Baker Salah A, Hennig Grant W, Ward Sean M, Sanders Kenton M
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
J Physiol. 2015 Apr 15;593(8):1945-63. doi: 10.1113/jphysiol.2014.287599. Epub 2015 Feb 23.
Platelet derived growth factor receptor α (PDGFRα(+) ) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα(+) cells generate Ca(2+) transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca(2+) transients in PDGFRα(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca(2+) transients evoked by nerve stimulation in PDGFRα(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFRα(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα(+) cells. PDGFRα(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions.
Interstitial cells, known as platelet derived growth factor receptor α (PDGFRα(+) ) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca(2+) transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20 Hz) caused Ca(2+) transients in enteric motor nerve processes and then in PDGFRα(+) cells shortly after the onset of stimulation (latency from EFS was 280 ms at 10 Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca(2+) fluorescence just after the initiation of Ca(2+) transients in PDGFRα(+) cells. Upon cessation of EFS, several fast Ca(2+) transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca(2+) transients evoked in PDGFRα(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca(2+) transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFRα(+) cells expressed gap junction genes, and gap junction uncouplers, 18β-glycyrrhetinic acid (18β-GA) and octanol blocked Ca(2+) transients in SMCs but not in neurons or PDGFRα(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα(+) cells by motor neurons. PDGFRα(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions.
结肠肌肉中的血小板衍生生长因子受体α（PDGFRα(+)）细胞由肠抑制性运动神经元支配。PDGFRα(+)细胞对外源性嘌呤产生Ca(2+)瞬变，这些反应被MRS - 2500阻断。在胆碱能和一氧化氮能成分被阻断的情况下，刺激肠神经元可在PDGFRα(+)和平滑肌细胞（SMC）中诱发Ca(2+)瞬变。MRS - 2500可消除对神经刺激的反应，在P2Y1受体基因失活的肌肉中未观察到这种反应。神经刺激在PDGFRα(+)细胞中诱发的Ca(2+)瞬变与电生理反应具有相同的时间特征。PDGFRα(+)细胞表达缝隙连接基因，抑制缝隙连接的药物可阻断SMC中的神经反应，但对神经纤维或PDGFRα(+)细胞无影响。PDGFRα(+)细胞直接由抑制性运动神经元支配，嘌呤能反应通过缝隙连接传导至SMC。
间质细胞，即血小板衍生生长因子受体α（PDGFRα(+)）细胞，与肠运动神经元的曲张体密切相关，并被认为介导胃肠道（GI）平滑肌中的嘌呤能超极化反应，但这一概念尚未在完整肌肉中得到直接证实。我们使用共聚焦显微镜监测外源性嘌呤或肠神经元电场刺激（EFS）诱发的小鼠结肠神经元和节后细胞中的Ca(2+)瞬变。EFS（1 - 20Hz）在肠运动神经纤维中引起Ca(2+)瞬变，然后在刺激开始后不久在PDGFRα(+)细胞中引起Ca(2+)瞬变（10Hz时EFS的潜伏期为280ms）。平滑肌细胞（SMC）中的反应通常是在PDGFRα(+)细胞中Ca(2+)瞬变开始后Ca(2+)荧光略有下降。EFS停止后，在SMC中观察到几个快速Ca(2+)瞬变（反弹兴奋）。在通过细胞内微电极记录的PDGFRα(+)细胞中由EFS诱发的Ca(2+)瞬变的时间特征与抑制性连接电位（IJP）之间发现了很强的相关性。EFS诱发的Ca(2+)瞬变和IJP被P2Y1拮抗剂MRS - 2500阻断，在P2ry1((-/-))小鼠中不存在。PDGFRα(+)细胞表达缝隙连接基因，缝隙连接解偶联剂18β - 甘草次酸（18β - GA）和辛醇可阻断SMC中的Ca(2+)瞬变，但对神经元或PDGFRα(+)细胞无影响。从SMC记录的IJP也被阻断。这些发现证明了运动神经元对PDGFRα(+)细胞的直接支配。PDGFRα(+)细胞是肠抑制性神经传递中嘌呤能神经递质的主要靶点。超极化反应通过缝隙连接传导至SMC。
