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Keywords:

  • apamin;
  • c-Kit;
  • interstitial cells of Cajal;
  • lower oesophageal sphincter;
  • nitric oxide;
  • Ws/Ws rat

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract  The distribution of interstitial cells of Cajal (ICC) and neurotransmission were investigated in lower oesophageal sphincter (LES) circular muscle strips from Sprague–Dawley (SD) rats, Ws/Ws mutant rats and their wild-type (+/+) siblings. Intramuscular c-Kit-positive cells, confirmed to be ICC-IM by electron microscopy, were observed throughout both muscle layers from SD and +/+ rats. In contrast, c-Kit-positive, ultrastructurally typical ICC-IM were absent in Ws/Ws. LES strips from Ws/Ws rats showed increased spontaneous contractile activity. Strips from SD and +/+ rats, responded to electrical neuronal stimulation with a relaxation that was in part L-NNA and in part apamin sensitive, followed by a contraction which was decreased by atropine. In Ws/Ws rats, similar to +/+ rats, neurally mediated relaxation was L-NNA and apamin sensitive and the contraction was decreased by atropine. We conclude that in the rat LES, relaxation is mediated by NO and an apamin-sensitive mediator, and contraction primarily by acetylcholine. Despite the absence of c-Kit-positive ICC, nerve–muscle interaction can be accomplished likely by diffusion of neurotransmitters to the smooth muscle cells. The lack of c-Kit-positive ICC is related to an increase in the basal tone and spontaneous contractile activity. The presence of fibroblast-like ICC in Ws/Ws rats might represent immature ICC whose possible functions need further investigation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The lower oesophageal sphincter (LES) acts as a barrier at the gastroesophageal junction. Basal tone of LES is primarily myogenic in origin and is modulated by a combination of hormonal factors and neurogenic mechanisms involving enteric and extrinsic innervation. Inhibitory enteric motor neurons are the final step of the inhibitory neural pathway to the LES, allowing swallowing-induced and -transient LES relaxations that allow for normal oesophageal emptying as well as gastroesophageal reflux and belching.1 It is well established that nitric oxide (NO) plays a primary role in LES relaxation in different species including humans.2–6 Other neurotransmitters have been proposed to share a role with NO in LES relaxation. These include an apamin-sensitive transmitter,7,8 probably ATP6 and vasoactive intestinal peptide (VIP).9 However, other authors suggest that VIP has a negligible role in LES relaxation.10,11 Acetylcholine is the main neurotransmitter in excitatory motor neurons.3,6,12 However, non-cholinergic excitatory neurotransmitters, possibly substance P, have also been described in the LES.13 In the rat, vagally evoked responses involve the release of several neurotransmitters, such as NO and a non-nitrergic inhibitory mediator, whereas acetylcholine mediates excitation acting on muscarinic receptors.14 In recent years, the search for specific roles for interstitial cells of Cajal (ICC) in innervation of gut smooth muscle has been intensifying, after almost a century of speculation based on morphological data starting with Cajal and his contemporaries and reflected on in a classical review on the innervation of the gastrointestinal tract by Gabella.15 Different subpopulations of ICC are now recognized. The most extensively studied are the ICC that are pacemaker cells16 but they are not prominent in the LES. The LES musculature has a rich network of intramuscular ICC. Daniel and Posey-Daniel17 shed light on potential roles of ICC-IM in the LES by demonstrating that 11–22% of the total population of nerve varicosities has a close association (<200 nm distance) with ICC-IM with 47% of the contacts <23 nm. A similar number of varicosities were close to smooth muscle cells but without contacts <23 nm. The remainder of associations involved separations between nerve and muscle >200 nm. These data showed a special innervation of ICC-IM and supported the hypothesis that neural responses to smooth muscle cells might be mediated by ICC. The proposed scheme suggested enteric nerves to be innervating both ICC and smooth muscle cells such that part of the overall response of the musculature was influenced by ICC activity. Mutant (W/Wv) mice which lack ICC-IM have provided further evidence. In the LES18 and fundus19 reductions in nitrergic and cholinergic neurotransmission were found and the conclusion was made that ICC-IM might provide the dominant nitrergic inhibitory and cholinergic excitatory input from nerve endings to smooth muscle. In contrast, in vivo studies showed that swallowing and vagal stimulation evoked a normal LES relaxation in W/Wv mice, suggesting that ICC-IM are not crucial to mediating nitrergic neurotransmission.20 The aim of the present study was to gain further insight by using a rat model with a similar mutation as the W/Wv mouse. The specific aims were to evaluate the Ws/Ws rat LES as a potential alternative model to the W/Wv mouse for lack of ICC-IM and subsequently to study and compare the responses to electrical nerve stimulation in wild-type and Sprague–Dawley (SD) rat LES, to deduce a possible role for ICC in neurotransmission. Preliminary data were presented at the XII Symposium on Neurogastroenterology and Motility, Cambridge (UK) 2004.21

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Tissue preparation

Male SD, wild-type (+/+) and mutant (Ws/Ws) rats (weighing 300–350 g) were used in the present study. Animals were fasted overnight (18 h) but allowed ad libitum access to water. Rats were killed by decapitation and bled. The entire stomach, including portions of the oesophagus and duodenum, was removed and placed in Krebs solution. The stomach and oesophagus were opened by an incision along the lesser curvature, continuing through the LES and the right side of the oesophagus. A second incision was made along the greater curvature from middle corpus to the apex of the fundus, revealing the junction between the oesophagus and the stomach. The LES was identified at the end of the oesophagus as the transition from striated to smooth muscle fibres in the longitudinal muscle as has been previously reported.14 The experimental procedure of this work was previously approved by the Ethics Committee of the Universitat Autònoma de Barcelona.

c-Kit immunohistochemistry

The proximal stomach and lower oesophagus were isolated and fixed in paraformaldehyde (4%) in phosphate-buffered saline (PBS) (pH 7.4) overnight before they were washed and kept in 30% sucrose in phosphate buffer (PB) (pH 7.4) for another overnight. The fixed tissues were then embedded in Tissue-Tek and frozen with isopentane emerged in liquid nitrogen. Frozen cross-sections of 8 μm in thickness were cut using a cryostat and mounted on coated slides. Endogenous peroxidase activity was blocked by incubation in 1% hydrogen peroxide in methanol for 25 min. Sections were then preincubated in 5% goat non-immune serum in PBS for 2 h before being incubated with a polyclonal antibody against c-Kit protein (Dakocytomation, Denmark) (DAKO rabbit anti-human 1 : 100 in 0.5% goat non-immune serum in PBS) at 4 °C overnight. Secondary immunoreactions were carried out with Vectastain ABC kits supplied by Vector Laboratories (Burlingame, CA, USA), in which there are biotinylated anti-rabbit IgG. 3,3′-diaminobenzidine (0.05%) plus 0.01% H2O2 in 0.05 mol L−1 Tris buffer saline (pH 7.6) was used as a peroxidase substrate. Control tissues were prepared in a similar manner, omitting c-Kit antibody from the incubation solution.

Electron microscopy

Tissues were fixed with 2% paraformaldehyde, 2.5% glutaraldehyde, 3% sucrose and 1.25 mmol L−1 CaCl2 in 0.05 mol L−1 cacodylate buffer (pH 7.4) at 4 °C overnight. They were then postfixed in 2% osmium tetroxide (OsO4) for 1 h, stained en bloc with 2% aqueous uranyl acetate for 40 min, dehydrated, infiltrated and embedded in Epon–Araldite resin. Ultrathin sections were cut and stained with lead citrate for 5 min before viewing with a transmission electron microscope (Jeol 1200EX Biosystem, Tokyo, Japan). Evidence is accumulating indicating plasticity in ICC, with ICC obtaining increased resemblance to fibroblast-like cells during an inflammatory insult,22 after obstruction23 and after ablation of enteric nerves.24 ICC with a similar ultrastructure were also encountered in the stomach of Ws/Ws rats and named ‘fibroblast-like ICC’.25 These cells are not presumed to be fibroblasts and differ from typical fibroblast in their structural location and special junctional contacts with other cells. In the present study, we use the nomenclature used by Komuro to describe fibroblast-like ICC.

Muscle bath studies

Full thickness preparations including the mucosa and both the circular and longitudinal muscle layers were obtained from the left-hand side of the LES. Strips (1 mm width and 3 mm length) were dissected parallel to the circular muscle fibres and placed in organ baths containing 15 mL of Krebs solution, constantly bubbled with 5% CO2 in O2. Changes in tone of the strips were measured using isometric force transducers (Model 03, Grass Instruments Co., Quincy, MA, USA) and recorded on a PC using the data acquisition software Acqknowledge 3.7.2 (Biopac Systems, Inc., CA, USA) at a sampling frequency of 25 Hz. Two strips from each animal were simultaneously studied. Strips were hung loosely, with no tension, in the organ bath for 20–30 min before the beginning of the study. Each strip was gently stretched up to 4.5–5 g and a stable tone was achieved after an equilibration period of 1 h. Spontaneous mechanical activity (area under curve, AUC, per minute) was measured in SD, wild-type +/+ and Ws/Ws LES strips.

Experimental procedure to study neuromuscular transmission

Electrical field stimulation (EFS) was applied by means of two platinum wire electrodes (10 mm apart) located parallel to LES strips. The electrodes were connected to an electrical stimulator (Model S88, Grass Instruments Co., Quincy, MA, USA) and a power booster (Stimu-Splitter II, Med-Lab Instruments, Loveland, CO, USA) to obtain identical and undistorted signals. Transmural EFS (pulses of 0.4 ms and 26 V, train duration 5 s at an increasing frequency from 0.3 to 20 Hz) were applied to LES strips.6 To study the inhibitory and excitatory neurotransmission, the response was evaluated after sequential addition of the nitric oxide synthase inhibitor L-NNA (10−3 mol L−1), a muscarinic antagonist atropine (10−6 mol L−1) and a small conductance Ca2+-activated K+ channel blocker apamin (10−6 mol L−1). This protocol allowed us to study simultaneously both the inhibitory and excitatory neurotransmission as previously described in several species including humans.3,6 In some Ws/Ws LES strips, KCl (10−2 mol L−1) was used to contract the preparation and to evaluate the effect of these drugs in EFS-induced response.

Solutions and drugs

The composition of the Krebs solution was (in mmol L−1) 138.5 Na+, 4.6 K+, 2.5 Ca2+, 1.2 Mg+, 125 Cl, 21.9 HCOinline image, 1.2 H2POinline image, 1.2 SOinline image and 11.5 glucose. KCl, apamin and L-NNA were obtained from Sigma-Aldrich Co. (Madrid, Spain). Atropine sulphate was obtained from Merck (Darmstadt, Germany). All drugs were dissolved in distilled water.

Data analysis

Tone was expressed in mN, and the spontaneous mechanical activity was evaluated by the area under the curve for 1 min. Relaxation was expressed as the percentage of LES tone at the end of the equilibration period. Contraction was expressed in mN. The number of experiments was represented by n (number of strips) and N (number of specimens). Data are expressed as mean ± SEM. Student's t-test was selected for comparisons, using the paired mode when appropriate. To characterize EFS responses, the comparison of frequency–response curves was performed using a two-way anova for repeated measures followed by a post hoc test (Bonferroni). A value of P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

c-Kit immunohistochemistry

c-Kit-positive ICC (ICC-IM) were distributed throughout the circular and longitudinal muscle layers in the LES of +/+ rats (N = 3, Fig. 1A, B). A similar distribution of ICC-IM was observed in SD rats (N = 3, data not shown). c-Kit-positive ICC (ICC-MP) were not found in the myenteric plexus region of +/+ rats. In the absence of the primary antibody (c-Kit), no immunoreactivity was found. In contrast to +/+ rats, no c-Kit immunolabelling was found in Ws/Ws rats (N = 3), neither in the circular nor in longitudinal muscle layers (Fig. 1C, D). These results show an absence of c-Kit-positive ICC in the muscle layers of the LES of Ws/Ws animals.

image

Figure 1.  Distribution of c-Kit immunoreactivity in the rat LES. (A, B) Wild-type (+/+) rat LES. (C, D) Ws/Ws LES. Numerous c-Kit-positive ICC were present within both circular (CM) and longitudinal (LM) muscle layers in wild-type rat LES. In contrast, there were almost no c-Kit-positive ICC in the Ws/Ws rat LES. Arrows in (B) show ICC-IM within CM and arrowheads show them in the septa. AP: Auerbach's plexus.

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Electron microscopy

To confirm the absence of ICC-IM (to exclude the possibility that only the kit protein and not ICC were absent), ultrastructural studies were carried out on four wild-type and four Ws/Ws rats. Fig. 2 shows typical ICC-IM in wild-type rats which were characterized by ultrastructural features26 including a high electron density, many mitochondria and rough endoplasmic reticulum in the cytoplasm, caveolae lining the membrane as well as gap junctional communication with smooth muscle cells (Fig. 2A and inset) and synapse-like contacts (20- to 25-nm interval space) with nerve varicosities (Figs 2C and 4A and inset). Gap junctions between ICC-IM were also identified (Fig. 2B and inset). In the Ws/Ws rats, ultrastructurally typical ICC-IM were not found. Instead, fibroblast-like ICC25 or fibroblast-like cells (which might have been fibroblast-like ICC, see below) were found (Figs 3A, B and 5). Fibroblast-like ICC occupied the usual position of ICC, they formed gap junction contacts with each other (Fig. 3B) and with smooth muscle cells (Fig. 3A and inset and Fig. 5). The fibroblast-like ICC were closely and directly apposed to nerve varicosities (Fig. 5), but synapse-like contacts as observed with ICC-IM were not found. The fibroblast-like ICC did not have caveolae nor a basal lamina. In most cases, the usual place for ICC appeared occupied by fibroblast-like cells. An important criterion to define a fibroblast-like ICC is its gap junctional connection with other fibroblast-like ICC or with adjacent smooth muscle cells. However, a single EM profile from an ultrathin section may not reveal all gap junctions and hence a positive identification is not always possible.

image

Figure 2.  Ultrastructure of wild-type (+/+) rat LES featuring gap junction contact with smooth muscle cells and synapse-like contact with nerve varicosities. An ICC-IM (ICC) with typical ICC ultrastructural feature makes a gap junctional connection (square and inset) with an adjacent smooth muscle cell (SM). N: nerve varicosities. An ICC-IM (ICC) cell body makes gap junctional contact (square and inset) with the process of another ICC-IM. Nearby are a nerve bundle (N) and a fibroblast (F). SM: smooth muscle cell. A synapse-like junction (arrow) between a nerve varicosity (N) and an ICC-IM (ICC). The latter is also closely associated with smooth muscle cells nearby (SM). All small arrows indicate caveolae lining the membrane of ICC-IM.

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image

Figure 4.  Comparison between contacts between nerve varicosities and ICC or smooth muscle cells in wild-type (+/+) rat LES. An enteric nerve varicosities (N) forms a synapse-like junctional connection (upper box and white arrow in inset) with the cell body of an ICC-IM (ICC); another varicosity (N) forms a membrane-to-membrane direct apposition (lower box and black arrow in inset) with a smooth muscle cell (SM). A nerve bundle with numerous varicosities passes through a muscle layer and a synapse-like junction (white arrow) between a nerve bundle (N) and an ICC process (ICC) can be seen as well as a close apposition (black arrow) between a varicosity of the same nerve bundle (N) and a smooth muscle cell (SM).

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image

Figure 3.  Ultrastructure of Ws/Ws rat LES. A fibroblast-like ICC (FLC); the cell body has the typical ultrastructure of fibroblasts with a high density of rough endoplasmic reticulum, it occupies the usual site of an ICC-IM and it is connected to a smooth muscle cell (SM) by a gap junction (square and inset). Gap junction (arrow) between two fibroblast-like ICC (FLC). SM: smooth muscle cell; N: nerves.

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image

Figure 5.  In the Ws/Ws rat LES, a fibroblast-like ICC (FLC), forms a direct apposition (white arrow) with an enteric nerve (N) and at the same time a gap-junction contact (black arrow) is present with a smooth muscle cells (SM). This configuration does not happen with typical fibroblasts but is a common feature in the Ws/Ws justifying the term fibroblast-like ICC as used by Komuro.

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Membrane-to-membrane appositions were present between enteric nerves and smooth muscle cells in both the wild-type and Ws/Ws rat LES (Fig. 4). In wild-type rats, the chance to see in any given field of view, close appositions between smooth muscle cells and enteric nerves with a distance of 100–300 nm is ∼ 60%. To see close contacts of 50-100 nm distance between smooth muscle cells and enteric nerves is ∼ 30%, compared to the chance to see synapse-like junctions between ICC and nerves which is ∼ 10%. In mutant rats, the chances to see the close contacts with distances 100–300 and 50–100 nm between nerves and smooth muscle cells were 75% and 25%. Close contacts (as close as 50 nm distance) between nerves and fibroblast-like ICC were rarely observed in both control and mutant rats due to the difficulty to identify all the fibroblast-like ICC. This is because they share the same ultrastructural features as regular fibroblasts except for the gap junctional contact with each other and/or with smooth muscle cells.

Tone and spontaneous mechanical activity in SD, +/+ and Ws/Ws mutants

After the initial stretch, tone decreased time dependently reaching a stable value after 3 min (Fig. 6A, B). The final tone reached by the strips, expressed as the percentage of the initial value, was markedly higher in Ws/Ws strips (60%) than in +/+ and SD and (40%) (anova, P < 0.0001, Fig. 6B). After an equilibration period of 1 h, Ws/Ws LES strips exhibited a higher stable tone (SD: 13.0 ± 1.9 mN; +/+: 14.8 ± 1.6 mN and Ws/Ws: 21.1 ± 2.5 mN, P < 0.05) and increased spontaneous contractile activity (SD: 28.0 ± 0.6 mN min−1; +/+: 33.6 ± 13.8 mN min−1 and Ws/Ws: 233.0 ± 66.8 mN min−1, P < 0.002) (Fig. 6C, D).

image

Figure 6.  Tracings (A and C) and measurements of tone after stretching (B), tension and spontaneous mechanical activity developed by SD, +/+ and Ws/Ws LES strips after the equilibration period (D).

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Neurotransmission in SD, +/+ and Ws/Ws rats

All SD LES strips responded to electrical nerve stimulation with a sharp relaxation during the electrical stimulus (‘on’ relaxation) followed by a contraction at the end of the stimulus (‘off’ contraction) (n = 8; N = 5). The amplitude of both responses was frequency dependent (Fig. 7). L-NNA (10−3 mol L−1) significantly inhibited the relaxation (anova, P < 0.0001) with significant effects from 1 to 20 Hz. Subsequent addition of atropine (10−6 mol L−1) caused an increase in EFS relaxation (anova, P < 0.0001). Apamin (10−6 mol L−1) completely abolished the non-nitrergic relaxation (anova, P < 0.0001). In relation to the ‘off’ contraction, L-NNA increased the response (anova, P < 0.001) with significant effects from 10 to 20 Hz. Subsequent addition of atropine reduced the ‘off’ contraction (anova, P < 0.001) and finally, apamin reduced the ‘off’ contraction (anova, P < 0.001) (Fig. 7). These results show that in SD rats, the inhibitory pathway involves a nitrergic and an apamin-sensitive mediator, whereas the excitatory pathway is mediated by acetylcholine and possibly another excitatory neurotransmitter.

image

Figure 7.  Effect of sequential addition of L-NNA, atropine and apamin in Sprague–Dawley LES strips. Top (A) show tracings and bottom (B) the frequency-related responses. Marks at the bottom of the tracings represent application of EFS.

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Similar to SD, all LES strips of wild-type (+/+) rats responded to EFS with a sharp relaxation during the electrical stimulus (‘on’ relaxation) followed by a contraction at the end of the stimulus (‘off’ contraction) (n = 9; N = 5). The amplitude of both responses was frequency dependent (Fig. 8). The EFS-induced relaxation was significantly inhibited by L-NNA (anova, P < 0.0001). Subsequent addition of atropine did not modify the EFS-induced relaxation and apamin abolished the non-nitrergic relaxation (anova, P < 0.0001). In relation to the ‘off’ contraction, L-NNA increased the amplitude (anova, P < 0.001). The subsequent addition of atropine markedly reduced the ‘off’ contraction (anova, P < 0.001); apamin did not modify the amplitude of the non-cholinergic contraction (Fig. 8). These results show that in +/+ rats, the inhibitory pathway involves a nitrergic and an apamin-sensitive component whereas the excitatory pathway is mainly mediated by acetylcholine.

image

Figure 8.  Effect of sequential addition of L-NNA, atropine and apamin in wild-type (+/+) LES strips. Top (A) show tracings and bottom (B) the frequency-related responses. Marks at the bottom of the tracings represent application of EFS.

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Ws/Ws LES strips responded to EFS with one of two different responses. Muscle preparations (66%) (n = 8 of 12 strips) responded to EFS with a sharp relaxation during the stimulus (‘on’ relaxation) followed by a contraction at the end of the stimulus (‘off’ contraction) (Fig. 9). This response was similar to those found in SD and wild-type (+/+) LES strips. The amplitude of both responses was frequency dependent. Muscle preparations (33%) (n = 4 of 12) responded with a frequency-dependent ‘off’ contraction (Fig. 9). Strips that responded to EFS with an ‘off’ contraction presented higher spontaneous contractile activity (448.1 ± 117.6 mN min−1) than strips with lower spontaneous contractile activity (125.5 ± 51.0 mNmin−1, P < 0.05) but did not show differences in basal tone (22.1 ± 2.8 mN vs 16.5 ± 2.8 mN, P = 0.27). When appropriate, to avoid a decrease in the basal tone and spontaneous contractions that might mask the muscle response, KCl (10−2 mol L−1) was added to the organ bath. Under these conditions, all the muscle preparations responded with an ‘on’ relaxation followed by an ‘off’ contraction (Fig. 9). Under these conditions the pharmacological characterization of the inhibition was carried out by sequential addition of L-NNA, atropine and apamin in Ws/Ws LES strips with KCl (n = 4; N = 3) and without the presence of KCl (n = 2; N = 2) (Figs 10 and 11). L-NNA inhibited the EFS relaxation (anova, P < 0.0001). Atropine increased the inhibitory response to nerve stimulation in some preparations but the average value of relaxation before and after atropine was not significantly different. Apamin abolished the non-nitrergic relaxation (anova, P < 0.0001). Regarding to the ‘off’ contraction, L-NNA increased its amplitude (anova, P < 0.001). The subsequent addition of atropine markedly reduced the ‘off’ contraction (anova, P < 0.001) and apamin did not modify the amplitude of the non-cholinergic contraction (Fig. 11). Fig. 10 shows similar effects of the drugs in both experimental conditions (with and without KCl): L-NNA reduced the relaxation and increased the contraction, subsequent addition of atropine reduced the contraction and, finally, the addition of apamin abolished the non-nitrergic relaxation. Altogether, these results show that also in Ws/Ws rats, the inhibitory pathway involves a nitrergic and an apamin-sensitive component, whereas the excitatory pathway is mainly mediated by acetylcholine.

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Figure 9.  Tracings showing the two patterns of EFS responses and the effect of KCl in Ws/Ws LES strips. 66% of the strips displayed a relaxation followed a contractions that was enhanced in presence of KCl (Top) and 33% of the strips exhibited high spontaneous activity that unmask the relaxatory effect of EFS that was revealed by KCl (bottom). Marks at the bottom of the tracings represent application of EFS.

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Figure 10.  Effect of sequential addition of L-NNA, atropine and apamin in Ws/Ws LES strips under basal conditions and with the presence of KCl. Right panel shows tracings under basal conditions and with the presence of KCl, left panel shows graphic representation that includes strips of both experimental conditions. Marks at the bottom of the tracings represent application of EFS.

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Figure 11.  Effect of sequential addition of L-NNA, atropine and apamin in Ws/Ws LES strips.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The present study shows that in wild-type (+/+) and Ws/Ws rats, two major inhibitory neurotransmitters mediate relaxation in the LES: nitric oxide and an apamin-sensitive component that might be ATP.6,27 The major excitatory component is cholinergic but another neurotransmitter might be present. These results are similar to those found in the LES of SD rats14 (present study) and other species including humans.3,7,8 The wild-type rats also show a typical distribution of c-Kit-positive ICC-IM throughout both muscle layers. Unlike the small intestine and colon, c-Kit-positive cells were not found within the myenteric plexus region of the rat (present study) and mouse LES.18 This observation is similar to the distribution of ICC in the murine fundus.28 In the mutant Ws/Ws rats, c-Kit-positive cells were absent in the LES similar to the lack of c-Kit-positive cells in sphincters of W/Wv mice.18 Consistently, in a previous report, the c-Kit messenger RNA-expressing cells were not detected in the stomach of Ws/Ws rats and decreased to 7% compared with the control in the ileum showing that Kit expression is dramatically reduced in mutant animals.29 These data establish the Ws/Ws rat as a model for the investigation of potential roles of ICC-IM in innervation.

In the present study, the response to electrical stimulation of intrinsic nerves in Ws/Ws animals was similar to that found in control animals in most preparations studied: an ‘on’ relaxation followed by an ‘off’ contraction was recorded. The relaxation was blocked in part by L-NNA indicating that nitric oxide released by nerves in response to EFS relaxed smooth muscle cells. The inhibition by apamin indicates that ATP is likely mediating part of the relaxation.6 Contractile activity evoked by EFS of the enteric nerves was in part blocked by atropine indicating that cholinergic nerves were activated and smooth muscle responded by contraction. Hence, electrical stimulation of the enteric nerves caused release of nitric oxide, ATP and acetylcholine which caused muscle responses similar to control tissue apparently not hindered by the absence of ICC. Notably, in 33% of the strips, nerve stimulation caused only an excitatory response that could be interpreted as an impairment of the inhibitory pathway. However, when the tone was raised by KCl, nerve stimulation caused a nitrergic and an apamin-sensitive relaxation and a cholinergic contraction. These results show that in the rat LES, the lack of relaxation found in basal conditions was not due to the absence of ICC but might be related to the difference in excitability of smooth muscle cells (see below). Hence, when all enteric nerves are stimulated by electrical stimulation that involves evoking TTX-sensitive Na-mediated action potentials, neurotransmitters reach smooth muscle cells with little hindrance. This supports the parallel innervation hypothesis, not only for apamin-sensitive30 but also for nitrergic and cholinergic innervation. Obviously, EFS will not be representative of all physiological ways of nerve stimulation. ICC have a special innervation31 and certain physiological stimuli might specifically activate nerves associated with ICC and in this way may modify smooth muscle function. Or, a physiological stimulus, as suggested by Daniel and Posey-Daniel,17 might activate nerves that innervate both ICC and smooth muscle cells and the current experimental protocols might not be sensitive enough to deduce the influence of ICC on the ultimate smooth muscle response. In any case, innervation of ICC-IM will affect ICC functions, such as propagation of electrical events through the musculature32 which will have a major impact on smooth muscle function. Consistent with the present study are data from the colon of Ws/Ws rats where NO-mediated relaxation is present while in this area of the gastrointestinal tract ICC-IM are extremely reduced33 and data on the internal anal sphincter from W/Wv mice where NO-mediated relaxation was also present.34 The present results are consistent with structural data that suggest parallel innervation to both ICC and smooth muscle cells in the LES (present study).17,20

Of note is that the absence of ICC is not necessarily equal to the complete loss of a cellular structure. The c-Kit mutation appears to allow embryonic development of ICC but results in arrested maturation after birth.35 This may result in c-Kit-negative, fibroblast-like cells as reported in the present study and also noted in earlier studies on the small intestine and stomach.35,36 Fibroblast-like ICC may also appear in response to injury because of obstruction23 or inflammation.22 Whether or not these cells can partially perform the functions of ICC cannot be deduced from the present study. It is highly unlikely that they can perform as normal ICC as they lack the c-Kit protein, caveolae and a basal lamina. Apamin-sensitive innervation could potentially be mediated by fibroblast-like cells which have been shown to carry the SK3 channel that might have mediated ATP-induced relaxation.37,38 Absence of c-Kit immunoreactivity can also result from the appearance of c-Kit-negative ICC as observed in the human deep muscular plexus.39 However, in the Ws/Ws rat LES, no ultrastructural typical ICC-IM were observed.

The differences in mechanical activity between LES strips from Ws/Ws rats and wild-type rats need further investigation. At the moment, the primary result of the c-Kit gene mutation (in relation to the gut musculature) is thought to be the absence of ICC. Functional changes found in the musculature might be the result of absence of ICC and/or other direct effects of the kit mutation not yet discovered and/or compensatory mechanisms developed as a result of loss of ICC. LES muscle strips from control animals with c-Kit-positive ICC (SD and +/+ rats) reached a stable tone (40% of the initial tone, ∼20 mN) without spontaneous activity. Similarly, strips from human or porcine LES do not appear to have spontaneous contractile activity.3,6 In contrast, LES strips from Ws/Ws rats showed higher tone (60% of the initial tone, ∼30 mN) and presented with spontaneous contractile activity. This was not expected because in vivo, in W/Wv mice, the LES is hypotensive compared with control animals.20 This may be associated with a role for c-Kit-positive ICC in the regulation of smooth muscle excitability. The lack of c-Kit-positive ICC might depolarize smooth muscle cells40 and increase the probability of opening of calcium channels leading to spontaneous contractions. Consistent with this is the observation that intestinal strips and whole intestine segments from W/Wv mice, which lack ICC at the Auerbach's plexus, display spontaneous rhythmic activity41 as well as colon preparations from the Ws/Ws rat.42 On the other hand, it has been speculated that c-Kit deficiency may affect the function and development of smooth muscle independently of the ICC-IM deficiency;43 spontaneous motility could be caused by abnormalities in K+ and/or Ca2+ channels. Another possibility is that the presence of fibroblast-like ICC causes smooth muscle depolarization causing increases in tone and spontaneous motility. The nature of the spontaneous contractions and potential abnormalities in smooth muscle of Ws/Ws mutant animals needs further investigation.

We conclude that nitric oxide, and an apamin-sensitive mediator, possibly ATP, are the main inhibitory neurotransmitters in the rat LES. Smooth muscle contraction in the rat LES is mainly mediated by acetylcholine. Ws/Ws animals lack c-Kit-positive ICC but electrically stimulated neurotransmitter (TTX sensitive) release causes normal muscle responses. This supports the notion of parallel innervation where major pathways of innervation are non-synaptic muscle innervation involving all major neurotransmitters and synaptic ICC innervation. Close apposition contacts between nerve varicosities and smooth muscle cells are numerous (present study)17,20 supporting direct muscle innervation. The lack of c-Kit-positive ICC may be related to an increase in muscle tone and spontaneous contractions. The presence of fibroblast-like ICC warrants further investigation related to abnormalities in ICC development and to plasticity of ICC during injury and disease.22,24

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Grant support was obtained from the Ministerio de Ciencia y Tecnología (SAF2003-05830 and BFU2006-05055/BFI), Fundació de Gastroenterologia Dr Francisco Vilardell, the Departament d'Universitats, Recerca i Societat de la Información (SGR2005) and the Canadian Institutes for Health Research. The authors thank Mrs Sonia Roca, Mònica Porras and Anna Maria Alcántara for their technical support.

References

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  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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