Diadenosine tetraphosphate activates P2Y1 receptors that cause smooth muscle relaxation in the mouse colon.

P2Y1 receptors play an essential role in inhibitory neuromuscular transmission in the gastrointestinal tract. The signalling pathway involves the opening of small conductance calcium activated potassium-channels (Kca2 family) that results in smooth muscle hyperpolarization and relaxation. Inorganic polyphosphates and dinucleotidic polyphosphates are putative neurotransmitters that potentially act on P2Y1 receptors. A pharmacological approach using both orthosteric (MRS2500) and allosteric (BPTU) blockers of the P2Y1 receptor and openers (CyPPA) and blockers (apamin) of Kca2 channels was used to pharmacologically characterise the effect of these neurotransmitters. Organ bath and microelectrodes were used to evaluate the effect of P1,P4-Di (adenosine-5') tetraphosphate ammonium salt (Ap4A), inorganic polyphosphates (PolyP) and CyPPA on spontaneous contractions and membrane potential of mouse colonic smooth muscle cells. PolyP neither modified contractions nor membrane potential. In contrast, Ap4A caused a concentration-dependent inhibition of spontaneous contractions reaching a maximum effect at 100 µM Ap4A response was antagonised by MRS2500 (1 µM), BPTU (3 µM) and apamin (1 µM). CyPPA (10 µM) inhibited spontaneous contractions and this response was antagonised by apamin but it was not affected by MRS2500 or BPTU. Both CyPPA and Ap4A caused smooth muscle hyperpolarization that was blocked by apamin and MRS2500 respectively. We conclude that Ap4A but not PolyP activates P2Y1 receptors causing smooth muscle hyperpolarization and relaxation. Ap4A signalling causes activation of Kca2 channels through activation of P2Y1 receptors. In contrast, CyPPA acts directly on Kca2 channels. Further studies are needed to evaluate if dinucleotidic polyphosphates are released from inhibitory motor neurons.

Mecanismos responsables de la relajación neuromuscular en el tracto gastrointestinal / Mechanisms responsible for neuromuscular relaxation in the gastrointestinal tract

El sistema nervioso entérico (SNE) es responsable de la génesis de los patrones motores que aseguran un correcto tránsito intestinal. Las neuronas entéricas se clasifican en aferentes, interneuronas y motoneuronas, que pueden a su vez ser excitatorias, causando contracción, o inhibitorias, provocando la relajación de la musculatura lisa. Los mecanismos de relajación muscular son claves para entender procesos fisiológicos como la relajación de los esfínteres, la acomodación gástrica o la fase descendente del reflejo peristáltico. El óxido nítrico (NO) y el ATP o una purina relacionada son los principales neurotransmisores inhibitorios. Las neuronas nitrérgicas sintetizan NO a partir del enzima nNOS. El NO difunde a través de la membrana celular uniéndose a su receptor, la guanilil ciclasa, y activando posteriormente una serie de mecanismos intracelulares que provocan finalmente una relajación muscular. El ATP actúa como neurotransmisor inhibitorio junto con el NO y el receptor de membrana purinérgico P2Y1 ha sido identificado como elemento clave para entender cómo el ATP relaja la musculatura intestinal. Aunque probablemente ningún clínico duda de la importancia del NO en la fisiopatología motora digestiva, la relevancia de la neurotransmisión purinérgica es aparentemente mucho menor puesto que el ATP no ha sido todavía asociado a una disfunción motora concreta. El objetivo de esta revisión es mostrar el funcionamiento de ambos mecanismos de relajación para poder establecer las bases fisiológicas de posibles disfunciones motoras asociadas a la alteración de la relajación intestinal (AU)

The enteric nervous system (ENS) is responsible for the genesis of motor patterns ensuring an appropriate intestinal transit. Enteric neurons are classified into afferent, interneuron, and motoneuron types, with the latter two being further categorized as excitatory or inhibitory, which cause smooth muscle contraction or inhibition, respectively. Muscle relaxation mechanisms are key for the understanding of physiological processes such as sphincter relaxation, gastric accommodation, or descending peristaltic reflex. Nitric oxide (NO) and ATP or a related purine represent the primary inhibitory neurotransmitters. Nitrergic neurons synthesize NO through nNOS enzyme activity. NO diffuses across the cell membrane to bind its receptor, namely, guanylyl cyclase, and then activates a number of intracellular mechanisms that ultimately result in muscle relaxation. ATP acts as an inhibitory neurotransmitter together with NO, and the purinergic P2Y1 membrane receptor has been identified as a key item in order to understand how ATP may relax intestinal smooth muscle. Although, probably, no clinician doubts the significance of NO in the pathophysiology of digestive motility, the relevance of purinergic neurotransmission is apparently much lower, as ATP has not been associated with any specific motor dysfunction yet. The goal of this review is to discuss the function of both relaxation mechanisms in order to establish the physiological grounds of potential motor dysfunctions arising from impaired intestinal relaxation (AU)

BPTU, an allosteric antagonist of P2Y1 receptor, blocks nerve mediated inhibitory neuromuscular responses in the gastrointestinal tract of rodents.

P2Y1 receptors mediate nerve mediated purinergic inhibitory junction potentials (IJP) and relaxations in the gastrointestinal (GI) tract in a wide range of species including rodents and humans. A new P2Y1 antagonist, with a non-nucleotide structure, BPTU, has recently been described using X-ray crystallography as the first allosteric G-protein-coupled receptor antagonist located entirely outside of the helical bundle. In this study, we tested its effect on purinergic responses in the gastrointestinal tract of rodents using electrophysiological and myographic techniques. BPTU concentration dependently inhibited purinergic inhibitory junction potentials and inhibition of spontaneous motility induced by electrical field stimulation in the colon of rats (EC50 = 0.3 µM) and mice (EC50 = 0.06 µM). Mechanical inhibitory responses were also concentration-dependently blocked in the stomach of both species. Compared to MRS2500, BPTU displays a lower potency. In the rat colon nicotine induced relaxation was also blocked by BPTU. BPTU also blocked the cessation of spontaneous contractility elicited by ADPßS and the P2Y1 agonist MRS2365. We conclude that BPTU is a novel antagonist with different structural and functional properties than nucleotidic antagonists that is able to block the P2Y1 receptor located at the neuromuscular junction of the GI tract.

Mechanisms responsible for neuromuscular relaxation in the gastrointestinal tract.

The enteric nervous system (ENS) is responsible for the genesis of motor patterns ensuring an appropriate intestinal transit. Enteric neurons are classified into afferent, interneuron, and motoneuron types, with the latter two being further categorized as excitatory or inhibitory, which cause smooth muscle contraction or inhibition, respectively. Muscle relaxation mechanisms are key for the understanding of physiological processes such as sphincter relaxation, gastric accommodation, or descending peristaltic reflex. Nitric oxide (NO) and ATP or a related purine represent the primary inhibitory neurotransmitters. Nitrergic neurons synthesize NO through nNOS enzyme activity. NO diffuses across the cell membrane to bind its receptor, namely, guanylyl cyclase, and then activates a number of intracellular mechanisms that ultimately result in muscle relaxation. ATP acts as an inhibitory neurotransmitter together with NO, and the purinergic P2Y1 membrane receptor has been identified as a key item in order to understand how ATP may relax intestinal smooth muscle. Although, probably, no clinician doubts the significance of NO in the pathophysiology of digestive motility, the relevance of purinergic neurotransmission is apparently much lower, as ATP has not been associated with any specific motor dysfunction yet. The goal of this review is to discuss the function of both relaxation mechanisms in order to establish the physiological grounds of potential motor dysfunctions arising from impaired intestinal relaxation.

α,ß-meATP mimics the effects of the purinergic neurotransmitter in the human and rat colon.

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 1 µM. In samples from human colon, EFS-induced inhibition of contractions was inhibited by either MRS2500 or apamin (1 µM) but not by iso-PPADS. In both species, α,ß-meATP, a stable analog of ATP, caused inhibition of spontaneous contractions. α,ß-meATP effect was concentration-dependent (EC50: 2.7 µM rat, 4.4 µM human) and was antagonized by either MRS2500 or apamin but unaffected by P2X antagonists. ATP, ADP, ß-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,ß-meATP is due to P2Y1 receptor activation leading the opening of sKca channels. Accordingly, α,ß-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.

Dynamics of inhibitory co-transmission, membrane potential and pacemaker activity determine neuromyogenic function in the rat colon.

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

Purinergic neuromuscular transmission is absent in the colon of P2Y(1) knocked out mice.

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. ß-Nicotinamide adenine dinucleotide (ß-NAD, 1 mm) partially mimicked the effect of MRS2365. We conclude that P2Y(1) receptors mediate purinergic neurotransmission in the gastrointestinal tract and ß-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.