Marcel Jiménez

Evaluation of oesophageal mucosa integrity by the intraluminal impedance technique

Background Oesophageal intraluminal impedance is currently used for assessment of reflux in gastro-oesophageal reflux disease (GORD). Oesophageal mucosa integrity may have a key role in heartburn perception in non-erosive reflux disease (NERD). Severe erosive oesophagitis is associated with low impedance baseline. We hypothesised that impedance baseline measurements could be used to evaluate changes in oesophageal mucosa integrity in man. Methods We measured oesophageal impedance baseline before, during and after acid perfusion in rabbits and healthy subjects. Transepithelial resistance (TER) was determined and dilated intercellular spaces (DIS) were assessed in isolated rabbit oesophageal mucosa. Impedance baseline was measured retrospectively at different levels of the oesophagus in impedance-pH recordings from asymptomatic volunteers and patients with GORD. Results In healthy subjects and rabbits, impedance baseline dropped dramatically during perfusion of control solution (pH 7.2) but after perfusion, impedance recovered. In rabbits, after perfusion with saline pH 1.5 and 1.0 impedance values remained a 39.1±7.0% and 63.9±6.5% (p<0.05) lower respectively. There was a positive correlation between in vivo basal impedance and in vitro TER values (r=0.72, p=0.0021). Tissue showed no erosions but both acidic solutions induced DIS. In healthy subjects, after perfusion with saline pH 2.0 and 1.0 the impedance baseline remained lower a 21.9±6.5% and 52.7±5.0%, (p<0.0001) respectively. Patients with GORD have a lower impedance baseline than healthy volunteers at the distal oesophagus. Conclusions Impedance baseline measurements might be used to evaluate the status of the oesophageal mucosa and to study the role of the impaired mucosal integrity in acid-induced heartburn in healthy volunteers and in patients with GORD.

The gaseous mediator, hydrogen sulphide, inhibits in vitro motor patterns in the human, rat and mouse colon and jejunum

Abstract  Hydrogen sulphide (H2S) 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 H2S donor) caused a concentration‐dependent inhibition of spontaneous motor complexes (MCs) (IC50 121 μmol L−1 in the colon and 150 μmol L−1 in the jejunum). This inhibitory effect of NaHS on MCs was (i) unaffected by tetrodotoxin (TTX), capsaicin, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonate and N‐nitro‐l‐arginine suggesting a non‐neural effect and (ii) significantly reduced by apamin 3 μmol L−1. NaHS concentration‐dependently inhibited the spontaneous motility in strips from human colon (IC50 261 μmol L−1) and rat colon (IC50 31 μmol L−1). The inhibitory effect of NaHS on colonic strips was (i) unaffected by the neural blocker TTX (1 μmol L−1) with IC50 183 μmol L−1 for the human colon and of 26 μmol L−1 for the rat colon and (ii) significantly reduced by glybenclamide (10 μmol L−1), apamin (3 μmol L−1) and TEA (10 mmol L−1) with IC50 values of 2464, 1307 and 2421 μmol L−1 for human strips, and 80, 167 and 674 μmol L−1 for rat strips respectively. We conclude that H2S 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.

Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO

The rat colonic circular muscle displays cyclic episodes of myenteric potential oscillations (MPOs), each of them associated with a spontaneous contraction. Nifedipine 1 μM abolished both MPOs and their associated contractions. TTX (1 μM) increased the amplitude and frequency of spontaneous contractions. Electrical field stimulation (EFS) induced a non‐adrenergic non‐cholinergic (NANC) inhibitory junction potential (IJP), with two phases: an initial fast hyperpolarization (characterized by IJP amplitude) and a sustained hyperpolarization (characterized by IJP duration). Sodium nitroprusside (10 μM) hyperpolarized and abolished spontaneous contractions even in presence of TTX or 1 μM apamin. ATP (100 μM) also hyperpolarized and abolished spontaneous contractions but its effects were decreased by TTX and abolished by apamin. Suramin (100 μM) or apamin reduced the amplitude of the IJPs, but did not affect their duration. Incubation with L‐NOARG (1 mM) reduced the duration but not the amplitude of the IJPs. In presence of L‐NOARG plus suramin or L‐NOARG plus apamin, both duration and amplitude of the IJPs were reduced but a residual IJP could still be recorded. We conclude that the mechanical and electrical cyclic activity of the rat colonic circular muscle is modulated but not originated by the enteric nervous system and involves L‐type calcium channel activity. EFS induces release of NANC inhibitory neurotransmitters which hyperpolarize and relax smooth muscle cells. Both ATP and NO are involved in IJP generation: ATP is responsible for the first phase of the IJPs involving activation of apamin‐sensitive potassium channels, whereas NO initiates the second phase which is independent of the activation of such channels.

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.

Two Independent Networks of Interstitial Cells of Cajal Work Cooperatively with the Enteric Nervous System to Create Colonic Motor Patterns

Normal motility of the colon is critical for quality of life and efforts to normalize abnormal colon function have had limited success. A better understanding of control systems of colonic motility is therefore essential. We report here a hypothesis with supporting experimental data to explain the origin of rhythmic propulsive colonic motor activity induced by general distention. The theory holds that both networks of interstitial cells of Cajal (ICC), those associated with the submuscular plexus (ICC–SMP) and those associated with the myenteric plexus (ICC–MP), orchestrate propagating contractions as pacemaker cells in concert with the enteric nervous system (ENS). ICC–SMP generate an omnipresent slow wave activity that causes propagating but non-propulsive contractions (“rhythmic propagating ripples”) enhancing absorption. The ICC–MP generate stimulus-dependent cyclic depolarizations propagating anally and directing propulsive activity (“rhythmic propulsive motor complexes”). The ENS is not essential for both rhythmic motor patterns since distention and pharmacological means can produce the motor patterns after blocking neural activity, but it supplies the primary stimulus in vivo. Supporting data come from studies on segments of the rat colon, simultaneously measuring motility through spatiotemporal mapping of video recordings, intraluminal pressure, and outflow measurements.

Motility patterns and distribution of interstitial cells of Cajal and nitrergic neurons in the proximal, mid- and distal-colon of the rat.

Abstract  The aim of this work was to study the patterns of spontaneous motility in the circular and longitudinal muscle strips and to characterize the distribution of c‐kit positive interstitial cells of Cajal (ICCs) and nitrergic neurons (nNOS) in the proximal, mid‐ and distal‐colon of Sprague–Dawley rats. We described two types of spontaneous contractions: high frequency (HF) and low frequency (LF) contractions, which were recorded in the presence of tetrodotoxin, suggesting a non‐neurogenic origin. Regional differences were found in the motility patterns depending on the muscle layer and on the part of the colon studied. Muscle strips without submuscular plexus (SMP) showed only LF contractions. The density of ICCs was of the same magnitude along the extent of the colon: about 90–120 cells mm−2 at Auerbachs plexus (AP) and 50–60 cells mm−2 at the SMP. nNOS positive cells were found at the level of the AP and the major density was found in the mid‐colon. Electrical field stimulation abolished LF but did not affect HF contractions. Our results indicate that HF contractions are due to the ICC network found associated with the submuscular plexus (ICC–SMP). The origin of LF contractions is still unknown.

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Abstract.Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present.

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.

Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon.