Wunderlich J E, Needleman B J, Chen Z, Yu J G, Wang Y, Grants I, Mikami D J, Melvin W S, Cooke H J, Christofi F L
Department of Anesthesiology, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA.
Am J Physiol Gastrointest Liver Physiol. 2008 Feb;294(2):G554-66. doi: 10.1152/ajpgi.00500.2007. Epub 2007 Dec 13.
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 Ca(2+) levels in submucosal plexus (SMP) from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal fluo-4 AM Ca(2+) imaging. A 3-s fiber tract stimulation (FTS) was used to elicit a synaptic Ca(2+) response. Short-circuit current (I(sc) = chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or electrical field stimulation was used to study I(sc) responses. Ca(2+) imaging was done in 1,222 neurons responding to high-K(+) depolarization from 61 surgical cases. FTS evoked synaptic Ca(2+) responses in 62% of recorded neurons. FTS caused frequency-dependent Ca(2+) responses (0.1-100 Hz). FTS Ca(2+) responses were inhibited by Omega-conotoxin (70%), hexamethonium (50%), TTX, high Mg(2+)/low Ca(2+) (< or = 100%), or capsaicin (25%). A P2Y(1) receptor (P2Y(1)R) antagonist, MRS-2179 or PLC inhibitor U-73122, blocked FTS responses (75-90%). P2Y(1)R-immunoreactivity occurred in 39% of vasoactive intestinal peptide-positive neurons. The selective adenosine A(3) receptor (AdoA(3)R) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IBMECA) caused concentration- and frequency-dependent inhibition of FTS Ca(2+) responses (IC(50) = 8.5 x 10(-8) M). The AdoA(3)R antagonist MRS-1220 augmented such Ca(2+) responses; 2-Cl-IBMECA competed with MRS-1220. Knockdown of AdoA(1)R 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 I(sc) responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in human enteric nervous system (huENS). The P2Y(1)/Galphaq/PLC/inositol 1,3,5-trisphosphate/Ca(2+) signaling pathway, N-type Ca(2+) channels, nicotinic receptors, and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory AdoA(3)R inhibit nucleotide or cholinergic transmission in the huENS.
基于在啮齿动物中的研究发现，我们试图验证嘌呤能对突触传递的调节作用是否发生在人类肠道中。使用蔡司LSM激光扫描共聚焦fluo - 4 AM钙成像技术，对来自Roux - Y手术标本的黏膜下神经丛（SMP）内神经元游离钙离子水平进行时间序列分析。采用3秒的纤维束刺激（FTS）来引发突触钙反应。在通量室中记录黏膜 - SMP中的短路电流（I(sc)=氯离子分泌）。使用扩张反射或电场刺激来研究I(sc)反应。对来自61例手术病例的1222个对高钾去极化有反应的神经元进行了钙成像。FTS在62%的记录神经元中引发了突触钙反应。FTS引起了频率依赖性的钙反应（0.1 - 100 Hz）。FTS钙反应被ω - 芋螺毒素（70%）、六甲铵（50%）、TTX、高镁/低钙（≤100%）或辣椒素（25%）抑制。P2Y(1)受体（P2Y(1)R）拮抗剂MRS - 2179或PLC抑制剂U - 73122阻断了FTS反应（75 - 90%）。P2Y(1)R免疫反应性出现在39%的血管活性肠肽阳性神经元中。选择性腺苷A(3)受体（AdoA(3)R）激动剂2 - 氯 - N(6)-(3 - 碘苄基)腺苷 - 5'-N - 甲基羧酰胺（2 - Cl - IBMECA）引起了浓度和频率依赖性的FTS钙反应抑制（IC(50)=8.5×10(-8) M）。AdoA(3)R拮抗剂MRS - 1220增强了这种钙反应；2 - Cl - IBMECA与MRS - 1220竞争。用8 - 环戊基 - 3 - N-(3 - {[3-(4 - 氟磺酰基)苯甲酰基]-氧基}-丙基)-1 - N - 丙基 - 黄嘌呤敲低AdoA(1)R并不能阻止2 - Cl - IBMECA的作用。MRS - 1220使TTX敏感的扩张I(sc)反应增强了31%。Roux - Y患者的SMP是研究人类肠神经系统（huENS）突触传递的合适模型。P2Y(1)/Gαq/PLC/肌醇1,3,5 - 三磷酸/钙信号通路、N型钙通道、烟碱受体和外在神经参与了huENS中的神经传递。抑制性AdoA(3)R抑制了huENS中的核苷酸或胆碱能传递。
