Sealfon S C, Gillo B, Mundamattom S, Mellon P L, Windle J J, Landau E, Roberts J L
Department of Neurology, Mount Sinai School of Medicine, New York, New York 10029.
Mol Endocrinol. 1990 Jan;4(1):119-24. doi: 10.1210/mend-4-1-119.
The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and N-Ac-D-Na1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.
通过向非洲爪蟾卵母细胞中注射从大鼠垂体以及源自转基因小鼠的促性腺激素细胞系αT3中分离出的RNA，对啮齿动物促性腺激素释放激素（GnRH）受体进行了表征。注射150 - 200 ng RNA后3至4天，通过电压钳记录发现，93%的卵母细胞对10⁻⁷ M GnRH有反应。注射垂体RNA后获得的平均内向电流为620 ± 88 nA（n = 22），注射αT3 RNA后为1415 ± 598（n = 4）。注射垂体RNA后，能够引发剂量依赖性电流的GnRH阈值浓度为3 × 10⁻⁹ M GnRH。卵母细胞的GnRH受体反应可被[D - Phe2,6,Pro3]GnRH和N - Ac - D - Na1, D - αD - Me, pCl - Phe2, D - Arg6, D - Ala10 - NH2]GnRH拮抗，而D - Ser(But)6,Pro9 - N - 乙酰胺GnRH（布舍瑞林）可引发该反应。通过电压斜坡测定，在注射垂体RNA和细胞系RNA的卵母细胞中，GnRH产生的电流的反转电位分别为 - 22.5 ± 1.0 mV（n = 7）和 - 25.6 ± 3.3 mV（n = 3），这与氯离子反转电位一致。通过细胞内注射乙二醇双(2 - 氨基乙醚) - N,N,N',N'-四乙酸（EGTA），GnRH受体反应几乎完全消除，但在无钙灌流液中施加配体时不受影响。细胞内注射肌醇1,4,5 - 三磷酸可模拟GnRH引发的反应。为了确定GnRH受体mRNA的大小，通过蔗糖梯度对αT3 RNA进行了大小分级分离。大于28S核糖体峰的一个级分诱导了最大的GnRH反应。因此我们发现，注射来自适当来源RNA的卵母细胞对GnRH产生电生理反应，该反应依赖于细胞内钙动员，不依赖于细胞外钙，并且可能由肌醇1,4,5 - 三磷酸介导。
