María Teresa Martín

Purinergic neuromuscular transmission is absent in the colon of P2Y1 knocked out mice

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 P2Y1 receptors are responsible for purinergic neurotransmission in the gastrointestinal tract. •  In the present study we used a genetically modified mouse in which P2Y1 receptors had been knocked out. •  P2Y1‐deficient mice had functional nitrergic neurotransmission but purinergic neurotransmission was absent. •  The present work confirms the hypothesis demonstrating that P2Y1 receptors mediate the purinergic component of the smooth muscle relaxation in the gastrointestinal tract.

Correlation between cyclical epithelial barrier dysfunction and bacterial translocation in the relapses of intestinal inflammation.

Background: Although several factors have been implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanisms underlying the recurrent relapses have not yet been clarified. We hypothesized that epithelial barrier dysfunction, associated with intestinal motor disturbances, could play a key role in exacerbation of inflammatory illness due to an increased uptake of luminal antigen and bacterial translocation. Methods: Indomethacin administration to rats induced a long‐lasting oscillation of active and quiescent phases of inflammation associated with phases of hypo and hypermotility. Rats selected at either active or quiescent phase and from 2 to 60 days after indomethacin treatment were used. Short‐circuit current; conductance and HRP flux were evaluated in small intestinal segments mounted in Ussing Chambers. Enterocyte endosomes containing HRP and ultrastructural damage were assessed by electron microscopy. Bacterial translocation was determined by cultures from mesenteric lymph nodes. Results: Rats with induced enteritis in both phases demonstrated a long‐lasting increase of epithelial paracellular permeability. In contrast, transcellular permeability was only disturbed during the active phases, coinciding with bacterial translocation and the increase in inflammatory parameters. Furthermore, although mithochondrial damage was observed throughout the inflammatory state, alterations were worse during the active phase. Conclusions: The sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa, and hence, be the basis for chronicity. By contrast, transcellular permeability only increases during the active phases, when hypomotility and bacterial translocation are also present, suggesting this factor may play a critical role in the course of acute relapses in IBD.

P2Y1 receptors mediate inhibitory neuromuscular transmission in the rat colon

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.

Purinergic and nitrergic neuromuscular transmission mediates spontaneous neuronal activity in the rat colon

Nitric oxide (NO) and ATP mediate smooth muscle relaxation in the gastrointestinal tract. However, the involvement of these neurotransmitters in spontaneous neuronal activity is unknown. The aim of the present work was to study spontaneous neuromuscular transmission in the rat midcolon. Microelectrode experiments were performed under constant stretch both in circular and longitudinal directions. Spontaneous inhibitory junction potentials (sIJP) were recorded. Tetrodotoxin (1 microM) and apamin (1 microM) depolarized smooth muscle cells and inhibited sIJP. N(omega)-nitro-l-arginine (l-NNA, 1 mM) depolarized smooth muscle cells but did not modify sIJP. In contrast, the P2Y(1) antagonist MRS-2500 (1 microM) did not modify the resting membrane potential (RMP) but reduced sIJP (IC(50) = 3.1 nM). Hexamethonium (200 microM), NF-023 (10 microM), and ondansetron (1 microM) did not modify RMP and sIJP. These results correlate with in vitro (muscle bath) and in vivo (strain gauges) data where l-NNA but not MRS-2500 induced a sustained increase of spontaneous motility. We concluded that, in the rat colon, inhibitory neurons regulate smooth muscle RMP and cause sIJP. In vitro, the release of inhibitory neurotransmitters is independent of nicotinic, P2X, and 5-hydroxytryptamine type 3 receptors. Neuronal NO causes a sustained smooth muscle hyperpolarization that is responsible for a constant inhibition of spontaneous motility. In contrast, ATP acting on P2Y(1) receptors is responsible for sIJP but does not mediate inhibitory neural tone. ATP and NO have complementary physiological functions in the regulation of gastrointestinal motility.

P2Y(1) knockout mice lack purinergic neuromuscular transmission in the antrum and cecum.

Background  Pharmacological studies using selective P2Y1 antagonists, such as MRS2500, and studies with P2Y1−/− knockout mice have demonstrated that purinergic neuromuscular transmission is mediated by P2Y1 receptors in the colon. The aim of the present study was to test whether P2Y1 receptors are involved in purinergic neurotransmission in the antrum and cecum.

Characterization of Housing-Related Spontaneous Variations of Gut Microbiota and Expression of Toll-Like Receptors 2 and 4 in Rats

Gut microbiota has been suggested as a key component of gut homeostasis, affecting immune responses within the gut. We determined changes in intestinal commensal bacteria and expression of toll-like receptors (TLR) 2 and 4 in rats bred under microbiologically controlled conditions (barrier), under standard conditions (conventional), and in barrier animals adapted to standard conditions (barrier/conventional). Cecal microbiota was analyzed by plate culture, and fluorescence in situ hybridization and microbial profiles were assessed by terminal restriction fragment length polymorphism. Cecal expression of TLR-2 and TLR-4 was determined by reverse transcription polymerase chain reaction (PCR). Total number of cecal bacteria was similar in the three groups. However, the barrier group showed a higher number of strict anaerobic bacteria (Bacteroides spp. and Clostridium spp.) while Bifidobacterium spp. were scarce. Re-housing the barrier-bred rats into conventional conditions led to a microbiota with intermediate characteristics between the barrier and conventional groups. Richness of the cecal microbial ecosystem was similar in the three groups, although a relative time-dependent variation, with highest homogeneity in the barrier group, was observed. Expression levels of TLR-2 and TLR-4 had no clear correlation with the microbiota. These results show that the relative composition of the cecal microbiota in rats varies spontaneously with changes in the environmental conditions, with minor impact in the expression of TLR-2 and TLR-4. These observations might be important in the understanding of variability in animal responses, particularly to immune-related stimuli, when assessed in the context of the environmental/microbiological conditions.

Otilonium bromide inhibits muscle contractions via L‐type calcium channels in the rat colon

Abstract  The aim of this study is to evaluate in vitro the effect of otilonium bromide (OB) on the mechanical and electrical activities of the rat colonic smooth muscle using muscle bath, microelectrodes and patch‐clamp techniques. Otilonium bromide dose dependently inhibited the spontaneous activity (logIC50 ± SE: −5.31 ± 0.05). This effect was not modified by TTX (10−6 mol L−1). Cyclic depolarizations were abolished by OB (10−4 mol L−1). Electrical field stimulation induced inhibitory junction potentials (IJPs) followed by a depolarization with superimposed spikes causing a contraction. In the presence of OB (10−4 mol L−1) IJPs were recorded, but spikes and contractions were abolished. Otilonium bromide (3 × 10−6 mol L−1) inhibited inward current obtained in isolated cells (amphotericin perforated patch technique). The otilonium‐sensitive current amplitude was maximal (75pA) around 0 mV. The effect of different doses of OB was tested by depolarizing cells from −70 mV to 0 mV. OB dose dependently inhibited the inward current with an EC50 of 885 nmol L−1. Abolishment of the otilonium‐sensitive current by 3 × 10−6 mol L−1 nifedipine confirmed that it was an L‐type Ca2+ current. Our results show that OB inhibits the spontaneous and triggered muscular contractions. This effect is produced by the inhibition of muscular action potentials carried by L‐type calcium current, confirming the spasmolytic properties of OB.

Potential role of the gaseous mediator hydrogen sulphide (H2S) in inhibition of human colonic contractility.

BACKGROUND Hydrogen sulphide (H2S) is an endogenous signalling molecule that might play a physiologically relevant role in gastrointestinal motility. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are two enzymes responsible for H2S production. d,l-Propargylglycine (PAG) is a CSE inhibitor whereas both aminooxyacetic acid (AOAA) and hydroxylamine (HA) are CBS inhibitors. The characterization of H2S responses and its mechanism of action are crucial to define H2S function. METHODS Human colonic strips were used to investigate the role of H2S on contractility (muscle bath) and smooth muscle electrophysiology (microelectrodes). NaHS was used as a H2S donor. RESULTS Combination of PAG and AOAA depolarized the smooth muscle (5-6mV, n=4) and elicited a transient increase in tone (260.5±92.8mg, n=12). No effect was observed on neural mediated inhibitory junction potential or relaxation. In the presence of tetrodotoxin 1μM, NaHS concentration-dependently inhibited spontaneous contractions (EC50=329.2μM, n=18). This effect was partially reduced by the guanylyl cyclase inhibitor ODQ 10μM (EC50=2.6μM, n=12) and by l-NNA 1mM (EC50=1.4mM, n=8). NaHS reversibly blocked neural mediated cholinergic (EC50=2mM) and tachykinergic (EC50=5.7mM) contractions. NaHS concentration-dependently reduced the increase in spontaneous mechanical activity (AUC) induced by carbachol (EC50=1.9mM) and NKA (EC50=1.7mM AUC). CONCLUSIONS H2S might be an endogenous gasomediator regulating human colonic contractility. Its inhibitory effect is observed at high concentrations and could be mediated by a direct effect on smooth muscle with a possible synergistic effect with NO, as well as by an interaction with the cholinergic and tachykinergic neural mediated pathways.

Melatonin as a modulator of the ileal brake mechanism

Objective The gastrointestinal tract represents the most important extrapineal source of melatonin. Intestinal melatonin release is induced by the ileal passage of nutrients and could play a part in the control of postprandial gut motility. The specific aim of this study was to determine the putative role of melatonin in the “ileal brake” reflex, an important mechanism released by ileal lipids that regulates the gastric emptying of chyme. Material and methods Under general anaesthesia rats were fitted with ileal cannula exteriorized at the back of the neck. After a 1-week recovery, experiments were performed in conscious fasted animals. Rats were fed by gavage 1.5 ml casein hydrolyse plus 0.05% phenol red and either saline or Intralipid were continuously infused (2 ml/h) into the ileum. Gastric emptying was measured 50 min after ingestion by gastric lavage and determination of phenol red by spectrophotometry. The effects of melatonin (1 mg/kg) and melatonin antagonist S-22153 (dose–response study 0.2–25 mg/kg) were tested versus vehicle in paired experiments at 1-week intervals. Results Ileal infusion of lipids delayed gastric emptying. During ileal infusion of lipids, melatonin antagonist S-22153, but not melatonin, potentiated the delay in gastric emptying induced by the ileal brake mechanism. The inhibition of gastric emptying induced by S-22153 was dose related. Neither melatonin nor S-22153 had noticeable effects on gastric emptying during ileal infusion of saline. Conclusions Our data suggest that melatonin, released in response to ileal lipids, exerts a modulatory influence that decreases the inhibitory effects of the ileal brake on gastric emptying of nutrients.

Ileal brake failure in streptozotocin‐induced diabetic rat

Background: Diabetes mellitus frequently alters gastrointestinal function, but the pathophysiology of the diabetic gut has not been fully elucidated. Our aim was to characterize the enterogastric modulation of gastric emptying in an experimental model of diabetic rat and to determine the putative consequences of impaired regulation on glycaemic control. Methods: Studies were performed in streptozotozin‐induced diabetic and control groups of male Sprague‐Dawley rats. In rats fitted with chronic ileal cannulae, gastric emptying of a peptide meal was measured during ileal infusion of either lipids (ileal brake) or saline. The influence of the ileal brake mechanism on blood glucose levels after oral administration of a glucose solution was also evaluated. Results: Diabetic rats exhibited a precipitous gastric emptying (80% ± 3% versus 57% ± 3% in controls; P < 0.05). Ileal lipids delayed gastric emptying in control (38 ± 4%; P < 0.05 versus ileal saline) but not in diabetic animals (77 ± 5%; N.S. versus ileal saline). As the ileal brake contributes to the management of postprandial blood glucose levels (114 ± 4.9 mg/dL after ileal lipids versus 134 ± 7.8 mg/dL after ileal saline in control rats; P < 0.05), the failure of this mechanism in diabetic rats worsens glycaemic control after feeding (455 ± 20.4 mg/dL after ileal lipids versus 399 ± 8.7 mg/dL after ileal saline; P < 0.05). Conclusion: Experimental diabetes impairs the ileal brake mechanism and disturbs gastric emptying. These abnormalities may contribute to difficult glycaemic control.