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

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.

Figure 1

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.

Figure 2

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 3

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.

Figure 4

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

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

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.

Figure 7

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.