Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BKCa channels through IP3 pathway
Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BKCa channels through IP3 pathway
复制标题
催产素通过 IP3 途径打开 BKCa 通道，使培养的十二指肠肌间固有初级传入神经元超极化
DOI：
10.1111/j.1471-4159.2012.07702.x
复制
发表时间：
2012-05-01
影响因子：
4.7
通讯作者：
Liu, Chuanyong
中科院分区：
文献类型：
--
作者：
Che, Tongtong;Sun, Hui;Liu, Chuanyong
催产素（OT）在临床上对肠道运动很重要，并组成性地降低十二指肠收缩力。内源性初级传入神经元（intrinsic primary afferent neurons，IPANs）是蠕动反射通路的第一级神经元，其生理分类为AH细胞。我们着手研究这种抑制作用是否由IPAN介导，并确定参与这种作用的离子通道和细胞内信号转导途径。从大鼠十二指肠纵肌肌间神经丛（LMMP）标本中分离肌间神经元，培养1624 h后进行全细胞电生理记录，并根据其电生理特性鉴定AH细胞。采用钙离子成像技术测定神经元胞浆内钙离子浓度（[Ca~（2+）] i）。采用ELISA法测定LMMP中IP 3的浓度和LMMP分泌的OT。催产素受体（OTR）和大电导钙激活钾（BKCa）通道，以及OT和IPAN标志物钙结合蛋白28 K的表达，肌间神经丛神经元上的本地化使用双免疫染色技术。我们发现，管理OT（10 - 7至10 - 5 M）的剂量依赖性超极化的静息膜电位和增加总外向电流。OTR拮抗剂阿托西班或BKCa通道阻断剂伊比利亚毒素（IbTX）阻断OT的作用，表明BKCa通道开放导致外向电流增加。OTR和BKCaa亚基在LMMP的肌间神经元亚群上共表达。NS 1619（10 - 5M，BKCa通道激活剂）增加外向电流，类似于OT的作用。OT管理也增加[Ca 2 +] i和OT诱发的外向电流显着衰减毒胡萝卜素（10 - 6 M）或氯化镉。OT对BKCa电流的作用也可被IP_3受体拮抗剂2-APB（10~（-4）M）或PLC抑制剂U_（73122）（10~（-5）M）阻断。OT（10 - 6 M）也增加了LMMP内的IP3浓度。阿托西班可增强OT的自发分泌和KCl诱导分泌。多数OT阳性细胞也呈钙结合蛋白28K阳性。总之，我们得出结论，OT通过OTR-PLC-IP3-Ca2+信号通路激活BKCa通道使肌间IPAN超极化。OT可能通过自分泌和负反馈方式调节IPAN介导的ENS反射。
Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 1624 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca2+ concentration ([Ca2+]i) of isolated neurons was measured using calcium imaging. The concentration of IP3 in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BKCa) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10-7 to 10-5 M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BKCa channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BKCa channel opening. OTR and the BKCaa subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10-5 M, a BKCa channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca2+]i and the OT-evoked outward current was significantly attenuated by thapsigargin (10-6 M) or CdCl2. The effect of OT on the BKCa current was also blocked by pre-treatment with the IP3 receptor antagonist 2-APB (10-4 M) or the PLC inhibitor U73122 (10-5 M). OT (10-6 M) also increased the IP3 concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BKCa channels via the OTR-PLC-IP3-Ca2+ signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner.