These individuals contributed equally.
Changes in neuromuscular transmission in the W/Wv mouse internal anal sphincter
Article first published online: 10 NOV 2011
© 2011 Blackwell Publishing Ltd
Neurogastroenterology & Motility
Volume 24, Issue 1, pages e41–e55, January 2012
How to Cite
Duffy, A. M., Cobine, C. A. and Keef, K. D. (2012), Changes in neuromuscular transmission in the W/Wv mouse internal anal sphincter. Neurogastroenterology & Motility, 24: e41–e55. doi: 10.1111/j.1365-2982.2011.01806.x
- Issue published online: 21 DEC 2011
- Article first published online: 10 NOV 2011
- Received: 13 May 2011 Accepted for publication: 1 October 2011
- fibroblast-like cells;
- internal anal sphincter;
- interstitial cells of Cajal;
- membrane potential
- Top of page
- Materials and Methods
- Supporting Information
Background Intramuscular interstitial cells of Cajal (ICC-IM) have been shown to participate in nitrergic neuromuscular transmission (NMT) in various regions of the gastrointestinal (GI) tract, but their role in the internal anal sphincter (IAS) is still uncertain. Contractile studies of the IAS in the W/Wv mouse (a model in which ICC-IM numbers are markedly reduced) have reported that nitrergic NMT persists and that ICC-IM are not required. However, neither the changes in electrical events underlying NMT nor the contributions of other non-nitrergic neural pathways have been examined in this model.
Methods The role of ICC-IM in NMT was examined by recording the contractile and electrical events associated with electrical field stimulation (EFS) of motor neurons in the IAS of wildtype and W/Wv mice. Nitrergic, purinergic, and cholinergic components were identified using inhibitors of these pathways.
Key Results Under NANC conditions, purinergic and nitrergic pathways both contribute to EFS-induced inhibitory junction potentials (IJPs) and relaxation. Purinergic IJPs and relaxation were intact in the W/Wv mouse IAS, whereas nitrergic IJPs were reduced by 50–60% while relaxation persisted. In the presence of L-NNA (NOS inhibitor) and MRS2500 (P2Y1 receptor antagonist), EFS gave rise to cholinergic depolarization and contractions that were abolished by atropine. Cholinergic depolarization was absent in the W/Wv mouse IAS while contraction persisted.
Conclusions & Inferences ICC-IM significantly contributes to the electrical events underlying nitrergic and cholinergic NMT, whereas contractile events persist in the absence of ICC-IM. The purinergic inhibitory neural pathway appears to be independent of ICC-IM.
Intramuscular interstitial cells of Cajal (ICC-IM) have been shown to be located in close proximity to nerves throughout most of the gastrointestinal (GI) tract1 including the murine internal anal sphincter (IAS).2 A variety of functional and morphologic studies have provided evidence that ICC-IM participate in nitrergic and cholinergic neuromuscular transmission (NMT) in various regions of the GI tract.3–6 Nonetheless, the role of these cells in NMT remains controversial.7–9
ICC express the receptor tyrosine kinase Kit. In Kit mutant animal models ICC numbers are greatly reduced in some portions of the GI tract.10,11 These models have frequently been used to assess the role of ICC in mediating NMT. Immunohistochemical studies examining the presence of ICC in the Kit mutant W/Wv mouse IAS have yielded conflicting results with one study suggesting a complete absence of ICC12, whereas another reported some ‘faintly stained’ ICC at the submucosal edge.13 We recently re-examined this issue in the mouse IAS and found that ICC are absent from the myenteric edge (ICC-My) of both wildtype (WT) and W/Wv mice, whereas stellate-shaped submucosal ICC (ICC-SM) are present in both. In contrast, ICC-IM are present in WT, but not in W/Wv mice.2
Studies of the W/Wv mouse IAS have also examined nerve-evoked relaxations and the in vivo rectoanal inhibitory reflex (RAIR).12,13 One study reported that the RAIR was unchanged12, whereas the other reported that the RAIR was reduced while nerve-evoked relaxations were intact.13 The latter group suggested that the reduction in RAIR could be due to the contribution of ICC to the afferent limb of the response.13 In spite of these differences, both studies concluded that ICC did not appear to be necessary for nitrergic inhibitory NMT in the mouse IAS.12,13 However, the electrical events underlying NMT were not examined in these studies, nor were possible changes in other non-nitrergic neural pathways. There is evidence that both purines such as ATP and peptides such as VIP contribute to inhibitory NMT in the mouse and rat IAS.14–16 It is therefore possible that changes could occur in inhibitory NMT in the W/Wv mouse IAS that were not detected using previous methodologies.
This study examines the role of ICC-IM in enteric NMT in the mouse IAS in more depth by determining if there are differences in electrical and contractile events underlying the actions of various neurotransmitters in WT vs W/Wv mice. To do this, we used the selective P2Y1 receptor antagonist, MRS2500, and the nitric oxide synthase (NOS) inhibitor, L-NNA and measured membrane potential and contractile responses to activation of motor neurons under NANC conditions. Cholinergic NMT was also examined in the absence of atropine. Purines and NO were found to contribute to both the electrical and mechanical events underlying inhibitory NMT in the IAS, whereas excitatory NMT was largely due to acetylcholine (ACh). Our results suggest that ICC-IM generate cholinergic depolarization and 50–60% of the nitrergic IJP, whereas contractile events persist in the absence of ICC-IM. Purinergic electrical and contractile events were independent of ICC-IM. A preliminary report of this work has been published in abstract form.17
Materials and Methods
- Top of page
- Materials and Methods
- Supporting Information
Mice used for these studies were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experiments and procedures were approved by the Institutional Animal Use and Care Committee at the University of Nevada. C57BL/6 and W/Wvmice (30–90 days old; Jackson Laboratory, Bar Harbor, MN, USA) were killed with isoflurane (Baxter, Deerfield, IL, USA) followed by cervical dislocation. The rectoanal region was removed by dissecting away overlying tissue and was pinned in a dissecting dish containing cold Krebs bicarbonate solution (KRB) of the following composition (in mmol L−1): NaCl 118.5, KCl 4.7, CaCl2 2.5, MgCl2 1.2, NaHCO3, 23.8, KH2PO4, 1.2, dextrose, 11.0. This solution was maintained at a pH of 7.4 at 37 °C by bubbling to equilibrium with 95%O2 to 5%CO2. The distal extremity of the IAS was identified, and is referred to in this study as the anal verge. Overlying skeletal muscle, glands, and mucosa were carefully dissected away before carrying out experiments.
Muscle strips were attached with sutures to a stable mount and to a Gould strain gauge and immersed in tissue baths containing 15 mL of oxygenated KRB solution maintained at 37 °C. A basal tension of 0.5 g was applied to muscle strips. Tissues were equilibrated during the first 60 min after mounting. Applied tension declined to approximately 0.2–0.3 g during this equilibration period. All experiments were performed in the presence of 10 μmol L−1 guanethidine. Data were stored and analyzed by computer using a data acquisition program (AcqKnowledge; Biopac systems Inc., Goleta, CA, USA). Neural responses were evaluated by the application of electrical field stimulation (EFS) using a Grass S48 stimulator (1–5 Hz at 15 V and 0.3 ms pulse duration for 1 min) as was determined by previous experiments. Platinum inoculation loops (3 mm diameter, separated from one another by 1 cm) were used as stimulating electrodes. Muscle strips were threaded through both loops. The muscle was attached at the bottom to a stable mount and at the top to a tension transducer. EFS gave rise to TTX-(1 μmol L−1) sensitive neural responses.
Contraction analysis Spontaneous contractions were evaluated using AcqKnowledge software. To determine phasic contractile amplitude, a threshold value was selected (in grams) that bisected a group of phasic contractions to be measured. The program determines the peak and trough values for every phasic contraction that passes through the threshold on both rising and falling phases. As fast phasic contractions (40–60 cpm) were usually superimposed upon a slower phasic rhythm (2–4 cpm), fast phasic contractile amplitude was averaged for three periods during the peak of the slow rhythm (‘peak phasic’) and three periods during the trough of the slow rhythm (‘trough phasic’). Mean values for each group were then reported (see Figure S1). The amplitude of the slow rhythm (‘slow phasic’) was determined by subtracting the trough value during minimum contraction from the trough value during peak contraction. Tone was defined as the mean trough value during minimum contraction. Total spontaneous contraction was determined by summing together peak phasic contractile amplitude plus slow rhythm plus tone (see Figure S1 and Table 1). Spontaneous contractile activity was calculated as a percentage of the contraction induced by 60 mmol L−1 KCl. Passive tension was determined at the end of the experiment with 10 μmol L−1 sodium nitroprusside (SNP) and 1 μmol L−1 nifedipine and was subtracted from spontaneous contractile amplitudes. Nerves were stimulated for 60 s, and the area of contraction during the initial 10 s and the remaining 50 s of electrical field stimulation (EFS) was quantitated using AcqKnowledge software. The initial 10 s contractile response was determined for comparison to electrical responses. Contractile area during EFS was compared with control area (‘Pre-Stimulus’) and expressed per 10 s. Statistical analysis was performed using one-way anova followed by a post hoc Dunn’s or Tukey test. Data are expressed as mean ± SE, and values were considered significantly different when P < 0.05.
|Mouse||1) Peak phasic||2) Trough phasic||3) Slow phasic||4) Tone||5) Total phasic (1 + 3)||6) Total spontaneous (1 + 3 + 4)|
|WT (g)||0.22 ± 0.03||0.12 ± 0.02||0.06 ± 0.01||0.23 ± 0.03||0.30 ± 0.04||0.51 ± 0.04|
|WT (% Max)||8.3 ± 0.9||4.2 ± 0.7||4.3 ± 0.6||11.9 ± 2.0||12.6 ± 1.5||24.5 ± 2.6|
|W/Wv (g)||0.2 ± 0.03||0.1 ± 0.02||0.1 ± 0.01||0.27 ± 0.04||0.28 ± 0.04||0.58 ± 0.05|
|W/Wv (% Max)||9.7 ± 0.5||5.6 ± 0.5||2.9 ± 0.4||11.9 ± 2.4||12.6 ± 0.5||24.5 ± 2.3|
Muscle strips were pinned to the base of an electrophysiologic chamber with the submucosal surface facing upwards. Smooth muscle cell (SMC) impalements were made with glass microelectrodes (filled with 3 mol L−1 KCl; tip resistances ranging from 90 to 150 MΩ) as previously described.14 Nerves were stimulated (0.05–5 Hz; 70 V; 0.2 ms) via platinum electrodes (1 cm length) placed 1 mm away from the muscle at both sides along its entire length. The stimulus parameters for these experiments differ from the isolated tissue bath experiments, since the electrode configuration differed. TTX-sensitive neural responses were elicited under these stimulation parameters. To maintain impalements and slow wave activity, experiments were carried out after superfusing tissue preparations with the myosin light chain kinase inhibitor wortmannin (25 μmol L−1) for 20 min followed by a 60-min washout period as previously described.18 This treatment permanently blocked contraction while electrical activity recovered to that of untreated tissues, i.e., Em, slow waves and spikes were very similar to those reported for the mouse IAS in a previous study in the absence of wortmannin.14
Analysis of intracellular measurements Resting membrane potential (Em) was determined as the average value of Em between slow waves. Slow wave amplitude, rise time, and frequency were calculated using AcqKnowledge software. The amplitude of the composite inhibitory junction potentials (IJP) elicited with 5 Hz for 10 s was measured as the most negative potential occurring during three different time periods; i.e., 0.3–1.3 s, 5–6 s, and 9–10 s, following the initiation of EFS. This was done, so that the value of Em between slow waves could be selected.
Atropine sulfate, N (ω)-nitro-L-arginine (L-NNA), guanethidine, sodium nitroprusside (SNP) tetrodotoxin (TTX), and nifedipine were all purchased from Sigma-Aldrich (Saint Louis, MO, USA). Wortmannin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MRS2179 and MRS2500 were purchased from Tocris Bioscience (Ellisville, MO, USA). Atropine, L-NNA, guanethidine, SNP, MRS2179, and MRS2500 were dissolved in de-ionized water. Nifedipine and wortmannin were dissolved in ethanol and DMSO respectively.
- Top of page
- Materials and Methods
- Supporting Information
Basic electrical properties and inhibitory junction potentials in the wildtype mouse IAS
Electrical activity in the wildtype (WT) mouse IAS was measured using intracellular recording techniques. Oscillations in membrane potential (Em) were present that we have referred to as ‘slow waves’ (Fig. 1A; Figure S2A). The average value of Em between slow waves was −49.1 ± 0.5 mV (n = 17). Slow waves in the IAS did not have an initial rapid depolarizing phase as described for other GI regions (e.g., 434 mV/sec upstroke in canine antrum,19). Rather, they slowly depolarized (i.e., 10–80 mV/sec maximum rate of rise) reaching peak after 0.4–0.7 s. The frequency of slow waves was relatively constant (38.2 ± 1.5 cpm, n = 17) whereas amplitude was quite variable; often exhibiting a ‘waxing and waning’ pattern (see Figure S2A and20,21). Spikes (260–800 mV/sec) of variable amplitude were often superimposed upon slow waves (see Fig. 1A, Figures S2A and S3A) Peak depolarization during the slow wave (including the spike) was determined for each tissue and averaged 26.0 ± 1.4 mV (n = 28).
A single shot electrical stimulus produced an inhibitory junction potential (IJP) averaging 22.0 ± 1.1 mV (n = 11). There was no further increase in IJP amplitude with repetitive stimulation (5 Hz, 10 s; atropine and guanethidine present throughout). At low frequencies of electrical field stimulation (EFS; 0.1–1 Hz) Em following peak hyperpolarization returned at least to the ‘resting’ value, and in some cases, this was followed by a spike (e.g., Fig. 1A). In contrast, at 5 Hz EFS individual hyperpolarizations fused together, and rebound was confined to the end of the stimulus train, although it was larger in amplitude and longer in duration than with a single shock (Fig. 1A). IJP amplitude during 5 Hz EFS was measured during three different time periods (i.e., 0.3–1.3, 5–6 and 9–10 s) to quantitate its time course. The initial peak hyperpolarization quickly declined in amplitude to a sustained level of hyperpolarization that persisted for the remainder of the stimulus train. At the end of stimulation, rebound depolarization occurred with superimposed slow waves and spikes (Fig. 1A,C,D).
Role of purinergic and nitrergic pathways in generation of inhibitory junction potentials in the wildtype mouse IAS
To investigate the contribution of P2Y1 receptors to generation of IJPs, we first compared the effects of two different P2Y1 receptor antagonists, i.e., MRS2179 and MRS2500 (Fig. 1B,C) as MRS2500 has been reported to be a more potent blocker of P2Y1 receptors in rat colon than MRS2179.22 In the presence of L-NNA (100 μmol L−1), MRS2179 (10 μmol L−1), reduced IJPs elicited with a single stimulus from 23.0 ± 1.2 mV to 8.0 ± 1.6 mV (n = 5; Fig. 1Ba), whereas MRS2500 (1 μmol L−1) abolished the single IJP (Fig. 1Bb). When MRS2179 (10 μmol L−1) and MRS2500 (1 μmol L−1) were tested on composite IJPs elicited with 5 Hz EFS for 10 s, IJPs and rebound depolarizations were reduced by either P2Y1 blocker. However, there was still a residual IJP remaining following combined addition of MRS2179 with L-NNA (Fig. 1Ca), whereas combined MRS2500 and L-NNA abolished the composite IJP (Fig. 1Cb). These data indicate that the IJP is composed of both purinergic and nitrergic components and supports the previous study of rat colon indicating that MRS2500 is a more potent P2Y1 antagonist than MRS2179.22
To further examine the time course and amplitude of purinergic IJPs elicited with repetitive EFS (5 Hz, 10 s), we carried out additional experiments in which L-NNA was applied before MRS2500. Peak IJP amplitude was unchanged in the presence of L-NNA, whereas there was a small but significant reduction in the sustained component (Fig. 1D,E). In contrast to P2Y1 blockers, rebound was not reduced by L-NNA. Interestingly, the remaining purinergic IJP also consisted of two phases, i.e., an initial peak hyperpolarization followed by rapid repolarization to a smaller sustained hyperpolarization. As this study was primarily focused upon the role of ICC-IM in nitrergic NMT, the remaining electrical measurements were carried out by first blocking the purinergic IJP with MRS2500, so that the nitrergic IJP could be examined in isolation.
Basal contractile properties and responses to stimulation of inhibitory nerves in the wildtype mouse IAS
Contractile activity in the WT mouse IAS was measured in strips of muscle in isolated tissue baths. Muscles developed spontaneous contractile activity over a 20–30 min period of time following immersion in the bath. The mean amplitude of peak spontaneous contraction (‘total spontaneous’ contraction) was 24.5 ± 2.6% of the maximum contraction elicited with 60 mmol L−1 KCl (see Fig. 2A, Table 1 and Figure S1; n = 7). Spontaneous contraction consisted of both phasic and tonic components as defined in the methods section (see also Figure S1). Phasic contractions were further divided into a fast rhythm (56.7 ± 2.8 cpm) superimposed upon a slow rhythm (3.5 ± 0.2 cpm) (see Figure S2C). The amplitude of fast phasic contractions superimposed upon the peak of the slow rhythm (‘peak phasic’) was approximately twice as large as the fast phasic contractions occurring during the trough of the slow rhythm (‘trough phasic’) (see Table 1, n = 7). Finally, the amplitude of total phasic activity (12.6 ± 1.5% Max) was not different from the amplitude of tone (11.9 ± 2.0% Max).
Low frequency EFS gave rise to brief relaxations followed by rebound contractions (e.g., 1 Hz, Fig. 2A), whereas at 5 Hz, there was continuous relaxation during EFS and rebound contraction at the termination of EFS (Fig. 2A). Rebound contraction with 5 Hz EFS was greater than with a single shock. The contractile patterns for lower (1 Hz) and higher (5 Hz) frequency EFS were therefore very similar to the patterns of junction potentials at lower and higher frequency EFS (Fig. 1A).
Role of purinergic and nitrergic pathways in nerve-evoked relaxations in the wildtype mouse IAS
To examine the contribution of nitrergic and purinergic pathways to nerve-evoked relaxation, we compared the effects of MRS2500 (1 μmol L−1) and L-NNA (100 μmol L−1). Neither blocker significantly reduced the initial 10 s or the subsequent 50 s of relaxation (5 Hz EFS 60 s; guanethidine and atropine present throughout) (Fig 2B,C middle traces). However, rebound contraction was greatly reduced by MRS2500 (Fig. 2B) and increased by L-NNA (Fig. 2C). We have previously noted this effect in the BALB/c mouse IAS.14 In contrast, when both blockers were added, relaxation was abolished during the initial 10 s of EFS suggesting that both nitrergic and purinergic pathways contribute to this phase of the response. After an additional 10 s of EFS, a slowly developing relaxation commenced that reached maximum by the end of the 60 s stimulus train (Fig. 2B,C right traces). Thus, contractile area from 10 to 60 s (50 s) was significantly less than control (Fig. 2D), suggesting that a third neurotransmitter is released with longer stimulus periods. This hypothesis was further supported by addition of the neurotoxin TTX (1 μmol L−1, n = 3) that abolished the remaining ultraslow relaxation.
Comparison of basal electrical activity and inhibitory junction potentials in the wildtype and W/Wv mouse IAS
To examine the role of ICC-IM in the electrical events underlying inhibitory NMT in the IAS, we first characterized the basal electrical activity of the W/Wv mouse IAS. Em was 4 mV more negative in W/Wv than in WT mice (−53.2 ± 0.5 mV, n = 14 W/Wv mice vs−49.1 ± 0.5 mV, n = 17 WT mice, P < 0.05). In addition, peak depolarization during the slow wave was 7 mV greater (33.3 ± 0.9 mV, n = 20 for W/Wv mice vs 26.0 ± 1.4 mV, n = 28 for WT mice, P < 0.05), whereas slow wave frequency was not different (43.0 ± 2.4 cpm, n = 14 for W/Wv mice vs 38.2 ± 1.5 cpm, n = 17 for WT). Slow waves waxed and waned in W/Wv mice as they did in WT mice (Figure S2A,B) with rapidly rising spikes superimposed upon some slow waves (Figure S3A)
To examine the electrical events underlying NMT, IJPs were elicited with 5 Hz EFS for 10 s in the W/Wv mouse IAS (guanethidine and atropine present throughout) and compared with those of the WT mouse. Control IJPs in the W/Wv mouse were not significantly different from WT (Fig. 3A left traces and B) including the presence of rebound at the end of the stimulus train (Fig. 3Ab). In contrast, following MRS2500 addition (which did not depolarize Em in either WT or W/Wv mice), the remaining hyperpolarization was 50–60% smaller in W/Wv than in WT mice (Fig. 3A middle traces and B) suggesting that ICC-IM contribute to the generation of nitrergic IJPs. Nonetheless, slow waves were still greatly reduced or abolished with EFS in the W/Wv mouse IAS. Application of a single shock of EFS in the presence of MRS2500 gave rise to a significant nitrergic IJP in WT mice (4.5 ± 0.6 mV, n = 11), but not in W/Wv mice (n = 12, see Figure S3) further confirming a role for ICC-IM in generating nitrergic IJPs. Combined addition of MRS2500 with either L-NNA or the guanylate cyclase inhibitor ODQ (10 μmol L−1) eliminated all remaining electrical changes evoked with EFS in both animals (Fig. 3A right traces and B), confirming that the MRS2500-independent IJP is due to the nitrergic pathway, and that guanylate cyclase is required. L-NNA depolarized four of nine WT muscles (3.9 ± 0.8 mV, n = 4), whereas no W/Wv muscles depolarized (n = 8).
Comparison of basal contractile activity and nerve-evoked relaxations in the wildtype and W/Wv mouse IAS
As described for WT mice, spontaneous contractions developed in W/Wv mice over a 20–30 min time period following immersion in the tissue bath and consisted of both phasic and tonic components (Figure S2D). The fast phasic (53.9 ± 1.7 cpm, n = 7 W/Wvvs 56.7 ± 2.8 cpm, n = 14 in WT IAS) and slow phasic (3.3 ± 0.2 cpm, n = 7 W/Wv, 3.5 ± 0.2 cpm, n = 7 WT) contractile frequencies were not different between W/Wv and WT mice, nor were mean amplitudes of total spontaneous contractile activity (i.e., 24.5 ± 2.3%, n = 7 for W/Wvvs 24.5 ± 2.6%, n = 7 for WT mice, see Table 1). In addition, there were no significant differences in the various components contributing to overall spontaneous contractile activity (see Table 1).
To investigate whether nerve-evoked relaxations differ in the absence of ICC-IM, we repeated contractile measurements in the W/Wv mouse IAS. Relaxations elicited with 5 Hz EFS (60 s; guanethidine and atropine present throughout) were compared with those of WT mice. Under control conditions, nerve-evoked relaxations were not significantly different between animals either during the first (10 s) or second (10–60 s) time period of EFS (Fig. 4A, left traces and B). In addition, MRS2500 did not significantly reduce relaxation in either WT or W/Wv mice (Fig. 4A, middle traces and B), whereas rebound contractions were reduced in both. As noted for WT mice (Fig. 2B–D), combined addition of MRS2500 with either L-NNA or ODQ completely abolished relaxation in the W/Wv mouse IAS during the first 20–30 s of EFS, whereas some relaxation developed with longer periods, again indicating the presence of a third inhibitory neurotransmitter (Fig. 4A,B). Neither MRS2500 nor MRS2500 in combination with either L-NNA or ODQ increased spontaneous contractile activity in WT and W/Wv mice. Although not seen during the period of contraction shown in Fig. 4, contractile activity always returned to the control amplitude 3–5 min after termination of EFS in both WT and W/Wv mice.
Differences in nerve-evoked depolarizations in the wildtype and W/Wv mouse IAS
In previous studies, we have shown that excitatory motor innervation is sympathetic in the monkey, but not in the mouse IAS.23 For this study, we therefore examined whether cholinergic nerves serve as excitatory motor neurons in the mouse IAS and whether this pathway is modified in the W/Wv mouse IAS. Atropine was omitted from the initial bathing conditions, and nerves were repetitively stimulated (5 Hz EFS, 10 s; guanethidine present throughout). The composite IJP was not significantly different between WT and W/Wv mice (Fig. 5A, 1st traces and B) nor was rebound depolarization. In contrast, following addition of MRS2500, the remaining hyperpolarization elicited with EFS was significantly smaller in W/Wv than in WT mice (Fig. 5A, 2nd traces and B), again suggesting that ICC-IM are important for generation of the nitrergic IJP. As observed in the WT mouse, MRS2500 reduced or abolished rebound depolarization (Fig. 5Ab) The most significant difference noted between animals was the appearance of a slowly developing depolarization in WT mice with combined MRS2500 and L-NNA (Fig. 5Aa, 3rd trace and Ba). This depolarization was blocked by atropine, confirming its cholinergic nature (Fig. 5Aa, 4th trace and B). In contrast, slow depolarization was not observed in the W/Wv mouse IAS (Fig. 5Ab, 3rd trace and Bb), suggesting that ICC-IM are required for generation of the cholinergic depolarization.
Comparison of nerve-evoked contractions in the wildtype and W/Wv mouse IAS
The role of ICC-IM in cholinergic NMT was also examined by comparing contractile responses to EFS (5 Hz, 60 s) in WT and W/Wv mice in the absence of atropine (guanethidine present throughout). Relaxations elicited with EFS were not significantly different between animals (Fig. 6A, 1st traces and B) nor were they different from relaxations elicited in the presence of atropine. However, rebound contraction was at least twice as large in the absence of atropine (compare Figs 4A and 6A). Responses in WT and W/Wv mice differed in that MRS2500 significantly reduced nerve-evoked relaxation during both the initial 10 s and the subsequent 50 s of EFS in W/Wv mice (Fig. 6Ab, 2nd trace and Bb), but not in WT mice (Fig. 6Aa, 2nd trace and Ba) suggesting that the strength of nitrergic NMT was reduced in the W/Wv mouse. Following combined addition of MRS2500 and L-NNA EFS gave rise to a large contractile response in both WT and W/Wv mice that approximately doubled spontaneous contractile activity (Fig. 6A, 3rd traces and B). This contraction was abolished with atropine confirming its cholinergic origin (Fig. 6A, 4th traces and B). There was no significant difference in the nerve-evoked contraction between animals (Fig. 6B), suggesting that ICC-IM independent pathways play an important role in the generation of cholinergic motor responses in this muscle.
- Top of page
- Materials and Methods
- Supporting Information
The role of ICC-IM in NMT was investigated in the mouse IAS by comparing the contractile and electrical responses to stimulation of motor neurons in WT and W/Wv mice. Nitrergic and cholinergic junction potentials were reduced or abolished in the W/Wv mouse IAS, whereas nitrergic and cholinergic contractile effects were unchanged. In contrast, purinergic hyperpolarization and relaxation were unchanged in the W/Wv mouse IAS. These data suggest that ICC-IM participate in the generation of nitrergic and cholinergic junction potentials, but not in purinergic NMT. Other ICC-IM independent pathways also play an important role in nitrergic relaxation and cholinergic contraction.
Basic contractile and electrical properties of wildtype and W/Wv mice
Spontaneous contractile activity in the mouse IAS consisted of fast and slow phasic events superimposed upon tone. Interestingly, the pattern and amplitude of this activity was not significantly different between WT and W/Wv mice. However, in the W/Wv mouse, Em was 4 mV more negative, whereas slow waves (SW) were 7 mV larger in amplitude. It is possible that the greater polarization of Em gives rise to the larger SW. A similar relationship between Em and SW amplitude has previously been reported in other GI muscles (e.g., 24). The lack of difference in contractile activity in WT and W/Wv mice (see Table 1) may reflect a balance between the more polarized Em and the larger SW in the W/Wv mouse IAS.
The persistence of SW in the W/Wv mouse is significant because SW are usually attributed to ICC.25 Although ICC-IM are absent from the W/Wv mouse IAS, ICC-SM persist.2 Thus, it is possible that ICC-SM give rise to SW in the mouse IAS as previously reported for the mouse colon.26 The greater polarization of Em in the W/Wv mouse IAS suggests that ICC-IM play a role in setting ‘resting’ Em. ICC-IM in mouse GI muscles have been reported to express the Ca2+ activated Cl− channel ANO1. These channels give rise to depolarizing currents27,28, and we have confirmed using immunohistochemical techniques that ANO1 is expressed in ICC of the mouse IAS (Cobine and Keef, unpublished observation). Thus, Em may be more negative in the W/Wv mouse IAS because of loss of inward ANO1 currents generated by ICC-IM.
The frequency of SW was somewhat less than the frequency of phasic contractions in both WT and W/Wv mice. The most likely explanation for this difference is that electrical and contractile recordings sample different populations of cells, i.e., the microelectrode records Em from a single cell, whereas the contractile recording represents the summed activity of the entire cross section of the tissue. Spikes (which are especially important for the generation of phasic contractions) tend to be local events that do not conduct over large distances.29 In addition, the waxing and waning pattern observed in this muscle (see Figure S2) is commensurate, with SW being generated by multiple sites.30 It is also possible that subtle differences in the recording conditions used for contractile and microelectrode measurements contributed to the apparent differences between SW and phasic contractile frequency in these muscles.
Neuromuscular transmission in the wild type mouse IAS
The mouse IAS exhibits ongoing SW, rhythmic (‘phasic’) contractions, and tone that are all reduced or abolished by inhibitory motor neurons. Nitrergic and purinergic NMT each contribute to nerve-evoked relaxation and hyperpolarization, but the time course of junction potentials elicited by these two pathways differ. Repetitive EFS (5 Hz) gives rise to a biphasic response consisting of an initial peak hyperpolarization that is almost entirely purinergic in nature followed by rapid repolarization to a more sustained level of hyperpolarization. NOS blockade with L-NNA significantly reduced the sustained, but not the peak hyperpolarization, although the remaining IJP was still biphasic. The L-NNA-resistant IJP was entirely abolished by the P2Y1 blocker MRS2500 (see Fig. 1D) suggesting that purinergic NMT is comprised of two phases, i.e. a large rapidly desensitizing phase and a smaller more sustained phase. This observation is similar to the rat IAS15, whereas in non-rodents, the purinergic IJP is more transient.31
Using the more potent P2Y1 receptor antagonist MRS2500, we were able to isolate and characterize the nitrergic IJP. This was not possible in our previous study that used MRS2179 as purinergic NMT contaminated the nitrergic response.14 MRS2179 has since been shown to be a significantly less potent and efficacious inhibitor of P2Y1 receptors in rat colon22, and we have confirmed this difference in the mouse IAS (see Fig. 1B,C). MRS2500 (1 μmol L−1) has also been shown to be an effective antagonist of the purinergic IJP in the rat IAS.15 The nitrergic IJP reached peak hyperpolarization within 1 s of EFS at 5 Hz. It was smaller in amplitude than the purinergic IJP, and remained relatively constant during 10 s of EFS. Thus, the nitrergic pathway contributes to the sustained phase of hyperpolarization, whereas peak hyperpolarization is determined by the purinergic pathway. A similar conclusion has been reported for IJPs in the rat IAS.15
A single shock of EFS also generated transient relaxations in the IAS, whereas the relaxation with repetitive EFS (5 Hz) was sustained. Interestingly, the response to 5 Hz EFS was not reduced by selective blockade of either nitrergic or purinergic NMT. However, when both pathways were simultaneously blocked, relaxation was completely abolished for ∼20 s EFS (5 Hz) indicating that this component of the response is mediated by both pathways. The fact that each pathway independently produced the same amount of relaxation suggests that they are of similar ‘strength’ to one another. However, they clearly do not add together in a straightforward manner.
The redundant nature of relaxations elicited by the nitrergic and purinergic pathways may be due to their shared ability to hyperpolarize Em. Rhythmic contractions and tone in the mouse IAS are blocked by the L-type Ca2+ channel (Cav) blocker nifedipine14,23 and by the Katp channel opener pinacidil (Keef and Cobine, unpublished observation) suggesting that Cav and Em play a critical role in regulating contractile activity in the IAS. Cav is a voltage dependent conductance with a relatively discrete threshold potential for channel activation. At potentials negative to this value, almost all Cav channels are closed, whereas at potentials positive to this value, a steep relationship between Em and Cav activity exists. In GI muscles, this voltage-dependence of Cav in turn gives rise to a ‘mechanical threshold’ for electromechanical coupling with values between −55 and −40 mV reported for various GI muscles.32,33 In the mouse IAS Em averages −49 mV and both purinergic and nitrergic pathways hyperpolarize Em by at least 9 mV (see Fig. 1E), bringing Em to at least −58 mV. This potential is negative to the range of values listed above for mechanical threshold. Once Cav are closed, further hyperpolarization will not produce a further reduction in calcium entry. Thus, nitrergic and purinergic pathways may be ‘non-additive’ in nature, because each hyperpolarizes Em below the mechanical threshold.
Differences in interstitial cell populations in the W/Wv mouse IAS
In a previous study of the mouse IAS, we compared the distribution of ICC and ‘fibroblast-like cells’ (FLC) in the IAS of WT (C57BL/6) and W/Wv mice along with their relationship to nitrergic nerves.2 The term FLC has been proposed to denote common features shared between these cells in the GI tract and true fibroblasts.34,35 Recent studies have shown that FLC express high levels of the receptor tyrosine kinase PDGFRα+.2,36,37 As a result of this, we and others have referred to intramuscular FLC as PDGFRα+-IM.2,36,38 Several studies have suggested that PDGFRα+-IM may participate in inhibitory NMT in the GI tract.2,36,39,40 In the mouse IAS, we found that ICC-IM and PDGFRα+-IM were each distributed throughout the thickness of the muscle layer and formed close associations with nitrergic nerves and with each other.2 Thus, ICC-IM, PDGFRα+-IM, and smooth muscle cells (SMC) all represent viable candidates for participation in NMT in the mouse IAS. The present study compares the electrical and contractile events associated with NMT in WT and W/Wv mice to further explore the role of ICC-IM in NMT.
Changes in nitrergic neuromuscular transmission in the W/Wv mouse IAS
Previous studies have concluded that ICC are not involved in nitrergic NMT as nerve-evoked relaxations were not reduced in the W/Wv mouse IAS.12,13 Our data confirm this observation. However, given the non-additive nature of nitrergic and purinergic NMT in the mouse IAS, it is possible that a significant reduction in nitrergic NMT could occur without a change in the overall relaxation response. To further address this question, we selectively isolated the nitrergic IJP and relaxation in the W/Wv mouse IAS using MRS2500. Under these conditions, the IJP evoked with 5 Hz EFS was reduced by 50–60% in the W/Wv mouse (see Figs 3 and 5), whereas the response to a single shock of EFS was absent (see Figure S3B). In addition, L-NNA depolarized Em in 44% of WT mice, but did not depolarize any muscles in W/Wv mice; again implicating a role for ICC-IM in mediating NO effects. Finally, in the absence of atropine, we found that MRS2500 significantly reduced relaxation in W/Wv, but not WT mice, suggesting that the nitrergic pathway is less effective at opposing the actions of cholinergic nerves when ICC-IM are absent (see Fig. 6Ab,Bb).
Taken together, these data suggest that ICC-IM significantly contribute to the generation of nitrergic IJPs and possibly to relaxation. This conclusion is in agreement with some studies of other GI regions.3,4 However, studies of the W/Wv mouse lower esophageal sphincter (LES) have suggested that nitrergic NMT is compromised in this animal because of changes in calcium handling by the smooth muscle sarcoplasmic reticulum rather than to a loss of ICC-IM.41 The changes reported for NMT in the W/Wv LES differ significantly from those we observed for the W/Wv IAS including: (i) cholinergic depolarization persisted in the W/Wv LES, but not in the W/Wv IAS and (ii) nitrergic IJP amplitude varied widely between animals in the W/Wv LES whereas nitrergic IJPs were consistently reduced in the W/Wv IAS (see Fig. 3B). These substantial differences make it difficult to extrapolate between studies.
Besides hyperpolarizing Em, the nitrergic pathway was also associated with blockade of SW. Blockade was sometimes complete (e.g., Figs 1A,Cb and 5Aa) and sometimes partial (e.g., Fig. 3Aa). Interestingly, this effect persisted in the W/Wv mouse IAS (e.g., Fig. 3Ab). SW and contraction in the mouse IAS are also blocked by Cav antagonists14,23 indicating that SW are an important source of calcium for contraction (see review25 for related discussion). The continued blockade of SW during nitrergic NMT in the W/Wv mouse IAS therefore provides a plausible explanation for why most nitrergic relaxation persists.
In summary, these data suggest that ICC-IM play a significant role in the generation of nitrergic IJPs in the mouse IAS (see Fig. 7). However, approximately 40–50% of the nitrergic IJP elicited with 5 Hz EFS still remains in the W/Wv mouse and SW are still blocked. Taken together, our results suggest that other cell types also contribute to generation of the electrical events underlying nitrergic NMT in the IAS, particularly when trains of stimuli are applied. Both SMC and PDGFRα+-IM represent viable candidates for this role (Fig. 7). Finally, pharmacomechanical coupling mechanisms such as a decrease in myofilament sensitivity may also contribute to relaxation.
Purinergic neuromuscular transmission is not different in the W/Wv mouse IAS
The peak hyperpolarization elicited with 5 Hz EFS was not different in WT and W/Wv mice, and this component was abolished by MRS2500 (see Fig. 3B) suggesting that ICC-IM are not required for generation of the purinergic IJP (Fig. 7). This conclusion is in agreement with several studies of other GI muscles.39,41,42 In addition, ‘resting’ Em in both WT and W/Wv mice was unaffected by addition of the P2Y1 antagonist MRS2500, suggesting that ‘resting’ Em is not modulated by basal purine release. A similar conclusion was reached for the rat IAS.15 An alternative cellular candidate for purinergic NMT in the IAS is PDGFRα+-IM. This cell has a number of features supporting a role in purinergic NMT including: (i) association with intramuscular nerves,2,39,43 (ii) the presence of gap junctions with SMC,35,39,44,45 (iii) the expression of two proteins critical for purinergic NMT, i.e., small conductance calcium activated potassium channels, SK3 (Kcnn3), and P2Y1 (P2ry1) receptors36,45–48, and (iv) the ability to generate large Ca2+-activated, apamin-sensitive K+ currents in response to purines, whereas SMC in the same preparation do not.36 Thus, we propose PDGFRα+-IM as possible mediators of purinergic NMT in the mouse IAS. SMC have been reported to express P2Y1 receptors15, making these cells candidates as well (Fig. 7).
Changes in cholinergic neuromuscular transmission in the W/Wv mouse IAS
The neural pathways involved in excitatory NMT in the IAS are species dependent. In dog, cat, sheep, monkey, and humans, extrinsic sympathetic nerves play the predominant role as excitatory motor neurons, whereas in smaller mammals such as rabbit and mouse, this is not the case (e.g.,23,49). We have now characterized excitatory NMT in the mouse IAS. EFS (5 Hz) gave rise to contraction and a small (i.e., ∼5 mV), slowly developing depolarization in the presence of guanethidine, MRS2500, and L-NNA. As both depolarization and contraction were abolished by atropine, they are due to cholinergic excitatory motor neurons. In contrast, depolarization was absent from the W/Wv mouse IAS. Thus, ICC-IM appear to be important for generation of cholinergic depolarization in this muscle (Fig. 7) as previously described in several other studies.4,5 However, although depolarization was absent in the W/Wv mouse IAS, cholinergic contraction at 5 Hz EFS remained unchanged. This result does not preclude a role for depolarization in the generation of cholinergic contraction, but it does suggest that other Em-independent pathways such as calcium release from the sarcoplasmic reticulum and an increase in myofilament sensitivity to calcium (for reviews see25,50) must also play important roles in cholinergic contraction.
Purinergic and nitrergic independent relaxation is not different in the W/Wv mouse IAS
Contractile and electrical responses were abolished following blockade of purinergic, nitrergic, and cholinergic pathways during 10 s of EFS@5 Hz. However, relaxation started to develop in these muscles after 20–30 s of EFS and by 60 s, tone had usually declined to near baseline. Interestingly, this slowly developing relaxation also reversed slowly following termination of EFS, taking several minutes to return to the prestimulus level. This relaxation was abolished by TTX, indicating its neural origin. The nature of this slower component was not further investigated in this study, but we have obtained preliminary evidence that it is due to VIP as it is abolished by VIP6-28 (10 μmol L−1).51 It is of interest that, like purinergic NMT, the slow relaxation remained unchanged in the W/Wv mouse IAS, suggesting that this pathway is also independent of ICC-IM. Although non-cholinergic excitatory NMT was not observed in the mouse IAS, it is possible that it was masked by purinergic and nitrergic independent inhibitory NMT.
In summary, we have re-examined the hypothesis that ICC-IM are not required for nitrergic NMT in the mouse IAS.12,13 Our results provide evidence that ICC-IM do indeed contribute to generation of nitrergic IJPs, but that other ICC-IM independent pathways are also important. In addition, we could find no role for ICC-IM in either purinergic NMT or in the slowly developing relaxation that accompanies longer duration EFS when adrenergic, cholinergic, purinergic, and nitrergic pathways are blocked. Given these significant redundancies in neural inhibitory pathways, it is not surprising that some investigators have reported an intact RAIR in the W/Wv mouse.12 Our results also suggest that ICC-IM are required for generation of cholinergic depolarization, but that other ICC-IM independent pathways also play an important role in cholinergic contraction. As PDGFRα+-IM persist in the W/Wv mouse IAS, both PDGFRα+-IM and SMC are viable cellular candidates for aspects of NMT that persist in the absence of ICC-IM.
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This work was supported by grant DK078736 to KDK. The authors thank Dr Kenton Sanders for critical evaluation of the manuscript. AMD performed most intracellular microelectrode recordings and some data analysis; CAC performed and analyzed all contractile experiments and analyzed most microelectrode recordings. She also contributed to the writing of this paper; KDK was the principal investigator of this study including intellectual development, data analysis, and writing the paper. She also participated in collecting some of the contractile and intracellular data.
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- 17Comparison of the morphological and functional properties of the internal anal sphincter in wild type mice (C57BL/6) and mice containing the reduced function Kit allele (Wv). Neurogastroenterol Motil 2010; 22(Suppl. 1): 86., , , , , .
- 26Pacemaker activity from submucosal interstitial cells of Cajal drives high-frequency and low-amplitude circular muscle contractions in the mouse proximal colon. Neurogastroenterol Motil 2004; 16: 621–7., , .
- 51Peptidergic inhibitory neuromuscular transmission in the mouse internal anal sphincter. Neurogastroenterol Motil 2011; 23(Suppl. 1): 49., , , , .
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Figure S1. Calculation of spontaneous contractile activity in the mouse IAS. Spontaneous contractile activity was composed of tone and phasic contractions. Spontaneous contractions were divided into six components, each depicted with an arrow and an associated number. These included: peak phasic contraction (1), trough phasic contraction (2), slow phasic contractions (3), tone (4), total phasic contraction (5) and total spontaneous contraction (6). Mean values (±SE) for each component in grams of contraction and percentage of the maximum contraction elicited with 60 mmol L−1 KCl are shown in the table below (see also Table 1 in results section). There were no significant differences in spontaneous contractile activity between WT (n = 7) and W/Wv (n = 7) mice.
Figure S2. Spontaneous electrical and contractile patterns in the WT and W/Wv mouse IAS. Similar fast and slow patterns of electrical and contractile activity were observed in WT and W/Wv mice. Shown are examples of two different electrical patterns in WT (A) and W/Wv (B) mice. Slow wave activity was present in all traces, but in the left traces (a) slow wave amplitude was organized into a ‘waxing and waning’ pattern whereas in the right traces (b) slow waves periodically gave rise to spikes. Examples of two different contractile patterns are also shown for WT (C) and W/Wv (D) mice. The level of zero active contraction (determined with 10 μmol L−1 SNP and 1 μmol L−1 nifedipine) is indicated with a dotted line. All muscles exhibited tone and rapid and slower frequency phasic contractions. The slower rhythm was usually organized into discrete oscillations occurring at approximately 2–4 cpm (Ca, Da) whereas in other cases it was less consistent.
Figure S3. Comparison of spontaneous and evoked electrical events in the WT and W/Wv mouse IAS. (A) Superimposed traces from a cell exhibiting a waxing and waning pattern in either the WT (left) or the W/Wv (right) mouse IAS. Smaller amplitude slow waves (black and red) rose slowly to peak, whereas larger slow waves gave rise to a spike (green and blue). The maximum dV/dt (mV/s) for each event is indicated in the corresponding color. (B) Comparison of inhibitory junction potentials (IJPs) elicited with a single shot (ss) of electrical field stimulation (EFS) in the WT (top) and W/Wv (bottom) mouse IAS. IJPs were present in both muscles in the absence of MRS2500 (left). However, following MRS2500 addition, a 7 mV slow IJP was observed in the WT (top, right) but not the W/Wv (bottom, right) mouse IAS. Maximum dV/dts for the numbered depolarizations are: 1: 596 mV/s, 2: 311 mV/s, 3: 473 mV/s, 4: 449 mV/s, 5: 25 mV/s, 6: 260 mV/s, 7: 31 mV/s, 8: 48 mV/s.
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