SEARCH

SEARCH BY CITATION

Keywords:

  • aganglionic bowel;
  • Hirschsprung’s disease, smooth muscle

Abstract

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

Background  Studies on animal models of Hirschsprung’s disease (HD) suggest that L-type Ca2+ channels are down-regulated in the aganglionic bowel segment, however, this has yet to be confirmed in HD patients. The objective of this study was to test the hypothesis that L-type Ca2+ current density is decreased in smooth muscle cells (SMC) obtained from the aganglionic bowel segment of patients with HD in comparison with those from the ganglionic segment.

Methods  Smooth muscle cells were freshly isolated from colon samples obtained from HD patients undergoing pull-through surgery. L-type Ca2+ currents were recorded using the perforated patch configuration of the whole cell voltage clamp technique and the expression levels of CACNA1C transcripts (which encode L-type Ca2+ channels) in the ganglionic and aganglionic bowel segments were compared using real-time quantitative PCR.

Key Results  All SMC displayed robust currents that had activation/inactivation kinetics typical of L-type Ca2+ current, were inhibited by nifedipine and enhanced by the L-type Ca2+ channel agonists FPL 64176 and Bay K 8644. Moreover, FPL 64176 activated currents were also inhibited by nifedipine. However, there was no significant difference in L-type Ca2+ current density, CACNA1C subunit expression or sensitivity to the pharmacological agents noted above, between SMC isolated from the ganglionic and aganglionic regions of the HD colon.

Conclusions & Inferences  In contrast to studies on genetic animal models of HD, L-type Ca2+ currents are not down-regulated in the aganglionic bowel segment of HD patients and are therefore unlikely to account for the impaired colonic peristalsis observed in these patients.


Introduction

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

Hirschsprung’s disease (HD) is a congenital condition characterized by the absence of enteric ganglia and proliferation of extrinsic nerve fibers in the distal colon leading to impaired peristalsis and functional obstruction of this area. The pathophysiology of HD is complex and is known to be associated with mutations in several genes and loci particularly those encoding for RET tyrosine kinase and the Endothelin receptor B (EDNRB) which are important for the normal migration of neural crest cells to the intestine during development.1 However, several studies have suggested that the behavior of the aganglionic bowel segment may also involve changes in the activity of smooth muscle cells (SMC) in this region. For example, Kubota et al. showed that SMC of the aganglionic segment were electrically quiescent and spontaneous action potentials, with a low frequency, were only observed in a few cells.2 This was in contrast to recordings made from the ganglionic region in which all SMC developed oscillatory changes in membrane potential.2 Spencer et al. found that endothelin type B receptor-deficient mice (Ednrbs-l/Ednrbs-l) which develop congenital rectal aganglionosis, and are thus used as model to study human HD, had sporadic and asynchronous Ca2+ waves between the longitudinal and circular muscle layers of the colon, leading to disrupted peristalsis.3 The exact reasons for the changes in behavior reported in these studies were not elucidated, however, it is well recognized that actions potentials and Ca2+ waves in colonic smooth muscle are dependent on Ca2+ influx via L-type Ca2+ channels.4,5 L-type Ca2+ channels are voltage gated and act as the major route for Ca2+ influx to activate the contractile machinery in intestinal smooth muscle.6 Interestingly, several studies on genetic animal models of HD have now reported reduced contractile responses of the aganglionic bowel segment to application to raised extracellular K+ solution7,8 consistent with a down-regulation of L-type Ca2+ channels in the aganglionic HD colon. Furthermore, Nakatsuji et al. found that the transcriptional expression of the α1c subunit of the L-type Ca2+ channel (now referred to as CACNA1C) was reduced in the aganglionic segment of the distal colon in endothelin type B receptor null rats (ETB (−/−)R in comparison with wild type controls.8

Despite these findings there has been no systematic evaluation of the activity of L-type Ca2+ channels in HD patients. In this study we have now, for the first time, provided a comprehensive characterization of the L-type Ca2+ current in SMC freshly isolated from the ganglionic and aganglionic bowel segments in HD patients.

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 contribution
  10. References

Tissue collection

Ethical approval was received, prior to the start of the project, from the Ethics Committee of Our Lady’s Children’s Hospital Crumlin, Dublin. Resected tissue was obtained immediately from HD patients at the time of pull-through surgery. Normal control colon specimens were obtained at the time of colostomy closure from patients treated for imperforate anus. In total, samples from nine HD patients and two controls were used in this study. All HD samples were short segment cases. Informed consent was obtained from parents/guardians of all patients included in this study. Hirschsprung’s disease patients ranged in age from 2 to 37 months (median 5 months) and colostomy closure patients aged in range from 4 to 62 months (median 9.5 months). The resected segment was divided by a consultant pathologist into ganglionic, and aganglionic regions, based on intra-operative biopsy results. All tissue segments were immediately immersed in ice-cold Kreb’s solution for transportation to the laboratory.

Cell isolation

Specimens were opened longitudinally and pinned in a Petri dish with a Sylgard elastomer lined base with the luminal side facing upwards. The mucosa was removed by sharp dissection, exposing the underlying smooth muscle layers. Strips of muscle, ∼0.5 cm in width were cut into 1 mm3 pieces and stored in Hanks Ca2+-free solution for 30 min before being incubated in dispersal medium containing [per 5 ml of Ca2+-free Hanks solution (see Solutions and drugs)]: 15 mg collagenase (Sigma type 1A; Sigma, Arklow, Ireland), 1.0 mg protease (Sigma type XXIV; Sigma), 10 mg bovine serum albumin (Sigma), and 10 mg trypsin inhibitor (Sigma) for 10–15 min at 37 °C. Tissue was then transferred to Ca2+-free Hanks solution and stirred for a further 15–30 min to release single SMC. These cells were plated in Petri dishes containing 100 μmol L−1 Ca2+ Hank’s solution and stored at 4 °C for use within 8 h.

Patch clamp recordings

Currents were recorded with the perforated patch configuration of the whole cell patch clamp technique.9 This circumvented the problem of current rundown encountered using the conventional whole cell configuration. The cell membrane was perforated using the antibiotic amphotericin B (600 μg mL−1; Sigma). Patch pipettes were initially front filled by dipping into pipette solution and then back filled with the amphotericin B containing solution. Pipettes were pulled from borosilicate glass capillary tubing (1.5 mm outer diameter, 1.17 mm inner diameter; Harvard Apparatus Ltd, Kent, England) to a tip of diameter approximately 1–1.5 μm and resistance of 2–4 MΩ. Voltage clamp commands were delivered via an Axopatch 1D patch clamp amplifier (Molecular Devices, Sunnydale, CA, USA) connected to a Digidata 1440A AD/DA converter (Molecular Devices) interfaced to a computer running pClamp software (Molecular Devices). During experiments, the cell under study was continuously superfused with Hanks’ solution by means of a close delivery system consisting of a pipette (tip diameter 200 μm) placed approximately 300 μm away. This could be switched, with a dead-space time of around 5 s, to a solution containing a drug. All experiments were carried out at 35–37 °C.

Solutions and drugs

The solutions used were of the following composition (mmol L−1): Hanks’: 130 Na+, 5.8 K+, 135 Cl, 4.16 HCO3, 0.34 HPO32−, 0.44 H2PO4−, 1.8 Ca2+, 0.9 Mg2+, 0.4 SO42−, 10 dextrose, 2.9 sucrose, 10 HEPES, pH adjusted to 7.4 with NaOH; Ca2+-free Hanks’ solution (for cell dispersal): NaCl (125), KCl (5.36), glucose,10 sucrose (2.9), NaHCO3 (15.5), KH2PO4 (0.44), Na2HPO4 (0.33), N-[2-Hydroxyethylpiperazine]-N′-[2-ethanesulfonic acid] (HEPES; 10), pH adjusted to 7.4 with NaOH. Krebs’ solution: NaCl (120), KCl (5.9), NaHCO3 (1.2), glucose (5.5) CaCl2 (12.5), MgCl2,6 pH maintained at 7.4 by bubbling with 95% O2–5% CO2. Perforated patch solution: CsCl (133), MgCl2 (1.0), EGTA (0.5), HEPES, (10) pH adjusted to 7.2 with CsOH. Drugs used were nifedipine (Abcam, Cambridge, UK), FPL 64176 (Tocris Bioscience, Bristol, UK) and Bay K 8644+ (Sigma).

Statistics

Summary data are presented as the mean ± SEM. Statistical differences in experiments were compared using Students paired t-test, or one-way anova as appropriate, taking the P < 0.05 level as significant. Throughout, n refers to the number of cells in each experimental series. In each case these were obtained from a minimum of three different colon samples.

Molecular biology

Tissue samples were stored at −20 °C in RNAlater (QIAGEN, Valencia, CA, USA) until use. Immediately prior to isolation of the RNA, tissue samples were transferred to a 1.5 mL tube, snap frozen in liquid nitrogen, and pulverized to yield a dry powder. RNA was isolated from these samples using the RNeasy mini kit (QIAGEN), with DNase treatment included, and eluted with RNase-free water. RNA concentration was determined using a nanodrop spectrophotometer (Thermo scientific, Waltham, MA, USA) and the purified RNA was then stored at −80 °C. Prior to cDNA synthesis, the RNA was denatured for 5 min at 70 °C and then transferred to ice. RNA was reverse transcribed using the Superscript VILO cDNA synthesis kit (Invitrogen, Life Technologies Ltd, Paisley, UK) according to the manufacturers’ instructions. Real-time quantitative PCR (qPCR) was performed on a QUANTICA real-time PCR system (TECHNE, Bibby Scientific Ltd, Staffordshire, UK) using the SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies Ltd, Paisley, UK) with CACNA1C gene-specific primers (forward 5′-CCCCGAAACATGAGCATGCCC-3′ and reverse 5′-CGCCAGTAGCGGCTGAACTTTGAC-3′). Each primer was designed to span a particular exon–exon boundary present in all major CACNA1C transcripts. Quantitect human GAPDH primers (QIAGEN) were used for amplification of GAPDH as a reference gene for use in sample normalization. The cycling conditions were as follows: an initial 10-min denaturation at 95 °C was followed by 40 cycles of: denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 68 °C for 1 min. Tissue samples were analyzed in triplicate. In addition, no-template controls were included for both primer sets. Dilutions of normal control human smooth muscle tissue cDNA were incorporated on every plate to generate standard curves for use in subsequent analyses. After real-time qPCR acquisition, a dissociation curve (50–95 °C) was obtained. Subsequent analysis of the individual melting curves allowed us to verify the specificity of GAPDH and CACNA1C primer sets. Using CACNA1C primers, we were able to demonstrate the quality of the RNA isolated from our tissue samples. Thus, the average Cp values obtained using RNA from a sample in which CACNA1C is highly expressed, FirstChoice Human Brain Reference RNA (Ambion, Life Technologies Ltd, Paisley, UK), and RNA isolated from our paired ganglionic and aganglionic tissue samples were similar in value (27.72 ± 0.15 and 27.95 ± 0.63, respectively).

Results

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

Enzymatic dispersal of all colon tissues yielded isolated SMC. The capacitance of each of these cells (calculated by integrating the capacitive current evoked by small hyperpolarizing and depolarizing voltage steps, and dividing by the amplitude of the voltage change) averaged 49.7 ± 4.7 pF in 3 SMC isolated from control tissues, 62.7 ± 4 pF in 22 SMC isolated from the ganglionic region of the HD bowel, and 60 ± 3.4 pF in 19 SMC from the aganglionic region of the HD bowel. There was no significant difference in the mean values between any groups (P > 0.05). To study L-type Ca2+ currents, SMC were voltage clamped at a holding potential of −80 mV and subjected to a series of test potentials ranging from −80 to +50 mV, for a duration of 500 ms and at 10 mV increments. The pipette solution contained Cs+ to minimize contaminating outward K+ currents. This protocol evoked transient inward currents, which peaked within 20 ms in each group of cells. Representative examples of currents recorded from SMC isolated from control, ganglionic, and aganglionic tissue specimens are shown in Fig. 1A–C, respectively. A summary plot of the peak current density against potential (I-V plot) for the full range of test potentials in each cell type is shown in Fig. 1D. Current density was calculated by dividing the peak inward current, at each potential, by the capacitance value of that cell. The inward currents activated at potentials positive to −40 mV, peaked between 0 and +10 mV and reversed at +40 mV. These properties are typical of those expected for L-type Ca2+ currents. Moreover, there was no significant difference in the mean peak current density in each group of cells. For example, the mean peak current density evoked by a step to +10 mV was, −1.84 ± 0.5 pA/pF in control, non-HD, ganglionic SMC (n = 3 cells from two patients), −2.2 ± 0.35 pA/pF in ganglionic SMC (n = 22 cells from seven patients), and −2.0 ± 0.2 pA/pFin aganglionic SMC (n = 19 cells from seven patients).These data were taken from a total of nine HD patients. In five of these patients we were able to record L-type Ca2+ currents from equal numbers of SMC isolated from both the ganglionic and aganglionic regions of each patient. The mean peak current density in the ganglionic SMC (n = 14 cells from five patients) was −1.67 ± 0.24 pA/pF compared to −1.73 ± 0.19 pA/pF in 14 aganglionic SMC taken from the same five patients. There was no significant difference between these groups (P > 0.05, paired t-test).

image

Figure 1.  (A–C) Representative whole cell, perforated patch recordings showing families of inward currents evoked from, control, ganglionic and aganglionic colonic smooth muscle cells (SMC), respectively. (D) Summary current – voltage (IV) plots for each cell type.

Download figure to PowerPoint

However, it should be noted that the small number of controls used in this series of experiments (n = 3) precludes a definitive comparison of L-type Ca2+ currents between controls and HD patients.

Next, we compared the voltage dependent activation and inactivation properties of the currents in cells isolated from the ganglionic and aganglionic segments from HD patients. Activation curves were constructed from the IV relationships by first calculating conductance, G, from: G = I/(VmErev), where Vm is the test potential and Erev is the reversal potential for the current. The conductance was then normalized to the maximum value (Gmax) and plotted as G/Gmax against the test potential. Summary activation curves are shown in Fig. 2A, where it can be seen that there was no significant difference in the voltage dependence of activation between ganglionic and aganglionic SMC (< 0.05). The mean V1/2 for activation in ganglionic SMC was −15 ± 0.9 mV (= 15), compared to −13 ± 0.4 mV (n = 10), in SMC isolated from the aganglionic region. A two-pulse protocol was used to assess the steady-state inactivation properties of L-ICa. The protocol consisted of holding the cell at −60 mV and stepping to conditioning potentials ranging from −100 to +10 mV for 2 s before stepping to a test potential of 0 mV. Mean inactivation data for all cells were well fitted with a Boltzmann function of the form:

  • image

where I/Imax is the normalized current, V1/2 is the membrane potential at which half inactivation occurs, and K−1 is the maximum slope factor. Summary inactivation plots are shown in Fig. 2A. The V1/2 for currents evoked from ganglionic SMC was −37 ± 0.7 mV and the mean slope factor was 5.8 ± 0.6 mV (n = 6). Similar values were obtained from cells isolated from the aganglionic region, thus the mean inactivation V1/2 was −36 ± 0.6 mV and the mean slope factor was 5.3 ± 0.5 mV (n = 9). These values were not significantly different (> 0.05) and are in the normal range for L-type Ca2+ current in smooth muscle.10

image

Figure 2.  (A) Summary plots showing the voltage dependent activation kinetics of L-type Ca2+ currents in ganglionic and aganglionic smooth muscle cells (SMC). (B) Summary plots showing the steady-state voltage-dependent inactivation in SMC isolated from the ganglionic and aganglionic regions of the HD colon.

Download figure to PowerPoint

To confirm that the inward currents described above were in fact L-type Ca2+ current, we examined their sensitivity to the classical L-type Ca2+ channel blocker, nifedipine. Families of currents were evoked from SMC isolated from aganglionic and ganglionic regions and subjected to the IV protocol described in Fig. 1, in the absence and presence of nifedipine (1 μmol L−1). Representative currents evoked from ganglionic SMC before and during addition of nifedipine are shown in Fig. 3A,B, respectively. Summary data from six cells (Fig. 3C) show that nifedipine greatly reduced the current amplitude across the voltage range (P < 0.05). For example, at 0 mV nifedipine reduced the peak inward current from −127 ± 34 to −16 ± 11 pA. Similar inhibitory effects of nifedipine were observed in aganglionic SMC, as can be seen by the representative traces in Fig. 3D,E and the summary plot in Fig. 3F. Nifedipine reduced the mean peak inward current in ASMC, evoked by a step to 0 mV, from −108 ± 18 to −5 ± 14 pA (n = 6).

image

Figure 3.  (A) and (B) Representative membrane currents evoked from ganglionic SMC, before and during exposure to 1 μmol L−1 nifedipine. (C) Summary IV plot of currents recorded before (control) and during exposure to 1 μmol L−1 nifedipine. (D) and (E) Representative membrane currents evoked from aganglionic SMC, before and during exposure to 1 μmol L−1 nifedipine. (F) Summary IV plot of currents recorded before (control) and during exposure to 1 μmol L−1 nifedipine.

Download figure to PowerPoint

In Fig. 4 we examined the effect of the L-type Ca2+ channel agonist FPL 64176 (300 nmol L−1) on inward currents evoked from GSMC and ASMC to confirm that they were mediated by L-type Ca2+ channels. FPL 64176 is a calcium channel modulator specific for the L-type family of voltage-gated calcium channels, which prolongs the opening of single calcium channels during depolarization and slows channel closing upon repolarization.11,12 Control inward currents in both GSMC (Fig. 4A) and ASMC (Fig. 4D) were greatly enhanced by 300 nmol L−1 FPL (Fig. 4B,E, respectively). This was accompanied by much slower inactivation of the current in both groups of cells. For example, the τ of inactivation, at 0 mV, in GSMC and ASMC significantly increased from mean values of 22.5 ± 2.7 to 141.8 ± 42.2 ms and 25 ± 6.9 to 109.9 ± 23.8 ms, respectively (P < 0.05), such that the currents were not fully inactivated at the end of the 500 ms pulse. The FPL evoked currents were also blocked by nifedipine (Fig. 4C,G), confirming that they were L-type Ca2+ currents. Summary IV plots, showing the effect of FPL and nifedipine in ganglionic and aganglionic SMC are shown in Fig. 4D,H, respectively. In ganglionic SMC the mean peak inward current increased in amplitude and the voltage at which current peaked shifted negatively by 20 mV. Peak inward current, measured at 0 mV, increased from −96 ± 19 to −194 ± 39 pA (n = 10; P < 0.05) in these cells. The large FPL-induced inward current was reduced to −4 ± −10.9 pA (n = 6; P < 0.05) following application of 1 μmol L−1 nifedipine. Similar results were achieved in aganglionic SMC, thus FPL increased the peak inward current at 0 mV from −118 ± 13 pA to −161 ± 21 pA (n = 11; P < 0.05). The FPL-induced inward current was reduced to −3 ± 7 pA (n = 7; P < 0.05) following application of 1 μmol L−1 nifedipine. Similar enhancement of the currents was also observed with application of 1 μmol L−1 Bay K 8644 (+) which increased the mean peak inward current amplitude evoked by a step to 0 mV by 95% in 5 GSMC (P < 0.05) and 97% in 8 ASMC (P < 0.05, data not shown).

image

Figure 4.  (A–C) Representative L-type Ca2+ currents recorded from a smooth muscle cell isolated from the ganglionic segment of the HD bowel under control conditions (A), in the presence of the L-type Ca2+ channel opener, FPL 64176 (300 nmol L−1, B) and in the presence of FPL 64176 and nifedipine (1 μmol L−1, C). (D) Summary IV plot for 6 ganglionic SMC under the conditions described above. (E–G) Representative L-type Ca2+ currents recorded from a smooth muscle cell isolated from the aganglionic segment of the HD bowel under control conditions (E), in the presence of the L-type Ca2+ channel opener, FPL 64176 (300 nmol L−1, F) and in the presence of FPL 64176 and nifedipine (1 μmol L−1, G). (H) Summary IV plot for aganglionic SMC (n = 6) under the conditions described above.

Download figure to PowerPoint

Nakatsuji et al. showed that the transcriptional levels of CACNA1C, the gene that encodes for L-type Ca2+ channels, were decreased in the aganglionic segment of the colon in EDNRB gene deficient rats, a commonly used animal model to study HD.8 To test if a similar expression pattern occurred in HD patients, real-time quantitative PCR experiments were performed to compare CACNA1C levels in smooth muscle taken from the ganglionic and aganglionic bowel segments in patients suffering from HD. The suitability of GAPDH as a reference gene was investigated by analysis of Cp variation in paired samples. No significant difference was observed in mean Cp value (mean ± SEM) between ganglionic (24.74 ± 0.26) and aganglionic (25.31 ± 0.43) samples (= 5). To confirm our conclusion, REST 2009 software was used to analyze the same data set. This analysis confirmed that expression of GAPDH in the aganglionic segment is not different to that in the ganglionic segment. GAPDH was thus concluded to be a suitable reference gene for the normalization of CACNA1C expression. CACNA1C expression levels in paired ganglionic and aganglionic samples are compared in Fig. 5. For five paired samples, the mean values (mean ± SEM) were 2.282 ± 0.7, for ganglionic tissue, and 2.234 ± 0.5, for aganglionic tissue (reported as fold change relative to normal tissue). A paired t-test analysis of the data set indicated that there was no significant difference between the ganglionic and aganglionic samples (P = 0.9456).

image

Figure 5.  The expression levels of CACNA1C in paired ganglionic and aganglionic tissue samples were normalized using GAPDH. CACNA1C expression was then expressed as the fold change relative to normal tissue control sample. The mean values for the sample set (n = 5) were 2.282 ± 0.7, for ganglionic tissue, and 2.234 ± 0.5, for aganglionic tissue. There was no significant difference in the expression levels of CACNA1C between the ganglionic and aganglionic HD bowel segments.

Download figure to PowerPoint

Discussion

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

L-type Ca2+ channels play a key role in the excitability of colonic smooth muscle and have been characterized in several species including dog, cat, rabbit, and human, 13–17 where they are involved in the generation of spontaneous action potentials and Ca2+ waves.4,5 L-type Ca2+ currents in colonic smooth muscle are known to be diminished in several pathophysiological conditions. For example, Kinoshita et al. showed that L-type Ca2+ current density was reduced in myocytes isolated from the colons of rats which had been treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis18 and Liu et al. showed that the expression of L-type Ca2+ channels was reduced during ethanol-acetic acid induced inflammation of the canine colon.19 Several studies have suggested that altered expression or behavior of L-type Ca2+ channels could also be a causative factor in the functional obstruction of the aganglionic bowel observed in patients with HD. For example, Won et al. reported that the intestines of endothelin type B receptor null rats [ETB (−/−)R], which are used as an animal model for the study of HD, produced a greater increase in force when stimulated by raised extracellular K+ solution, consistent with a change in L-type Ca2+ channel activity.20 However, an earlier study by Ikadai et al. demonstrated that the response to raised extracellular K+ solutions was decreased in the rat aganglionic colon.7 Similar findings were reported by Nakajitsu et al. who showed that the peak level of force development in the aganglionic distal colon of EDNRB (−/−) rats in response to high K+ solution (60 mmol L−1) was less than half of that recorded in wild type controls.8 Moreover, this study found that the transcriptional expression of the α1c subunit of the L-type Ca2+ channel (now referred to as CACNA1C) was also reduced in the aganglionic segment of EDNRB (−/−) rats compared with controls. Therefore, although these studies suggest that L-type Ca2+ channels could be implicated in the pathogenesis of HD, there is little consensus on the matter. A feature of the studies noted above is that they were all performed on tissues taken from animal models and although L-type Ca2+ currents have been recorded from adult human colonic myocytes,16 there have been no previous investigations of L-type Ca2+ channels in patients who suffer from HD. In this study, we set out to investigate this issue directly by performing a comprehensive pharmacological, electrophysiological, and molecular characterization of the L-type Ca2+ current in SMC isolated from both the ganglionic and aganglionic segments of the HD bowel.

The findings of this study provide robust evidence for the existence of L-type Ca2+ channels in SMC isolated from both the ganglionic and aganglionic bowel. Thus, the currents had activation/inactivation kinetics typical of the L-type calcium current, were inhibited by nifedipine and enhanced by the L-type Ca2+ channel agonists FPL 64176 and Bay K 8644. Moreover, the currents activated by FPL 64176 were also inhibited by nifedipine. These data are consistent with those previously reported by Xiong et al.17 who characterized L-type Ca2+ in adult human colonic myocytes. However, they conflict with the other studies on genetic animal models of HD noted above, which predicted a change in the activity of L-type Ca2+ channels in the aganglionic bowel of HD patients. Therefore, although the understanding of the etiology of HD has been greatly enhanced by various genetic animal models,1 the data reported in this study suggest that findings in these models does not always correlate with those in HD patients, highlighting the importance of patient orientated research into HD. At present we cannot definitively explain why the results obtained in this study did not correlate with those in the animal models described above. However, it is worth pointing out that although the human and HD animal model (Ednrbs-l/Ednrbs-l mice and EDNRB (−/−) rats) tissues were aganglionic, this may be due to different underlying factors. For example, the animal models display distal colonic aganglionosis because endothelin 3 and its receptor EDNRB are needed to prevent the premature differentiation of crest-derived cells that would lead to aganglionosis. However, human HD has been associated with mutations in more than 10 genes, and those involving endothelin 3 or its receptor, endothelin receptor B (EDNRB), account for only 5–10% of all HD cases.21 Therefore, the reduced L-type Ca2+ channel expression, observed in EDNRB deficient rodents, may only correlate with a small subset of human HD cases associated with mutations in endothelin 3 or EDNRB genes. Mutations in EDNRB genes are also associated with other syndromic conditions, such as Shah Waardenburg syndrome.22 Therefore, in the future it will be interesting to test if patients with this syndrome or with HD associated with EDNRB mutations do have differences in L-type Ca2+ channel expression, as predicted by these animal models.

In conclusion, this study confirms that L-type Ca2+ currents are present in the ganglionic and aganglionic segments of the HD bowel, but in contrast to previous studies on genetic animal models of HD, they are not down-regulated and are therefore unlikely to account for the lack of peristalsis in the narrowed aganglionic bowel segment in HD.

Funding

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

This work was sponsored by the Children’s Medical and Research Foundation, Our Lady’s Children’s Hospital, Dublin.

Author contribution

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

RL, EB, and TW performed the research; MH, KT, NM, and GS designed the research study; AMO, PP, MH, KT, NM, and GS contributed essential reagents or tools, and analyzed the data; GS wrote 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 contribution
  10. References
  • 1
    Burzynski G, Shepherd IT, Enomoto H. Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung’s disease. Neurogastroenterol Motil 2009; 21: 11327.
  • 2
    Kubota M, Suita S, Kamimura T, Ito Y, Szurszewski JH. Electrophysiological properties of the aganglionic segment in Hirschsprung’s disease. Surgery 2002; 131(1 Suppl.): S28893.
  • 3
    Spencer NJ, Bayguinov P, Hennig GW et al. Activation of neural circuitry and Ca2+ waves in longitudinal and circular muscle during CMMCs and the consequences of rectal aganglionosis in mice. Am J Physiol Gastrointest Liver Physiol 2007; 292: G54655.
  • 4
    Ward SM, Sanders KM. Upstroke component of electrical slow waves in canine colonic smooth muscle due to nifedipine-resistant calcium current. J Physiol 1992; 455: 32137.
  • 5
    Hennig GW, Smith CB, O’Shea DM, Smith TK. Patterns of intracellular and intercellular Ca2+ waves in the longitudinal muscle layer of the murine large intestine in vitro. J Physiol 2002; 543: 23353.
  • 6
    Bolton TB, Prestwich SA, Zholos AV, Gordienko DV. Excitation-contraction coupling in gastrointestinal and other smooth muscles. Annu Rev Physiol 1999; 61: 85115.
  • 7
    Ikadai H, Shimizu K, Nakajyo S, Imamichi T, Sasaki Y, Urakawa N. Responses of narrow segments of intestines in the congenital aganglionosis rat to various stimulants. Nihon HeikatsukinGakkaiZasshi 1982; 18: 34761.
  • 8
    Nakatsuji T, Ieiri S, Masumoto K, Akiyoshi J, Taguchi T, Suita S. Intracellular calcium mobilization of the aganglionic intestine in the endothelin B receptor gene-deficient rat. J Pediatr Surg 2007; 42: 166370.
  • 9
    Rae J, Cooper K, Gates P. Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 1991; 37: 1526.
  • 10
    McDonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation and modulation of calcium channels in cardiac, skeletal and smooth muscle cells. Physiol Rev 1994; 742: 365507.
  • 11
    Baxter AJG, Dixon J, Ince F, Manners CN, Teague SJ. Discovery and synthesis of methyl 2,5-dimethyl-4-[2-(phenylmethyl)-benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176) and analogues: the first examples of a new class of calcium channel activator. J Med Chem 1993; 36: 273944.
  • 12
    Triggle D. Pharmacology of CaV1 (L-type) calcium channels. In: McDonough SI, ed. Calcium Channel Pharmacology. New York: Kluwer Academic/Plenum Publishing, 2004: 2172.
  • 13
    Langton PD, Burke EP, Sanders KM. Participation of Ca currents in colonic electrical activity. Am J Physiol 1989; 257(3 Pt 1): C45160.
  • 14
    Rich A, Kenyon JL, Hume JR, Overturf K, Horowitz B, Sanders KM. Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells. Am J Physiol 1993; 264(3 Pt 1): C74554.
  • 15
    Bielefeld DR, Hume JR, Krier J. Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon. Pflugers Arch 1990; 415: 67887.
  • 16
    Mayer EA, Loo DD, Snape WJ Jr, Sachs G. The activation of calcium and calcium-activated potassium channels in mammalian colonic smooth muscle by substance P. J Physiol 1990; 420: 4771.
  • 17
    Xiong Z, Sperelakis N, Noffsinger A, Fenoglio-Preiser C. Ca2+ currents in human colonic smooth muscle cells. Am J Physiol 1995; 269(3 Pt 1): G37885.
  • 18
    Kinoshita K, Sato K, Hori M, Ozaki H, Karaki H. Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-kappaB inhibitors in Crohn’s colitis model. Am J Physiol Gastrointest Liver Physiol 2003; 285: G48393.
  • 19
    Liu X, Rusch NJ, Striessnig J, Sarna SK. Down-regulation of L-type calcium channels in inflamed circular smooth muscle cells of the canine colon. Gastroenterology 2001; 120: 4809.
  • 20
    Won KJ, Torihashi S, Mitsui-Saito M et al. Increased smooth muscle contractility of intestine in the genetic null of the endothelin ETB receptor: a rat model for long segment Hirschsprung’s disease. Gut 2002; 50: 35560.
  • 21
    Amiel J, Lyonnet S. Hirschsprung disease, associated syndromes, and genetics: a review. J Med Genet 2001; 38: 72939.
  • 22
    Pingault V, Ente D, Dastot-Le Moal F, Goossens M, Marlin S, Bondurand N. Review and update of mutations causing Waardenburg syndrome. Hum Mutat 2010; 31: 391406. (Review).