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. 2014 Dec 8;593(Pt 2):403–414. doi: 10.1113/jphysiol.2014.273540

Dominant role of interstitial cells of Cajal in nitrergic relaxation of murine lower oesophageal sphincter

Dieter Groneberg 1, Eugen Zizer 2, Barbara Lies 1, Barbara Seidler 3, Dieter Saur 3, Martin Wagner 2, Andreas Friebe 1,✉
PMCID: PMC4303385  PMID: 25630261

Abstract

Oesophageal achalasia is a disease known to result from reduced relaxation of the lower oesophageal sphincter (LES). Nitric oxide (NO) is one of the main inhibitory transmitters. NO-sensitive guanylyl cyclase (NO-GC) acts as the key target of NO and, by the generation of cGMP, mediates nitrergic relaxation in the LES. To date, the exact mechanism of nitrergic LES relaxation is still insufficiently elucidated. To clarify the role of NO-GC in LES relaxation, we used cell-specific knockout (KO) mouse lines for NO-GC. These include mice lacking NO-GC in smooth muscle cells (SMC-GCKO), in interstitial cells of Cajal (ICC-GCKO) and in both SMC/ICC (SMC/ICC-GCKO). We applied oesophageal manometry to study the functionality of LES in vivo. Isometric force studies were performed to monitor LES responsiveness to exogenous NO and electric field stimulation of intrinsic nerves in vitro. Cell-specific expression/deletion of NO-GC was monitored by immunohistochemistry. Swallowing-induced LES relaxation is strongly reduced by deletion of NO-GC in ICC. Basal LES tone is affected by NO-GC deletion in either SMC or ICC. Lack of NO-GC in both cells leads to a complete interruption of NO-induced relaxation and, therefore, to an achalasia-like phenotype similar to that seen in global GCKO mice. Our data indicate that regulation of basal LES tone is based on a dual mechanism mediated by NO-GC in SMC and ICC whereas swallow-induced LES relaxation is mainly regulated by nitrergic mechanisms in ICC.

Key points.

  • Nitric oxide (NO) is an important inhibitory neurotransmitter in the gastrointestinal tract. Oesophageal achalasia may result from impairment of nitrergic relaxation.

  • Smooth muscle cells (SMCs) have been accepted to be the major targets for neuronal NO to mediate relaxation. However, besides SMCs, the receptor for NO, NO-sensitive guanylyl cyclase (NO-GC), has been shown in interstitial cells of Cajal (ICC).

  • Using cell-specific knockout mice, this study shows that NO-GC in SMC and ICC modulates lower oesophagus sphincter tone in vitro and in vivo.

  • More importantly, NO-GC in ICC possesses a dominant role in mediating swallowing-induced relaxation. Lack of functional nitrergic signalling, thus, results in deficits in relaxation of the lower oesophagus sphincter as seen in achalasic patients.

Introduction

Achalasia is characterized by impaired relaxation of the lower oesophageal sphincter (LES), dysregulation of oesophageal peristalsis and, consequently, progressive problems in swallowing. This dysfunction is a consequence of degeneration or loss of myenteric neurons thought to be caused by viral infections, autoimmunity, inheritance or hormones (Goldblum et al. 1996; Raymond et al. 1999; Zarate et al. 2006; Richter, 2010; Boeckxstaens et al. 2014). The resulting imbalance between contraction and relaxation leads to an elevated pressure of the LES and is usually ascribed to unchanged activity of cholinergic neurons (contraction) with decreased inhibitory neuronal activity (relaxation). Nitric oxide (NO) was shown to be the main inhibitory neurotransmitter in LES in many species, including humans and mice (Tottrup et al. 1991; Yamato et al. 1992; Mearin et al. 1993). NO synthase (NOS) inhibitors can abolish or reduce swallow-induced LES relaxation in vivo (Yamato et al. 1992; Xue et al. 1996; Konturek et al. 1997). Studies in mice deficient in neuronal NOS (nNOS) support the significance of NO for normal LES relaxation (Kim et al. 1999; Sivarao et al. 2001), which is corroborated by the fact that LES tissue of patients with achalasia was shown to lack NOS (Mearin et al. 1993).

NO-sensitive guanylyl cyclase (NO-GC) is the main target for NO. NO is known to activate the enzyme by binding to the prosthetic haem moiety and to induce the production of the second messenger cGMP (Friebe & Koesling, 2003). NO-GC has been shown to mediate smooth muscle relaxation in vascular, gastrointestinal (GI) and lower urinary tract tissue. Consequently, the deletion of NO-GC in the murine system has been shown to lead to a divergent phenotype, including systemic hypertension and GI dysmotility (Mergia et al. 2006; Friebe et al. 2007; Buys et al. 2012) and confirmed the exclusive role for NO-GC as NO receptor. Regarding the GI tract, general deletion of NO-GC was shown to prevent NO-induced relaxation of fundus, duodenum, jejunum and colon. In vivo, this lack of nitrergic relaxation resulted in a reduced GI motility. Surprisingly, smooth muscle cell-specific deletion of NO-GC (SMC-GCKO) did not prevent nitrergic relaxation in different GI segments and gut motility was unaffected (Groneberg et al. 2011). In addition to SMC, interstitial cells of Cajal (ICC) are thought to be involved in the mediation of nitrergic signals (Daniel & Posey-Daniel, 1984; Sanders & Ward, 1992; Burns et al. 1996; Zhu & Huizinga, 2008; Klein et al. 2013). Very recently, we were able to show that the concerted action of SMC and ICC is necessary for nitrergic relaxation of the murine fundus as only concomitant deletion of NO-GC in both cell types led to a loss of NO-induced responses (Groneberg et al. 2011, 2013).

The aim of this study was to determine (1) whether the dual mechanism for nitrergic relaxation shown previously in fundus also accounts for the LES, and (2) which of the two cell types, SMC or ICC, is responsible for the regulation of basal tone and swallow-induced relaxation of the LES. We used organ bath experiments to monitor nitrergic relaxation of LES from several cell-specific GCKO models and correlated these in vitro data to oesophageal manometry recorded in vivo. Our results clearly show the presence of a dual mechanism of nitrergic relaxation via SMC and ICC in LES. In addition, our data indicate that NO-GC in both SMC and ICC is responsible for the regulation of basal sphincter tone whereas NO-GC in ICC, but not in SMC, appears to mediate swallowing-induced relaxation.

Methods

Ethical approval

All experiments were conducted in accordance with German legislation on the protection of animals and approved by the local animal care committee.

Animals

Mice (C57BL/6 and mixed C57BL/6-SV129 background) were housed in standard mouse cages (267 × 207 × 140 mm; maximally three animals/cage) with woodchip bedding material and under conventional laboratory conditions (constant room temperature (22°C), humidity level (55%), a 12 h light/12 h dark cycle (lights on at 06.00 h) and standard rodent diet (Altromin, Lage, Germany) and water ad libitum. Animals of either sex were killed aged 8–16 weeks by isoflurane overdose and tissues were isolated. A total of 117 animals were used.

Generation of cell-specific knockout mice

SMC-GCKO and ICC-GCKO mice carry a floxed exon (exon 10 of the ß1 subunit of NO-GC; Friebe et al. 2007) and are transgenic for the inducible Cre recombinase in SMC (SMMHC-CreERT2; Wirth et al. 2008); and ICC (cKIT-CreERT2; Klein et al. 2013). SMC/ICC-GCKO mice were generated by crossing SMC-GCKO with ICC-GCKO mice. Mice from all three KO lines aged 6–8 weeks were injected with tamoxifen (1 mg i.p.) on five consecutive days to remove the floxed exon. Fifty days after the last tamoxifen injection, deletion of NO-GC in SMC was considered complete (Groneberg et al. 2011). In LES ICC, deletion efficiency of NO-GC 50 days after tamoxifen was estimated >95% in n = 3 sections. Thus, for experiments with cell-specific KO tissue, we used animals >50 days after tamoxifen treatment. In every experiment, respective tamoxifen-injected heterozygous littermates were used as controls.

Preparation of murine tissues and isometric force studies

Isometric force studies were performed as described (Groneberg et al. 2011). Briefly, animals were killed by isoflurane anaesthesia and cervical dislocation. Mice were opened, LES was isolated and transferred to Krebs–Henseleit solution bubbled with 95% O2/5% CO2. LES rings were mounted longitudinally on fixed segment support pins in two four-chamber myographs (Myograph 610; Danish Myo Technology, Aarhus, Denmark) containing 5 ml Krebs–Henseleit solution. Resting tension was set to 3 mN. Rings were pre-contracted with carbachol (CCh; 1 μm). Relaxation was induced with DEA-NO or 8-Br-cGMP as indicated.

Electric field stimulation (EFS) was applied to LES rings precontracted with CCh (1 μm) using two platinum wire electrodes (5 mm distance; maximal voltage 0.5 ms, 1–4 Hz, 10 s). The ratio of the 4 Hz relaxation without and with the NO-GC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was taken for analysis.

For normalization, IBMX (100 μm) was added at the end of each experiment to determine maximal relaxation. In case of NO-GC sensitization, strips were pre-incubated with the NO-GC sensitizer Bay 41-2272 (0.1 μm) for 20 min.

Immunohistochemistry

Immunohistochemical analyses were performed as previously described (Groneberg et al. 2011). Briefly, mice were killed by cervical dislocation and tissues were prefixed with 2% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, incubated overnight with 20% sucrose and then snap-frozen in liquid nitrogen. Cryosections (50 μm) were cut, air-dried and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4 for 7 min. Incubation was performed overnight with a homemade antibody against the ß1 subunit of NO-GC raised in rabbit (1:800) (Friebe et al. 2007) together either with a fluorescein isothiocyanate-conjugated mouse anti-α-smooth muscle actin antibody (1:500, clone 1A4; Sigma-Aldrich, Munich, Germany), rat antiplatelet-derived growth factor receptor alpha (PDGFRα) antibody (1:200, clone APA5; eBioscience, Frankfurt, Germany) or a rat anti-c-kit antibody (1:400, clone ACK4; Linaris, Wertheim-Bettingen, Germany). The rabbit antibody was detected with an Alexa 555-conjugated antirabbit IgG antibody raised in donkey (1:800; Invitrogen, Darmstadt, Germany); the rat antibodies were detected with an Alexa 488-conjugated donkey antirat IgG antibody (1:400; Invitrogen) for 1 h. The sections were mounted in Mowiol and were evaluated using a confocal microscope (Leica TCS Sp5, Leica Biosystems, Wetzlar, Germany). Confocal images were obtained from digital composites of Z-series scans of 20–28 optical sections through a depth of 20 μm or from single slices using the software Leica LAS AF.

Oesophageal manometry

Oesophageal manometry was performed as described previously (Zizer et al. 2010). Briefly, mice were anaesthetized with pentobarbital (50 mg kg–1 i.p.). Oesophageal manometry was performed using a customized catheter (Dentsleeve International, Ltd, Ontario, Canada; perfusion pressure 10 kPa) connected to a perfusion pump (Mui-Scientific, Ontario, Canada) via pressure transducers (Alpine Biomed GmbH, Langenfeld, Germany) and 0.01 ml resistor catheters (Dentsleeve International, Ltd). The transducers were connected to a Polygraf-ID recorder (Medtronic GmbH, Meerbusch, Germany), and pressure data were evaluated using the Polygraf NET-software (Medtronic Polygram Net, version 4.2.95.80; Medtronic GmbH). The catheter was introduced antegrade and fixed with the very distal opening in the LES area. Swallows were induced by a mechanical irritation and activity was monitored for 30 min.

Individual statistical analyses

For calculation of statistical tests, GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA) was used. Mann–Whitney U test were used for the data (Figs 2B and D and 4A–D). In Figs 5B and 6B and C, all groups were compared using the Kruskal–Wallis test. If P > 0.05 for the global test, two groups were compared by Mann–Whitney U test (Fig. 6B and C).

Figure 2.

Figure 2

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Lack of NO-induced relaxation in LES from SMC/ICC-GCKO mice

A, representative original traces of organ bath experiments showing DEA-NO-induced relaxation in LES from control (upper trace) and SMC/ICC-GCKO animals (lower trace) after pre-contraction with 1 μm CCh. B, quantitative analysis of experiments from control, ICC-GCKO, SMC-GCKO and SMC/ICC-GCKO animals. Data shown are means ± SEM of n = 5–10 per genotype. **P < 0.01 starting at 0.1, 0.3 and 0.03 μm for SMC-GCKO, ICC-GCKO and SMC/ICC-GCKO, respectively, by Mann–Whitney U test. C, representative original traces of EFS-induced relaxation of CCh-precontracted (1 μm) LES from control, ICC-GCKO, SMC-GCKO and SMC/ICC-GCKO animals. EFS was repeated after the addition of 10 μm ODQ. D, quantitative analysis of 4 Hz-induced relaxations. Data shown are means ± SEM of n = 4–9 per genotype. EFS-induced relaxation of LES rings in the absence and presence of 10 μm ODQ reached statistical significance for control (***P = 0.0005), SMC-GCKO (**P = 0.0079) and ICC-GCKO (*P = 0.0317). No statistical significance was reached for SMC/ICC-GCKO (n.s., P = 0.2) by Mann–Whitney U test. CCh, carbachol; DEA-NO, 2-(N,N-diethylamino)-diazenolate-2-oxide.diethylammonium salt; EFS, electric field stimulation; ICC-GCKO, interstitial cells of Cajal-specific guanylyl cyclase knockout; LES, lower oesophageal sphincter; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SMC-GCKO, smooth muscle cell-specific guanylyl cyclase knockout.

Figure 4.

Figure 4

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Sensitization of NO-induced relaxation by the NO-GC stimulator Bay 41-2272

Lower oesophageal sphincter rings from control (A), SMC-GCKO (B), ICC-GCKO (C) and SMC/ICC-GCKO (D) were precontracted with 1 μm carbachol and then relaxed with increasing concentrations of DEA-NO in the absence or presence of the haem-dependent NO-GC stimulator Bay 41-2272. Data shown are means ± SEM of n = 3–10 per genotype. *P < 0.05 from 0.03 to 0.3 μm (control), at 1 μm (SMC-GCKO) and from 0.03 to 1 μm (ICC-GCKO) and **P < 0.01 at 3 to 10 μm (SMC/ICC-GCKO) by Mann–Whitney U test. No statistical significance was reached in either comparison (P = 0.19–0.92, Mann–Whitney U test) for SMC/ICC-GCKO. DEA-NO, 2-(N,N-diethylamino)-diazenolate-2-oxide.diethylammonium salt; ICC-GCKO, interstitial cells of Cajal-specific guanylyl cyclase knockout; NO, nitric oxide; NO-GC, nitric oxide-sensitive guanylyl cyclase; SMC-GCKO, smooth muscle cell-specific guanylyl cyclase knockout.

Figure 5.

Figure 5

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Lower oesophageal sphincter relaxation to 8-bromoguanosine 3′,5′-cyclic monophosphate

Lower oesophageal sphincter rings from control, SMC-GCKO, ICC-GCKO and SMC/ICC-GCKO were precontracted with 1 μm CCh and then relaxed with 10 μm 8-bromoguanosine 3′,5′-cyclic monophosphate. IBMX (100 μm) was administered to achieve maximal relaxation. A, representative original traces, B, quantitative analysis. Data shown are means ± SEM of n = 4–5 per genotype. No statistical significance was reached for all groups by Kruskal–Wallis test (P = 0.73). CCh, carbachol; ICC-GCKO, interstitial cells of Cajal-specific guanylyl cyclase knockout; SMC-GCKO, smooth muscle cell-specific guanylyl cyclase knockout.

Figure 6.

Figure 6

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Oesophageal manometry of basal and swallow-induced LES tone

A, representative original traces of pressure recordings in the LES high-pressure zone and 8 mm above the LES. Arrow indicates induction of swallow-induced LES relaxation. B, statistical analysis of LES basal and relaxed pressures. Data shown are mean ± SEM of n = 5–6 per genotype. Statistical significance was seen for basal vs. relaxed tone in control and SMC-GCKO (**P = 0.008 and 0.002, respectively). No statistical difference was reached for basal vs. relaxed tone in ICC-GCKO and SMC/ICC-GCKO (n.s.; P = 0.151 and 0.132, respectively). Comparison of basal tone between control and KO strains reached significance for SMC-GCKO, ICC-GCKO and SMC/ICC-GCKO (**P = 0.004, 0.008 and 0.004, respectively). Analyses were performed by Mann–Whitney U test. C, relative LES relaxation. No statistical significance was reached for control vs. SMC-GCKO (P = 0.33). *P = 0.016 for control vs. ICC-GCKO and *P = 0.017 for control vs. SMC/ICC-GCKO by Mann–Whitney U test. ICC-GCKO, interstitial cells of Cajal-specific guanylyl cyclase knockout; LES, lower oesophageal sphincter; SMC-GCKO, smooth muscle cell-specific guanylyl cyclase knockout.

Materials

8-Br-cGMP, DEA-NO and ODQ, were purchased from Axxora (Lörrach, Germany). Bay 41-2272, CCh, IBMX and tamoxifen were from Sigma (Taufkirchen, Germany). Mowiol was purchased from Roth (Karlsruhe, Germany).

Results

Immunohistochemical analysis of nitric oxide-sensitive guanylyl cyclase expression in lower oesophagus sphincter

The identity of NO-GC-expressing cells in the murine LES has not been shown yet; therefore, we first performed immunohistochemistry on LES from control mice using an antibody against the ß1 subunit being the mandatory subunit for both NO-GC isoforms together with an antibody against alpha smooth muscle actin. Figure 1A shows strong NO-GC staining in interstitial cells and vascular SMC whereas only faint staining was detected in SMCs of the tunica muscularis. To determine the identity of the NO-GC-positive interstitial cells, we used antibodies against the tyrosine-protein kinase Kit (c-kit) and PDGFRα as markers for ICC and fibroblast-like cells (FLC), respectively. Figure 1B shows co-localization of NO-GC and c-kit immunoreactivity indicating weak NO-GC expression in ICC of the control LES. Much stronger staining for NO-GC, however, was detected in FLC as indicated by the co-localization with PDGFRα (Fig. 1C).

Figure 1.

Figure 1

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Immunohistochemical analysis of NO-GC expression in lower oesophageal sphincter from control mice

A, lower oesophageal sphincter from control mice was co-stained with specific antibodies against NO-GC (red) and α-smooth muscle actin (green). NO-GC expression in vascular smooth muscle cells, interstitial cells and smooth muscle cells of the tunica muscularis are indicated by arrows, asterisks and arrowheads, respectively. Localization of NO-GC in c-kit-positive interstitial cells of Cajal (B) and PDGFRα-positive fibroblast-like cell (C) as single slice and composite image, respectively. LMM, lamina muscularis mucosae; NO-GC, nitric oxide-sensitive guanylyl cyclase; PDGFRα, platelet-derived growth factor receptor alpha; α-SMA, alpha smooth muscle actin; TM, tunica muscularis.

Nitric oxide-induced relaxation of lower oesophagus sphincter

We then performed organ bath experiments to measure NO-induced relaxation of LES precontracted with CCh (1 μm). LES from control mice showed a concentration-dependent relaxation in response to the NO donor DEA-NO (Fig. 2A and B). Deletion of NO-GC in ICC (ICC-GCKO) or SMC (SMC-GCKO) only partially impaired NO-induced relaxation (Fig. 2B). However, when compared to LES from control animals, the concentration–response curves were shifted to the right indicating a reduced responsiveness in both KO strains. Double deletion of NO-GC in SMC and ICC (SMC/ICC-GCKO) abolished NO-induced relaxation. However, only at very high NO concentrations (>1 μm) a minor NO-induced relaxation was detected.

To investigate the effect of endogenously released NO, we used EFS in the absence and presence of the NO-GC inhibitor ODQ. LES rings were precontracted with CCh (1 μm). LES from control, SMC-GCKO and ICC-GCKO animals showed an EFS-induced relaxation. Preincubation with ODQ led to a significant reduction in relaxation (46, 43 and 48% for the three genotypes, respectively). In contrast, EFS relaxation of LES from SMC/ICC-GCKO was much less than that of the other genotypes and was not influenced by ODQ indicating abolished effect of endogenous NO. The residual EFS-induced relaxation suggests the release of yet another inhibitory neurotransmitter, possibly vasoactive intestinal polypeptide (VIP) or a purine such as ATP or β-nicotinamide adenine dinucleotide (see Discussion).

Taken together, our results demonstrate that NO, in analogy to the murine fundus, acts by a dual pathway involving SMC and ICC to induce LES relaxation.

Verification of genomic deletion of nitric oxide-guanylyl cyclase in lower oesophagus sphincter from smooth muscle cell/interstitial cells of Cajal-guanylyl cyclase knockout animals

The immunohistochemical analysis of the LES from SMC/ICC-GCKO showed deletion of NO-GC in SMCs of the tunica muscularis and blood vessels (Fig. 3A). Positive NO-GC staining remained only in cells negative for alpha smooth muscle actin. Staining with the c-kit antibody revealed effective deletion also in ICC (>95%; Fig. 3B). As expected, the use of an antibody against PDGFRα revealed preserved co-staining with NO-GC in SMC/ICC-GCKO LES (Fig. 3C).

Figure 3.

Figure 3

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Immunohistochemical analysis of NO-GC expression in the lower oesophageal sphincter from smooth muscle cell/interstitial cells of Cajal-specific guanylyl cyclase knockout mice

A, lower oesophageal sphincter from smooth muscle cell/interstitial cells of Cajal-specific guanylyl cyclase knockout mice animals was co-stained with specific antibodies against NO-GC (red) and α-smooth muscle actin (green). Absence of NO-GC in c-kit-positive interstitial cells of Cajal (B) but preserved NO-GC expression in PDGFRα-positive fibroblast-like cell (C). LMM, lamina muscularis mucosae; NO-GC, nitric oxide-sensitive guanylyl cyclase; PDGFRα, platelet-derived growth factor receptor alpha; α-SMA, alpha smooth muscle actin; TM, tunica muscularis.

Sensitisation of nitric oxide-induced relaxation by the nitric oxide-guanylyl cyclase stimulator Bay 41-2272

To prove that NO-induced relaxation was mediated by cGMP we repeated the DEA-NO-induced relaxation experiments in the presence of the compound Bay 41-2272. Binding of Bay 41-2272 to a so far unknown regulatory site potentiates NO stimulation. Bay 41-2272 sensitized NO-GC towards NO as shown by the leftward shift of the relaxation responses in LES from control, SMC-GCKO and ICC-GCKO mice (Fig. 4A–C). These data indicate that the DEA-NO-induced relaxation was indeed mediated by cGMP in either SMC or ICC. Combined deletion of NO-GC in both SMC and ICC led to a loss of NO-induced relaxation (Fig. 4D) confirming the dual mechanism shown in Fig. 2B.

8-Br-cGMP-induced signal transduction by cGMP-dependent protein kinase and beyond

To exclude a possible change in signalling downstream of NO-GC we treated precontracted LES with the membrane-permeable cGMP analog 8-Br-cGMP (10 μm). As can be seen in Fig. 5, cGMP led to similar relaxation of LES from all genotypes (control, SMC-GCKO, ICC-GCKO and SMC/ICC-GCKO). We therefore exclude a change in downstream signalling to compensate for the loss of NO-GC in the different cell types.

Oesophageal manometry

To demonstrate the in vivo significance of our ex vivo findings, we determined LES tone of the different KO animals by using oesophageal manometry in anaesthetized mice. Original traces indicating the basal tone and the swallow-induced response of the LES high pressure zone in control, SMC-GCKO, ICC-GCKO and SMC/ICC-GCKO mice are shown in Fig. 6A. Figure 6B shows the statistical analysis of the pressure recordings: The basal LES tone of control animals was 5.7 ± 0.5 mmHg. Mechanical irritation of the tongue led to reflexive swallowing and decreased the pressure in the high-pressure zone to 2.2 ± 0.4 mmHg. Deletion of NO-GC in SMC and ICC led to increases in basal tone and in swallow-induced pressure compared to control animals (SMC-GCKO: 10.0 ± 0.5 mmHg and 5.0 ± 0.6 mmHg; ICC-GCKO: 11.4 ± 1.3 mmHg and 8.2 ± 1.6 mmHg; as well as SMC/ICC-GCKO: 14.8 ± 1.4 mmHg and 10.4 ± 1.5 mmHg for basal and relaxed tone, respectively).

Comparison of the relative responses between the different KO strains indicated a prominent role of ICC for swallow-induced relaxation (Fig. 6C). Deletion of NO-GC in SMC did not alter swallow-induced relaxation significantly compared to control LES (49.6% ± 5.4% vs. 62.3% ± 4.6%) whereas lack of NO-GC in ICC significantly reduced this response (29.0% ± 6.8%). This reduction was similarly found in SMC/ICC-GCKO (31.8% ± 5.3%). Therefore, we conclude that basal LES tone is dependent on NO-GC in both ICC and SMC whereas nitrergic LES relaxation is predominantly mediated via ICC.

Discussion

NO has been shown to be the main inhibitory neurotransmitter responsible for relaxation of the LES (Murray et al. 1991). NO generated by nNOS in inhibitory neurons results in the synthesis of cGMP by NO-GC in several target cells. NO-GC expression was detected in SMC, ICC, neurons and, recently, in FLC in various parts of the GI tract (Iino et al. 2008; Groneberg et al. 2011). The individual function of NO-GC in SMC and ICC has been addressed by our group recently by using global and cell-specific KO mice (Groneberg et al. 2013). In fact, we were able to provide evidence for a dual regulatory pathway for nitrergic relaxation of the murine fundus (Fig. 7): Generally, fundus contraction is initiated by muscarinic receptor activation on both ICC and SMC (Ward et al. 2000; Zhu et al. 2011). Relaxation may be induced directly in SMC by reduction of the acetylcholine-induced calcium increase, and additionally in ICC in which cGMP results in the impairment of the ICC-based contractile signal by a yet unknown mechanism. Our model can easily explain several pathophysiological or KO settings, including the achalasic situation in which nNOS expression is absent thus leading to an unopposed cholinergic contraction (Mearin et al. 1993) (Fig. 7; see below).

Figure 7.

Figure 7

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Dual mechanism for nitrergic lower oesophageal sphincter relaxation via ICC and SMC

Basal lower oesophageal sphincter tone is mediated by both ICC and SMC whereas swallow-induced tone is mediated mainly by ICC. See text for further explanations. ACh, acetylcholine; GCKO, guanylyl cyclase knockout; ICC, interstitial cells of Cajal; NO, nitric oxide; nNOS, neuronal nitric oxide synthase; SMC, smooth muscle cell.

In this report, we show that this dual pathway model generally fits to the data obtained with the LES: in vitro, NO-induced relaxation was reduced and, in vivo, basal LES tone was significantly increased in SMC-GCKO and ICC-GCKO animals. The double KO, lacking NO-GC in both SMC and ICC, showed an even more pronounced phenotype indicating additivity of the effects: NO-induced relaxation in vitro was abolished and basal LES tone even further increased in vivo. These data clearly show that both SMC and ICC contribute to NO-mediated signalling and the regulation of basal tone.

Our major finding is the dominant role of ICC regarding LES relaxation in vivo. There has been a long-lasting controversy on the question whether SMC or ICC act as primary targets of NO released from enteric neurons (Ward et al. 1998; Sivarao et al. 2001; Huizinga et al. 2008; Goyal & Chaudhury, 2010; Zhang et al. 2010). Our previous report indicated that in murine fundus both cells participate in nitrergic signalling but we were unable to determine the individual extents. In LES, deletion of NO-GC in SMC had no statistically significant effect on swallow-induced relaxation when compared to controls. Apparently, NO-GC in SMC is mainly involved in the regulation of basal tone. Deletion of NO-GC in ICC, however, strongly reduced swallow-induced relaxation, and this reduction was not augmented by the additional deletion in SMC. To our knowledge, this is the first time that nitrergic function in a specific organ can be quantitatively attributed to ICC.

In all genotypes, LES was able to relax upon swallow induction. Opening of the LES is also apparent by the fact that all KO animals feed normally and have a normal life span. Presence of swallow-induced relaxation but absence of NO-based EFS-induced relaxation (presence of ODQ) seen in the SMC/ICC-GCKO suggest a second transmitter to act, probably VIP or a purine. To our surprise, LES from SMC-GCKO had a slightly higher (although not statistically significant) swallow-induced reduction in LES pressure than those from the other strains (5.0 ± 0.6 mmHg vs. 3.5 ± 0.3, 3.2 ± 0.8 and 4.5 ± 0.5 mmHg for control, ICC-GCKO and SMC/ICC-GCKO animals, respectively). At higher basal pressure, however, the force needed to overcome the higher basal pressure in the LES of the KO strains is probably generated by the striated muscle of the oesophagus.

LES function in mouse models has been investigated earlier by Sivarao et al. (2001). They determined LES pressures in W/Wv mice, which lack ICC-IM in stomach and sphincters, and in nNOS-deficient mice. Both mouse lines were expected to suffer from a lack of nitrergic responses and, therefore, to share a common phenotype. In fact, W/Wv mice had no achalasia-like phenotype: Rather, the LES of W/Wv mice were hypotensive and swallow-induced relaxation was unperturbed. In contrast, the LES of nNOS-KO mice was hypertensive with reduced relaxation. The hypertensive phenotype of nNOS-KO LES is understandable by the preserved contractile function of acetylcholine via ICC and SMC with concomitant lack of NO (Fig. 7). In W/Wv mice, contraction is reduced due to the lack of muscarinic agonism on ICC (probably being the primary contractile signal). The contraction induced by activation of muscarinic receptors on SMC, however, can be easily counteracted by activation of NO-GC in the same cell.

It is necessary to mention that both W/Wv and nNOS-KO mice are suboptimal models for the investigation of nitrergic signalling. Cell-specific KO strains for nNOS are lacking. The enzyme is also located in neurons of the CNS and can therefore be expected to influence the peripheral phenotype. In addition, the nNOS-KO used in previous studies still contains residual NOS activity stemming from alternatively spliced mRNA (Huber et al. 1998). The W/Wv mouse has the disadvantage that it lacks only certain populations of ICC in different regions of the GI tract; despite its mosaic pattern, the deletion of the ICC leads to a reduction in both relaxant and contractile responses. In addition, the nitrergic component of inhibitory junction potentials in response to EFS was shown to vary considerably in W/Wv LES (Zhang et al. 2010). With this in mind, cell-specific strategies as used in this report can be expected to be more adequate to unravel the ICC/SMC dispute.

Deletion of NO-GC in ICC alone or in both SMC and ICC reduced but did not abolish LES relaxation in vivo. This residual relaxation may be based on other transmitters known to participate in LES relaxation including VIP or ATP (or a related purine) (Goyal et al. 1980; Farre et al. 2006). As NO-GC deletion in ICC is not quantitative (Groneberg et al. 2013), minor expression of NO receptors may account for the residual response. Alternatively, NO may signal through FLC still expressing NO-GC. The role of FLC in nitrergic signalling still awaits clarification. In vitro, we only see a very tiny residual relaxation in SMC/ICC-GCKO LES, indicating a minor role of this novel cell type. It has to be kept in mind, however, that NO-GC in FLC may have a facilitatory role only seen in a setting where the communication between all cells is unperturbed. FLC-specific KO mice may help to solve this question.

In summary, using cell-specific KO mice, we were able to discriminate the roles of SMC and ICC in the maintenance of LES tone in vitro and in vivo. Our data clearly show a dominant role of ICC in swallow-induced nitrergic LES relaxation. For the future, it will be of interest to discriminate ICC-and SMC-specific functions in nitrergic relaxation of other parts of the GI tract.

Glossary

8-Br-cGMP

8-bromoguanosine 3′,5′-cyclic monophosphate

CCh

carbachol

DEA-NO

2-(N,N-diethylamino)-diazenolate-2-oxide.diethylammonium salt

EFS

electric field stimulation

FLC

fibroblast-like cell

GCKO

guanylyl cyclase knockout

GI

gastrointestinal

IBMX

3-isobutyl-1-methylxanthine

ICC

interstitial cells of Cajal

ICC-GCKO

ICC-specific guanylyl cyclase knockout

LES

lower oesophageal sphincter

NO

nitric oxide

NO-GC

nitric oxide-sensitive guanylyl cyclase

nNOS

neuronal nitric oxide synthase

ODQ

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one

PDGFRα

platelet-derived growth factor receptor alpha

SMC

smooth muscle cell

SMC-GCKO

smooth muscle-specific guanylyl cyclase knockout

VIP

vasoactive intestinal polypeptide

Additional information

Competing interests

None.

Author contributions

D.G. and A.F. provided the conception and design of the experiments. D.G., B.L., E.Z., M.W. and A.F. were responsible for the collection, analysis and interpretation of data. The critical mouse models were provided by B.S., D.S. and A.F. Drafting the article or revising it critically for important intellectual content was done by D.G. and A.F.

Funding

The work was supported by Deutsche Forschungs-gemeinschaft (FR 1725/1-3 and FR 1725/1-5).

Acknowledgments

The excellent technical help of Linda Kehrer is gratefully acknowledged. We are grateful to Stefan Offermanns, Max-Planck-Institut Bad Nauheim, for the kind donation of the SMMHC-CreERT2 mouse strain.

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