Adenosine 5′‐diphosphate‐ribose is a neural regulator in primate and murine large intestine along with β‐NAD⁺

Abstract

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...

Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut

“…However, responses to Up4A were much greater than responses to ADP, suggesting that ADP formation cannot explain the relaxation responses to Up4A. In contrast to Up4A, the relaxation to ADP likely is mediated by receptors in addition to P2Y1R (17).…”

“…Finally, as is consistent with the notion that the smooth muscle relaxation is mediated by P2Y1R and is coupled to the activation of SK channels (13-15), we found that the relaxation response to Up4A in the colon, hyperpolarization responses in intact muscles, and outward currents activated in PDGFRα + cells were inhibited by SK-channel blockers. Up4A was more potent in inducing relaxation of colonic muscles than the well-known P2Y1R agonists ADP and ATP, the recently discovered neurotransmitter candidates NAD + and ADPR (14,15,17), and AMP and adenosine. The responses to even brief applications (i.e., 15-30 s) of Up4A were greater than the responses to equal concentrations of ATP and ADP, suggesting that the weaker effect of ATP and ADP cannot be attributed to ATP and ADP degradation to inactive (or less active) metabolites.…”

“…A reverse-phased gradient liquid chromatography system equipped with a fluorescence detector was used to detect 1,N 6 -etheno nucleotides and nucleosides as described previously (14,15,17). The formation of UTP, UDP, and UMP from eUp4A was detected in the same samples using a DAD (260 nm) connected in sequence to the FLD.…”

“…Enteric neural regulation of GI motility includes motor neurotransmission mediated by inhibitory neurons releasing purines that act via P2Y1 receptors (P2Y1Rs) (13)(14)(15)(16)(17) and apaminsensitive small-conductance Ca 2+ -activated K + (SK) channels (13,14,18,19). ATP (20), NAD + (14,15,21), and adenosine 5′-diphosphate ribose (ADPR) (17) activate P2Y1R and SK channels and might be inhibitory neurotransmitters in the colon (22).…”

Mechanisms responsible for neuromuscular relaxation in the gastrointestinal tract

“…Each of these has greater or lesser affinity for the different purine receptors subtypes (31). It was recently hypothesized that β-NAD (β-nicotinamide adenine dinucleotide), ADPR (ADP ribose) or Up4A (uridine adenosine tetraphosphate) might be (even instead of ATP) the purinergic neurotransmitter in the GI tract (46)(47)(48)(49). However, some experimental data have nuanced these results (43,50-52).…”