Indirect evidence suggests that ATP is a neurotransmitter involved in inhibitory pathways in the neuromuscular junction in the gastrointestinal tract. The aim of this study was to characterize purinergic inhibitory neuromuscular transmission in the human colon. Tissue was obtained from colon resections for neoplasm. Muscle bath, microelectrode experiments, and immunohistochemical techniques were performed. 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) was used as a selective inhibitor of P2Y(1) receptors. We found that 1) ATP (1 mM) and adenosine 5'-beta-2-thiodiphosphate (ADPbetaS) (10 microM), a preferential P2Y agonist, inhibited spontaneous motility and caused smooth muscle hyperpolarization (about -12 mV); 2) MRS 2179 (10 microM) and apamin (1 microM) significantly reduced these effects; 3) both the fast component of the inhibitory junction potential (IJP) and the nonnitrergic relaxation induced by electrical field stimulation were dose dependently inhibited (IC(50) approximately 1 microM) by MRS 2179; 4) ADPbetaS reduced the IJP probably by a desensitization mechanism; 5) apamin (1 microM) reduced the fast component of the IJP (by 30-40%) and the inhibitory effect induced by electrical field stimulation; and 6) P2Y(1) receptors were localized in smooth muscle cells as well as in enteric neurons. These results show that ATP or a related purine is released by enteric inhibitory motoneurons, causing a fast hyperpolarization and smooth muscle relaxation. The high sensitivity of MRS 2179 has revealed, for the first time in the human gastrointestinal tract, that a P2Y(1) receptor present in smooth muscle probably mediates this mechanism through a pathway that partially involves apamin-sensitive calcium-activated potassium channels. P2Y(1) receptors can be an important pharmacological target to modulate smooth muscle excitability.

Hydrogen sulphide (H 2 S) has been recently proposed as a transmitter in the brain and peripheral tissues. Its role in the gastrointestinal tract is still unknown despite some data which suggest an involvement mediating smooth muscle relaxation. The aim of this study was to investigate the effect of this gas on intestinal segments from mouse jejunum and colon, and muscular strips from the human and rat colon. In isolated segments of mouse colon and jejunum, bath applied sodium hydrogen sulphide (NaHS) (a H 2 S donor) caused a concentration-dependent inhibition of spontaneous motor complexes (MCs) (IC 50 121 lmol L human strips, and 80, 167 and 674 lmol L )1 for rat strips respectively. We conclude that H 2 S strongly inhibits in vitro intestinal and colonic motor patterns. This effect appears to be critically dependent on K channels particularly apamin-sensitive SK channels and glybenclamide-sensitive K (ATP) channels.

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 M), but not the P2Y receptor agonist adenosine 5Ј-O-2-thiodiphosphate (10 M), was able to cause a sustained relaxation. N G -nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2Ј-deoxy-N 6 -methyl adenosine 3Ј,5Ј-diphosphate tetraammonium salt (MRS 2179) (10 M), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 M). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 M), MRS 2179 (1 M), and NF023 (10 M). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).ATP; IJP; smooth muscle; rundown; purinergic receptors THE IDENTITY OF THE INHIBITORY neurotransmitter(s) involved in nonadrenergic, noncholinergic (NANC) inhibitory neurotransmission is still being debated, but ATP and nitric oxide (NO) are presently recognized as two of the major inhibitory mediators in the gastrointestinal (GI) tract. Nitrergic neurons mediate lower esophageal sphincter relaxation after swallowing (34), and vagally induced gastric (11) and pyloric (2) relaxation regulate colonic transit time (26) and mediate the rectoanal inhibitory reflex (28). Fewer studies have been published on the putative role of ATP as a neurotransmitter mediating these physiological functions. ATP and NO might participate in the regulation of gastroduodenal motility in rats, (17) and ATP might participate in vagally induced gastric relaxation (4) and intrinsic gastric adaptive relaxation in mice (12). These (and other) data suggest that these neurotransmitters have important physiological functions; however, smooth muscle relaxation might have different properties (i.e., tonic vs. phasic) that should be better characterized. The release of inhibitory neurotransmitter(s) at the neuromuscular junction is the final step for smooth muscle relaxation. Inhibitory junction potentials (IJPs) have been well c...

Background and purpose: To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon. Experimental approach: Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors. Key results: Strips developed weak spontaneous rhythmic contractions (3.67 ± 0.49 g, 2.54 ± 0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 mM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1-100 mM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by N G -nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 mM) or the P2Y 1 receptor antagonist MRS 2179 (10 mM) and were unaffected by the P2X antagonist NF279 (10 mM) or a-chymotrypsin (10 U mL À1 ). Amplitude of on-and off-contractions was reduced by atropine (1 mM) and the selective NK 2 receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH 2 (1 mM). MRS 2179 reduced the amplitude of EFS on-and off-contractions without altering direct muscular contractions induced by ACh (1 nM-1 mM) or substance P (1 nM-10 mM). Conclusions and implications: Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y 1 receptors through apaminsensitive K þ channels. EFS-induced on-and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK 2 receptors. Prejunctional P2Y 1 receptors might modulate the activity of excitatory EMNs. P2Y 1 and NK 2 receptors might be therapeutic targets for colonic motor disorders.

Key points• Neural-mediated relaxation occurs in the gastrointestinal tract. To accomplish this function, two neurotransmitters, ATP or a related purine and nitric oxide, are released by inhibitory motorneurons.• The type of purinergic receptor is still under debate but previous data using a classical pharmacological approach (receptor agonists and antagonists) suggested that P2Y 1 receptors are responsible for purinergic neurotransmission in the gastrointestinal tract.• In the present study we used a genetically modified mouse in which P2Y 1 receptors had been knocked out.• P2Y 1 -deficient mice had functional nitrergic neurotransmission but purinergic neurotransmission was absent.• The present work confirms the hypothesis demonstrating that P2Y 1 receptors mediate the purinergic component of the smooth muscle relaxation in the gastrointestinal tract.Abstract Purinergic and nitrergic co-transmission is the dominant mechanism responsible for neural-mediated smooth muscle relaxation in the gastrointestinal tract. The aim of the present paper was to test whether or not P2Y 1 receptors are involved in purinergic neurotransmission using P2Y 1 −/− knock-out mice. Tension and microelectrode recordings were performed on colonic strips. In wild type (WT) animals, electrical field stimulation (EFS) caused an inhibitory junction potential (IJP) that consisted of a fast IJP (MRS2500 sensitive, 1 μM) followed by a sustained IJP (N ω -nitro-L-arginine (L-NNA) sensitive, 1 mM). The fast component of the IJP was absent in P2Y 1 −/− mice whereas the sustained IJP (L-NNA sensitive) was recorded. In WT animals, EFS-induced inhibition of spontaneous motility was blocked by the consecutive addition of L-NNA and MRS2500. In P2Y 1 −/− mice, EFS responses were completely blocked by L-NNA. In WT and P2Y 1 −/− animals, L-NNA induced a smooth muscle depolarization but 'spontaneous' IJP (MRS2500 sensitive) could be recorded in WT but not in P2Y 1 −/− animals. Finally, in WT animals, 1 μM MRS2365 caused a smooth muscle hyperpolarization that was blocked by 1 μM MRS2500. In contrast, 1 μM MRS2365 did not modify smooth muscle resting membrane potential in P2Y 1 −/− mice. β-Nicotinamide adenine dinucleotide (β-NAD, 1 mM) partially mimicked the effect of MRS2365. We conclude that P2Y 1 receptors mediate purinergic neurotransmission in the gastrointestinal tract and β-NAD partially fulfils the criteria to participate in rodent purinergic neurotransmission. The P2Y 1 −/− mouse is a useful animal model to study the selective loss of purinergic neurotransmission.

BACKGROUND AND PURPOSEThe role of hydrogen sulphide (H2S) as a putative endogenous signalling molecule in the gastrointestinal tract has not yet been established. We investigated the effect of D,L-propargylglycine (PAG), an inhibitor of cystathionine g-lyase (CSE), amino-oxyacetic acid (AOAA) and hydroxylamine (HA), inhibitors of cystathionine b-synthase (CBS) on rat colonic motility. EXPERIMENTAL APPROACHImmunohistochemistry, H2S production, microelectrode and organ bath recordings were performed on rat colonic samples without mucosa and submucosa to investigate the role of endogenous H2S in motility. KEY RESULTSCSE and CBS were immunolocalized in the colon. H2S was endogenously produced (15.6 Ϯ 0.7 nmol·min -1 ·g -1 tissue) and its production was strongly inhibited by PAG (2 mM) and AOAA (2 mM). PAG (2 mM) caused smooth muscle depolarization and increased spontaneous motility. The effect was still recorded after incubation with tetrodotoxin (TTX, 1 mM) or N w -nitro-Larginine (L-NNA, 1 mM). AOAA (2 mM) caused a transient (10 min) increase in motility. In contrast, HA (10 mM) caused a 'nitric oxide-like effect', smooth muscle hyperpolarization and relaxation, which were antagonized by 1H- [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, 10 mM). Neither spontaneous nor induced inhibitory junction potentials were modified by AOAA or PAG. CONCLUSIONS AND IMPLICATIONSWe demonstrated that H2S is endogenously produced in the rat colon. PAG and AOAA effectively blocked H2S production. Our data suggest that enzymatic production of H2S regulates colonic motility and therefore H2S might be a third gaseous inhibitory signalling molecule in the gastrointestinal tract. However, possible non-specific effects of the inhibitors should be considered. AbbreviationsAOAA, amino-oxyacetic acid; AUC, area under the curve; CBS, cystathionine b-synthase; CSE, cystathionine g-lyase; D-AP5, D-(-)-2-amino-5-phosphonopentanoic acid; EFS, electrical field

Background and purpose: Inhibitory junction potentials (IJP) are responsible for smooth muscle relaxation in the gastrointestinal tract. The aim of this study was to pharmacologically characterize the neurotransmitters [nitric oxide (NO) and adenosine triphosphate (ATP)] and receptors involved at the inhibitory neuromuscular junctions in the rat colon using newly available P2Y1 antagonists. Experimental approach: Organ bath and microelectrode recordings were used to evaluate the effect of drugs on spontaneous mechanical activity and resting membrane potential. IJP and mechanical relaxation were studied using electrical field stimulation (EFS). Key results: N w -nitro-L-arginine (L-NNA) inhibited the slow component of the IJP and partially inhibited the mechanical relaxation induced by EFS. MRS2179, MRS2500 and MRS2279, all selective P2Y1 receptor antagonists, inhibited the fast component of the IJP without having a major effect on the relaxation induced by EFS. The combination of both L-NNA and P2Y1 antagonists inhibited the fast and the slow components of the IJP and completely blocked the mechanical relaxation induced by EFS. Sodium nitroprusside caused smooth muscle hyperpolarization and cessation of spontaneous motility that was prevented by oxadiazolo[4,3-a]quinoxalin-1-one. Adenosine 5′-O-2-thiodiphosphate, a preferential P2Y agonist, hyperpolarized smooth muscle cells and decreased spontaneous motility. This effect was inhibited by P2Y1 antagonists. Conclusions and implications:The co-transmission process in the rat colon involves ATP and NO. P2Y1 receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y1 receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y1 receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.

Inhibitory purinergic neuromuscular transmission in the human colon was pharmacologically assessed by the use of new P2Y(1) receptor antagonists MRS2179, MRS2279, and MRS2500. The rank order of potency of the P2Y(1) antagonists is MRS2500 > MRS2279 > MRS2179. We found that β-NAD partially fulfills the criteria to be considered an inhibitory neurotransmitter in the human colon, but the relative contribution of each purine (ATP/ADP vsβ-NAD) requires further studies.