may be an increase in the concentration of an inhibitory component in myenteric neurons during EFS that can cause increased relaxation in smooth muscles. We found that NCX2 HET does not show enhanced amplitude of EFS-induced relaxation under NANC conditions, suggesting that enhanced relaxation in NCX2 HET is associated with a decrease in the concentration of an excitatory component. Consistent with this suggestion, attenuation of EFS-induced contraction in the presence of l-NNA in NCX2 HET is consistent with a decrease in the concentration of an excitatory component. Therefore, we exclude the possibility that NCX2 HET causes increased release of inhibitory NANC mediators such as NO. However, gastrointestinal tract motility can be regulated by sensitivity to mediators of the smooth muscle cells in addition to the release of mediators from myenteric neurons. In fact, the immunohistochemical results indicated the presence of NCX-positive cells in the smooth muscles as well as in the myenteric plexus. To determine whether the release of mediators from myenteric neurons can regulate smooth muscle cells, we used NOR-1, an NO donor, to relax distal colonic smooth muscles. In NCX1 HET and NCX2 HET, the magnitudes of NOR-1-induced relaxation were similar to that of WT. Hence, we can exclude the possibility that NCX deficiency enhances the sensitivity of the smooth muscle cells to inhibitory NANC mediators such as NO. How does NCX2 HET decrease the concentration of the excitatory component? There are at least two possible explanations. One possibility is the decreased release of excitatory mediators such as ACh from neurons. The second possibility is attenuated sensitivity to excitatory mediators such as ACh on the smooth muscle cells. Carbachol induces contraction in the distal colonic smooth muscles. In the present study, the amplitude of carbachol-induced contraction was not significantly altered in NCX1 HET and NCX2 HET relative to WT, suggesting that enhanced relaxation in NCX2 HET is not associated with attenuated sensitivity of the smooth muscle cells to ACh. As expected, NCX2 HET displayed decreased release of ACh in the distal colon segments. These results suggest that NCX2 regulates colonic motility by altering ACh release onto the myenteric neurons of the distal colon.

Experiments in which l-NNA was added under NANC conditions indicated that the NO component accounts for approximately 40% of the relaxations in WT, whereas it accounts for approximately 70% of the relaxations in NXC1 HET and NCX2 HET. However, EFS-induced relaxations under NANC conditions are almost identical among WT, NCX1 HET, and NCX2 HET. The likely explanation is that inhibitory NANC mediators other than NO are decreased, rather than the existence of an increased NO component in NCX1 HET and NCX2 HET.

We chose to use the NCX inhibitor YM-244769 because the mechanism of action of NCX has not been fully characterized in the gastrointestinal tract. YM-244769 is a potent NCX inhibitor that has very little effect on other molecules and receptors.31 It has also been shown to inhibit Ca2+ influx without affecting Ca2+ efflux from NCX1, NCX2 and NCX3.31 The use of this inhibitor demonstrated that the effect of NCX blockade leads to enhanced EFS-induced relaxation, unaltered EFS-induced relaxation under NANC conditions, and unaltered ACh-induced contraction. These observations are consistent with the observation in NCX2 HET, although 1 μmol L−1 YM-244769 (the concentration used in this study) inhibits NCX1, NCX2, and NCX3.31 Elevated intracellular Ca2+ concentrations are essential for neurotransmitter release, including the release of ACh. These results are consistent with the possibility that NCX2 contributes to Ca2+ influx, but not efflux, during neurotransmitter release in response to EFS, as NCX1 HET and NCX2 HET display different phenotypes of distal colon motility. Also consistent with this explanation is the well-known fact that Ca2+ entry via voltage-gated Ca2+ channels is essential for neurotransmitter release.33 However, Ca2+ can be made available from sources other than the extracellular space. Ca2+ release from internal stores, including the SR and mitochondria, is also important for neurotransmitter release.34 Recently, it was found that Ca2+ entry via Ca2+/H+ antiporters can contribute to neurotransmitter release.35 In rat cortical neurons, it has been reported that Ca2+ entry via NCX contributes to neurotransmitter release.36 In contrast, Ca2+ influx through NCX has been suggested to occur in pathophysiological states, as Ca2+ influx through NCX increases substantially when intracellular Na+ rises. This may occur as a result of Na+/K+ ATPase inhibition37 or sodium hydrogen exchanger (NHE)-mediated Na+ increase.38 The promotion of Ca2+ influx through NCX has been suggested to occur in heart failure39 and ischemia, which are typically associated with increases in intracellular Na+ levels.40 In combination with the previous evidence, these results suggest that Ca2+ influx through NCX2 may contribute to physiological Ca2+ transients during neurotransmitter release and/or action potentials.

Off-contraction is mainly caused by non-cholinergic mechanisms, because there is off-contraction under the NANC condition (Figs 2D and 3D). In our results of WT, off-contraction under the NANC condition is smaller than that in the normal condition (Figs 2D and 3D). Moreover, YM-244769 attenuated off-contraction (Fig. 7D). Off-contraction in the WT under the NANC condition is similar to that in the presence of YM-244769 (Figs 3D and 7D). These results suggest that approximately 20% of the off-contraction would depend on ACh, and the possibility that YM-244769 may decrease the release of ACh.

In conclusion, the current study shows that NCX2 plays an important role in smooth muscle motility in the mouse distal colon. NCX2-heterozygote knockout causes changes in ACh release in the myenteric neurons of the mouse distal colon. The NCX-heterozygote knockout mouse could be a valuable tool for delineating the mechanisms of NCX action on gastrointestinal tract motility. The current study may provide information useful in identifying therapeutic targets. NCX inhibitors are an important consideration in the design of therapeutic agents in cardiac muscle, vascular smooth muscle, and nerve fibers.41,42 It would be interesting to investigate the potency of the NCX inhibitor in human colonic tissue.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References

YTA & TT designed the research study; KN & YTA performed the research and analyzed the data; SK, IK & TI contributed the knockout mice for the study; HN analyzed the data; TI contributed essential reagents; YTA wrote the paper. All authors approved the final version of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Funding
  8. Disclosure
  9. Author contributions
  10. References