摘要
目的·用电生理学方法证明RIN-14B细胞是一种可用的肠嗜铬细胞(enterochromaffin cells, EC)模型;明确Nav1.3通道在其兴奋性调控中的作用。方法·在传代培养的RIN-14B细胞中,用电流钳记录静息电位,观察给予河豚毒素(tetrodotoxin,TTX)和Nav1.3阻断剂ICA-121431后电位变化;用电压钳记录Na~+电流,观察给予TTX和ICA-121431后电流变化。结果·RIN-14B细胞静息膜电位约为-60 mV,在去极化刺激下发放动作电位。TTX可以完全阻断动作电位,而ICA-121431可以浓度依赖性地抑制动作电位。钠通道的激活电流和失活电流被TTX完全阻断,激活电流被ICA-121431浓度依赖性抑制。结论·RIN-14B细胞是一种可兴奋细胞,TTX敏感型的钠通道介导其动作电位的发放,Nav1.3参与调控其兴奋性,与EC特征相似;说明RIN-14B细胞是一种良好的EC模型。
Objective · To provide electrophysiological evidence for RIN-14B cells as an useful model of enterochromaffin cells(EC) and to study the role of Nav1.3 channel in the control of its excitability. Methods · Resting membrane potential was recorded and the effects of TTX and ICA-121431 were examined by current-clamp in cultured RIN-14B cells. The effects of TTX and ICA-121431 on Na+ current of RIN-14B cells were examined by voltageclamp. Results · RIN-14B cells had a resting potential around-60 mV and fired action potentials when stimulated with depolarizing current pulses. The action potential was completely blocked by TTX and inhibited by ICA-121431 in a dose-dependent manner. TTX blocked activation and inactivation of sodium current. In addition ICA-121431 dose-dependently inhibited activation of Na~+ current. Conclusion · The action potential of RIN-14B cells is induced by TTX-sensitive sodium channel and the excitability is controlled by Nav1.3. These results suggest RIN-14B cells are similar to EC and it may be a good model of EC.
引文
[1]Gershon MD.5-Hydroxytryptamine(serotonin)in the gastrointestinal tract[J].Curr Opin Endocr Diabetes Obes,2013,20(1):14-21.
[2]Mawe GM,Hoffman JM.Serotonin signalling in the gut:functions,dysfunctions and therapeutic targets[J].Nat Rev Gastroenterol Hepatol,2013,10(8):473-486.
[3]Spiller R.Serotonin and GI clinical disorders[J].Neuropharmacology,2008,55(6):1072-1080.
[4]Bellono NW,Bayrer JR,Leitch DB,et al.Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways[J].Cell,2017,170(1):185-198.
[5]Eijkelkamp N,Linley JE,Baker MD,et al.Neurological perspectives on voltage-gated sodium channels[J].Brain,2012,135(9):2585-2612.
[6]Dib-Hajj SD,Black JA,Waxman SG.Voltage-gated sodium channels:therapeutic targets for pain[J].Pain Med,2009,10(7):1260-1269.
[7]Xie MX,Yang J,Pang RP,et al.Bulleyaconitine A attenuates hyperexcitability of dorsal root ganglion neurons induced by spared nerve injury:the role of preferably blocking Nav1.7 and Nav1.3 channels[J].Mol Pain,2018,14:1744806918778491.
[8]Strege PR,Knutson K,Eggers SJ,et al.Sodium channel Na V 1.3 is important for enterochromaffin cell excitability and serotonin release[J].Sci Rep,2017,7(1):15650.
[9]Hampton T.Organoids reveal clues to gut-brain communication[J].JAMA,2017,318(9):787-788.
[10]Schulze A,Hartung P,Schaefer M,et al.Transient receptor potential ankyrin1(TRPA1)channel activation by the thienopyridine-type drugs ticlopidine,clopidogrel,and prasugrel[J].Cell Calcium,2014,55(4):200-207.
[11]Kalbe B,Schlimm M,Mohrhardt J,et al.Helional induces Ca2+decrease and serotonin secretion of QGP-1 cells via a PKG-mediated pathway[J].J Mol Endocr,2016,57(3):201-210.
[12]Li?án-Rico A,Ochoa-Cortes F,Zuleta-Alarcon A,et al.UTP-gated2018-09-30signaling pathways of 5-HT release from BON cells as a model of human enterochromaffin cells[J].Front Pharmacol,2017,8:429.
[13]Wu SV,Yuan PQ,Lai J,et al.Activation of type 1 CRH receptor isoforms induces serotonin release from human carcinoid BON-1N cells:an enterochromaffin cell model[J].Endocrinology,2011,152(1):126-137.
[14]Nozawa K,Kawabata-Shoda E,Doihara H,et al.TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells[J].Proc Natl Acad Sci U S A,2009,106(9):3408-3413.
[15]Suzawa S,Takahashi K,Shimada T,et al.Carbonyl stress-induced5-hydroxytriptamine secretion from RIN-14B,rat pancreatic islet tumor cells,via the activation of transient receptor potential ankyrin 1[J].Brain Res Bull,2016,125:181-186.
[16]Tsuchiya K,Kubota K,Ohbuchi K,et al.Transient receptor potential ankyrin 1agonists improve intestinal transit in a murine model of postoperative ileus[J].Neurogastroenterol Motil,2016,28(12):1792-1805.
[17]Hatakeyama Y,Takahashi K,Tominaga M,et al.Polysulfide evokes acute pain through the activation of nociceptive TRPA1 in mouse sensory neurons[J].Mol Pain,2015,11(1):24.
[18]Wang F,Knutson K,Alcaino C,et al.Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces[J].J Physiol,2017,595(1):79-91.
[19]McCormack K,Santos S,Chapman ML,et al.Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels[J].Proc Natl Acad Sci U S A,2013,110(29):E2724-E2732.
[20]Hains BC,Saab CY,Klein JP,et al.Altered sodium channel expression in second-order spinal sensory neurons contributes to pain after peripheral nerve injury[J].J Neurosci,2004,24(20):4832-4839.
[21]Su S,Shao J,Zhao Q,et al.MiR-30b attenuates neuropathic pain by regulating voltage-gated sodium channel Nav1.3 in rats[J].Front Mol Neurosci,2017,10:126.
