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

Abstract

Key points beta-Nicotinamide adenine dinucleotide (beta-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 alpha (PDGFR alpha(+)cells). Measuring metabolism of 1,N-6-etheno-NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFR alpha(+)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 PDGFR alpha(+)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 PDGFR alpha(+)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. Prior studies suggest that beta-nicotinamide adenine dinucleotide (beta-NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of beta-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 alpha (PDGFR alpha(+)cells). We examined possible specialized functions of the NEJ in beta-NAD metabolism by determining the degradation of 1,N-6-etheno-NAD (eNAD) in colonic tunica muscularis of wild-type,Cd38(-/-),Nt5e(-/-),Enpp1(-/-)andCd38(-/-)/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. PDGFR alpha(+)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 > PDGFR alpha(+)cells. Our data show that extracellular metabolism of beta-NAD in the colon is mediated by multiple enzymes with cell-specific expression.