生理学报, Aug. 2001,
53 (4): 311～315
Acta Physiologica Sinica
研 究 快 报
非洲爪蟾卵母细胞GABAB和GABAC受体介导的电流反应
杨青２, 李之望１, 魏劲波２
(１华中科技大学同济医学院实验医学研究中心,
武汉 430030;２武汉大学医学院生理教研室, 武汉 430071)
摘要: 实验应用双电极电压箝技术, 在具有滤泡膜的非洲爪蟾(Xenopus
laevis)卵母细胞上记录到γ-氨基丁酸(γ-aminobutyric acid, GABA)-激活电流。 此GABA-激活电流的特点及有关GABA受体类型的研究和分析如下:
(1) 在35.5% (55/155)的受检细胞外加GABA可引起一慢的浓度依赖性的外向电流。(2) GABAA受体的选择性拮抗剂bicuculline
(10－５ mol/L)对GABA (10－５ mol/L)引起的外向电流无阻断作用(n=6)。(3)GABAB受体的选择性拮抗剂2-hydroxysaclofen
(10－４ mol/L)能将GABA (10－５ mol/L)引起的外向电流可逆性地转变为内向电流, 后者又可被GABAC受体的选择性拮抗剂I4AA (10－５
mol/L)所消除(n=6)。(4) GABAB受体的特异性激动剂baclofen可引起部分(20%, 12/60)受检细胞产生一慢的浓度依赖性的外向电流。3×10－６、
3×10－５ 及3×10－４ mol/L 2-hydroxysaclofen分别阻断baclofen (10－５ mol/L)-激活电流(6.3±3.2)%,
(44.1±2.2)%及(86.0±1.6)%(n=6)。(5) baclofen激活电流的I-V曲线显示逆转电位在－96.8±7.2 mV左右, 此电流可分别被TEA
(5 mmol/L)和BaCl2 (2 mmol/L)所阻断。以上结果提示: 在非洲爪蟾的卵母细胞上存在内源性GABAB和GABAC受体, GABAB受体介导的为外向电流,
而GABAC受体介导的为内向电流。
关键词: 非洲爪蟾卵母细胞; GABAB受体; GABAC受体;
baclofen; 2-hydroxysaclofen; I4AA;
双电极电压箝
学科分类号: Q424
Current responses mediated by endogenous GABAB and GABAC receptors in Xenopus oocytes
YANG
Qing２, LI Zhi-Wang１，, WEI Jin-Bo２
(１Research
Center of Experimental Medicine, Tongji Medical College,Huazhong University of
Science & Technology, Wuhan 430030; ２Department of Physiology, Medical College
of Wuhan University, Wuhan 430071)
Abstract: The properties of GABA-activated current in Xenopus
oocytes and its underlying mechanism were studied using the two-electrode
voltage-clamp technique. External application of GABA (10－１０～10－３ mol/L) induced
a concentration-dependent outward current in a proportion of oocytes (35.5%,
55/155). Selective GABAA receptor antagonist bicuculline (10－５ mol/L) did not
block the GABA-activated current (n=6). However, 2-hydroxysaclofen (10－４
mol/L), a GABAB receptor antagonist,
reversed the GABA-activated outward current to an inward current (n=9),
which was abolished completely by
application of I4AA (10－５ mol/L), a GABAC receptor selective antagonist
(n=6). In 20% (12/60) of oocytes,
application of baclofen (10－１０～10－４ mol/L), a GABAB receptor agonist,
also induced a concentration-dependent outward current 2-Hydroxysaclofen at the
concentrations of 3×10－６, 3×10－５ and 3×10－４ mol/L blocked the baclofen(10－５ mol/L)-activated outward current
by (6.3±3.2)%, (44.1±2.2)%, and (86.0±1.6)%, respectively (n=6). The reversal
potential for baclofen-activated current was around －96.8±7.2 mV (n=6), and the
baclofen-activated current could be blocked by TEA (n=5) and Ba2+ (n=5). These
results suggest that there exist endogenous GABA receptors, GABAB receptors
mediating a slow and sustained outward current and GABAC receptors mediating an
inward current in follicular Xenopus oocytes.
Key words: Xenopus oocytes; GABAB
receptor; GABAC receptor; baclofen; 2-hydroxysaclofen; I4AA; two-electrode
voltage-clamp recording
非洲爪蟾是一种在南非生长的爪蟾(South-African
clawed toad), 其卵母细胞目前已被广泛用于表达膜通道和受体蛋白基因。同时, 卵母细胞本身也存在一些内源性受体和通道。Kusano等［1］曾报道在具有滤泡膜的非洲爪蟾卵母细胞(follicular
oocytes of Xenopus laevis)上存在乙酰胆碱(ACh)、 儿茶酚胺(CA)、 多巴胺(DA)等内源性受体; 另外, Lotan等［2］和King等［3］先后证明在这种卵母细胞上也存在有嘌呤受体。但是,
Kusano等在具有滤泡膜的非洲爪蟾卵母细胞上未记录到γ-氨基丁酸(GABA)激活电流; 顾全保等［4］证明, 在除去滤泡膜的(defolliculated)中华大蟾蜍卵母细胞上,
较高浓度的GABA也诱导不出膜电流。最近, 我们应用双电极电压箝技术, 在具有滤泡膜的非洲爪蟾卵母细胞上记录到了内源性的GABA激活电流。
本实验的目的是观察非洲爪蟾卵母细胞GABA激活电流的特点,
并分析介导此种电流的GABA受体的类型。
1材料和方法
1.1 卵母细胞标本的制备实验用非洲爪蟾35只(由中国科学院上海细胞生物学研究所提供),
每只非洲爪蟾可循环取卵4～5次。选择一只6周内未被取卵的非洲爪蟾, 冰浴麻醉约30 min, 待其四肢活动丧失后, 将其置于手术器械盘上。在腹部外侧皮肤上作一约1
cm的切口, 然后在肌肉层也剪一同样大小的切口, 拉出子宫瓣, 剪下1～3个卵叶, 将其浸泡于盛有Barth氏液的培养皿中。在体视显微镜下用游丝镊从卵叶分离出单个卵母细胞。将单个卵母细胞置于容积为1
ml的浴槽内, 记录膜电流反应, 浴槽内以Ringer氏液持续灌流。室温保持在20～24℃。Ringer氏液的成分为(mmol/L): NaCl 95,
KCl 1, MgCl2 1, CaCl2 1, HEPES 5。Barth氏液的成分为(mmol/L): NaCl 88, KCl 1, NaHCO3
2.4, Ca(NO3)2 0.32, MgSO4 0.81, CaCl2 0.41, HEPES 5, 庆大霉素100 IU/ml。所有溶液的pH值用1
mol/L NaOH调到7.6。
1.2 电生理记录采用双电极电压箝记录方法。电压电极与电流电极的尖端分别为3～5
μm和5～7 μm, 两电极内分别充以0.15和3 mol/L KCl溶液, 其电阻分别为5～10 和0.1～1 MΩ。参比电极用Ag/AgCl电极与浴槽相连。选择膜静息电位大于-35
mV的卵母细胞进行记录。所用仪器为OC-725 B & C型电压箝放大器(Warner Co, USA) 。箝制电压置于-50 mV。用二道生理记录仪(LMS2B,
成都仪器厂)记录膜电流反应。
1.3 药物 包括bicuculline(Sigma), I4AA
(Sigma), 2-hydroxysaclofen(RBI), baclofen(RBI), GABA (上海试剂三厂), TEA (上海试剂一厂), BaCl2(开封东大化工集团有限公司试剂厂)等。所用药品均用Ringer氏液配制。
1.4 资料分析GABA-激活电流值与baclofen激活电流值均以mean±SE表示。资料采用t检验进行统计学分析。浓度-效应曲线用下列公式进行拟合:
Y=Emax/［1+(Kd/C)n］, 式中, C和Y分别代表浓度和效应, Emax代表最大反应效应, Kd代表受体的解离常数, n为Hill系数, 即浓度-效应曲线的斜率,
由R/(Rmax-R)对GABA浓度(mol/L)作图而求得。
2结果
２.１ 带有滤泡膜的卵母细胞对GABA的电流反应及GABA受体的类型
在部分受检的带有滤泡膜的卵母细胞(35.5%, 55/155)外加GABA (10－１０～10－３ mol/L)可引起一外向电流。此外向电流幅值小但时程较长(98.8±21.2
s, n=55), 具有明显的浓度依赖性(图1), EC50为1.5×10－7 mol/L。
图1.GABA激活外向电流的浓度-效应关系
Fig. 1.Concentration-response
relationship for GABA-activated outward current. A. Sequential current traces demonstrate the currents
responding to different concentrations of GABA (10－１０～10－５mol/L). The
horizontal bar under each current record indicates the period of drug
application. All records were obtained from the same oocyte. B. In the concentration-response curve,
each point represents the mean±SE of GABA-activated currents of 5～8 oocytes
(HP=-50 mV). All GABA-activated currents were normalized to the peak current
induced by 10－５ mol/L GABA (see symbol marked with asterisk). The curve is a good fit for the data to
the logistic equation Y=Emax/［1+(Kd/C)n］, where C is the concentration of GABA,
Y is the fraction of the maximum value, and Kd, the dissociation constant of
the GABA receptors. The curve was drawn according to the equation described
above assuming Hill coefficient (n)=0.42±0.03. Inset graph is plotted to assess
the slope of the dose-response curve for GABA.
GABA引起的外向电流不被GABAA受体选择性拮抗剂bicuculline
(10－５ mol/L)所阻断(n=6)(图2A)。在应用了GABAB受体选择性拮抗剂2-hydroxysaclofen (10－４ mol/L)后不仅完全消除了GABA
(10－５ mol/L)引起的外向电流, 而且还将其转变为内向电流, 这种阻断作用是可逆性的, 在冲洗之后外向电流可以完全恢复(n=9)(图2B)。
进一步的实验观察到: 当GABA引起的外向电流被2-hydroxysaclofen翻转成内向电流之后,
如再加GABAC受体拮抗剂I4AA (10－５ mol/L), 则内向电流最终被完全取消(n=6)(图2C)。
２．2 Baclofen引起的卵母细胞外向电流
为了证实上述GABA引起的外向电流是由于GABAB受体激活所致, 我们在具有滤泡膜的卵母细胞外加GABAB受体激动剂baclofen(10－１０～10－４
图2.介导GABA引起的膜电流反应的受体类型
Fig. 2.Subtypes of GABA receptors
medicating GABA-activated currents in Xenopus oocytes. A. Sequential current traces responded
to GABA (10－５ mol/L) from left to right: control, coapplication of GABA and
bicuculline (10－５ mol/L) and washout (n=6). B. GABA (10－５ mol/L)-activated outward current (left) was
reversed to an inward current by 2-hydroxysaclofen (10－４ mol/L) (middle) and
washout (n=9). C. GABA (10－５
mol/L)-activated outward current (upper row, left) was reversed by
2-hydroxysaclofen (10－４ mol/L) to an inward current (upper row, right), which
could be abolished completely by I4AA (10－５ mol/L) (lower row, left) and
washout(lower row, right)(n=6).
图3.Baclofen激活电流的浓度-效应曲线
Fig. 3.Concentration-response
relationship for baclofen-activated current. In the concentration-response curve for baclofen-activated
currents, each point represents the mean±SE of 4～6 oocytes (HP=-50 mV). The
curve was generated according to the equation Y=Emax/［1+(Kd/C)n］ assuming Hill
coefficient (n)=0.42±0.05. Inset
graph was plotted to assess the slope of the dose-response curve for baclofen.
mol/L), 也可引起一明显的外向电流。此种外向电流与GABA引起的外向电流相似并具有明显的浓度依赖性,
图3示为baclofen引起的外向电流的浓度-效应关系曲线, 由此曲线可以看出其EC50为6×10－８ mol/L。此外向电流可分别被3×10－６、 3×10－５及3×10－４
mol/L 2-hydroxysaclofen阻断(6.3±3.2)%, (44.1±2.2)%, (86.0±1.6)%(n=6)。
2．3 Baclofen激活电流的电流-电压关系
为了探讨baclofen引起的外向电流的离子机制, 我们对其进行了电流－电压关系分析。从图4的I-V曲线可以看出, 其外向电流基本呈线性,
翻转电位值为－96.8±7.2 mV (n=5, 图4A)。进一步的实验证明TEA (5 mmol/L)和BaCl2 (2 mmol/L)均可将baclofen引起的外向电流完全阻断(n=5,
图4B、 C)。
图4.Baclofen (10－６
mol/L)激活电流的电流-电压关系
Fig. 4.Current-voltage relationship
for baclofen-activated currents.
A. In the I-V curve each point represents the mean±SE from 4～6
oocytes. All current amplitudes
were normalized to the current amplitude at -50 mV (see symbol marked with
asterisk). B and C. Baclofen (10－６
mol/L)-activated currents (B and C, left) were abolished completely by TEA (5
mmol/L)(B, right) and Ba2+ (2 mmol/L)(C, right).
3讨论
非洲爪蟾卵母细胞是一种巨大的细胞, 直径约为1～1.2
mm, 其外包绕着一层粘蛋白的基质, 称为卵黄膜(vitelline membrane), 起着维持卵母细胞形状和强度的作用。再外一层为多细胞构成的滤泡膜(follicle
cell layer), 这些细胞彼此间联系为电耦联, 而滤泡膜与卵母细胞间联系为缝隙连接(gap junction)。以往, 无论是在带有滤泡膜［1～3］或去除滤泡膜［4］的卵母细胞标本上的研究报道,
均未记录到GABA引起的膜电流反应。本实验加10－１０～10－３ mol/L GABA于具有滤泡膜的卵母细胞, 发现在部分受检细胞(35.5%,
55/155)可引起一慢的浓度依赖性的外向电流, 而选择性GABAA受体拮抗剂bicuculline不能取消此种GABA引起的外向电流, 因而排除了此电流与GABAA受体有关的可能性。进一步的实验应用GABAB受体的拮抗剂saclofen后,
此外向电流不仅被消除, 而且翻转成为内向电流。后者又可为GABAC受体拮抗剂I4AA所完全阻断。另一方面, 当加入GABAB受体激动剂baclofen时, 也可引起一个能被saclofen阻断的外向电流,
在saclofen阻断之后并无内向流出现。这些实验结果清楚地证明了卵母细胞上存在GABAB和GABAC受体。
GABAB受体属G蛋白耦联受体超家族。编码GABAB受体的cDNA目前已克隆成功(GABABR1a & b［5］、 GABABR2［6,７］)。而且只有当GABABR1和GABABR2二者一起形成异二聚体(heterodimer)时,
才对GABA具有亲和性［5～8］。已知GABAB受体激活后经G蛋白耦联至相应的效应蛋白, 其功能依细胞种类和部位不同而有所不同, 在中枢神经元的突触后膜主要是K+电导增加［9,10］;
在外周神经元突触前膜部位则是使电压门控性Ca2+通道受到抑制［11］。 近年来有资料证明在中枢神经元也存在此种现象［12］。
GABAC受体是对bicuculline和baclofen均不敏感的一类GABA受体, 属配基门控受体超家族, 其ρ1和ρ2亚基已被克隆出来。ρ1亚基仅在视网膜表达;
ρ2亚基除视网膜外, 在嗅球、 小脑、 中脑、 皮层、 海马及基底神经节等部位也有表达。对GABAC受体的生理及药理特性的了解主要来自对视网膜的研究, 它也是通过开启氯离子通道产生抑制效应,
但其介导的Cl－离子电流和GABAA受体介导的电流相比较缓慢、 持久, 仅有微弱的失敏现象。至于本实验观察到的GABAC受体介导的内向电流的离子机制, 尚有待进一步研究。
实验中还观察了GABA或baclofen所引起的卵母细胞的外向电流是否与K+电导改变有关。因为baclofen平衡电位为－96.8±7.2mV,
此数值接近于K+平衡电位值, 说明GABA/baclofen引起的外向电流主要是由K+所携载的。本实验应用TEA和Ba2+均可阻断baclofen引起的外向电流的结果,
更证实了此外向电流是K+电导增大所致。因而推测GABAB受体激活后, 是经G蛋白直接地, 也可能经胞内信号转导而间接地控制K+通道。至于所耦联的G蛋白的种类和细胞信号转导的途径,
则有待进一步的探讨。卵母细胞上何以存在GABAB和GABAC受体, 在生理情况下其配基的来源以及生理意义何在, 均有待于进一步研究阐明。
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Received 2001-05-22Accepted
2001-07-12
This work was supported by the
National Natural Science
Foundation of China (No.39870259).
*Corresponding author. Tel:
027-83692340; Fax:027-83693761; E-mail:zhiwangli@yahoo.com