Diana Gallego

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.

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.

P2Y1 receptors mediate inhibitory neuromuscular transmission and enteric neuronal activation in small intestine

Abstract  There is increasing evidence that adenosine 5′‐triphosphate or a related purine plays a crucial role in smooth muscle relaxation and enteric synaptic neurotransmission. Accordingly, the aim of the present work is to investigate the role P2Y1 receptors in purinergic inhibitory neurotransmission (pig ileum) and enteric neuronal activation in the small intestine (guinea‐pig ileum). Using contractility measurements, micro‐electrode recordings and Ca2+ imaging we found that (i) adenosine 5′‐Ο‐2‐thiodiphosphate (ADPβS) (10 μmol L−1) caused smooth muscle relaxation and hyperpolarization that was antagonized by MRS2179 (10 μmol L−1) a P2Y1 receptor antagonist and apamin (1 μmol L−1); (ii) electrical field stimulation (EFS) caused a non‐nitrergic inhibitory junction potential (IJP) and relaxation that was antagonized by MRS2179 (10 μmol L−1); (iii) P2Y1 receptors were immunolocalized in smooth muscle cells and enteric neurons; (iv) superfusion of ADPβS (1 μmol L−1) induced Ca2+ transients in myenteric neurons that were inhibited by MRS2179 (1 μmol L−1), but not by tetrodotoxin (1 μmol L−1); and (v) EFS induced calcium transients were partially inhibited by MRS2179 (1 μmol L−1). We conclude that in the small intestine purinergic neuromuscular transmission responsible for the IJP and non‐nitrergic relaxation is mediated by P2Y1 receptors located in smooth muscle cells. Functional P2Y1 receptors are also present in guinea‐pig myenteric neurons. Therefore, P2Y1 receptors might be an important pharmacological target to modulate gastrointestinal functions.

Nitrergic and purinergic mechanisms evoke inhibitory neuromuscular transmission in the human small intestine

Inhibitory neuromuscular transmission in the human colon is due to nitrergic and purinergic (P2Y1‐mediated) inputs. The aim of this study was to determine the mechanisms of neuromuscular transmission in different regions of the human small intestine.

Purinergic neuromuscular transmission in the gastrointestinal tract; functional basis for future clinical and pharmacological studies.

Nerve‐mediated relaxation is necessary for the correct accomplishment of gastrointestinal (GI) motility. In the GI tract, NO and a purine are probably released by the same inhibitory motor neuron as inhibitory co‐transmitters. The P2Y1 receptor has been recently identified as the receptor responsible for purinergic smooth muscle hyperpolarization and relaxation in the human gut. This finding has been confirmed in P2Y1‐deficient mice where purinergic neurotransmission is absent and transit time impaired. However, the mechanisms responsible for nerve‐mediated relaxation, including the identification of the purinergic neurotransmitter(s) itself, are still debatable. Possibly different mechanisms of nerve‐mediated relaxation are present in the GI tract. Functional demonstration of purinergic neuromuscular transmission has not been correlated with structural studies. Labelling of purinergic neurons is still experimental and is not performed in routine pathology studies from human samples, even when possible neuromuscular impairment is suspected. Accordingly, the contribution of purinergic neurotransmission in neuromuscular diseases affecting GI motility is not known. In this review, we have focused on the physiological mechanisms responsible for nerve‐mediated purinergic relaxation providing the functional basis for possible future clinical and pharmacological studies on GI motility targeting purine receptors.

Mechanisms of action of otilonium bromide (OB) in human cultured smooth muscle cells and rat colonic strips.

The pharmacological properties of otilonium bromide (OB) have been investigated using different experimental models, techniques, and conditions, and consequently, the results are not always easy to compare. The aim of the present work was to investigate the pharmacological properties of OB in human cultured colonic smooth muscle cells (HCSMCs), which is the main target of the drug ‘in vivo’. Rat colonic strips were used to confirm the pharmacological properties.

In vitro motor patterns and electrophysiological changes in patients with colonic diverticular disease

PurposeThe underlying mechanism responsible for motility changes in colonic diverticular disease (DD) is still unknown. In the present study, our aim was to investigate the structural and in vitro motor changes in the sigmoid colon of patients with DD.MethodsMuscle bath, microelectrodes and immunohistochemical techniques were performed with samples obtained from the left and sigmoid colon of patients with DD and compared with those of patients without DD.ResultsThe amplitude and area under the curve of the spontaneous rhythmic phasic contractions were greatly reduced in patients with DD whereas their frequency and tone remained unaltered. Electrical field stimulation induced a neurally mediated, enhanced ON-contraction (amplitude) in patients with DD and increased the duration of latency of OFF-contractions. The resting membrane potential of smooth muscle cells was hyperpolarized and the amplitude of the inhibitory junction potential was increased in patients with DD. In contrast, no significant histological differences were observed in patients with DD as smooth muscle (circular and longitudinal layers), interstitial cells of Cajal, glial cells and myenteric neurons densities remained unaltered.ConclusionsSigmoid strips from patients with asymptomatic DD showed an altered motor pattern with reduced spontaneous motility and enhanced neurally mediated colonic responses involving both excitatory and inhibitory motor pathways. No major neural and muscular structural elements were detected at this stage of the disease. These findings could be valuable in understanding the pathophysiology of this prevalent digestive disease.

Enteric motor pattern generators involve both myogenic and neurogenic mechanisms in the human colon.

Coordinated motor activity is required to develop the major functions of the colon, which are: 1-absorption of water, electrolytes, bile salts, short-chain fatty acids and other bacterial metabolites, 2-storage of colonic contents and 3-propulsion of fecal material (Christensen, 1991). Interstitial cells of Cajal (ICCs) generate spontaneous pacemaker currents which are conducted to smooth muscle cells (SMCs) causing rhythmic contractile patterns (Rumessen et al., 1993; Huizinga et al., 1995). Even though in vitro experiments disrupt enteric neural pathways crucial to develop a variety of in vivo colonic motor patterns and rule out any influence of extrinsic innervation, they are useful to better understand the mechanisms underlying colonic motility. Accordingly, the aim of this article is to summarize myogenic and neurogenic activities described in the human colon, hypothesize about how these mechanisms might be related and propose a new concept, enteric motor pattern generators, for this interplay.