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Extracellular metabolism of the enteric inhibitory neurotransmitter β-nicotinamide adenine dinucleotide (β-NAD) in the murine colon.

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β-Nicotinamide adenine dinucleotide (β-NAD) is a key inhibitory neurotransmitter in the colon. The neuroeffector junction in the gut consists of enteric motor neurons and SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα⁺ cells). Measuring metabolism of 1,N⁶ -etheno-NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFRα⁺ cells with HPLC-FLD, we report that (1) in tissues, eNAD is degraded to eADP-ribose, eAMP and e-adenosine (eADO) by CD38, ENPP1 and NT5E, (2) with SMCs and PDGFRα⁺ cells, eNAD is metabolized to eADO by ENPP1 and NT5E, (3) eNAD is not metabolized by ICC, (4) NT5E is expressed chiefly by SMCs and moderately by PDGFRα⁺ cells, (5) SIP cells are not the primary location of CD38. These data argue that the duration and strength of purinergic neurotransmission can be modulated by targeting multiple enzymes with specialized cellular distribution in the colon.

Abstract

Prior studies suggest that β-nicotinamide adenine dinucleotide (β-NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of β-NAD at the neuroeffector junction (NEJ) is likely to be necessary for terminating inhibitory neurotransmission and may also produce bioactive metabolites. The enteric NEJ consists of enteric neurons and postjunctional cells of the SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα⁺ cells). We examined possible specialized functions of the NEJ in β-NAD metabolism by determining the degradation of 1,N⁶ -etheno-NAD (eNAD) in colonic tunica muscularis of wild-type, Cd38^-/- , Nt5e^-/- , Enpp1^-/- and Cd38^-/- /Nt5e^-/- mice and in SIP cells from mice expressing cell-specific fluorescent reporters purified by fluorescence activated cell sorting (FACS). We measured eNAD and its metabolites eADP-ribose (eADPR), eAMP and e-adenosine (eADO) from tissues and sorted SIP cells using liquid chromatography. eNAD exposed to colonic muscularis of wild-type mice produced eADPR, eAMP and eADO. CD38 mediated the conversion of eNAD to eADPR, whereas ENPP1 mediated degradation of eNAD and eADPR to eAMP. NT5E (aka CD73) was the primary enzyme forming eADO from eAMP. PDGFRα⁺ cells and SMCs were involved in production of eADO from eNAD, and ICC were not involved in extracellular metabolism of eNAD. CD38 mediated the eNAD metabolism in whole tissues, but CD38 did not appear to be functionally expressed by SMCs or ICC. NT5E was expressed in SMCs > PDGFRα⁺ cells. Our data show that extracellular metabolism of β-NAD in the colon is mediated by multiple enzymes with cell-specific expression.

SUBMITTER: Durnin L

PROVIDER: S-EPMC7793634 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 ?-nicotinamide adenine dinucleotide (?-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here ?-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons.A small-volume superfusion assay and high-pressure liquid chromatography with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of ?-NAD, ATP, and metabolites. Postjunctional responses to nerve stimulation, ?-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of ?-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts were assayed by reverse transcription polymerase chain reaction.In contrast to ATP, overflow of ?-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by the neurotoxins, tetrodotoxin, and ?-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous ?-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179), and (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 (MRS 2500). ?-NAD activated nonselective cation currents in SMCs, but failed to activate outward currents.?-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.

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Extracellular metabolism of the enteric inhibitory neurotransmitter β-nicotinamide adenine dinucleotide (β-NAD) in the murine colon.

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