Víctor Gil

Autonomous University of Barcelona, Cerdanyola del Vallès, Catalonia, Spain

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Publications (19)74.51 Total impact

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    ABSTRACT: 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 is crucial to define H2S function.
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    ABSTRACT: AimATP and nitric oxide (NO) are released from enteric inhibitory motor neurons and are responsible for colonic smooth muscle relaxation. However, how frequency of neural stimulation affects this co-transmission process and the post-junctional responses has not been systematically characterized in the human colon.Methods The dynamics of inhibitory co-transmission were studied using different protocols of electrical field stimulation (EFS) to characterize the inhibitory junction potentials (IJP) and the corresponding relaxation in colonic strips obtained from 36 patients.ResultsSingle pulses elicited a fast IJP (IJPfMAX=-27.6±1.6mV), sensitive to the P2Y1 antagonist MRS2500 1μM, that ran down with frequency-increase leaving a residual hyperpolarization at high frequencies (IJPf∞=-3.7±0.6 mV). Accordingly, low frequencies of EFS caused purinergic transient relaxations that cannot be maintained at high frequencies. Addition of the P2Y1 agonist MRS2365 10μM during the purinergic rundown did not cause any hyperpolarization. PKC, a putative P2Y1 desensitizator, was able to reduce the amplitude of the IJPf when activated but the rundown was not modified by PKC inhibitors. Frequencies higher than 0.60±0.15Hz were needed to evoke a sustained nitrergic hyperpolarization that progressively increased reaching IJPs∞= -13±0.4mV at high frequencies and leading to a sustained inhibition of spontaneous motility.Conclusion Changes in frequency of stimulation possibly mimicking neuronal firing will post-junctionally determine purinergic vs nitrergic responses underlying different functional roles. NO will be responsible for sustained relaxations needed in physiological processes such as storage while purinergic neurotransmission evoking sharp transient relaxations will be dominant in processes such as propulsion.This article is protected by copyright. All rights reserved.
    Acta Physiologica 10/2014; DOI:10.1111/apha.12408 · 4.25 Impact Factor
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    ABSTRACT: The purine receptor involved in inhibitory responses in the gastrointestinal tract has been recently identified. P2Y1 receptor activation mediates the fast component of the inhibitory junction potential (IJPf) and the non-nitrergic relaxation. The aim of the present work has been to investigate which purinergic agonist better mimics endogenous responses. We used different agonist and antagonist of P2 receptors. Contractility and microelectrode experiments were used to compare the effects of exogenously added purines and electrical field stimulation (EFS)-induced nerve mediated effects in rat and human colonic strips. In rat colon, the IJPf and EFS-induced inhibition of contractions were concentration-dependently inhibited by the P2Y1 antagonist MRS2500 but not by iso-PPADS or NF023 (P2X antagonists) up to 1μM. In samples from human colon, EFS-induced inhibition of contractions was inhibited by either MRS2500 or apamin (1μM) but not by iso-PPADS. In both species, α,β-meATP, a stable analogue of ATP, caused inhibition of spontaneous contractions. α,β-meATP effect was concentration-dependent (EC50: 2.7μM rat, 4.4μM human) and was antagonised by either MRS2500 or apamin but unaffected by P2X antagonists. ATP, ADP, β-NAD and ADP-ribose inhibited spontaneous contractions but did not show the same sensitivity profile to purine receptor antagonists as EFS-induced inhibition of contractions. The effect of α,β-meATP is due to P2Y1 receptor activation leading the opening of sKca channels. Accordingly, α,β-meATP mimics the endogenous purinergic mediator. In contrast, exogenously added putative neurotransmitters do not exactly mimic the endogenous mediator. Quick degradation by ecto-nuclease or different distribution of receptors (junctionally vs extrajunctionally) might explain these results.
    European Journal of Pharmacology 07/2014; DOI:10.1016/j.ejphar.2014.06.048 · 2.68 Impact Factor
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    ABSTRACT: Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types including smooth muscle cells (SMC), interstitial cells of Cajal (ICC) and fibroblast-like cells. Up to date the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO) and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP resulting in a stronger and a prolonged hyperpolarization in fundus and colon SMC, respectively. Thus, NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from colon clearly reveal the nitrergic IJP to be biphasic resulting from individual inputs of ICC and SMC.
    AJP Gastrointestinal and Liver Physiology 05/2014; 307(1). DOI:10.1152/ajpgi.00082.2014 · 3.74 Impact Factor
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    ABSTRACT: Interaction of different neuromyogenic mechanisms determines colonic motility. In rats, cyclic depolarizations and slow waves generate myogenic contractions of low frequency (LF) and high frequency (HF), respectively. Interstitial cells of Cajal (ICC) located near the submuscular plexus (SMP) generate slow waves. Inhibitory junction potential (IJP) consists on a purinergic fast (IJPf) followed by a nitrergic slow (IJPs) component leading to relaxation. In the present study, we characterized (1) the dynamics of purinergic-nitrergic inhibitory co-transmission and (2) its contribution on prolonged inhibition of myogenic activity. Different protocols of electrical field stimulation (EFS) under different pharmacological conditions were performed to characterize electrophysiological and mechanical responses. Smooth muscle cells (SMCs) in tissue devoid of ICC-SMP had a resting membrane potential (RMP) of -40.7 ± 0.7 mV. Single pulse protocols increased purinergic and nitrergic IJP amplitude in a voltage-dependent manner (IJPfMAX = -26.4 ± 0.6 mV, IJPsMAX = -6.7 ± 0.3 mV). Trains at increasing frequencies enhanced nitrergic (k = 0.8 ± 0.2 s, IJPs∞ = -15 ± 0.5 mV) whereas they attenuated purinergic responses (k = 3.4 ± 0.6 s,IJPf∞ = -8.9 ± 0.6 mV). In tissues with intact ICC-SMP, the RMP was -50.0 ± 0.9 mV and nifedipine insensitive slow waves (10.1 ± 2.0 mV, 10.3 ± 0.5 cpm) were recorded. In these cells, (1) nitrergic and purinergic responses were reduced and (2) slow waves maintained their intrinsic frequency and increased their amplitude under nerve-mediated hyperpolarization. Based on the co-transmission process and consistent with the expected results on RMP, prolonged EFS caused a progressive reduction of LF contractions whereas HF contractions were partially insensitive. In conclusion, inhibitory neurons modulate colonic spontaneous motility and the principles determining post-junctional responses are (1) the frequency of firing that determines the neurotransmitter/receptor involved, (2) the transwall gradient and (3) the origin and nature of each myogenic activity.
    Pflügers Archiv - European Journal of Physiology 03/2014; 466(12). DOI:10.1007/s00424-014-1500-8 · 3.07 Impact Factor
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    ABSTRACT: 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. Human cultured colonic smooth muscle cells were studied using the calcium imaging technique. Microelectrodes and muscle bath experiments were performed in rat colonic strips. Otilonium bromide (OB) concentration dependently inhibited nifedipine-sensitive calcium transients induced by KCl (EC50 = 3.6 μM) and BayK8644 (EC50 = 4.0 μM). All the following experiments were performed in the presence of nifedipine. In HCSMC, carbachol-induced calcium transients were inhibited by OB (EC50 = 8.4 μM). Carbachol evoked 1-a smooth muscle depolarization (10 mV) that was antagonized by 100 μM OB; and 2-a contraction that was inhibited by OB (EC50 = 13.0 μM). 'Non-nitrergic (L-NNA 1 mM) non-purinergic (MRS2500 1 μM)' conditions were used to elicit endogenous excitatory responses. Electrical field stimulation caused 1-an atropine-sensitive excitatory junction potential that was inhibited by OB (EC50 = 8.9 μM) and 2-an atropine-sensitive contraction that was inhibited by OB (EC50 = 7.3 μM). In HCSMC, neurokinin A (NKA) and CaCl2 induced calcium transients that were inhibited by OB (NKA: EC50 = 11.7 μM; CaCl2 : EC50 = 17.5 μM). Otilonium bromide causes inhibition of L-/T-type calcium channels, muscarinic, and tachykininergic responses that acting together explain the pharmacological properties of the compound.
    Neurogastroenterology and Motility 08/2013; 25(12). DOI:10.1111/nmo.12206 · 3.42 Impact Factor
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    ABSTRACT: PURPOSE: The 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. METHODS: Muscle 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. RESULTS: The 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. CONCLUSIONS: Sigmoid 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.
    International Journal of Colorectal Disease 05/2013; 28(10). DOI:10.1007/s00384-013-1716-7 · 2.42 Impact Factor
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    ABSTRACT: Background  Pharmacological studies using selective P2Y(1) antagonists, such as MRS2500, and studies with P2Y(1) (-/-) knockout mice have demonstrated that purinergic neuromuscular transmission is mediated by P2Y(1) receptors in the colon. The aim of the present study was to test whether P2Y(1) receptors are involved in purinergic neurotransmission in the antrum and cecum. Methods  Microelectrode recordings were performed on strips from the antrum and cecum of wild type animals (WT) and P2Y(1) (-/-) mice. Key Results  In the antrum, no differences in resting membrane potential and slow wave activity were observed between groups. In WT animals, electrical field stimulation elicited a MRS2500-sensitive inhibitory junction potential (IJP). In P2Y(1) (-/-) mice, a nitrergic IJP (N(ω) -nitro-l-arginine-sensitive), but not a purinergic IJP was recorded. This IJP was equivalent to the response obtained in strips from WT animals previously incubated with MRS2500. Similar results were obtained in the cecum: 1- the purinergic IJP (MRS2500-sensitive) recorded in WT animals was absent in P2Y(1) (-/-) mice 2- nitrergic neurotransmission was preserved in both groups. Moreover, 1- spontaneous IJP (MRS2500-sensitive) could be recorded in WT, but not in P2Y(1) (-/-) mice 2- MRS2365 a P2Y(1) agonist caused smooth muscle hyperpolarization in WT, but not in P2Y(1) (-/-) animals, and 3- β-NAD caused smooth muscle hyperpolarization both in WT and P2Y(1) (-/-) animals. Conclusions & Inferences  1- P2Y(1) receptor is the general mechanism of purinergic inhibition in the gastrointestinal tract, 2- P2Y(1) (-/-) mouse is a useful animal model to study selective impairment of purinergic neurotransmission and 3- P2Y(1) (-/-) mouse might help in the identification of purinergic neurotransmitter(s).
    Neurogastroenterology and Motility 01/2013; 25(3). DOI:10.1111/nmo.12060 · 3.42 Impact Factor
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    ABSTRACT: BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2) S) is an endogenous gaseous signalling molecule with putative functions in gastrointestinal motility regulation. Characterization of H(2) S effects on colonic motility is crucial to establish its potential use as therapeutic agent in the treatment of colonic disorders. EXPERIMENTAL APPROACH: H(2) S effects on colonic motility were characterized using video recordings and construction of spatio-temporal maps. Microelectrode and muscle bath studies were performed to investigate the mechanisms underlying H(2) S effects. Sodium hydrosulphide (NaHS) was used as H(2) S source. KEY RESULTS: Rhythmic propulsive motor complexes (RPMCs) and ripples were observed in colonic spatio-temporal maps. Serosal addition of NaHS concentration-dependently inhibited RPMCs. In contrast, NaHS increased amplitude of the ripples without changing their frequency. Therefore, ripples became the predominant motor pattern. Neural blockade with lidocaine inhibited RPMCs, which were restored after administration of carbachol. Subsequent addition of NaHS caused RPMCs inhibition. Luminal addition of NaHS did not modify motility patterns. NaHS inhibited cholinergic excitatory junction potentials, carbachol-induced contractions and hyperpolarized smooth muscle cells but did not modify slow wave activity. CONCLUSIONS AND IMPLICATIONS: H(2) S is able to modulate colonic motility inhibiting propulsive contractile activity and enhancing the amplitude of ripples, promoting mixing. Muscle hyperpolarization and inhibition of neurally-mediated cholinergic responses contribute to the inhibitory effect on propulsive activity. H(2) S effects are not related to changes in the frequency of slow wave activity originating in the ICC network located near the submuscular plexus. Luminal H(2) S does not modify colonic motility probably because of epithelial detoxification.
    British Journal of Pharmacology 01/2013; DOI:10.1111/bph.12100 · 4.99 Impact Factor
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    ABSTRACT: Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 μM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 μM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 μM), ODQ (10 μM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 μM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 μM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 μM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.
    AJP Gastrointestinal and Liver Physiology 05/2012; 303(3):G412-23. DOI:10.1152/ajpgi.00040.2012 · 3.74 Impact Factor
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    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.
    The Journal of Physiology 02/2012; 590(Pt 8):1943-56. DOI:10.1113/jphysiol.2011.224345 · 4.54 Impact Factor
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    ABSTRACT: 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.
    Frontiers in Neuroscience 08/2011; 5:93. DOI:10.3389/fnins.2011.00093
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    ABSTRACT: In the present study, we further characterize the purinergic receptors mediating the inhibitory junction potential (IJP) and smooth muscle relaxation in the human colon using a new, potent and selective agonist (MRS2365), and antagonists (MR2279 and MRS2500) of the P2Y(1) receptor. The P2Y(12) antagonist AR-C66096 was tested as well. Using this pharmacological approach, we tested whether β-nicotinamide adenine dinucleotide (β-NAD) fulfilled the criteria to be considered an inhibitory neurotransmitter in the human colon. We carried out muscle bath and microelectrode experiments on circular strips from the human colon and calcium imaging recordings on HEK293 cells, which constitutively express the human P2Y(1) receptor. Both the fast component of IJP and non-nitrergic relaxation was concentration-dependently inhibited by MRS2279 and MRS2500. This antagonism was confirmed in HEK293 cells. However, AR-C66096 did not modify either inhibitory response. Adenosine 5'-Ο-2-thiodiphosphate and MRS2365 caused a smooth muscle hyperpolarization and transient inhibition of spontaneous motility that was antagonized by MRS2279 and MRS2500. β-Nicotinamide adenine dinucleotide inhibited the spontaneous motility (IC(50) = 3.3 mmol L(-1) ). Nevertheless, this effect was not antagonized by high concentrations of P2Y(1) antagonists. 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.
    Neurogastroenterology and Motility 05/2011; 23(8):792-e338. DOI:10.1111/j.1365-2982.2011.01725.x · 3.42 Impact Factor
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    ABSTRACT: The 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 γ-lyase (CSE), amino-oxyacetic acid (AOAA) and hydroxylamine (HA), inhibitors of cystathionine β-synthase (CBS) on rat colonic motility. Immunohistochemistry, 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. CSE and CBS were immunolocalized in the colon. H₂S was endogenously produced (15.6 ± 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 µM) or N(ω) -nitro-L-arginine (L-NNA, 1 mM). AOAA (2 mM) caused a transient (10 min) increase in motility. In contrast, HA (10 µM) caused a 'nitric oxide-like effect', smooth muscle hyperpolarization and relaxation, which were antagonized by 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 10 µM). Neither spontaneous nor induced inhibitory junction potentials were modified by AOAA or PAG. We 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 ight be a third gaseous inhibitory signalling molecule in the gastrointestinal tract. However, possible non-specific effects of the inhibitors should be considered.
    British Journal of Pharmacology 04/2011; 164(2b):485-98. DOI:10.1111/j.1476-5381.2011.01431.x · 4.99 Impact Factor
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    ABSTRACT: 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. Rat IAS strips were studied using organ baths, microelectrodes, and immunohistochemistry. Internal anal sphincter strips developed active myogenic tone (0.31 g), enhanced and stabilized by prostaglandin F(2α) (PGF2α). L-NNA (1 mmol L(-1)) depolarized SMC and increased tone but did not modify sIJP. In contrast, the specific P2Y(1) 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. P2Y(1) receptors were immunolocalized in the circular SMC of IAS. The effects of inhibitory MNs on rat IAS are mediated by a functional co-transmission process involving nitrergic and purinergic pathways through P2Y(1) receptors with specific and complementary roles on the control of tone, sIJP, and hyperpolarization and relaxation of IAS following stimulation of inhibitory MNs.
    Neurogastroenterology and Motility 10/2010; 23(1):e11-25. DOI:10.1111/j.1365-2982.2010.01602.x · 3.42 Impact Factor
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    ABSTRACT: 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.
    AJP Gastrointestinal and Liver Physiology 07/2010; 299(1):G158-69. DOI:10.1152/ajpgi.00448.2009 · 3.74 Impact Factor
  • Gastroenterology; 05/2010
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    ABSTRACT: 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 P2Y(1) antagonists. 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). N(omega)-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 P2Y(1) 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 P2Y(1) 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-alpha]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 P2Y(1) antagonists. The co-transmission process in the rat colon involves ATP and NO. P2Y(1) receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y(1) receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y(1) receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.
    British Journal of Pharmacology 11/2009; 158(6):1641-52. DOI:10.1111/j.1476-5381.2009.00454.x · 4.99 Impact Factor
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    ABSTRACT: 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 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), 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 microM). 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 microM), MRS 2179 (1 microM), and NF023 (10 microM). 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).
    AJP Gastrointestinal and Liver Physiology 08/2008; 295(3):G522-33. DOI:10.1152/ajpgi.00510.2007 · 3.74 Impact Factor

Publication Stats

247 Citations
74.51 Total Impact Points

Institutions

  • 2008–2015
    • Autonomous University of Barcelona
      • Department of Cellular Biology, Physiology and Immunology
      Cerdanyola del Vallès, Catalonia, Spain
  • 2013
    • McMaster University
      • Department of Medicine
      Hamilton, Ontario, Canada
  • 2012
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain
  • 2010
    • Instituto de Salud Carlos III
      Madrid, Madrid, Spain