Dual purinergic synaptic transmission in the human enteric nervous system

Abstract

Based on findings in rodents, we sought to test the hypothesis that purinergic modulation of synaptic transmission occurs in the human intestine. Time series analysis of intraneuronal free Ca2+ levels in submucosal plexus (SMP) from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal fluo-4 AM Ca2+ imaging. A 3-s fiber tract stimulation (FTS) was used to elicit a synaptic Ca2+ response. Short-circuit current ( Isc = chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or electrical field stimulation was used to study Isc responses. Ca2+ imaging was done in 1,222 neurons responding to high-K+ depolarization from 61 surgical cases. FTS evoked synaptic Ca2+ responses in 62% of recorded neurons. FTS caused frequency-dependent Ca2+ responses (0.1–100 Hz). FTS Ca2+ responses were inhibited by Ω-conotoxin (70%), hexamethonium (50%), TTX, high Mg2+/low Ca2+ (≤100%), or capsaicin (25%). A P2Y1 receptor (P2Y1R) antagonist, MRS-2179 or PLC inhibitor U-73122, blocked FTS responses (75–90%). P2Y1R-immunoreactivity occurred in 39% of vasoactive intestinal peptide-positive neurons. The selective adenosine A3 receptor (AdoA3R) agonist 2-chloro- N6-(3-iodobenzyl)adenosine-5′- N-methylcarboxamide (2-Cl-IBMECA) caused concentration- and frequency-dependent inhibition of FTS Ca2+ responses (IC50 = 8.5 × 10−8 M). The AdoA3R antagonist MRS-1220 augmented such Ca2+ responses; 2-Cl-IBMECA competed with MRS-1220. Knockdown of AdoA1R with 8-cyclopentyl-3- N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1- N-propyl-xanthine did not prevent 2-Cl-IBMECA effects. MRS-1220 caused 31% augmentation of TTX-sensitive distension Isc responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in human enteric nervous system (huENS). The P2Y1/Gαq/PLC/inositol 1,3,5-trisphosphate/Ca2+ signaling pathway, N-type Ca2+ channels, nicotinic receptors, and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory AdoA3R inhibit nucleotide or cholinergic transmission in the huENS.