Section of Endocrinology, Department of Medicine, University of Chicago, Chicago, Ill., USA.
Neuroendocrinology. 2012;96(1):68-80. doi: 10.1159/000335985. Epub 2012 Feb 14.
Kisspeptin plays an important role in puberty and subsequent fertility by activating its receptor, G-protein-coupled receptor 54 (GPR54), and increasing cytoplasmic free Ca(2+) concentration (Ca(2+)) and gonadotropin-releasing hormone (GnRH) secretion in GnRH neurons. Yet the mechanism by which kisspeptin increases Ca(2+) in GnRH neurons remains to be fully elucidated. In other neurons, voltage-gated Ca(2+) channel (VGCC) activity has been shown to be inversely related to Ca(2+). We used whole-cell patch-clamp recording to examine the effects of kisspeptin-10 (KP-10) on VGCC activity evoked by step depolarizations in GnRH neurons in brain slices from pubertal male GnRH-green fluorescent protein transgenic mice. Prolonged (>30 s) KP-10 application inhibited Ca(2+) currents. The GPR54 antagonist peptide 234, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, substitution of Ba(2+) for Ca(2+), the calmodulin antagonists calmidazolium and trifluoperazine, the phospholipase C inhibitor edelfosine, the canonical transient receptor potential (TRPC) channel and inositol 1,4,5-trisphosphate receptor (IP(3)R) antagonist 2-APB, the TRPC channel antagonist BTP2 and the endoplasmic reticulum Ca(2+)-ATPase blocker cyclopiazonic acid each prevented inhibition. The IP(3)R antagonists caffeine (10 µM), heparin and intracellular 2-APB prevented inhibition to a lesser extent. The ryanodine receptor (RyR) antagonists ryanodine and dantrolene prevented inhibition, and the RyR agonist caffeine (30 mM) mimicked the effects of KP-10 on Ca(2+) currents. Our results suggest that kisspeptin induces Ca(2+) influx through TRPC channels and Ca(2+) release via IP(3)Rs and RyRs, and that this is followed by Ca(2+)/CaM-dependent inhibition of VGCCs.
Kisspeptin 通过激活其受体 G 蛋白偶联受体 54（GPR54），增加细胞浆游离钙浓度 (Ca(2+)) 和促性腺激素释放激素 (GnRH) 在 GnRH 神经元中的分泌，在青春期和随后的生育能力中发挥重要作用。然而， kisspeptin 增加 GnRH 神经元中 Ca(2+) 的机制仍有待充分阐明。在其他神经元中，已表明电压门控钙通道 (VGCC) 活性与 Ca(2+) 呈负相关。我们使用全细胞膜片钳记录技术，在青春期雄性 GnRH-绿色荧光蛋白转基因小鼠脑片的 GnRH 神经元中，通过阶跃去极化来检测 kisspeptin-10 (KP-10) 对 VGCC 活性的影响。长时间 (>30 s) KP-10 应用抑制 Ca(2+) 电流。GPR54 拮抗剂肽 234、1,2-双(2-氨基苯氧基)乙烷 N,N,N',N'-四乙酸螯合细胞内 Ca(2+)、用 Ba(2+) 替代 Ca(2+)、钙调蛋白拮抗剂氯丙嗪和三氟拉嗪、磷脂酶 C 抑制剂埃德尔福辛、经典瞬时受体电位 (TRPC) 通道和肌醇 1,4,5-三磷酸受体 (IP(3)R) 拮抗剂 2-APB、TRPC 通道拮抗剂 BTP2 和内质网 Ca(2+)-ATP 酶阻滞剂环匹阿尼酸均可阻止抑制。IP(3)R 拮抗剂咖啡因 (10 µM)、肝素和细胞内 2-APB 对抑制的抑制作用较小。ryanodine 受体 (RyR) 拮抗剂 ryanodine 和 dantrolene 可阻止抑制，ryanodine 激动剂咖啡因 (30 mM) 模拟了 KP-10 对 Ca(2+) 电流的作用。我们的结果表明， kisspeptin 通过 TRPC 通道诱导 Ca(2+) 内流，并通过 IP(3)Rs 和 RyRs 释放 Ca(2+)，随后是 Ca(2+)/CaM 依赖性 VGCC 抑制。
