Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that ␤-nicotinamide adenine dinucleotide (␤-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of ␤-NAD mimics the endogenous neurotransmitter better than ATP. Levels of ␤-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of ␤-NAD depends on the frequency of nerve stimulation. ␤-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or -conotoxin GVIA. ␤-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous ␤-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to ␤-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2,4-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. ␤-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.enteric nervous system ͉ gastrointestinal motility ͉ P2Y receptor ͉ purinergic neurotransmission ͉ interstitial cells of Cajal

Background & Aims An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5’-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K+ channels. In murine colon ß-nicotinamide adenine dinucleotide (ß-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here ß-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. Methods A small-volume superfusion assay and HPLC with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of ß-NAD, ATP and metabolites. Postjunctional responses to nerve stimulation, ß-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of ß-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts and proteins were assayed by RT-PCR. Results In contrast to ATP, overflow of ß-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by neurotoxins, tetrodotoxin and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous ß-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, PPADS, MRS2179 and MRS2500. ß-NAD activated non-selective cation currents in SMCs, but failed to activate outward currents. Conclusions ß-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current.

Key points• Normal colonic motility is regulated by excitatory and inhibitory motor neurons, and previous studies have shown that both components of neural regulation are important for normal propulsion of colonic contents.• Inhibitory neural control consists of two main components, and the major neurotransmitters have been identified as nitric oxide and purines; we investigated the nature of the receptors responsible for purine inhibitory motor control of the colon using mice with P2Y1 receptors deactivated.• Inhibitory control of the colon by purine neurotransmitters was dramatically decreased in these animals and transit of fecal pellets was delayed.• Inhibitory responses to purine neurotransmission and exogenous β-NAD, a neurotransmitter candidate, were completely abolished in P2Y1 receptor knockouts.• These studies demonstrate the importance of purinergic neural regulation of colonic motility and suggest this form of neural regulation depends upon P2Y1 receptors to receive and transduce inhibitory neural signals.Abstract Activation of enteric inhibitory motor neurons causes inhibitory junctional potentials (IJPs) and muscle relaxation in mammalian gastrointestinal (GI) muscles, including humans. IJPs in many GI muscles are bi-phasic with a fast initial hyperpolarization (fIJP) due to release of a purine neurotransmitter and a slower hyperpolarization component (sIJP) due to release of nitric oxide. We sought to characterize the nature of the post-junctional receptor(s) involved in transducing purinergic neural inputs in the murine colon using mice with genetically deactivated P2ry1. Wild-type mice had characteristic biphasic IJPs and pharmacological dissection confirmed that the fIJP was purinergic and the sIJP was nitrergic. The fIJP was completely absent in P2ry1 −/− mice and the P2Y1 receptor antagonist MRS2500 had no effect on electrical activity or responses to electrical field stimulation of intrinsic nerves in these mice. Contractile experiments confirmed that purinergic responses were abolished in P2ry1 −/− mice. Picospritzing of neurotransmitter candidates (ATP and its primary metabolite, ADP) and β-NAD (and its primary metabolite, ADP-ribose, ADPR) caused transient hyperpolarization responses in wild-type colons, but responses to β-NAD and ADPR were completely abolished in P2ry1 −/− mice. Hyperpolarization and relaxation responses to ATP and ADP were retained in colons of P2ry1 −/− mice. Video imaging revealed that transit of fecal pellets was significantly delayed in colons from P2ry1 −/− mice. These data demonstrate the importance of purinergic neurotransmission in regulating colonic motility and confirm pharmacological experiments suggesting that purinergic neurotransmission is mediated via P2Y1 receptors.

Chemical signaling in autonomic neuromuscular transmission involves agents that function as neurotransmitters and/or neuromodulators. Using high performance liquid chromatography techniques with fluorescence and electrochemical detection we observed that, in addition to ATP and norepinephrine (NE), electrical field stimulation (EFS, 4 -16 Hz, 0.1-0.3 ms, 15 V, 60 -120 s) of isolated vascular and non-vascular preparations co-releases a previously unidentified compound with apparent nucleotide or nucleoside structure. Extensive screening of more than 25 nucleotides and nucleosides followed by detailed peak identification revealed that ␤-nicotinamide adenine dinucleotide (␤-NAD) is released in tissue superfusates upon EFS of canine mesenteric artery (CMA), canine urinary bladder, and murine urinary bladder in the amounts of 7.1 ؎ 0.7, 26.5 ؎ 4.5, and 15.1 ؎ 3.2 fmol/mg of tissue, respectively. Smaller amounts of the ␤-NAD metabolites cyclic adenosine 5-diphosphoribose (cADPR) and ADPR were also present in the superfusates collected during EFS of CMA (2.5 ؎ 0.9 and 5.8 ؎ 0.8 fmol/mg of tissue, respectively), canine urinary bladder (1.8 ؎ 0.5 and 9.0 ؎ 6.0 fmol/mg of tissue, respectively), and murine urinary bladder (1.4 ؎ 0.1 and 6.2 ؎ 2.4 fmol/mg of tissue, respectively). The three nucleotides were also detected in the samples collected before EFS (0.2-1.6 fmol/mg of tissue). Exogenous ␤-NAD, cADPR, and ADPR (all 100 nM) reduced the release of NE in CMA at 16 Hz from 27.8 ؎ 6.0 fmol/mg of tissue to 15.5 ؎ 5.0, 12 ؎ 3.0, and 10.0 ؎ 4.0 fmol/mg of tissue, respectively. In conclusion, we detected constitutive and nerve-evoked overflow of ␤-NAD, cADPR, and ADPR in vascular and non-vascular smooth muscles, ␤-NAD being the prevailing compound. These substances modulate the release of NE, implicating novel nucleotide mechanisms of autonomic nervous system control of smooth muscle.Chemical signaling constitutes a major mechanism in neuroeffector transmission in the central and peripheral nervous systems. Postganglionic nerve terminals, in particular, characteristically release multiple factors upon action potential, a process referred to as plurichemical neurotransmission (1) or co-transmission (2). The evidence is particularly strong for postganglionic sympathetic nerves, which have been shown to co-release ATP, NE, 1 and neuropeptide Y, factors that presently fulfill transmitter criteria (3-6). Postganglionic parasympathetic nerve terminals, on the other hand, release acetylcholine and ATP (2). In addition, neural control of effector cells involves molecules that implement potent excitatory or inhibitory actions at the neuroeffector junction in the absence of stringent evidence for transmitter function. These effects, collectively referred to as neuromodulation, are usually poorly understood, often due to lack of potent and selective antagonists (6); nonetheless, neuromodulation is an important mechanism, as is neurotransmission (7, 8).We report here that postganglionic nerve terminals in several smooth muscle preparatio...

Key points• Normal gastrointestinal activity depends upon orderly movement of nutrients and wastes through the alimentary canal. These movements require coordinated contractions of the muscular wall and regulation by excitatory and inhibitory motor neurons of the enteric nervous system. • We examined the nature of candidate purine neurotransmitters (ATP and β-NAD) and their metabolites (ADP and ADP-ribose) and the effects of these compounds on electrical and mechanical responses of colonic muscles.• After release, ATP and β-NAD were rapidly degraded to ADP and ADP-ribose, suggesting that inhibitory neural responses may include actions of primary transmitters and metabolites.• Metabolites of both neurotransmitter candidates elicited responses similar to responses to inhibitory nerve stimulation. However, only ADP-ribose had pharmacology that mimicked the effects of the endogenous inhibitory neurotransmitter.• These results help us better understand neural regulation of colonic motility and provide new insights about how defects in neural responses might lead to motility disorders such as constipation.Abstract Adenosine 5 -triphosphate (ATP) has long been considered to be the purine inhibitory neurotransmitter in gastrointestinal (GI) muscles, but recent studies indicate that another purine nucleotide, β-nicotinamide adenine dinucleotide (β-NAD + ), meets preand postsynaptic criteria for a neurotransmitter better than ATP in primate and murine colons. Using a small-volume superfusion assay and HPLC with fluorescence detection and intracellular microelectrode techniques we compared β-NAD + and ATP metabolism and postjunctional effects of the primary extracellular metabolites of β-NAD + and ATP, namely ADP-ribose (ADPR) and ADP in colonic muscles from cynomolgus monkeys and wild-type (CD38 +/+ ) and CD38 −/− mice. ADPR and ADP caused membrane hyperpolarization that, like nerve-evoked inhibitory junctional potentials (IJPs), were inhibited by apamin. IJPs and hyperpolarization responses to ADPR, but not ADP, were inhibited by the P2Y1 receptor antagonist (1R,2S,4S,5S)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0] hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500). Degradation of β-NAD + and ADPR was greater per unit mass in muscles containing only nerve processes than in muscles also containing myenteric ganglia. Thus, mechanisms for generation of ADPR from β-NAD + and for termination of the action of ADPR are likely to be present near sites of neurotransmitter release. Degradation of β-NAD + to ADPR and other metabolites appears to be mediated by pathways besides CD38, the main NAD-glycohydrolase in mammals. Degradation of β-NAD + and ATP were equal in colon. ADPR like its precursor, β-NAD + , mimicked the effects L. Durnin and S. J. Hwang contributed equally to this study. of the endogenous purine neurotransmitter in primate and murine colons. Taken together, our observations support a novel hypothesis in which multiple purines contribute to enteric inhibitory regula...