生理学报Acta Physiologica Sinica, August

研究论文

铃蟾肽介导的豚鼠肠系膜下神经节非胆碱能迟慢兴奋性突触后电位

孔德虎*, 王刚, 王宏梅,柯道平,胡金兰, 祝延, 黄振信

安徽医科大学生理学教研室神经生理实验室, 合肥 230032

摘要: 应用细胞内记录技术, 对铃蟾肽(bombesin, BOM)在豚鼠离体肠系膜下神经节(inferior mesenteric ganglion, IMG)非胆碱能兴奋性突触传递中的作用进行了研究。重复电刺激突触前结肠神经, 有74.3% (52/70) IMG细胞可诱发迟慢兴奋性突触后电位(ls-EPSP)。在可引出ls-EPSP 的细胞中, 22% (4/18)细胞同时对BOM和SP敏感。用BOM持续灌流IMG, 可明显抑制对BOM敏感细胞的ls-EPSP, 对BOM不敏感细胞的ls-EPSP则无影响, 且BOM受体与SP受体间无交叉脱敏。BOM受体阻断剂tyr4[D-phe12]bombesin能明显可逆性地抑制BOM敏感细胞的ls-EPSP和去极化, 但对BOM不敏感细胞则无影响。研究结果提示, BOM可能是介导豚鼠IMG 细胞ls-EPSP的一种递质。

关键词: 铃蟾肽; P物质; 迟慢兴奋性突触后电位; 肠系膜下神经节; 豚鼠

中图分类号: Q421

Bombesin-mediated non-cholinergic late slow excitatory postsynaptic potentials in guinea pig inferior mesenteric ganglion in vitro

KONG De-Hu*, WANG Gang, WANG Hong-Mei, KE Dao-Ping, HU Jin-Lan, ZHU Yan, HUANG Zhen-Xin

Laboratory of Neurophysiology, Department of Physiology, Anhui Medical University, Hefei 230032

Abstract: The effect of bombesin (BOM) on non-cholinergic excitatory synaptic transmission of the guinea pig inferior mesenteric ganglion (IMG) was investigated by intracellular recording. Repetitive stimulation of the colon nerves (1 ms, 25 Hz, 4 s) elicited a burst of action potentials, which was followed by a long-lasting depolarization in 74.3% (52/70) of the IMG neurons. The depolarization was not blocked by nicotinic (d-tubocurarine, 100 μmol/L) and muscarinic (atropine, 1 μmol/L) antagonists, but was eliminated in a low Ca2+/high Mg2+ Krebs solution, indicating that the depolarization was due to the release of non-cholinergic transmitters. Superfusing the ganglia with BOM (10 μmol/L, 1 min) induced a slow depolarization in 66.5% (109/164) neurons tested. The BOM response was not appreciably changed in low Ca2+/high Mg2+ Krebs solution (n=6, P>0.05), suggesting that BOM depolarized the neurons by acting directly on the postsynaptic membrane rather than via a release of other endogenous depolarizing substances. In a total of 102 cells that exhibited late slow excitatory postsynaptic potential (ls-EPSP), superfusion of the ganglia with BOM produced a membrane depolarization in 82 neurons (80%), while the remaining 20 cells (20%) exhibited no response to BOM. In 18 neurons with ls-EPSP, 4 (22%) neurons were sensitive to both BOM and SP; 6 (33%) and 5 (28%) neurons were only sensitive to BOM and SP, respectively. The remaining 3 (17%) neurons were insensitive to both BOM and SP. Membrane resistance (Rm) had no apparent change in 47.3%, 59.5 % of the neurons tested during the ls-EPSP (n=55) and BOM depolarization (n=84), respectively, but had a marked decrease in 38.2%, 27.4%, and a marked increase in the remaining 14.5%, 13.1% of the neurons. However, when the Rm change accompanying ls-EPSP was compared with that accompanying BOM depolarization (n=20) in the same neuron, the changes in Rm were always parallel. Moreover, ls-EPSP (n=6) and BOM depolarization (n=8) were all augmented by conditioning hyperpolarization. The extrapolated values of the reversal potentials of ls-EPSP and BOM depolarization were －46.0±8.0 and －50.0±7.0 mV (n=8, P>0.05), respectively. In 14 BOM-sensitive neurons, a ls-EPSP was elicited by repetitive colon nerve stimulation. Superfusion of BOM (10 μmol/L) in these cells initially caused a large depolarization and then membrane potential gradually subsided to resting level in the continuous presence of BOM. Stimulation of the presynaptic nerves at this time failed to elicit a detecable ls-EPSP in 2 neurons and induced a much smaller one in 10 cells, while the ls-EPSP in the remaining 2 neurons was not appreciably affected. On the other hand, prolonged superfusion of BOM had no effect on the amplitude and duration of ls-EPSP in 6 BOM-insensititive neurons studied (P>0.05). The amplitude and duration of SP-induced depolarization were not altered by prolonged superfusion of BOM (n=4, P>0.05). Superfusion of tyr4[D-phe12]bombesin (1 μmol/L, 10－15 min), a BOM receptor antagonist, did not cause any noticeable changes in passive membrane properties nor block nicotinic f-EPSPs, but markedly supressed (n=5) or completely abolished (n=11) BOM depolarization in all 16 neurons tested. Similarly, tyr4[D-phe12]bombesin partially or completely antagonized the ls-EPSP in 9 out of a total of BOM sensitive neurons (n=11). The ls-EPSP elicited in the remaining two neurons was insignificantly affected by this drug. However, following 10－20 min of wash with Krebs solution the ls-EPSP was reversed. In contrast, superfusion of the ganglia with tyr4[D-phe12]bombesin did not change the amplitude and duration (P>0.05) of ls-EPSP in 10 BOM-insensitive cells. Similarly, the amplitude and duration of SP-induced depolarization were not appreciably affected by tyr4[D-phe12]bombesin (n=6, P>0.05). In conclusion, our results indicate that BOM may be another transmitter mediating the ls-EPSP in the guinea pig IMG and that there is no cross-desensitization of BOM receptors and SP receptors.

Key words: bombesin; substance P; late slow excitatory postsynaptic potential; inferior mesenteric ganglion; guinea pig

我们及一些学者以往的研究表明, 重复电刺激突触前神经或扩张结肠可在豚鼠肠系膜下神经节(inferior mesenteric ganglion, IMG) 细胞诱发一种不同于胆碱能兴奋性突触后电位(fast excitatory postsynaptic potentials, f-EPSPs) 的非胆碱能慢去极化电位, 即迟慢兴奋性突触后电位(late slow EPSP, ls-EPSP)[1,2]。对其发生机制的分析, 先后有学者报道, 一些神经肽和单胺类递质如P物质(SP)[1]、 胆囊收缩素(CCK)[2]、 神经激肽A (NKA)[3]、 血管活性肠肽 (VIP)[2]、 血管升压素[4]和5-羟色胺 (5-HT)[5]等可能参与豚鼠IMG细胞ls-EPSP的形成。然而, 我们的研究提示, 尚有部分IMG细胞的ls-EPSP对上述神经递质并不敏感, 推测可能还有其它递质参与ls-EPSP的形成。最近我们发现, 铃蟾肽(bombesin, BOM)可易化大多数IMG细胞的突触传递, 免疫组织化学研究亦提示IMG中分布大量BOM免疫反应阳性纤维, 推测BOM可能作为一种递质参与豚鼠IMG细胞ls-EPSP的形成[6,7]。本研究应用离体神经节细胞内记录, 观察BOM对豚鼠IMG细胞ls-EPSP的影响, 分析其作用机制, 并比较BOM和已确定的介导ls-EPSP的递质SP间的相互关系。

1材料和方法

1.1 神经节标本制备

实验用豚鼠60只(200－300 g), 雌雄不拘。击豚鼠后脑致昏迷并经颈总动脉放血处死, 剖开腹腔, 摘取IMG及其相连的腹下神经(HGN)、 内脏神经(SN)和结肠神经(CN), 随即移入灌流-记录浴槽, 以95% O2与5% CO2饱和Krebs液(35℃, pH 7.4±0.05)持续恒速灌流(3 ml/min)[1]。 在灌流液中常规加入阿托品(浓度为1 μmol/L), 以阻断可能存在的M型胆碱能慢突触传递。有些实验还在灌流液中加入低钙(0.25 mmol/L)/高镁(12 mmol/L) Krebs液以阻断神经元的突触传递。

1.2 细胞内记录

在解剖显微镜下将内充3 mol/L KCl溶液、 尖端阻抗为30－60 MΩ玻璃微电极刺入IMG细胞。生物电信号经微电极引导, 输入微电极放大器(WPI-707A)后, 分别输送到双线示波器(SS-5702, IWATSU)及二道生理记录仪(LMS-2B型, 成都仪器厂)进行监测与记录。微电极放大器由三通道刺激器与隔离器(SS-8301, 南通电声总厂)提供方波直流电, 经微电极向细胞内注射超极化方波电流(0.05－0.4 nA), 可引起超极化电紧张电位, 测量其电位幅度, 并根据欧姆定律R=V/I即可算出细胞膜电阻。实验中, 经吸引电极持续4 s电刺激(1 ms, 25 V, 25 Hz) 突触前神经以诱发突触后电位。

1.3 药物

BOM、 SP、 tyr4[D-phe12]bombesin (BOM受体阻断剂)、 筒箭毒、 阿托品等药物均来源于Sigma公司, 这些试剂使用时分别用克氏液配成所需的浓度, 经浴槽内灌注给药(3 ml/min) 。

1.4 统计学处理

实验数据以mean±SE表示, 组间比较用Student 配对t检验。

2 结果

2.1 非胆碱能ls-EPSP和BOM去极化反应

重复电刺激突触前神经在60% (69/115)的IMG细胞上引出一串动作电位, 随后即出现缓慢持久的去极化反应(图1)。此去极化不被N型受体拮抗剂筒箭毒(d-TC, 100 μmol/L)阻断, 但低钙/高镁Krebs液灌流可消除这种反应 (n=20), 表明此反应可能是由某种非胆碱能神经递质的释放引起的。刺激结肠神经(colon nerves, CN) (n=70)、 腹下神经 (hypogastric nerves, HN)(n=33)和内脏神经(splanchnic nerves, SN) (n=12), ls-EPSP的出现频率分别为74.3%、 57.6%和50.0% (P>0.05)。重复电刺激CN (1 ms, 25 Hz, 4 s)所引出的ls-EPSP幅度为6.3±0.3 mV, 时程为98.8±5.9 s (n=70), 与刺激HN和SN引出ls-EPSP的幅度、 时程无明显差异。

用BOM灌流IMG (10 μmol/L, 1 min), 有66.5% (109/164)的细胞出现去极化反应, 幅度和时程分别为6.2±0.2 mV、 166±7.2 s。此反应在灌流低钙/高镁Krebs液时无显著改变(n=6, P>0.05), 提示BOM可能直接作用于突触后膜而非通过释放其它内源性物质间接发挥作用。在102个诱发ls-EPSP的细胞中, 用BOM灌流, 有82个细胞(80%) 产生去极化, 剩余20个细胞(20%) 对BOM无反应。在18个可引出ls-EPSP的细胞中, 发现4个(22%) 同时对BOM和SP敏感, 单独对BOM、 SP敏感的细胞分别为6个(33%)和5个(28%), 3个细胞(17%)对BOM和SP都不敏感。

刺激突触前神经诱导的ls-EPSP和BOM引起的去极化反应均不同程度地伴随膜电阻(Rm)的变化, 在刺激神经诱发ls-EPSP的55个细胞和用BOM灌流引起去极化的84个细胞中, Rm无明显变化的分别为 47.3%和59.5%; Rm显著减小的分别为38.2%和27.4%; Rm显著增加的则分别为14.5%和13.1%。在20个细胞中, 刺激突触前神经诱发的ls-EPSP和BOM引起的去极化反应, 膜电阻的变化基本一致(图1)。在膜电位向超极化方向变动时, ls-EPSP (n=6)和BOM 去极化 (n=8) 的幅度呈增大的趋势(图2)。经测定, ls-EPSP和BOM去极化的逆转电位分别是－46.0±8.0 mV和－50.0±7.0 mV (n=8, P>0.05)。

图1.刺激结肠神经诱发的ls-EPSP和BOM引起的去极化以及伴随的膜电阻变化

Fig. 1.Changes in membrane potential and Rm following repetitive nerve stimulation and BOM application. Rm changes were monitored by hyperpolarizing electrotonic potentials (small downward deflections) induced by hyperpolarizing pulse of 100 ms duration (not shown). Repetitive colon nerve stimulation (25 Hz, 4 s, arrow) elicited a burst of action potential and an ls-EPSP and the following superfusion of BOM evoked a slow depolarization.

A: Rm is constant;B: Rm decreased; C: Rm increased recorded from the same neuron. ↑, repetitive stimulation of CN; —, superfusion marker.

图2.条件超极化对BOM去极化、 ls-EPSP的影响

Fig. 2.Parallel increase in the amplitude of ls-EPSP and BOM depolarization upon conditioned membrane hyperpolarization. A and B show BOM depolarization and ls-EPSP respectively, from 2 different neurons. Membrane potential levels are indicated on the left part of each tracing.

图3.BOM受体失敏对ls-EPSP的影响

Fig. 3.Desensitization of BOM receptors and its effects on ls-EPSP.

A: Superfusion of BOM (10 μmol/L, 1 min) elicited a slow depolarization. Repetitive stimulation of the colon nerve (25 Hz, 4 s, arrow) elicited a burst of action potentials followed by an ls-EPSP.

B: After prolonged superfusion of BOM for 10 min, BOM depolarization was markedly attenuated, and the ls-EPSP also decreased in both duration and amplititude (P<0.05).

C: After the preparation was washed with Krebs solution, the ls-EPSP and BOM depolarization recovered towards the control level.

2.2 BOM和SP受体的失敏

在14 个对BOM敏感的细胞, 重复电刺激CN可诱发ls-EPSP。先用BOM (10 μmol/L)持续灌流IMG, 使去极化幅度逐渐减低最终消失, 此时再刺激突触前神经, 有2个细胞不能诱发ls-EPSP, 10个细胞引出的ls-EPSP明显变小(图3), 其余2个细胞仍能引出明显的ls-EPSP。灌流BOM前后, ls-EPSP的幅度分别为6.3±0.4和3.9±0.8 mV; 时程为91.2±11.4和52.4±7.6 s (n=14, P<0.05) 。6个BOM不敏感细胞在持续灌流BOM时, ls-EPSP的幅度和时程不受影响(P>0.05) , SP诱发的去极化反应的幅度和时程亦无明显变化 (n=4, P>0.05)(图4) 。同时还发现, 在SP敏感细胞(n=5) 重复电刺激突触前神经, 可诱发ls-EPSP, 用SP (1 μmol/L)持续灌流可消除或显著削弱ls-EPSP (图5)。在SP不敏感细胞(n=2)电刺激神经诱发的ls-EPSP不受持续灌流相同浓度SP的影响。持续灌流SP对BOM引起的去极化幅度和时程也无影响(n=4, P>0.05)(图6)。另有4个对BOM、 SP都不敏感的细胞, 灌流BOM、 SP对电刺激神经诱发的ls-EPSP均无影响。

图4.BOM受体失敏对SP去极化的影响

Fig. 4.Desensitization of BOM receptors and its effects on SP depolarization.

A: In SP-sensitive neurons superfusing SP (1 μmol/L, 1 min) induced a slow depolarization.

B: Superfusion of BOM also elicited a slow depolarization, indicating this neuron is sensitive to both BOM and SP.

C: After prolonged superfusion of BOM, the membrane potential gradually subsided.

D: Suferfusion of SP still resulted in a slow depolarization.

图5.SP受体失敏对ls-EPSP的影响

Fig. 5. Desensitization of SP receptors and its effects on ls-EPSP.

A: Superfusion of SP (1 μmol/L, 1 min) elicited a slow depolarization.

B: Repetitive stimulation of the colon nerve (25 Hz, 4 s, arrow) elicited a burst of action potentials followed by an ls-EPSP.

C: After a prolonged perfusion of SP for 10 min (not shown) the membrane potential gradually subsided.

D: Repetitive stimulation of colon nerve, the ls-EPSP decreased in both duration and amplititude

(P<0.05).

E: After the preparation was washed with Krebs solution, the ls-EPSP recovered towards the control level.

2.3 BOM受体拮抗剂对BOM去极化和ls-EPSP的影响

灌流BOM受体拮抗剂tyr4[D-phe12]bombesin (1 μmol/L) 10－15 min既不影响膜的被动电学特性, 也不能阻断N-AChR介导的f-EPSPs。

在16个被观察的细胞, 它可显著抑制(n=5)或完全消除(n=11)由BOM引起的去极化。在灌流tyr4[D-phe12]bombesin前后, BOM去极化幅度为6.2±0.7和3.8±0.7 mV; 时程为128.9±15.8 和64.6±11.5 s (n=11, P<0.05) 。同样, 在11个BOM敏感细胞中, 9个细胞(82%)的ls-EPSP被tyr4[D-phe12]bombesin部分或完全阻断(图7)。灌流tyr4[D-phe12]bombesin前后, ls-EPSP幅度为6.2±0.6和3.5±0.7 mV; 时程为98.9±14.8 和54.6±11.6 s (n=11, P<0.05)。用Krebs液持续冲洗10－20 min, ls-EPSP可逐渐恢复。在10个对BOM不敏感的细胞, 灌流tyr4[D-phe12]bombesin对ls-EPSP的幅度和时程无影响 (P>0.05) (图8), 对SP诱发的去极化反应的幅度和时程亦无影响 (n=6, P>0.05)。

图6.SP受体失敏对BOM去极化的影响

Fig. 6. Desensitization of SP receptors and its effects on BOM depolariztion.

A: In BOM-sensitive neurons superfusion of BOM (10 μmol/L, 1 min) induced a slow depolarization.

B: Superfusion of SP also elicited a slow depolarization, demonstrating this neuron is sensitive to both BOM and SP.

C: After prolonged superfusion of SP, the membrane potential gradually subsided.

D: Suferfusion of BOM still resulted in a slow depolarization.

图7.BOM受体阻断剂tyr4[D-phe12]bombesin对ls-EPSP的影响

Fig. 7. Inhibitory effect of tyr4[D-phe12]bombesin on ls-EPSP evoked by nerve stimulation.

A: Control, repetitive stimulation of colon nerve elecitied a burst of action potential followed by a ls-EPSP.

B: After prolonged superfusion of the ganglion with tyr4[D-phe12]bombesin, a BOM receptor antagonist, the passive membrane properties were not changed (not shown), but ls-EPSP was markedly suppressed.

C: The depressant effect of tyr4[D-phe12]bombesin was reversible after washing with Krebs solution.

图8.BOM受体阻断剂tyr4[D-phe12]bombesi对BOM不敏感细胞的ls-EPSP的影响

Fig. 8. No effect of tyr4[D-phe12]bombesin on ls-EPSP in a BOM-insensitive neurons. A: Superfusion of BOM (10 μmol/L, 1 min) did not induce a slow depolarization.

B: Repetitive stimulation of colon nerve elecited a burst of action potential followed by a ls-EPSP.

C: Superfusion of tyr4[D-phe12]bombesin for 15 min did not change the ls-EPSP.

3讨论

BOM及其受体广泛分布于哺乳动物外周组织和中枢神经系统。BOM的生物学作用正不断为人们所

认识, 如调节生物节律[8]、 影响摄食行为[9]、 参与应激反应[10]、 影响学习记忆[11] 以及肿瘤形成等多种生理和病理过程[12]。BOM及其受体也存在于肠神经系统 [7, 13, 14], 但对BOM在这些部位的功能和作用方式却少有报道。本文的结果提示, 在IMG细胞上存在BOM受体, BOM可能是介导ls-EPSP的一种递质。

重复电刺激突触前神经可在半数以上的IMG细胞上诱发ls-EPSP, 灌流BOM亦可引起部分IMG细胞去极化, 且在同一细胞上这两种不同反应伴随平行的Rm的改变。在细胞超极化时, 无论是电刺激神经诱发的ls-EPSP, 还是用BOM灌流引起的去极化反应都表现增大效应。这一结果提示Na+、 K+、 Ca2+电导不同程度的增强或相应的改变可能是诱发IMG细胞ls-EPSP和BOM引起的去极化反应的离子机制[6]。Woodruff等[15]报道, BOM兴奋视上核神经元是通过受体途径使细胞膜的K+电导降低和某种阳离子电导的增强实现的。 我们在豚鼠IMG发现, 降低灌流液中Na+浓度, BOM引起的去极化幅度随之降低, 推测在中枢神经系统和肠神经系统由BOM介导的去极化反应可能具有相似的离子机制, 但确切的机制尚需进一步证实。在本实验观察的细胞中, 随ls-EPSP或BOM去极化的发生, Rm表现为增大、 降低或不变, 我们推测, 在不同的神经元所诱发的ls-EPSP或BOM引起的去极化过程中, 膜电导的变化程度是不相同的。

用BOM持续灌流IMG可明显地抑制ls-EPSP, 这与长时程作用所致的受体失敏是一致的, 提示BOM通过与突触后膜相应的受体结合发挥作用; 但持续灌流BOM对SP引起的去极化和BOM不敏感细胞诱发的ls-EPSP却无影响, 说明BOM和SP分别通过各自的受体发挥作用, 两者之间并不存在交叉失敏。这与NKA和SP受体之间的相互作用不同, NKA也是一种介导豚鼠IMG细胞ls-EPSP的递质, Saria等[16]发现NKA受体失敏可显著地影响SP去极化和SP敏感细胞的ls-EPSP, 在NKA受体和SP受体间则存在明显的交互失敏, 因为两者同属速激肽家族。这些报道和我们的研究提示, 介导ls-EPSP的递质间可能在受体交互失敏上存在不同的作用模式, 进一步证明介导ls-EPSP的递质的多样性和彼此间相互作用的复杂性。鉴于tyr4[D-phe12]bombesin可明显地抑制BOM去极化和BOM敏感细胞的ls-EPSP, 对N-AChR介导的f-EPSPs和BOM不敏感细胞的ls-EPSP无明显影响, 提示BOM受体可能存在于对BOM敏感细胞上, 至于由何种亚型的BOM受体参与诱发ls-EPSP和BOM的去极化反应尚待进一步证实。

ls-EPSP形成的机制较为复杂, 介导其形成的递质种类不仅仅限于5-HT、 SP、 CCK、 NKA 、 VIP 、 vasopressin[1－5], 我们的研究表明BOM可能也是介导ls-EPSP的一种递质。将来可能还会发现其它新的递质参与ls-EPSP的形成。这些递质通过何种途径共同影响或调制ls-EPSP的形成, 以及它们之间复杂的交互作用的关系, 值得深入研究。

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