Virmani M A, Stojilković S S, Catt K J
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Endocrinology. 1990 May;126(5):2499-505. doi: 10.1210/endo-126-5-2499.
The mechanism by which gamma-aminobutyric acid (GABA) stimulates the release of LH was analyzed in cultured female rat pituitary cells. In 3-h incubations, GABA (1-100 microM) caused a dose-dependent increase in LH release, with the maximal response about 16% of that evoked by 10 nM GnRH. GABA action was independent of the GnRH receptor, since 1 microM GnRH antagonist [( N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10] GnRH), which completely inhibits GnRH action, did not affect the response to GABA. In studies on the effects of GABA receptor agonists and antagonists, 4,5,6,7-tetrahydoisoxazolo-[5,4-c]pyridin-3(2H)-one (THIP) and muscimol (GABAA agonists) gave similar response patterns, with the same maximal stimulation as GABA but much higher potencies. In contrast, the GABAB receptor agonist baclofen did not stimulate LH release. The GABAA receptor antagonist SR95531 caused dose-dependent inhibition of the LH-releasing effects of GABA and muscimol (10 microM), with complete blockade at 10 microM SR95531. T-Butylbicyclophosphorothionate, an inhibitor of the GABAA receptor-associated chloride channel, also dose-dependently reduced the releasing effect of 100 microM GABA. These results indicate that GABA action is mediated by the chloride channel-associated GABAA receptor. However, the other GABAA receptor antagonists, including bicuculline, picrotoxin, and strychnine, did not attenuate the LH-releasing effect of 100 microM GABA in concentrations up to 100 microM, suggesting that GABA action is mediated by nonclassical GABAA receptors. Incubation in the presence of nifedipine (1 microM) or in calcium-free medium inhibited the LH-releasing action of GABA, indicating that calcium influx through voltage-sensitive calcium channels (VSCC) is required for GABA-induced LH release. Such entry of Ca2+ would result from activation of VSCC by depolarization due to the increased Cl- conductance caused by GABAA receptor activation. In cell perfusion studies, the actions of GABA and muscimol were attenuated or abolished after repetitive stimulation, consistent with desensitization of the GABA receptors. These findings have demonstrated that the stimulation of LH release by GABA is independent of GnRH action, occurs via binding to nonclassical GABAA receptors, which rapidly desensitize, and is mediated by the activation of VSCC.
在培养的雌性大鼠垂体细胞中分析了γ-氨基丁酸（GABA）刺激促黄体生成素（LH）释放的机制。在3小时的孵育中，GABA（1 - 100μM）引起LH释放呈剂量依赖性增加，最大反应约为10 nM促性腺激素释放激素（GnRH）所诱发反应的16%。GABA的作用独立于GnRH受体，因为1μM的GnRH拮抗剂[（N - 乙酰 - D - 对氯苯丙氨酸1,2，D - 色氨酸3，D - 赖氨酸6，D - 丙氨酸10]GnRH，它能完全抑制GnRH的作用，但不影响对GABA的反应。在关于GABA受体激动剂和拮抗剂作用的研究中，4,5,6,7 - 四氢异恶唑并[5,4 - c]吡啶 - 3(2H) - 酮（THIP）和蝇蕈醇（GABAA激动剂）给出了相似的反应模式，与GABA有相同的最大刺激，但效力高得多。相反，GABAB受体激动剂巴氯芬不刺激LH释放。GABAA受体拮抗剂SR95531导致对GABA和蝇蕈醇（10μM）的LH释放作用呈剂量依赖性抑制，在10μM SR95531时完全阻断。GABAA受体相关氯离子通道的抑制剂叔丁基双环磷硫代酸酯也呈剂量依赖性降低100μM GABA的释放作用。这些结果表明GABA的作用是由与氯离子通道相关的GABAA受体介导的。然而，其他GABAA受体拮抗剂，包括荷包牡丹碱、印防己毒素和士的宁，在浓度高达100μM时并未减弱100μM GABA的LH释放作用，这表明GABA的作用是由非经典GABAA受体介导的。在硝苯地平（1μM）存在下孵育或在无钙培养基中孵育可抑制GABA的LH释放作用，表明通过电压敏感性钙通道（VSCC）的钙内流是GABA诱导LH释放所必需的。由于GABAA受体激活导致氯离子电导增加而引起的去极化会激活VSCC，从而导致Ca2+的这种内流。在细胞灌注研究中，GABA和蝇蕈醇的作用在重复刺激后减弱或消失，这与GABA受体的脱敏一致。这些发现表明GABA对LH释放的刺激独立于GnRH作用，通过与快速脱敏的非经典GABAA受体结合而发生，并由VSCC的激活介导。
