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The Journal of Physiology logoLink to The Journal of Physiology
. 2015 Aug 21;593(20):4589–4601. doi: 10.1113/JP270511

Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon

Barbara Lies 1, Katharina Beck 1, Jonas Keppler 1, Dieter Saur 2, Dieter Groneberg 1, Andreas Friebe 1,✉
PMCID: PMC4606533  PMID: 26227063

Abstract

Key points

  • Dysregulation of nitric oxide (NO) signalling is associated with GI motility dysfunctions like chronic constipation, achalasia or Hirschsprung's disease. The inhibitory effect of NO is mainly exerted via NO‐sensitive guanylyl cyclase (NO‐GC) which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC).

  • Here, we focus on the investigation of NO‐GC function in murine colon. Using cell‐specific knock‐out mice, we demonstrate that NO‐GC is expressed in myenteric ICC of murine colon and participates in regulation of colonic spontaneous contractions in longitudinal smooth muscle.

  • We report a novel finding that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCS in the murine proximal colon.

  • These results help in understanding possible pathological mechanisms involved in slowed colonic action and colonic inertia.

Abstract

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO‐sensitive guanylyl cyclase (NO‐GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide‐sensitive guanylyl cyclase (NO‐GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild‐type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle‐specific guanylyl cyclase knockout (SMC‐GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC‐specific guanylyl cyclase knockout (ICC‐GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (l‐NAME, ODQ) did not significantly affect contractions of GCKO and ICC‐GCKO colon whereas the arrhythmic contractile patterns of WT and SMC‐GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field‐stimulated neuronal NO release was similar in SMC‐GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.

Abbreviations

DEA‐NO

2‐(N,N‐diethylamino)‐diazenolate‐2‐oxide diethylammonium salt

EFS

electrical field stimulation

GCKO

guanylyl cyclase knockout

ICC

interstitial cells of Cajal

ICC‐GCKO

ICC‐specific guanylyl cyclase knockout

KO

knockout

NO

nitric oxide

NO‐GC

nitric oxide‐sensitive guanylyl cyclase

l‐NAME

N G‐nitro‐l‐arginine methyl ester

nNOS

neuronal nitric oxide synthase

ODQ

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

PKG

cGMP‐dependent protein kinase

SMC

smooth muscle cell

SMC‐GCKO

smooth muscle‐specific guanylyl cyclase knockout

TTX

tetrodotoxin

WT

wild‐type

Introduction

The control of intestinal motility is based on a complex interaction of neurogenic and myogenic processes. The underlying mechanisms have been investigated in several studies using various species such as guinea pig, rabbit, rat, mouse and dog (Keef et al. 1997; Pluja et al. 1999; Huizinga et al. 2011; Costa et al. 2013; Huizinga & Chen, 2014). The cyclic contractions that occur in most gastrointestinal (GI) smooth muscles derive from rhythmic fluctuations of the smooth muscle membrane potential termed slow waves. In this context, myenteric interstitial cells of Cajal (ICC‐MY) are accepted to play a crucial role in pacemaking (Thuneberg, 1982; Sanders et al. 2006; Huizinga et al. 2009). The W/Wv mouse, an animal model of selective ICC loss, has been used extensively to reveal the functional consequences of ICC depletion (Maeda et al. 1992; Ward et al. 1994; Huizinga et al. 1995). This model has a disrupted ICC‐MY network, and thereby offers an approach to examine the general mechanisms of motility. ICC‐MY have been demonstrated to coordinate and coherently propagate Ca2+ waves and phasic contractions in the longitudinal GI smooth muscle (Hennig et al. 2010). In addition, the ICC‐MY network was found to be electrically coupled to longitudinal SMCs as demonstrated by low resistance electrical pathways between ICC and SMCs (Cousins et al. 2003; Mitsui & Komuro, 2003).

Recently, colonic motor patterns have been convincingly shown to result from cooperation of ICC networks with the enteric nervous system (Huizinga et al. 2011), yet information on the pacemaking mechanism is still limited. Two ICC networks have been shown to generate slow waves, submucosal ICC (ICC‐SM) and ICC‐MY (Pluja et al. 2001; Sanders et al. 2006; Huizinga et al. 2011). In rat, it has been shown that longitudinal muscle of the colon is only affected by the pacemaking rhythm of ICC‐MY (Pluja et al. 2001). In addition, another class of ICC, the intramuscular ICC (ICC‐IM), has been demonstrated to be essential intermediates in GI neurotransmission (Burns et al. 1996; Ward et al. 1998; Groneberg et al. 2013; Lies et al. 2014).

Studies on different parts of the intestine from various animal species have implicated the participation of nitric oxide (NO) in the regulation of spontaneous contractions. NO is widely recognized as an important inhibitory neurotransmitter in the GI tract. Over the past years several studies have revealed that colonic contractile activity is suppressed (but not abolished) by basal NO release since inhibition of NO synthases led to increased spontaneous contractions and/or tone (Ward et al. 1992; Keef et al. 1997; Boeckxstaens et al. 1999). Whether these effects are mediated by ICC or SMCs, however, has not been addressed yet.

In previous studies we could show that both SMCs and intramusclar ICC (ICC‐IM) are generally capable of mediating nitrergic relaxation and inhibitory junction potentials of GI smooth muscle (Groneberg et al. 2013; Lies et al. 2014). Here, we focused on the role of nitrergic signalling in the modulation of murine colonic contractile activity. Comparison of wild‐type (WT) and guanylyl cyclase knockout (GCKO) mice showed that NO increases the frequency and reduces the mean amplitude of spontaneous contractions. Using cell‐specific knockout mice we show that this action of basally released neuronal NO occurs solely through ICC. Stimulation of neuronal NO release, however, is shown to directly act on NO‐GC in SMCs. These results imply differential functions of NO‐GC in ICC and SMCs in the murine colon which depend on the strength of the NO signal.

Methods

Ethical approval

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

Animals

Mice (C57BL/6 background) were housed in standard mouse cages (267 × 207 × 140 mm; maximally 3 animals per 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 either standard rodent diet or in the case of global GCKO fibre‐reduced diet (Altromin, Lage, Germany) and water available ad libitum). Animals of either sex were killed at an age of 8–16 weeks by cervical dislocation and tissues were isolated. A total of 81 animals were used.

Generation of SMC‐GCKO and ICC‐GCKO

Smooth muscle‐specific guanylyl cyclase knockout (SMC‐GCKO) and ICC‐specific guanylyl cyclase knockout (ICC‐GCKO) mice carry a floxed exon (exon 10 of the β1 subunit of NO‐GC) (Friebe et al. 2007) and are transgenic for the inducible Cre recombinase in SMCs (SMMHC‐CreERT2; Wirth et al. 2008) and ICC (cKIT‐CreERT2; Klein et al. 2013). Mice from both knockout lines aged 6–8 weeks were injected with tamoxifen (dissolved in Miglyol 812; 1 mg i.p.) on five consecutive days in order to remove the floxed exon. This protocol avoids high doses of tamoxifen reported to induce GI epithelial side effects (Huh et al. 2012). As previously shown, deletion of NO‐GC in GI smooth muscle was complete only after 50 days; therefore, all mice were analysed at least 50 days after the last tamoxifen injection (Groneberg et al. 2011). The efficacy of NO‐GC deletion in the SMC‐ and ICC‐GCKO has been shown previously (Groneberg et al. 2011, 2013).

Isometric force studies

Animals were killed by cervical dislocation. The abdomen was opened and the colon was quickly removed and transferred to Krebs–Henseleit solution (118 mm NaCl, 4.7 mm, KCl, 2.5 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 25 mm NaHCO3, pH 7.4, 7.5 mm glucose) bubbled with 95% O2–5% CO2. Proximal colon strips were mounted on fixed segment support pins in two four‐chamber myographs (Myograph 610; Danish Myo Technology, Aarhus, Denmark) containing 5 ml of Krebs–Henseleit solution. Contractions and responses to electrical field stimulation (EFS) of the longitudinal muscle layer were measured. Strips were allowed to equilibrate for at least 1 h. Spontaneous contractions were recorded and analysed. Influence of 2‐(N,N‐diethylamino)‐diazenolate‐2‐oxide diethylammonium salt (DEA‐NO, 10 μm), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μm), N G‐nitro‐l‐arginine methyl ester (l‐NAME, 200 μm) and tetrodotoxin (TTX, 1 μm) on spontaneous contractions was determined.

Electrical field stimulation

To examine the impact of endogenously released NO on spontaneous colonic contractions, electrical field stimulation (EFS; Multiplexing Pulse Booster, Ugo Basile, Comerio, Italy) was applied to colon strips via two platinum wire electrodes (5 mm distance; supramaximal voltage, 0.5 ms, 2 Hz). EFS was conducted in the absence of pre‐contracting agonists and without non‐adrenergic non‐cholinergic conditions. To measure the inhibition of contraction by endogenously released NO, colon strips were stimulated by an electric field in the absence and presence of ODQ (10 μm, 30 min pre‐incubation). The ratio of the 2 Hz contraction peaks before and after the addition of ODQ was taken for analysis.

Immunohistochemical analysis

Mice were killed by cervical dislocation and colon tissue was fixed with 4% paraformaldehyde in 0.1 mM phosphate buffer, pH 7.4. The tissue was subsequently cryoprotected using 20% sucrose in phosphate buffer and snap frozen. Cryosections (10 μm) were cut, air‐dried and incubated overnight with a homemade antibody against the β1 subunit of NO‐GC raised in rabbit (1:800) together with a rat anti‐ckit antibody (1:200; clone ACK4, Linaris, Wertheim‐Bettingen, Germany). The rabbit antibody was detected by an Alexa 555‐conjugated anti‐rabbit IgG antibody raised in donkey (1:800; Invitrogen, Darmstadt, Germany) and the rat antibody was detected with an Alexa 488‐conjugated donkey anti‐rat IgG antibody (1:400, Invitrogen, Darmstadt, Germany). Secondary antibodies were incubated in Antibody diluent either alone or in combination for 1 h. The sections were mounted in Mowiol and were evaluated using a confocal microscope equipped with appropriate filter sets for Alexa 555/Cy3 and FITC/Alexa 488.

For determination of the excision rate in ICC‐GCKO animals, we counted ckit‐positive cells and cells positive for both ckit and NO‐GC (n = 4, 3 sections, each).

Materials

DEA‐NO, ODQ, TTX were purchased from Axxora (Lörrach, Germany). l‐NAME and tamoxifen were from Sigma (Taufkirchen, Germany).

Statistical analysis

For calculation of statistical tests, GraphPad Prism 5.0 for Windows was used (GraphPad Software Inc., La Jolla, CA, USA). For comparison of independent variables, GCKO, SM‐GCKO, ICC‐GCKO and WT were compared by the Kruskal–Wallis test. If P was ≤0.05 for the global test, basal values (contraction duration and frequency) from GCKO, SM‐GCKO, ICC‐GCKO and WT were each compared with values after pharmacological treatment by the Mann–Whitney U test (Figs 3–5). In Figs 6 and 7, values (contraction duration and frequency) from GCKO, SM‐GCKO, ICC‐GCKO were each compared with values from WT by Mann–Whitney U test. Comparisons of individual groups were only reported if the global test reached significance. The individual statistical analyses for each figure are given in the figure legends. Data are expressed as means ± SEM.

Results

Effect of NO‐GC deletion on colonic spontaneous contractions in longitudinal smooth muscle

Proximal colon of WT mice showed an irregular contraction pattern with varying amplitudes (Fig. 1 A–C). Contractions occurred with a frequency of 4.3 ± 0.4 contractions per minute (cpm) and with an average duration of 8.8 ± 0.9 s per contraction. In contrast, GCKO colon showed a regular contraction pattern with uniform contraction amplitudes. In the absence of NO‐GC, single contractions were prolonged to 20.6 ± 1.8 s and the frequency was decreased to 1.9 ± 0.1 cpm. These results prove the involvement of NO‐GC in the regulation of spontaneous contractions in the colon.

Figure 1. Spontaneous contractions of longitudinal muscle .

Figure 1

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A, representative original tracings of spontaneous contractions of longitudinal smooth muscle strips from proximal colon of WT, GCKO, ICC‐GCKO and SMC‐GCKO mice. B and C, statistical analysis of spontaneous contractions between the genotypes. The data show means ± SEM of n = 6 per genotype (*P < 0.05, **P < 0.01 compared to WT). Frequency and duration of SMC‐GCKO contractions did not significantly differ from WT (P = 0.8357 and P = 0.7206, respectively).

Next, we used mice lacking NO‐GC specifically in either SMCs or ICC (Fig. 1 A–C). Interestingly, muscle strips from ICC‐GCKO mice showed a contraction pattern similar to that seen in GCKO muscle strips: spontaneous contractions were strictly periodic with equal amplitudes. The frequency of spontaneous contractions was 2.4 ± 0.4 cpm with an average duration of 16.9 ± 3.0 s for each contraction. The contraction pattern of SMC‐GCKO colon seemed more irregular than that of WT tissue; however, frequency (4.3 ± 0.4 cpm) and duration of each contraction (9.6 ± 1.5 s) were equal to those of WT colonic tissue. These data indicate a prominent role for NO‐GC in ICC. As the generation of the pacemaker activity has been assigned to myenteric ICC (ICC‐MY), neuronal NO is likely to modulate the contraction pattern via NO‐GC in ICC‐MY.

Expression of NO‐GC in colonic ICC‐MY

To prove NO‐GC expression in ICC‐MY of the proximal colon we used immunohistochemistry on WT, GCKO and ICC‐GCKO tissues. Figure 2 shows co‐staining of NO‐GC and ckit (a marker for ICC) in cells surrounding the myenteric plexus of WT colon. GCKO colon lacks NO‐GC‐specific staining. Induction of ICC‐specific Cre recombinase led to the almost complete absence of NO‐GC staining in ICC‐GCKO colon. These results provide evidence that NO‐GC is in fact expressed in ICC‐MY from murine colon. In addition, ckit‐Cre‐mediated deletion of NO‐GC occurs in 97% of all ICC; thus, ICC‐GCKO mice prove to be a reliable tool for the examination of nitrergic effects on ICC.

Figure 2. Immunohistochemical stainings of the myenteric plexus in proximal murine colon .

Figure 2

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Murine proximal colon of WT, ICC‐GCKO and GCKO mice were stained using antibodies against NO‐GC (red) and ckit (green). Yellow signals in the merge panel indicate co‐localization of NO‐GC and ckit.

Effect of TTX on spontaneous contractions in longitudinal smooth muscle

To evaluate the neuronal impact on spontaneous contractions, we used tetrodotoxin (TTX) to inhibit voltage‐dependent Na+ channels (Gershon, 1967; Narahashi, 1972). TTX (1 μm) reduced the contractile frequency of WT colon, increased the duration of single contractions and led to constant amplitudes (Fig. 3). In SMC‐GCKO tissue, TTX‐induced changes in contractility were similar to those seen in WT tissue. The spontaneous contractions of ICC‐GCKO and GCKO colon were not significantly reduced in frequency and duration. Figure 3 B–E shows the statistical analysis of contraction frequency and duration in the presence and absence of TTX. Reduction of contraction frequency was stronger in SMC‐GCKO (57.6 ± 4.4 %) than in WT (32.5 ± 5.8 %) tissue, which is probably based on the slightly higher contraction frequency under basal conditions (compare top and bottom traces in Figs 1 A and 3 A). The contraction duration (Fig. 3 D and E) was strongly increased by TTX in WT and SMC‐GCKO tissue (78.5 ± 15.3 % and 99.2 ± 27.3 %, respectively), whereas no significant change was observed in ICC‐GCKO and GCKO colon. This suggests that NO generally shortens individual contractions. Taken together, these results indicate spontaneous contractions to be non‐neurogenic. In addition, inhibitory and excitatory neurotransmitters are released under basal conditions ex vivo to modulate the contraction pattern without central nervous input.

Figure 3. Effect of TTX on spontaneous contractions in longitudinal smooth muscle .

Figure 3

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A, representative original tracings of spontaneous contractions from proximal colon of the different genotypes before and after the addition of 1 μm TTX. B–E, statistical analysis of the effect of TTX on spontaneous contractions. The TTX‐induced changes in frequency (B and C) and duration of each contraction (D and E) were evaluated. The data show means ± SEM of n = 6 per genotype (*P < 0.05, **P < 0.01 compared to WT). TTX did not induce significant changes in frequency and duration in colon from GCKO (P = 0.5113 and P = 0.4848) and ICC‐GCKO (P = 0.1275 and P = 0.1413) mice.

Effect of l‐NAME and ODQ on spontaneous contractions in longitudinal smooth muscle

In order to more specifically address the role of basal neuronal NO release in colon, we blocked the cascade, first by using the NOS inhibitor N G‐nitro‐l‐arginine methyl ester (l‐NAME). Figure 4 shows that addition of l‐NAME (200 μm) significantly reduced the contraction frequency and increased the duration of a single contraction in colon tissue from WT and SMC‐GCKO mice, whereas both frequency and duration were basically unchanged in ICC‐GCKO and GCKO colon. These results indicate basal NO release from nitrergic neurons to regulate colonic contractile activity via ICC. Use of the specific NO‐GC inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) corroborated our findings with l‐NAME: Fig. 5 shows that addition of ODQ (10 μm) caused the contraction pattern of WT and SMC‐GCKO colon to become regular as in GCKO colon. In ICC‐GCKO and GCKO colon, ODQ did not alter the contractile activity. These results indicate the effect of basal NO release to be mediated mainly via NO‐GC in ICC. Figure 4 B–E shows a detailed evaluation of frequency and duration of the spontaneous contractions for each genotype. ODQ reduced the frequency in both WT and SMC‐GCKO colon (45.0 ± 2.5 % and 36.9 ± 6.7 %, respectively). Furthermore, ODQ also increased the duration of a single contraction in these two genotypes (118.2 ± 15.1 % and 86.3 ± 20.8 %, respectively). In contrast, neither frequency nor duration of contractions was affected by ODQ in colon from GCKO and ICC‐GCKO mice. Taken together, these results show that inhibition of NO‐GC signalling in WT and SMC‐GCKO colon induces contraction patterns similar to those seen in GCKO and ICC‐GCKO colon.

Figure 4.

Figure 4

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Effect of l‐NAME on spontaneous contractions in longitudinal smooth muscle

A, representative original tracings of spontaneous contractions from proximal colon of the different genotypes before and after the addition of 200 μm l‐NAME. B–E, statistical analysis of the effect of l‐NAME on spontaneous contractions. The l‐NAME‐induced changes in frequency (B and C) and duration of each contraction (D and E) were evaluated. The data show means ± SEM of n = 6 per genotype (*P < 0.05, **P < 0.01 compared to WT). l‐NAME did not induce significant changes in frequency and duration in colon from GCKO (P = 0.6273 and P = 0.172) and ICC‐GCKO (P = 0.0542 and P = 0.1797) mice.

Figure 5. Effect of ODQ on spontaneous contractions in longitudinal smooth muscle .

Figure 5

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A, representative original tracings of spontaneous contractions from proximal colon of the different genotypes before and after the addition of 10 μm ODQ. B–E, statistical analysis of the effect of ODQ on spontaneous contractions. The ODQ‐induced changes in frequency (B and C) and duration of each contraction (D and E) were evaluated. The data show means ± SEM of n = 6 for WT, GCKO, ICC‐GCKO and n = 7 for SMC‐GCKO (*P < 0.05, **P < 0.01 compared to WT). ODQ did not induce significant changes in frequency and duration in colon from GCKO (P = 0.3982 and P = 0.212) and ICC‐GCKO (P = 0.173 and P = 0.3527) mice.

Acute effects of pharmacological NO and NO‐GC inhibition on spontaneous colonic contractions in longitudinal smooth muscle

Addition of the radical NO donor DEA‐NO (10 μm) suppressed spontaneous contractions in WT colon for approximately 10 min after which contractions started to slowly reappear (Fig. 6 A). Spontaneous contractions of GCKO colon were unaffected by DEA‐NO treatment. In colon from both cell‐specific knockout mice, DEA‐NO induced the suppression of the spontaneous contractions albeit for a shorter time period than in WT colon (approx. 6.0 and 5.3 min for ICC‐GCKO and SMC‐GCKO, respectively; Fig. 6 B). These data reveal that pharmacologically applied NO can transiently abolish spontaneous contractions in WT, SMC‐GCKO and ICC‐GCKO colon. Lack of response in GCKO tissue indicates NO‐GC to be the only mediator of NO‐induced effects in the colon. Moreover, NO‐GC in both SMCs and ICC is generally capable of mediating the inhibition of spontaneous contractions after pharmacological treatment. In previous studies, we demonstrated a dual mechanism of NO‐induced relaxation mediated by NO‐GC in SMCs and intramuscular ICC (ICC‐IM) in murine fundus. The data described here also suggest individual ICC‐ and SMC‐mediated nitrergic inhibitory mechanisms to exist in colon based on the suppression of spontaneous contractions in SMC‐ and ICC‐GCKO. Due to the fact that pharmacological NO can be assumed to be present ubiquitously, this set‐up does not allow us to determine whether ICC‐MY or ICC‐IM (or both) are involved.

Figure 6. Effect of the NO donor DEA‐NO on spontaneous contractions in longitudinal smooth muscle .

Figure 6

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A and B, representative original tracings showing the effect of DEA‐NO (10 μm; A) and ODQ (10 μm; B) on longitudinal muscle strips of proximal colon. C, statistical analysis of the time period of suppression of contractile activity after the addition of DEA‐NO (10 μm). Data show means ± SEM of n = 6 per genotype (*P < 0.05, **P < 0.01 compared to WT).

The acute effects of NO‐GC inhibition by ODQ are shown in Fig. 6 C. Besides leading to a regular contraction pattern in WT and SMC‐GCKO colon (as shown in Fig. 4), a short increase in tonic contraction was detected in WT and SMC‐GCKO colon directly following ODQ application. This may be due to a rebound effect of removing the inhibitory effect of NO. Both effects are not detected in ICC‐GCKO and global GCKO animals, which corroborates the basal release of NO acting via NO‐GC in ICC to influence phasic as well as tonic contraction.

Effect of endogenous NO released by electrical field stimulation

The use of pharmacological NO donors in our previous experiments can be assumed to lead to a widespread release of NO in the tissue. In order to evaluate the effects of local neuronal NO release, we used electrical field stimulation (EFS; 2 Hz, 10 s, supra‐maximal voltage) in the different knockout (KO) models (Fig. 7). EFS was applied in the absence of pre‐contracting agonists and adrenergic/cholinergic blockers. Thus, the expected EFS responses correspond to the combined effects of simultaneously released contractile and relaxant factors.

Figure 7. Effect of endogenous NO released by electrical field stimulation .

Figure 7

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A, representative mechanical recordings of EFS (2 Hz; 10 s; supra‐maximal voltage) in longitudinal smooth muscle from WT, GCKO, ICC‐GCKO and SMC‐GCKO colon in the absence and presence of ODQ (10 μm). B, statistical analysis of EFS‐induced on‐contraction. To quantify NO‐mediated inhibition of contraction, the ratio of the amplitude of the on‐contraction before and after addition of ODQ was determined. Data show means ± SEM, n = 6 per genotype (*P < 0.05, **P < 0.01, P = 0.6623 (ns) compared to WT).

In WT colon, EFS induced a short low amplitude on‐contraction which reversed to basal level during stimulation. Upon cessation of the EFS, an off‐contraction with higher amplitude than the on‐contraction was observed. EFS in GCKO colon led to a prolonged on‐contraction which did not return to baseline; consequently, only a small off‐contraction was measured. ICC‐GCKO colon showed an EFS response similar to that seen in WT colon. In SMC‐GCKO colon, EFS evoked a response as in GCKO tissue, but with a relatively high amplitude of the on‐contraction.

In order to identify the part of the response mediated by NO, EFS was repeated with the same tissues in the presence of ODQ (10 μm). Inhibition of NO‐GC resulted in a prolonged on‐contraction; the off‐contraction was only minor in all colon smooth muscle strips after EFS cessation due to the elevated contraction level (as seen in GCKO and SMC‐GCKO colon without ODQ). To quantify the inhibitory effect of NO, we determined the ratio of the on‐contraction with and without ODQ (Fig. 7 B). In WT colon, NO‐GC stimulation reduced the on‐contraction by 58.6 ± 4.6 %. Likewise, NO release in ICC‐GCKO colon led to a decreased on‐contraction (42.3 ± 7.1 % of the contraction seen in the presence of ODQ). The presence of ODQ did not significantly affect EFS‐induced contractions in GCKO and SMC‐GCKO colon. These results indicate that NO from enteric neurons limits the effect of simultaneously released contractile agonists via NO‐GC in SMCs. Taken together, the effects of basal NO release are mediated mainly by NO‐GC in ICC whereas stimulated NO release significantly affects NO‐GC in SMCs under the conditions chosen.

Discussion

GI motility is based on coordinated contraction and relaxation. It requires various motility patterns such as propulsive and non‐propulsive contractions, segmentation, pendulum movements and tonic sphincter contractions. The exact mechanisms underlying these movements are intricate and still need to be further explored to obtain a better understanding of GI motility disorders. In colon, the specific motor patterns that underlie motility include spontaneous contractions of smooth muscle which are independent of the central nervous system. Contractions of the circular smooth muscle are effective in mixing, turning over, and propulsion, whereas contractions of the longitudinal muscle shorten the length of the colon. Nitrergic neurotransmission is known to inhibit smooth muscle contraction, thus counteracting processes induced by excitatory transmitters. The exact impact of NO on longitudinal colonic smooth muscle has not been comprehensively addressed so far. Therefore, the major aim of the current study was to elucidate the influence of NO‐GC on spontaneous contractions of the colonic longitudinal smooth muscle using a tamoxifen‐inducible cell‐specific knockout strategy.

Spontaneous contractions in murine colon are non‐neurogenic and modulated by inhibitory and excitatory neurotransmitters. Our experiments with GCKO colon (i.e. global absence of NO‐GC) show that the colonic contractile pattern is regular with uniform contraction amplitudes. In WT, in contrast, the presence of NO‐GC interferes with this regular contraction rhythm leading to shorter contractions with varying amplitudes. This clearly demonstrates an important influence of NO/cGMP signalling on the rhythmicity of spontaneous contractions in murine colonic smooth muscle. As the regular rhythm can be restored by the blockade of NO signalling (l‐NAME, ODQ), we conclude that basally released NO interferes with the constant colonic pacemaker rhythm. This latter finding is in line with several studies reporting that basal NO release mediates continuous suppression of visceral smooth muscle activity and contractile tone in various species (Gillespie et al. 1989; Li & Rand, 1989; Boeckxstaens et al. 1990; Keef et al. 1997).

In accordance with these results, TTX led to a change in WT rhythmicity due to the inhibition of neural activity. Unexpectedly, TTX also induced a slight but non‐significant reduction of GCKO colonic rhythm, which was not observed in the presence of l‐NAME or ODQ. Dampening of the basal release of contractile agonists by TTX may well account for this phenomenon.

Using our cell‐specific knockout models, we were able to investigate the individual function of NO‐GC in ICC and SMCs. To our surprise, ICC‐GCKO colon showed a contractile motor pattern similar to that in GCKO colon whereas contractile behaviour in SMC‐GCKO colon was similar to that of WT. Thus, the effect of basal NO release on spontaneous colonic contractions is dependent on NO‐GC in ICC. The effect upon acute inhibition of NO‐GC by ODQ corroborates this notion: only with NO‐GC expressed in ICC (WT and SMC‐GCKO animals) was a transient tonic contraction is observed (Fig. 6 B). This indicates (1) that excitatory and inhibitory agonists are continuously released under basal conditions in colonic tissue and (2) that NO‐GC in ICC constantly counteracts the contractile influence. Immunohistochemistry revealed a dense network of NO‐GC‐positive ICC in the myenteric region (ICC‐MY) of the murine colon. As ICC‐MY have been found to be crucial for pacemaking (Torihashi et al. 1995), the physiological NO‐GC‐mediated inhibition of contractile function can be ascribed to this type of ICC. Although not available at the moment, an ICC‐MY‐specific KO model would be ideal to prove this hypothesis.

Addition of the NO donor DEA‐NO led to suppression of spontaneous contractions in WT as well as in both SMC‐ and ICC‐GCKO colon. Thus, both SMCs and ICC are involved in direct NO‐induced inhibition of colonic contractions. In previous studies with fundus tissue from our knockout mice, we described a dual mechanism of nitrergic relaxation involving SMCs and ICC (Groneberg et al. 2013). It is reasonable to assume a similar mechanism in colon by which activation of NO‐GC in either SMCs or ICC suppresses spontaneous contractions. Whereas NO‐GC signalling in SMCs will probably directly interfere with the contractile mechanism, NO‐GC stimulation will exert different effects in ICC‐IM and ICC‐MY. In ICC‐IM, NO influences SMCs electrotonically (Burns et al. 1996; Ward et al. 1998; Lies et al. 2014) whereas NO will affect pacemaking in ICC‐MY. Again, ICC‐subspecific KO models would allow us to describe the individual role of the two types of ICC.

Interestingly, NO release after EFS reduces the on‐contraction in ICC‐GCKO, which proves that endogenous NO acts directly via NO‐GC in SMCs. Moreover, deletion of NO‐GC in SMCs (SMC‐GCKO as well as GCKO animals) prevented this nitrergic inhibition. In conclusion, basal and stimulated NO release differentially affects three different cell types: in ICC‐MY, NO‐GC controls the rhythmic motor pattern whereas in ICC‐IM and SMCs, nitrergic signalling inhibits spontaneous contractions via control of the SMC membrane potential, probably by influencing contractile coupling.

Why should basal and stimulated release of NO act on three different cell types in murine colon? Fluctuations of the low basal NO concentrations may set the excitability of colonic smooth muscle by affecting the basal rhythmicity via ICC‐MY and by influencing the resting membrane potential via ICC‐IM. The relaxant effect resulting from acutely increased NO release acting directly on SMCs would then clearly depend on the degree of excitability the smooth muscle tissue was set to. In sum, nitrergic signalling could thereby influence frequency, duration and strength of contractions in order to modulate peristalsis as required.

Additional information

Competing interests

The authors have nothing to disclose.

Author contributions

Conception and design of the study: B.L. and A.F.; collection, assembly, analysis and interpretation of data: B.L., K.B., D.G., J.K. and A.F.; drafting of the manuscript or revising it critically for important intellectual content: B.L. and A.F. cKIT‐CreERT2‐mouse was provided by D.S.

Funding

The work was supported by Deutsche Forschungsgemeinschaft (FR 1725/1‐5).

Acknowledgements

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.

B. Lies and K. Beck contributed equally to this work.

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