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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. Methods Microelectrode recordings were performed on strips from the antrum and cecum of wild type animals (WT) and P2Y1−/− 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 P2Y1−/− mice, a nitrergic IJP (Nω-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 P2Y1−/− mice 2- nitrergic neurotransmission was preserved in both groups. Moreover, 1- spontaneous IJP (MRS2500-sensitive) could be recorded in WT, but not in P2Y1−/− mice 2- MRS2365 a P2Y1 agonist caused smooth muscle hyperpolarization in WT, but not in P2Y1−/− animals, and 3- β-NAD caused smooth muscle hyperpolarization both in WT and P2Y1−/− animals. Conclusions & Inferences 1- P2Y1 receptor is the general mechanism of purinergic inhibition in the gastrointestinal tract, 2- P2Y1−/− mouse is a useful animal model to study selective impairment of purinergic neurotransmission and 3- P2Y1−/− mouse might help in the identification of purinergic neurotransmitter(s).

Background 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 P2Y1 receptor. The P2Y12 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. Methods 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 P2Y1 receptor. Key Results 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 (IC50 = 3.3 mmol L−1). Nevertheless, this effect was not antagonized by high concentrations of P2Y1 antagonists. Conclusions & Inferences Inhibitory purinergic neuromuscular transmission in the human colon was pharmacologically assessed by the use of new P2Y1 receptor antagonists MRS2179, MRS2279, and MRS2500. The rank order of potency of the P2Y1 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.