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Keywords:

  • immunohistochemistry;
  • inhibitory junction potential;
  • nitric oxide;
  • P2Y1 receptors

Abstract

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

Background  The neurotransmitters mediating inhibitory pathways to internal anal sphincter (IAS) have not been fully characterized. Our aim was to assess the putative release of nitric oxide, purines and vasoactive intestinal peptide (VIP) from inhibitory motor neurons (MNs) and their role in the myogenic tone, resting membrane potential (RMP) of smooth muscle cells (SMC), spontaneous inhibitory junction potentials (sIJP), mechanical relaxation, and IJP induced by electrical field stimulation (EFS) or nicotine.

Methods  Rat IAS strips were studied using organ baths, microelectrodes, and immunohistochemistry.

Key Results  Internal anal sphincter strips developed active myogenic tone (0.31 g), enhanced and stabilized by prostaglandin F (PGF2α). l-NNA (1 mmol L−1) depolarized SMC and increased tone but did not modify sIJP. In contrast, the specific P2Y1 receptor antagonist MRS2500 (1 μmol L−1) did not modify the RMP or the basal tone but abolished sIJP. Electrical field stimulation and nicotine (10 μmol L−1) caused IAS relaxation (−45.9%VS−52.2%), partially antagonized by l-NNA (35%–45%,P ≤ 0.05) and fully abolished by MRS2500 (P ≤ 0.001). Electrical field stimulation induced a biphasic inhibitory junction potential (IJP), the initial fast component was selectively blocked by MRS2500 and the sustained slow component was blocked by l-NNA. Vasoactive intestinal peptide 6–28 (0.1 μmol L−1) or α-chymotrypsin (10 U mL−1) did not modify the RMP, sIJP, EFS-induced IJP, or relaxation. P2Y1 receptors were immunolocalized in the circular SMC of IAS.

Conclusions & Inferences  The effects of inhibitory MNs on rat IAS are mediated by a functional co-transmission process involving nitrergic and purinergic pathways through P2Y1 receptors with specific and complementary roles on the control of tone, sIJP, and hyperpolarization and relaxation of IAS following stimulation of inhibitory MNs.


Abbreviations:
EFS

electrical field stimulation

IAS

internal anal sphincter

IJP

inhibitory junction potential

IJP-f

fast component of the inhibitory junction potential

IJP-s

slow component of the inhibitory junction potential

MNs

motor neurons

RMP

resting membrane potential

sIJP

spontaneous inhibitory junction potentials

SD-sIJP

standard deviation of spontaneous inhibitory junction potentials

Introduction

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

The rectoanal inhibitory reflex is initiated by stimulation of rectal mechanoreceptors by rectal distension and descendent stimulation of intrinsic inhibitory motor neurons (MNs) in the internal anal sphincter (IAS).1In vitro studies on humans2,3 and pigs4,5 mainly attribute IAS relaxation to nitric oxide (NO) release. However, in species such as the rabbit,6 rat,7 guinea pig,8 and opossum9 other mediators such as ATP, vasoactive intestinal peptide (VIP), and/or CO are complementary inhibitory neurotransmitters which contribute to IAS relaxation. In addition, study of the biosynthetic enzymes for CO and NO in knockout mice shows that the physiological effects of VIP in the IAS are mediated by CO.10 Vasoactive intestinal peptide and NO synthase are co-localized in inhibitory neurons in the opossum IAS.11 We recently found that porcine IAS relaxation following stimulation of inhibitory MNs is mediated by neurotransmission of NO and ATP acting on P2Y1 receptor.5 However, the specific physiological role of each of these neurotransmitters on the inhibitory pathways to the IAS is not settled.

Hyperpolarization and inhibitory junction potentials (IJP) are the electrophysiological basis of mechanical relaxation. Spontaneous inhibitory junction potentials (sIJP) are small-amplitude, brief hyperpolarization of the smooth muscle cell (SMC) membrane which occur as a consequence of tonic release of inhibitory neurotransmitters.12 Evoked IJP by electrical field stimulation (EFS) have a fast followed by a slow component. Nitric oxide contributes to the sustained or slow phase of the transient hyperpolarization (usually called IJP-s) but not to the fast component (IJP-f).13 The study of ATP or a related purine as a putative inhibitory mediator has been difficult due to the lack of a proper pharmacological approach. Usually non-selective antag-onists such as suramine and/or pyridoxal-phosphate-6-azophenyl-2′, 4′-disulfonate (PPADS) have been used to antagonize the non-nitrergic component of the relaxation.8,14,15 Alternatively, apamin – sK(Ca) channel blocker – has been widely used as a tool to distinguish between the nitrergic and non-nitrergic inhibitory responses.7,8,16 However, sK(Ca) channels might be activated downstream by several neurotransmitters and can be expressed in neurons, SMC, and interstitial cells of Cajal.17 In addition, purines may contribute to inhibitory responses through both apamin-sensitive and insensitive pathways.18,19 MRS2179 is a P2Y1 receptor antagonist that we have used in the colon13,20,21 and small intestine.22 MRS2179 has also been used to characterize the contribution of P2Y1 receptors in IAS relaxation in mice14, sheep,23 and our previous studies on pigs.5 In these studies, high concentrations of up to 10 μmol L−1 are needed to observe mild effects on the IJP-f and relaxation suggesting other P2Y receptors might also participate. We have recently found that the new P2Y1 receptor antagonist MRS2500 has a greater potency and abolished the IJP-f at 1 μmol L−1 in the rat colon while MRS2179 only caused a 20% reduction.24

We have now used this new pharmacological tool to characterize the specific roles for NO and purines on the steady-state electrical and mechanical properties, on the electrical and mechanical responses following stimulation of inhibitory MNs of rat IAS and on the relationships between these physiological events.

Materials and methods

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

All experiments were approved by the Ethics Committee of the Universitat Autònoma de Barcelona. All animals were treated according the guidelines for the Care and Use of Laboratory Animals of this university.

Animals and tissue preparation

Male Sprague–Dawley rats (300–350 g) were kept at a constant temperature (19–21 °C) and humidity (60%), with a lighting cycle of 12 h light/12 h dark and given ad libitum access to water and food. Rats were killed by decapitation and bled. The rectoanal region was dissected, quickly removed, placed in carbogenated Krebs solution, opened along the longitudinal axis and pinned to a Sylgard base with the mucosa facing upward. The mucosal and submucosal layers were removed by sharp dissection. Circular smooth muscle strips from the IAS were obtained from the lower part of the anal canal at the level of the pectinate line as previously described.25

Morphological studies

For light microscopy and immunohistochemistry studies, the tissue was fixed in paraformaldehyde 4% in phosphat-buffered saline (pH 7.3), dehydrated, and embedded in paraffin. We used hematoxylin–eosin (H&E) stain and S100 (Polyclonal Rabbit Anti-Cow S-100; Dako, Glostrup, Denmark) performed with Dako Autostainer as previously described.5 For anti-HuD, immunohistochemistry tissue samples were fixed with 4% paraformaldehyde in 0.2 mol L−1 phosphate buffer. Paraffin sections were mounted on glass slides and kept in a cold place until processed. Slides were deparaffinized and rehydrated, then pretreated with NH4Cl 50 mmol L−1 pH 8.0 to reduce autofluorescence. Afterward, a standard blocking was performed with Triton X-100 0.1% + Tween 20 0.2% and goat serum 10% for 30 min. The incubation with the primary antibody H-300 (rabbit polyclonal antibody; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) 1 : 100 was performed overnight at 4 °C. After rinsing, the sections were incubated with the secondary antibody (Alexa Fluor 568 goat anti-rabbit, Invitrogen Ltd, Paisley, UK) at 1 : 200 for 1 h at room temperature. For P2Y1 immunohistochemistry we used primary antibody Anti P2Y1 (Alomone Labs Ltd, Jerusalem, Israel) 1 : 50 and performed as previously described.13

Mechanical studies in organ baths

Circular smooth muscle strips (7 mm long and 3 mm wide; mean weight 0.027 ± 0.002 g) from 28 specimens were placed between two parallel platinum wire electrodes, attached to force transducers and transferred to 10 mL organ baths containing Krebs solution (pH 7.4, 37 °C) bubbled with 5% CO2 in O2. Strips were stretched up to 2 g and equilibrated up to 90 min. To maintain a stable and enhanced IAS tone, prostaglandin F (PGF2α) at 1 μmol L−1 was added to the bath.14 Changes in tension of the strips were measured using isometric force transducers (Model 03 Force Transducer and Model 7 Series Polygraph, respectively; Grass Instruments Co., Quincy, MA, USA), and recorded on a computer using the data acquisition software AcqKnowledge® (MP100; Biopac Systems, Inc., Goleta, CA, USA). Electrical field stimulation pulses of 0.5 ms duration, frequency 0.5–20 Hz, 4 s trains at 10 V26 were delivered by an electrical stimulator Model S88 (Grass Instruments Co.) and a power booster (Stimu-Splitter II, Med-Lab Instruments, Loveland, CO, USA) in order to obtain six identical and undistorted stimuli. These electrical stimuli were simultaneously recorded with tension tracings through a synchronized transistor–transistor logic signal between the electrical stimulator Grass S88 and the computerized Biopac System in order to assess whether the mechanical responses occurred during (‘on’) or after (‘off’) the electrical stimulus.21

Intracellular microelectrode recording

Muscle strips were pinned to the base of a Sylgard coated chamber, submucosa side up, and continuously perfused with carbogenated Krebs solution at 37 ± 1 °C and equilibrated 1 h before recording. Circular SMC were impaled with glass microelectrodes filled with 3 m KCl (30–60 MΩ of resistance). Membrane potential was measured using standard electrometer Duo773 (WPI Inc., Sarasota, FL, USA). Tracings were displayed on an oscilloscope 4026 (Racal-Dana Ltd, Windsor, UK) and simultaneously digitalized (100 Hz) using PowerLab 4/30 system and Chart 5 software for Windows (all from ADInstruments, Castle Hill, NSW, Australia). Electrical stimulation (100 ms of train duration, 20 Hz, 0.3 ms pulse and increasing amplitude voltage of 5–50 V)22 was applied using two silver chloride plates placed perpendicular to the longitudinal axis of the preparation and 1.5 cm apart. Nifedipine (1 μmol L−1) was used to abolish the spontaneous contractions during electrophysiological experiments and obtain stable impalements.

Solutions and drugs

The composition of the Krebs solution was (in mmol L−1) glucose, 10.10; NaCl, 115.48; NaHCO3, 21.90; KCl, 4.61; NaH2PO4, 1.14; CaCl2, 2.50; and MgSO4, 1.16 (pH 7.3–7.4). The solution was bubbled with carbogen, 95% O2, and 5% CO2. The following drugs were used: atropine from Merck (Darmstadt, Germany), nifedipine, (PGF2α), Nω-nitro-l-arginine (l-NNA), VIP, VIP 6–28, α-chymotrypsin (α-CMT) and nicotine from Sigma–Aldrich Co (St. Louis, MO, USA). Tetrodotoxin (TTX) and (1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester diammonium salt (MRS2500) from Tocris (Bristol, UK). Stock solutions were made by dissolving drugs in distilled water except for nifedipine which was dissolved in 96% ethanol, and l-NNA which was dissolved in Krebs solution by sonication.

Experimental design

Morphological studies aimed to assess the nature and relationship of SMC of the rat IAS, and locate the enteric MNs and the purinergic P2Y1 receptors. Physiological studies: In organ bath studies we assessed the mechanical responses following stimulation of MNs by EFS or through nicotinic acetylcholine receptors (nAChRs) by nicotine (10 μmol L−1).5 A frequency-related curve on the effect of EFS on IAS strips was drawn in order to explore the responses (relaxation/contraction) induced by electrical stimulation of MNs. Electrical field stimulation responses were characterized by the neurotoxin TTX (1 μmol L−1), and antagonists of inhibitory neurotransmitters l-NNA (1 mmol L−1), MRS2500 (1 μmol L−1), VIP 6-28 (0.1 μmol L−1) and α-CMT (10 U mL−1). Excitatory responses were characterized by atropine (1 μmol L−1). Finally, nicotine responses were also characterized by l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1). In electrophysiological studies, the amplitude and the duration of EFS-induced IJP with single electrical stimuli were measured under control conditions and after infusion of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1), and in separate protocol, VIP 6-28 (0.1 μmol L−1) and α-CMT (10 U mL−1). In addition, longer electrical stimuli with pulses of 5 s duration, 50 V and 0.3 ms were also performed at 1 Hz (5 pulses) and 5 Hz (25 pulses) as previously described.20 The fast component was assessed by measuring the maximum amplitude of the IJP during the initial pulses and the amplitude of the slow component was assessed at 2.5 and 3.75 s after the beginning of the stimulus in control conditions and after l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1), and in separate protocol, VIP 6-28 (0.1 μmol L−1) and α-CMT (10 U mL−1). Finally, we also characterized the hyperpolarization induced by stimulation of inhibitory MNs by nicotine (10 μmol L−1) with the sequential addition of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1). The frequency of spontaneous IJP (sIJP) was also assessed by: (i) measuring the frequency distribution (0.5 mV bins) of the values of the membrane potential (30–60 s) as described by our group,27 and (ii) measuring the SD-sIJP (mV) as previously described.28 Resting membrane potential was considered the value with a highest probability of the frequency distribution.27

Data analysis and statistics

Basal tone was determined by averaging the tone of the strips during the last 5 min of the equilibration period, before the strips developed active myogenic tone. Active myogenic tone was defined as the tone that the strips developed spontaneously. Total tone also included the tone induced by the drugs used in the study. Relaxation of strips was expressed as a percentage of total tone while contraction was expressed in grams. Student’s t-test or one-way anova were selected for comparisons using the paired model when appropriate, and the effect of pharmacological agents on frequency–response curves was determined using two-way anova. For microeletrode experiments, differences in the RMP before and after infusion of different drugs were compared by one-way anova followed by the Bonferroni post hoc test. The differences between the amplitude and duration of the IJP before and after drug infusion were compared by two-way anova (drug and voltage), using GraphPad prism 4 (Version 4.01; GraphPad Software, San Diego, CA, USA). Number of animals was represented by n. Data were expressed as mean ± SEM. A P value <0.05 was considered statistically significant and non-significant.

Results

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

Histological studies

Hematoxylin–eosin stains showed two layers of SMC in the IAS: the circular layer, divided into discrete circular muscle bundles separated by connective tissue septa, and the longitudinal muscle layer (LM), an external and continuous thin layer following the LM of the rectum (Fig. 1A). The circular and LM layers are attached and the striated external anal sphincter is in direct contact with the external border of the LM (n = 6). Neural S100 and anti-HuD staining identified glial cells (Fig. 1C) and enteric MNs (Fig. 1D) in the ganglia of the myenteric plexus between LM and IAS fibers (n = 6). Positive P2Y1 receptor immunoreactivity was found in the SMC of circular and LM layers; a minor staining was also observed in the myenteric plexus (n = 4) (Fig. 1B).

image

Figure 1.  Morphological aspects of the internal anal sphincter in the rat. (A) Hematoxylin–eosin stain identifies the myenteric plexus between the circular and longitudinal smooth muscle layers (scale bar 50 μm). (B) Immunohistochemical localization of P2Y1 receptors (brown staining) in circular and longitudinal smooth muscle cells (scale bar 10 μm). (C) Glial cells with small nuclei and positive to S100 staining in the myenteric plexus (scale bar 30 μm), and (D) Positive anti-HuD neurons in a motor ganglion of the myenteric plexus (scale bar 30 μm). IAS, internal anal sphincter; LM, longitudinal muscle; MP, myenteric plexus; Gn, myenteric ganglion; PL pectinate line; SM, striated muscle.

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Physiological studies

Organ bath mechanical studies

Internal anal sphincter tone. After equilibration period, IAS strips developed an unstable active myogenic tone (0.31 ± 0.05 g, n = 20) and phasic contractile activity with a mean amplitude of 0.092 ± 0.014 g and frequency of 51.20 ± 1.6 contractions min−1. Prostaglandin F (1 μmol L−1) induced an intense and stable tonic contraction and significantly increased the amplitude of phasic activity (n = 14) (Table 1). The NOS inhibitor l-NNA (1 mmol L−1) strongly increased IAS tone and the amplitude of phasic activity (n = 8); in contrast, the P2Y1 receptor antagonist MRS2500 (1 μmol L−1) did not modify IAS tone (n = 10) or the phasic activity (Table 1). Following l-NNA or MRS2500, atropine did not significantly affect IAS tone (−2.41 ± 1.74%, n = 14, ns). The free calcium Krebs solution reduced IAS tone by −83.34 ± 3.4% (n = 10, < 0.001). In contrast, the neurotoxin TTX did not affect IAS tone nor spontaneous contractions (n = 3, data not shown).

Table 1.   Pharmacological characterization of nitrergic and purinergic control of IAS tone, phasic contractions, resting membrane potential and spontaneous inhibitory junction potentials
 Mechanical studiesElectrophysiological studies
Tone (g)Phasic contractionsRMP (mV)SD-sIJP (mV)
Amplitude (g)Frequency (contraction min−1)
  1. Values are mean ± SEM. IAS, internal anal sphincter; RMP, resting membrane potential; SD-sIJP, standard deviation of the membrane potential reflecting the presence of spontaneous IJP. Control values in mechanical experiments are obtained on addition of PGF2α. Paired Student’s t-test was used to assess the statistical significance of differences. *P < 0.05; **P < 0.01; ***P < 0.001; ns, non-significant.

Protocol 1
 CONTROL0.86 ± 0.160.14 ± 0.0651.00 ± 1.7−51.11 ± 2.150.33 ± 0.05
 +l-NNA (1 mmol L−1)1.67 ± 0.18***0.18 ± 0.02***52.02 ± 1.6ns−44.90 ± 2.53***0.34 ± 0.05ns
 +MRS 2500 (1 μmol L−1)1.24 ± 0.17ns0.17 ± 0.01ns52.50 ± 1.4ns−44.86 ± 2.46ns0.11 ± 0.03**
Protocol 2
 CONTROL0.99 ± 0.110.10 ± 0.0150.13 ± 1.4−50.94 ± 3.060.30 ± 0.05
 +MRS 2500 (1 μmol L−1)1.06 ± 0.1ns0.09 ± 0.01ns49.90 ± 1.3ns−51.1 ± 3.04ns0.02 ± 0.02*
 +l-NNA (1 mmol L−1)1.26 ± 0.11***0.15 ± 0.01***50.16 ± 1.5ns−45.04 ± 2.49**0.02 ± 0.01ns

Mechanical responses induced by electrical stimulation of enteric motor neurons. All IAS strips responded to EFS with a frequency dependent ‘on’ relaxation during electrical stimulus followed by an ‘off’ contraction at the end of the stimulus. Maximal ‘on’ relaxation was observed at 3 Hz (−45.90 ± 6.03% of total tone) and amplitude of ‘off’ contraction was maximal at 20 Hz (1.37 ± 0.17 g) (Fig. 2). Both EFS-‘on’ and ‘off’ responses were abolished by TTX (1 μmol L−1) (data not shown). The NOS inhibitor l-NNA (1 mmol L−1) significantly reduced EFS-‘on’ relaxation at 3 and 5 Hz by −28.93 ± 4.45% (< 0.05) and −38.96 ± 3.75% (<0.01), respectively (Fig. 2). Electrical field stimulation-‘off’ contraction was significantly enhanced by l-NNA at 10 and 20 Hz by 2.24 ± 0.47 and 3.21 ± 0.89 g, respectively (< 0.05). Sequential addition of the P2Y1 receptor antagonist MRS2500 (1 μmol L−1) fully blocked the residual non-nitrergic relaxation at all frequencies (< 0.001), and shifted the contractile ‘off’ response to an ‘on’ contraction during EFS. Atropine (1 μmol L−1) almost abolished EFS-‘on’ contraction by −91.63 ± 0.1% (n = 5, < 0.05) showing the major role of cholinergic neurons in excitatory neurotransmission in the rat IAS (Fig. 2B). In separate experiments, initial incubation of MRS2500 (1 μmol L−1) did not affect EFS-‘on’ relaxation at any frequency tested, and in contrast, significantly reduced the electrical ‘off’ contraction at 10 and 20 Hz by −55.56 ± 0.08% (< 0.05). Sequential addition of l-NNA (1 mmol L−1) completely blocked the non-purinergic relaxation at all frequencies (< 0.01), and shifted the EFS-‘off’ contraction to an ‘on’ contraction enhancing their amplitude by 1.84 ± 0.3 g (at 20 Hz, < 0.05). Finally, atropine (1 μmol L−1) almost blocked EFS-‘on’ contraction by −83.24 ± 0.1% (n = 7, < 0.01) (Fig. 2C).

image

Figure 2.  Pharmacologic characterization of neurotransmitters released during stimulation of internal anal sphincter enteric motor neurons by electrical field stimulation (EFS) (0.5–20 Hz) in mechanical study. (A) Representative tracing showing the response of rat internal anal sphincter strips pre-incubated with PGF2α (1 μmol L−1) to EFS (n = 11). And in both protocols simultaneous addition of (B) l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) or (C) MRS2500 (1 μmol L−1) and l-NNA (1 mmol L−1) was required to fully block EFS relaxation (n = 5 for B and n = 7 for C). Horizontal axis depicts the time schedule of the experiment during sequential addition of antagonists; drugs were incubated for 15 min before the following EFS. Frequencies are indicated below the tracing. White dots represent ‘on’ responses and black dots, ‘off’ responses. All values are expressed as mean ± SEM. * 0.05; ** 0.01; *** 0.001; ns, non-significant.

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Mechanical responses induced by stimulation of enteric motor neurons through nAChRs. In two independent sets of experiments on the same strips, we compared the mechanical responses induced by stimulation of MNs though nAChRs by nicotine with those caused by 3 Hz. Stimulation of enteric MNs by nicotine (10 μmol L−1) induced a sustained IAS relaxation of −52.15 ± 5.07% of total tone (n = 11) with 17.5 s to Emax (Figs 3 and 4). The NOS inhibitor l-NNA (1 mmol L−1) significantly reduced nicotine relaxation by −34.95 ± 3.07% (n = 4, < 0.05) and the sequential addition of MRS2500 (1 μmol L−1) completely blocked non-nitrergic relaxation, inducing a contraction of 0.8 ± 0.11 g (< 0.01) (Figs 3A and 4A). In contrast, initial addition of MRS2500 did not significantly affect nicotine relaxation while sequential addition of l-NNA fully blocked non-purinergic relaxation (< 0.01), inducing a contraction of 0.71 ± 0.2 g (< 0.01) (Figs 3B and 4B). This contraction was fully abolished by atropine (n = 8, < 0.05). Stimulation of enteric MNs by 3 Hz electrical stimuli caused IAS relaxation of −43.81 ± 4.17% of total tone followed by an ‘off’ contraction of 0.87 ± 0.08 g. l-NNA (1 mmol L−1) significantly reduced the EFS ‘on’ relaxation by −41.68 ± 6.8% (< 0.001) and significantly enhanced ‘off’ contraction by 0.44 ± 0.10 g (< 0.05); the sequential addition of MRS2500 (1 μmol L−1) fully abolished the residual non-nitrergic relaxation (< 0.001) and slightly reduced electrical contraction (n = 4, ns) (Fig. 4A). In contrast, when the initial antagonist was MRS2500, the amplitude of EFS-‘on’ relaxation and ‘off’-contraction was not affected; and further addition of l-NNA fully blocked non-purinergic residual relaxation (n = 4, < 0.001) (Fig. 4B). The morphology of EFS relaxation (sharp and sustained ‘on’ relaxation and sharp ‘off’ contraction) was not modified by either l-NNA or MRS2500 (Fig. 3).

image

Figure 3.  Representative mechanical tracings showing the responses induced by stimulation of enteric motor neurons by an isolated 3 Hz electrical stimuli or nicotine (10 μmol L−1) and pharmacological characterization with the sequential addition of (A) l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) or (B) MRS2500 (1 μmol L−1) and l-NNA (1 mmol L−1). Antagonists were incubated for 15 min. After the maximal relaxation induced by nicotine, strips were repeatedly washed.

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image

Figure 4.  Paired experiments comparing the mechanical relaxation induced by stimulation of inhibitory enteric motor neurons through nAChRs by nicotine (10 μmol L−1) and by maximal electrical stimulation by 3 Hz. Pharmacological characterizations of inhibitory responses by (A) l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) or (B) MRS2500 (1 μmol L−1) and l-NNA (1 mmol L−1) in independent protocols. Note that, following incubation of l-NNA and MRS2500, both nicotine and EFS induced a weak IAS contraction. Nicotine is represented by strip bar and 3 Hz EFS-‘on’ response by white bar while EFS-‘off’ response is represented by black bar. Drugs were added to the bath in sequential addition and incubated for 15 min, after nicotine produced maximal relaxation; strips were repeatedly washed, and allowed to equilibrate before the following protocol. All values are expressed as mean ± SEM. & 0.05; && 0.01; * 0.05; *** 0.001; ns, non-significant.

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Electrophysiological studies

Resting membrane potential and spontaneous inhibitory junction potentials. Circular SMC had a mean RMP of −51.05 ± 1.6 mV (n = 9) in control conditions. Spontaneous inhibitory junction potential were recorded in eight out of nine preparations, with variable amplitude (from less than 1 mV to 10 mV) and leading to a standard deviation of the RMP reflecting the sIJP (SD-sIJP) (n = 8) (Table 1). l-NNA (1 mmol L−1) depolarized circular SMC (n = 6) but did not modify sIJP. In contrast, the selective P2Y1 antagonist MRS2500 (1 μmol L−1) did not modify the RMP (n = 6) but inhibited sIJP (Table 1 and Fig. 5). Notice that l-NNA caused a shift to the right of the curve representing the bin distribution of the RMP, indicating a strong depolarization without modifying the frequency distribution of the spontaneous IJP (Fig. 5D). In contrast, MRS2500 reduced the frequency of distribution of the membrane potential indicating inhibition of sIJP without greatly modifying the RMP (Table 1 and Fig. 5D). Taken together, these data demonstrate that the RMP is mainly NO regulated whereas sIJP have a purinergic origin.

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Figure 5.  Representative mechanical recording of the (A) IAS tone and (B) intracellular microelectrode recording showing the effect of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1). (C) Representative microelectrode recordings showing the spontaneous IJP and the effect of the sequential addition of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) left and MRS2500 (1 μmol L−1) and l-NNA (1 mmol L−1) right on them. (D) Frequency distribution (0.5 mV bins) of the membrane potential (60 s recording) from a circular smooth muscle cell in control conditions and after the sequential addition of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) on the left and MRS2500 (1 μmol L−1) and l-NNA (1 mmol L−1) on the right. Note that l-NNA increased the IAS tone and depolarized smooth muscle cells while MRS2500 only blocked spontaneous IJP. Drugs were incubated for 15 min.

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Inhibitory junction potentials evoked by electrical stimulation of inhibitory MNs. Single EFS pulses caused an IJP-f followed by a more sustained response (IJP-s). In control experiments, the increase in the voltage of stimulation caused a progressive increase in the amplitude and duration of the IJP reaching transient hyperpolarization of −24.96 ± 2.26 mV and 1.94 ± 0.26 s at 50 V. l-NNA (1 mmol L−1) caused a significant reduction in the duration of the IJP without affecting its amplitude and further addition of MRS2500 (1 μmol L−1) completely blocked the IJP (Fig. 6). In experiments using longer pulses of electrical stimuli, EFS at 1 Hz (50 V during 5 s) elicited five consecutive single pulses (P1–P5). P1 induced an initial fast IJP of −15.31 ± 2.5 mV (n = 6) and the following pulses P2–P5 elicited a lesser response probably attributable to rundown as observed in other species. The EFS at 5 Hz (50 V during 5 s) elicited a transient initial fast hyperpolarization followed by a more sustained response, the amplitude of the fast IJP was −21.98 ± 2.05 mV (n = 6) and the measurements at 2.5 and 3.75 s were −17.19 ± 1.11 mV and −15.41 ± 0.9 mV, respectively. l-NNA did not affect either the amplitude of the IJP at 1 Hz (P1–P5) or the IJP-f at 5 Hz, but significantly inhibited the slow component of the IJP at 5 Hz measured at 2.5 and 3.75 s. Sequential addition of MRS2500 fully abolished the amplitude of the IJP elicited by the single pulses at 1 Hz and the IJP-f at 5 Hz (Fig. 7).

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Figure 6.  Intracellular microelectrode recording showing (A) a representative electrical field stimulation (EFS)-induced inhibitory junction potential (IJP) obtained with 50 V of EFS in control conditions and after incubation with l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1), and the three recordings superimposed, and (B) plot graphs showing the inhibitory effect of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) on the amplitude (left) and duration (right) of the EFS-induced IJP at 5, 10, 12, 15, 17, 20, 25, 30, and 50 V. Antagonists were incubated for 15 min. All values are expressed as mean ± SEM. * 0.05; ** 0.01; *** 0.001.

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Figure 7.  Intracellular microelectrode recordings showing (A) the EFS-induced IJP obtained after 5 Hz and 5 s stimulation (25 pulses) (bottom) and 1 Hz and 5 s stimulation (5 pulses: P1–P5) (top) in control conditions and after the sequential addition of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) and (B) histograms showing the response of the fast component and the sustained component measured at 2.5 and 3.75 s after the beginning of the stimuli of 5 Hz (top) and the response from each pulse (P1–P5) in each experimental condition (1 Hz frequency) (bottom). Antagonists were incubated for 15 min. All values are expressed as mean ± SEM. ** 0.01; *** 0.001.

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Hyperpolarization induced by stimulation of MNs through nAChRs. Nicotine (10 μmol L−1) induced a sustained hyperpolarization of −12.87 ± 1.47 mV (n = 9) partly antagonized by l-NNA (1 mmol L−1) by −46.28 ± 4.03% of control values (n = 3, < 0.01). Sequential addition of MRS2500 (1 μmol L−1) completely abolished the non-nitrergic hyperpolarization. When MRS2500 (1 μmol L−1) was used prior to l-NNA the hyperpolarization induced by nicotine was only reduced by −19.42 ± 12.68% of the control values (n = 3, ns) and was completely abolished when both drugs were added (< 0.001). In both experiments, nicotine caused a marked increase in sIJP; l-NNA did not affect the sIJP but MRS2500 completely abolished them (Fig. 8).

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Figure 8.  Intracellular microelectrode recordings showing (A) the pharmacological characterization of the hyperpolarization induced by nicotine (10 μmol L−1) in control conditions and after sequential incubation of l-NNA (1 mmol L−1) and MRS2500 (1 μmol L−1) (left) and MRS2500 (1 μmol L−1) and l-NNA (1 mmol L−1) (right) and (B) histograms showing the quantitative effects on resting membrane potential (RMP) (bottom) and the standard deviation (SD) of the RMP induced by the spontaneous IJPs before and after the sequential incubation of l-NNA and MRS2500, and MRS2500 and l-NNA. Nicotine was added by superfusion and antagonists were incubated for 15 min. All values are expressed as mean ± SEM. * 0.05; ** 0.01; *** 0.001.

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Role of VIP. Single concentrations of VIP (0.01 μmol L−1) caused a mechanical relaxation of -43.9 ± 5.1% total tone and an electrical hyperpolarization of −3.7 ± 0.46 mV (n = 4). Both VIP 6–28 (0.1 μmol L−1) and α-CMT (10 U mL−1) significantly antagonized VIP-induced relaxation (−79 ± 9.8% for α-CMT and −33 ± 6.3% for VIP 6–28), and completely blocked VIP-induced hyperpolarization.

During electrical stimulation of inhibitory MNs at 3 and 5 Hz, sequential addition of l-NNA (1 mmol L−1), decreased significantly (P ≤ 0.05) electrical-induced relaxation by 13.73 ± 9.2% (3 Hz) and 46.80 ± 10.5% (5 Hz). In contrast, neither VIP 6–28 [2.22 ± 6.9% (3 Hz) and 36.36 ± 10.21% (5 Hz)] nor α-CMT [21.52 ± 13.02% (3 Hz) and 2.22 ± 9.3% (5 Hz)] modified the non-nitrergic electrical relaxation. Finally, sequential addition of MRS2500 (1 μmol L−1) completely blocked non-nitrergic relaxation (P ≤ 0.05, n = 4) (Fig. 9).

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Figure 9.  Mechanical (A) and intracellular (B) representative tracings of the relaxation and hyperpolarization induced by exogenous addition of VIP in rat internal anal sphincter strips. Representative tracings of the blockade of the endogenous release of VIP by sequential addition of VIP 6–28 (0.01 μmol L−1) and α-CMT (10 U mL−1) by EFS-induced IJP (C) and EFS induced mechanical relaxation (D and E). Antagonists were incubated for 15 min.

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In electrophysiological studies, VIP (0.01 μmol L−1) induced a hyperpolarization of −3.7 ± 0.46 mV (n = 4) that was completely blocked by both VIP 6–28 (0.1 μmol L−1) and α-CMT (10 U mL−1) (P ≤ 0.001). In contrast, neither VIP 6-28 nor α-CMT affected the amplitude [(−20.71 ± 2.6 mV VS−20.54 ± 2.7 mV) for VIP 6-28 and (−22.33 ± 3.8 mV VS−23.36 ± 4.6 mV) for α-CMT] or the duration [(1.55 ± 0.09 s VS 1.62 ± 0.1) for VIP 6-28 and (1.62 ± 0.13 VS 1.52 ± 0.17) for α-CMT] of EFS-induced IJP at 50 V, (ns, n = 4). Finally, the RMP, the SD-sIJP and electrical responses at 1 and 5 Hz were unaffected by these VIP antagonists (ns, n = 4) (data not shown).

Discussion

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

In the present study we assessed the neurotransmitters involved in the mechanical and electrical inhibitory responses in the rat IAS and the relationships between these responses. We found that the inhibitory neurotransmission in rat IAS had two main components, one attributable to ATP or a related purine acting through P2Y1 receptors, and the second one to NO. We also found that each of these components had different physiological functions. Adenosine triphosphate mediated spontaneous IJP, the fast component of the inhibitory junction potential (IJP-f) and had no effect on the RMP or on basal mechanical tone. In contrast, NO released from the inhibitory MNs decreased the basal IAS tone and the RMP and mediated the slow component of the IJP. Thus, our mechanical and electrophysiological data suggest that these two neurotransmitters are co-released in resting conditions and following stimulation of inhibitory MNs and have specific and complementary roles in the inhibitory neurotransmission pathways to rat IAS.

The first part of our study was to characterize the IAS preparation by morphological and mechanical studies. The immunohistochemical studies found anti-HuD immunoreactivity in the neurons of the myenteric plexus, as described in guinea pig small intestine,29 and S100 stained both glial cells and MNs. Immunoreactivity for P2Y1 receptors was found in circular and longitudinal smooth muscle layers, and a minor staining was observed in the myenteric plexus. A similar distribution of P2Y1 receptors was described in the pig ileum,22 human colon,13 and in the mouse intestine.30

In our mechanical studies, circular IAS strips developed an active myogenic tone dependent on extracellular calcium and unaffected by neurotoxin TTX, as found in other species.5,9 We used PGF2aα to cause a direct precontraction of circular SMC, stabilize the IAS tone and facilitate the reproducibility of the mechanical responses.14,31 We also found that MRS2500 had no effect on IAS tone whereas l-NNA strongly enhanced it suggesting that continuous release of NO modulates myogenic tone. The electrical stimuli activated both inhibitory and excitatory MNs causing a mechanical ‘on’ relaxation during stimulus followed by an ‘off’ contraction at the end of the stimulus;5,21 both responses were blocked by TTX showing that they were induced by stimulation of enteric MNs. We observed maximal stimulation of inhibitory MNs at low frequencies (3–5 Hz) whereas maximal stimulation of excitatory MNs was obtained at high frequencies (20 Hz).5 We used the NO synthase inhibitor l-NNA and the new P2Y1 receptor antagonist MRS2500 to characterize the inhibitory neurotransmitters mediating IAS relaxation following electrical stimulation of MNs. We found, as expected, that l-NNA partially inhibited EFS relaxation but MRS2500 did not affect EFS relaxation when added to the bath prior to l-NNA. However, blockade of P2Y1 receptors was necessary to fully block EFS relaxation. Previous studies also observed that l-NNA reduced IAS relaxation by ∼50% in humans2,3 and several animal species;4,5,7,8,32 and purinergic antagonists such as apamin, PPADS and MRS2179, alone or combined, reduced but did not block the non-nitrergic relaxation.5,7,14 To our knowledge this is the first study that has used the new P2Y1 antagonist MRS2500 in the IAS, claimed to be more specific than previous antagonists.24,33 We believe that the full blockade of non-nitrergic relaxation by MRS2500, as shown in the present study, could be due to its greater potency and affinity on P2Y1 receptors. In parallel experiments we compared the effects of maximal stimulation of inhibitory MNs by 3 Hz EFS or through nAChRs by nicotine.5,7,34 We found IAS relaxation of similar magnitude in both cases and again l-NNA antagonized both relaxations; no effect was seen when MRS2500 was first added to the bath, and MRS2500 was again necessary to fully block the relaxation.

In the second part of our study we assessed the electrical correlates of our mechanical observations. We have recently proposed a method of quantitation of the neural inhibitory tone in the rat colon35 which includes both the quantitation of the RMP and spontaneous IJP.28 This ongoing release of inhibitory transmitters counteracts the myogenic tone as it causes smooth muscle hyperpolarization and relaxation. In the present study, l-NNA caused a strong IAS depolarization and increased tone; in contrast, MRS2500 did not affect either the membrane potential or the tone. This suggests that both NO and ATP or a related purine are ‘spontaneously’ released from inhibitory MNs but NO regulates the RMP and mechanical tone and ATP causes spontaneous IJP. When inhibitory MNs were stimulated by single EFS pulses, we observed a biphasic IJP with a fast component followed by a sustained component, a result consistent with that observed in other species such as the guinea pig,8 mouse IAS,14 and rat colon.36 In contrast, in the human colon13 and mouse cecum,37 single pulses failed to elicit a sustained component and a longer stimulus was needed to reveal the IJP-s.20 In this study we found that the slow component of the IJP was sensitive to l-NNA and therefore mediated by NO and the fast component of the IJP was insensitive to l-NNA and consequently mediated by other/s neurotransmitter/s as in the human colon,13 the pig ileum,22 and in the guinea pig small intestine.38 Electrical field stimulation at 5 Hz for 5 s also elicited a fast component followed by a sustained one, which has been previously observed in the mouse IAS.14 In contrast, pulses at 1 Hz for 5 s elicited an initial IJP of higher amplitude than the following ones, probably due to the rundown mechanism.22,39 When the more specific P2Y1 antagonist, MRS2500, was used, we found that it fully blocked the IJP-f elicited by single pulses, the IJP-f at longer pulses of 5 Hz, and the response at 1 Hz. Our data agree to some extent with recent results obtained on the mouse IAS where high doses of MRS2179 (10 μmol L−1) failed to completely abolish the IJP-f.14 We believe the differences between these results may be attributable to the lower affinity of MRS2179 for P2Y1 receptors as we found in the rat colon24 or to a different distribution of purinergic receptors between species. In the mouse gastrointestinal tract, both P2Y1 and P2Y2 have been immunolocalized in SMC30 and maybe other P2Y receptors could be accountable for the MRS2179-resistant part of the IJP-f. Finally, we found that stimulation of inhibitory MNs through nAChRs caused a sustained hyperpolarization of IAS smooth muscle cells and increased the spontaneous IJP. This hyperpolarization was significantly reduced by l-NNA but not modified by MRS2500, while the combination of both completely blocked it. In contrast, while l-NNA did not affect spontaneous IJP, MRS2500 completely abolished them.

Previous in vitro studies have reported a role for VIP in IAS relaxation in several species.9,11,23,40 We found that VIP produced a mechanical relaxation and a hyperpolarization; however, we were unable to demonstrate the endogenous release of VIP from inhibitory neurons as VIP 6-28 and α-CMT did not reduce EFS-induced IJP or mechanical relaxation in our experimental conditions. Nevertheless, a role for VIP in the IAS cannot be ruled out and, in other experimental conditions,41 VIP might participate in IAS relaxation.

In summary, our results clearly show a functional co-transmission for NO and purines with specific and complementary roles in the tonic and phasic effects of inhibitory motor pathways to rat IAS. Resting tone and membrane potential is mainly controlled by NO whereas purines mediate sIJP. Internal anal sphincter hyperpolarization and relaxation following stimulation of inhibitory enteric MNs in the rat IAS is caused by co-release of NO and a purine acting at P2Y1 receptors. In addition, we found a functional specialization between these two independent pathways: the purine acting through P2Y1 receptors may be the neurotransmitter responsible for phasic relaxations and NO responsible for the sustained tonic IAS relaxation. Our results also suggest that the contribution of NO is predominant in IAS relaxation and that both represent a ‘reserve’ physiological mechanism as abolition of one of these pathways (specially the purinergic one) does not prevent a strong IAS relaxation following stimulation of inhibitory MNs. Further studies are needed to establish the physiological and physiopathological significance of this functional specialization.

Acknowledgments

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

This study was supported by a grant from the Fundació de Gastroenterologia Dr Francisco Vilardell, the Fundació Salut del Consorci Santari del Maresme, the Departament d’Universitats, Recerca i Societat de la Informació (2009-SGR-708), the Fondo de Investigaciones Sanitarias (IF063678-1), the Ministerio de Ciencia e Innovación, (BFU2009-11118) and Ciberehd. Diana Gallego is also supported by the Centro de Investigación Biomédica en red de enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain.

The authors thank Prof Patri Vergara (Department of Physiology, UAB), Dra Maria José Fantova (Department of Pathology, CSdM), and Joan Antoni Fernández BVSc, MSc, PhD student for support and advice, and Lorena Gonzalez, Claudia Arenas, and Emma Martínez for the technical assistance in the experiments.

Author contributions

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

All authors of the study have contributed to all of the following:

1 Conception, design, analysis and interpretation of data.

2 Drafting the article and revising it critically for important intellectual content.

3 Final approval of the version to be published.

References

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