γ-氨基丁酸对离体大鼠黄体化颗粒细胞孕酮分泌的影响
作者：姜杰 倪江 李庆雷 边淑玲 张伟
单位：解放军第211医院 麻醉科, 哈尔滨 150080
关键词：γ-氨基丁酸；黄体化颗粒细胞；孕酮
基础医学与临床000620 摘要：用离体孵育方法，观察γ-氨基丁酸(GABA)对大鼠黄体化颗粒细胞孕酮分泌的影响，同时观察在PMSG、蝇覃醇、放线菌酮、forskolin作用下，GABA对黄体化颗粒细胞孕酮分泌的影响。结果显示GABA促进大鼠黄体化颗粒细胞孕酮分泌，蝇覃醇有同样作用。GABA减弱PMSG对颗粒细胞孕酮分泌的促进作用。GABA在放线菌酮作用下对颗粒细胞孕酮分泌无显著影响。GABA促进forskolin作用下的颗粒细胞孕酮分泌。提示GABA促进大鼠黄体化颗粒细胞孕酮分泌是通过结合到颗粒细胞上的GABA受体，可能与细胞内腺苷酸环化酶系统有关，而与蛋白质合成无关。
中图分类号：Q492 文献标识码：A
, http://www.100md.com
文章编号：1001-6325(2000)06-0067-02
Effect of GABA on progesterone secretion in rat luteinising granulosa cells in vitro
JIANG Jie NI Jiang LI Qing-le i et al
(Department of Anesthesiology, the 211th Hospital of PLA, Harbin 150080，China)
Abstract:It has been recently showed that GABA plays an important role in the regulation of ovarian function. The present study was to evaluate the effect of GABA on progesterone production using rat luteinising granulosa cells in vitro. The luteinising granulosa cells were cultured in KRBGAL for 1 h, the cells were treated with reagents for a further 2 h. The secretion of progesterone was assayed by radioimmunoassay. The results showed that GABA stimulated progesterone secretion ，GABA agonist muscimol had the same effect.GABA decreased PMSG induced progesterone secretion.Under the influence of cycloheximide, the effect of GABA on progesterone secretion had no significant difference. With the effect of GABA and forskolin , progesterone secretion had been increased. The results indicated that GABA stimulated progesterone secretion by rat luteinising granulosa cells in vitro and its effect was mediated by GABA receptor and adenylate cyclase-cAMP messenger system.
, http://www.100md.com
Key words：GABA；luteinising granulosa cell；progesterone
在中枢神经系统γ-氨基丁酸(GABA)抑制促性腺激素释放激素的释放，减少黄体生成素的释放，且抑制排卵[1]。GABA在哺乳动物的30多种外周组织里存在，大多数组织中GABA浓度很低，但大鼠卵巢和输卵管中含有高浓度的GABA[2]。在大鼠性周期中卵巢GABA含量在排卵后出现一个高峰，输卵管中GABA含量情前期最高[3]。本实验选用离体培养大鼠黄体化颗粒细胞模型，观察GABA对其孕酮(P)分泌的影响。
1 材料与方法
1.1 试剂
孕马血清促性腺激素(PMSG)、GABA、GABA受体激动剂蝇覃醇(muscimol)、蛋白质合成抑制剂放线菌酮(cycloheximide)、腺苷酸环化酶抑制剂forskolin、0.1%牛血清白蛋白葡萄糖-Krebs-Ringer碳酸缓冲液(KRBGAL)。
, http://www.100md.com
1.2 大鼠黄体化颗粒细胞悬液的制备及培养
选用28-30日龄健康雌性Wistar大鼠，每只皮下注射PMSG 100U，48h后断头处死动物，取出卵巢，用针刺破卵泡，用KRBGAL冲洗卵泡，将含颗粒细胞的KRBGAL离心10min(300×g)，弃上清向细胞团中加入KRBGAL，充分混匀制得颗粒细胞悬液。用KRBGAL稀释至6×105 cells/ml。预孵育60min后加入处理因素，再继续孵育120min。孕酮测定按放射免疫试剂盒说明操作， 用t检验检查显著性差异。
2 结果
2.1 GABA对大鼠黄体化颗粒细胞基础P分泌的影响
不同浓度的GABA均促进大鼠黄体化颗粒细胞基础P分泌，以4×10-6mol/L GABA促进作用最强(表1)。
, http://www.100md.com
表1 GABA对离体大鼠黄体化颗粒细胞基础孕酮分泌的影响
Table 1 The effect of GABA on basal progesterone secretion
by rat luteinising granulosa cells in vitro(n=9)
groupprogesterone(fmol/3×105 cells)
Control4039.37±404.85
GABA (mol/L)
4×10-84652.89±346.37*
, 百拇医药
4×10-74877.22±371.99*
4×10-65403.90±390.09**
4×10-55011.97±500.20*
* P<0.01 vs control; ** P<0.001 vs control
2.2 GABA对不同因素作用下的大鼠黄体化颗粒细胞P分泌的影响
4×10-6mol/L GABA、20 U PMSG、4×10-6mol/L蝇覃醇、1×10-3mol/L forskolin均明显促进大鼠黄体化颗粒细胞P分泌(P<0.001)。GABA和PMSG共同作用仍促进大鼠黄体化颗粒细胞P分泌，与PMSG组相比，P分泌量减少。GABA和forskolin共同作用亦可促进大鼠黄体化颗粒细胞P分泌，较forskolin组作用增强(P<0.001)。2×10-4mol/L放线菌酮抑制大鼠颗粒细胞P分泌，GABA 和放线菌酮共同作用与GABA组相比，P分泌量无明显差别(表2)。表2 GABA对不同因素作用下的大鼠黄体化
, 百拇医药
颗粒细胞孕酮分泌的影响
Table 1 The effect of GABA on progesterone
secretion under the influence of different
factors by rat luteinising granulosa cells
in vitro(n=9) group
progesterone(fmol/3×105 cells)
Control
4187.52±129.48
, 百拇医药
GABA
5305.83±320.54*
PMSG
8042.11±715.35*
GABA+PMSG
6048.96±290.39#
Muscimol
5674.12±401.52*
GABA+Muscimol
5418.96±529.27*
, 百拇医药
Cycloheximide
3508.75±243.91*
GABA+Cycloheximide
5079.91±319.31
Forskolin
6343.61±733.41*
GABA+Forskolin
8204.51±936.28**
* P<0.001 vs control; # P<0.001 vs PMSG
, http://www.100md.com
** P<0.001 vs Forskolin3 讨论
Shaeffer等[4]报道颗粒细胞上有GABA结合位点，本实验表明 GABA 促进大鼠黄体化颗粒细胞P分泌，GABA受体激动剂蝇覃醇可发挥同样作用，故推测GABA可能与颗粒细胞上的GABA受体结合而影响P分泌。本文发现PMSG促进大鼠颗粒细胞P分泌， GABA减弱PMSG对颗粒细胞P分泌的促进作用，由于颗粒细胞上有促性腺激素受体，推测PMSG和GABA与相应受体相互作用时可能相互影响。Gould等[5]报道雌鼠皮下注射放线菌酮后可使血清中孕酮水平降低。本文表明放线菌酮抑制大鼠颗粒细胞P分泌，GABA在放线菌酮影响下与GABA单独作用相比，对P分泌无影响，提示GABA对颗粒细胞P分泌的影响可能与蛋白质合成无关。研究表明cAMP参与卵巢功能的调节[6]， Forskolin能影响黄体细胞P分泌[7]。本实验用forskolin来增加细胞内cAMP 水平,结果表明内源性cAMP 水平的增高可明显提高颗粒细胞P分泌，提示GABA对大鼠黄体化颗粒细胞P分泌的促进作用可能与细胞内cAMP 系统有关。
, http://www.100md.com
参考文献：
[1] Kang SH, Seong JY, Cho S, et al. Acute increse of GABA ergic neurotransmission exerts a stimulatory effect on GnRH ovariectomized estrogen and progesterone-treated adult female rats[J]. Neuroendocrinology,1995,61:486-492.
[2] Erdo SL, Rosdy B, Szpokny L. Higher GABA concentrations in fallopian tube than in brain of the rat[J].b J Neurochem, 1982, 38:1174-1176.
[3] Louzan P, Gallardo MGP, Tramezzani JH. Gamma-aminobutyric acid in the genital tract of the rat during theoestrous cycle[J]. J Reprod Fert, 1986, 77: 499-524.
, 百拇医药
[4] Schaeffer JM and Hsweh AJW. Identification of r-aminobutyric acid and its binding sites in the rat ovary[J]. Life Sci, 1982, 30: 1599-1604.
[5] Gould KG, Fanlkaner JR, Pernikoff D. Alteration in circulating LH level and in cerrical mucus induced by mid-cycle progesterone in the chimpazees[J]. J Reprod Fert, 1984, 70: 379-385.
[6] Grazul-Bilska AT, Reynolds LP, Kirsch JD ,et al.Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of cyclic adenosine 3',5'-monophosphate[J]. Biol Reprod, 1996,54(3):538-545.
[7] Feng SM, Almond GW. Effects of LH, prostaglandin E2, 8-bromo-cyclic AMP and forskolin on progesterone secretion by pig luteal cells[J]. J Reprod Fertil,1998, 113(1):83-89.
收稿日期：1999-07-16
修回日期：1999-11-10, 百拇医药
单位：解放军第211医院 麻醉科, 哈尔滨 150080
关键词：γ-氨基丁酸；黄体化颗粒细胞；孕酮
基础医学与临床000620 摘要：用离体孵育方法，观察γ-氨基丁酸(GABA)对大鼠黄体化颗粒细胞孕酮分泌的影响，同时观察在PMSG、蝇覃醇、放线菌酮、forskolin作用下，GABA对黄体化颗粒细胞孕酮分泌的影响。结果显示GABA促进大鼠黄体化颗粒细胞孕酮分泌，蝇覃醇有同样作用。GABA减弱PMSG对颗粒细胞孕酮分泌的促进作用。GABA在放线菌酮作用下对颗粒细胞孕酮分泌无显著影响。GABA促进forskolin作用下的颗粒细胞孕酮分泌。提示GABA促进大鼠黄体化颗粒细胞孕酮分泌是通过结合到颗粒细胞上的GABA受体，可能与细胞内腺苷酸环化酶系统有关，而与蛋白质合成无关。
中图分类号：Q492 文献标识码：A
, http://www.100md.com
文章编号：1001-6325(2000)06-0067-02
Effect of GABA on progesterone secretion in rat luteinising granulosa cells in vitro
JIANG Jie NI Jiang LI Qing-le i et al
(Department of Anesthesiology, the 211th Hospital of PLA, Harbin 150080，China)
Abstract:It has been recently showed that GABA plays an important role in the regulation of ovarian function. The present study was to evaluate the effect of GABA on progesterone production using rat luteinising granulosa cells in vitro. The luteinising granulosa cells were cultured in KRBGAL for 1 h, the cells were treated with reagents for a further 2 h. The secretion of progesterone was assayed by radioimmunoassay. The results showed that GABA stimulated progesterone secretion ，GABA agonist muscimol had the same effect.GABA decreased PMSG induced progesterone secretion.Under the influence of cycloheximide, the effect of GABA on progesterone secretion had no significant difference. With the effect of GABA and forskolin , progesterone secretion had been increased. The results indicated that GABA stimulated progesterone secretion by rat luteinising granulosa cells in vitro and its effect was mediated by GABA receptor and adenylate cyclase-cAMP messenger system.
, http://www.100md.com
Key words：GABA；luteinising granulosa cell；progesterone
在中枢神经系统γ-氨基丁酸(GABA)抑制促性腺激素释放激素的释放，减少黄体生成素的释放，且抑制排卵[1]。GABA在哺乳动物的30多种外周组织里存在，大多数组织中GABA浓度很低，但大鼠卵巢和输卵管中含有高浓度的GABA[2]。在大鼠性周期中卵巢GABA含量在排卵后出现一个高峰，输卵管中GABA含量情前期最高[3]。本实验选用离体培养大鼠黄体化颗粒细胞模型，观察GABA对其孕酮(P)分泌的影响。
1 材料与方法
1.1 试剂
孕马血清促性腺激素(PMSG)、GABA、GABA受体激动剂蝇覃醇(muscimol)、蛋白质合成抑制剂放线菌酮(cycloheximide)、腺苷酸环化酶抑制剂forskolin、0.1%牛血清白蛋白葡萄糖-Krebs-Ringer碳酸缓冲液(KRBGAL)。
, http://www.100md.com
1.2 大鼠黄体化颗粒细胞悬液的制备及培养
选用28-30日龄健康雌性Wistar大鼠，每只皮下注射PMSG 100U，48h后断头处死动物，取出卵巢，用针刺破卵泡，用KRBGAL冲洗卵泡，将含颗粒细胞的KRBGAL离心10min(300×g)，弃上清向细胞团中加入KRBGAL，充分混匀制得颗粒细胞悬液。用KRBGAL稀释至6×105 cells/ml。预孵育60min后加入处理因素，再继续孵育120min。孕酮测定按放射免疫试剂盒说明操作， 用t检验检查显著性差异。
2 结果
2.1 GABA对大鼠黄体化颗粒细胞基础P分泌的影响
不同浓度的GABA均促进大鼠黄体化颗粒细胞基础P分泌，以4×10-6mol/L GABA促进作用最强(表1)。
, http://www.100md.com
表1 GABA对离体大鼠黄体化颗粒细胞基础孕酮分泌的影响
Table 1 The effect of GABA on basal progesterone secretion
by rat luteinising granulosa cells in vitro(n=9)
groupprogesterone(fmol/3×105 cells)
Control4039.37±404.85
GABA (mol/L)
4×10-84652.89±346.37*
, 百拇医药
4×10-74877.22±371.99*
4×10-65403.90±390.09**
4×10-55011.97±500.20*
* P<0.01 vs control; ** P<0.001 vs control
2.2 GABA对不同因素作用下的大鼠黄体化颗粒细胞P分泌的影响
4×10-6mol/L GABA、20 U PMSG、4×10-6mol/L蝇覃醇、1×10-3mol/L forskolin均明显促进大鼠黄体化颗粒细胞P分泌(P<0.001)。GABA和PMSG共同作用仍促进大鼠黄体化颗粒细胞P分泌，与PMSG组相比，P分泌量减少。GABA和forskolin共同作用亦可促进大鼠黄体化颗粒细胞P分泌，较forskolin组作用增强(P<0.001)。2×10-4mol/L放线菌酮抑制大鼠颗粒细胞P分泌，GABA 和放线菌酮共同作用与GABA组相比，P分泌量无明显差别(表2)。表2 GABA对不同因素作用下的大鼠黄体化
, 百拇医药
颗粒细胞孕酮分泌的影响
Table 1 The effect of GABA on progesterone
secretion under the influence of different
factors by rat luteinising granulosa cells
in vitro(n=9) group
progesterone(fmol/3×105 cells)
Control
4187.52±129.48
, 百拇医药
GABA
5305.83±320.54*
PMSG
8042.11±715.35*
GABA+PMSG
6048.96±290.39#
Muscimol
5674.12±401.52*
GABA+Muscimol
5418.96±529.27*
, 百拇医药
Cycloheximide
3508.75±243.91*
GABA+Cycloheximide
5079.91±319.31
Forskolin
6343.61±733.41*
GABA+Forskolin
8204.51±936.28**
* P<0.001 vs control; # P<0.001 vs PMSG
, http://www.100md.com
** P<0.001 vs Forskolin3 讨论
Shaeffer等[4]报道颗粒细胞上有GABA结合位点，本实验表明 GABA 促进大鼠黄体化颗粒细胞P分泌，GABA受体激动剂蝇覃醇可发挥同样作用，故推测GABA可能与颗粒细胞上的GABA受体结合而影响P分泌。本文发现PMSG促进大鼠颗粒细胞P分泌， GABA减弱PMSG对颗粒细胞P分泌的促进作用，由于颗粒细胞上有促性腺激素受体，推测PMSG和GABA与相应受体相互作用时可能相互影响。Gould等[5]报道雌鼠皮下注射放线菌酮后可使血清中孕酮水平降低。本文表明放线菌酮抑制大鼠颗粒细胞P分泌，GABA在放线菌酮影响下与GABA单独作用相比，对P分泌无影响，提示GABA对颗粒细胞P分泌的影响可能与蛋白质合成无关。研究表明cAMP参与卵巢功能的调节[6]， Forskolin能影响黄体细胞P分泌[7]。本实验用forskolin来增加细胞内cAMP 水平,结果表明内源性cAMP 水平的增高可明显提高颗粒细胞P分泌，提示GABA对大鼠黄体化颗粒细胞P分泌的促进作用可能与细胞内cAMP 系统有关。
, http://www.100md.com
参考文献：
[1] Kang SH, Seong JY, Cho S, et al. Acute increse of GABA ergic neurotransmission exerts a stimulatory effect on GnRH ovariectomized estrogen and progesterone-treated adult female rats[J]. Neuroendocrinology,1995,61:486-492.
[2] Erdo SL, Rosdy B, Szpokny L. Higher GABA concentrations in fallopian tube than in brain of the rat[J].b J Neurochem, 1982, 38:1174-1176.
[3] Louzan P, Gallardo MGP, Tramezzani JH. Gamma-aminobutyric acid in the genital tract of the rat during theoestrous cycle[J]. J Reprod Fert, 1986, 77: 499-524.
, 百拇医药
[4] Schaeffer JM and Hsweh AJW. Identification of r-aminobutyric acid and its binding sites in the rat ovary[J]. Life Sci, 1982, 30: 1599-1604.
[5] Gould KG, Fanlkaner JR, Pernikoff D. Alteration in circulating LH level and in cerrical mucus induced by mid-cycle progesterone in the chimpazees[J]. J Reprod Fert, 1984, 70: 379-385.
[6] Grazul-Bilska AT, Reynolds LP, Kirsch JD ,et al.Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of cyclic adenosine 3',5'-monophosphate[J]. Biol Reprod, 1996,54(3):538-545.
[7] Feng SM, Almond GW. Effects of LH, prostaglandin E2, 8-bromo-cyclic AMP and forskolin on progesterone secretion by pig luteal cells[J]. J Reprod Fertil,1998, 113(1):83-89.
收稿日期：1999-07-16
修回日期：1999-11-10, 百拇医药