Arroyo Armando, Kim Beomsu, Rasmusson Randall L, Bett Glenna, Yeh John
Department of Gynecology-Obstetrics, University at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York 14222, USA.
J Soc Gynecol Investig. 2006 Sep;13(6):442-50. doi: 10.1016/j.jsgi.2006.05.010. Epub 2006 Jul 31.
The current research was conducted to determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are expressed in gonadotropin-releasing hormone (GnRH) neurons in the female rat hypothalamus and immortalized GnRH neurons (GT1-7 cells).
Double-label fluorescence immunohistochemistry was used to colocalize HCN1-4 channels and GnRH in GnRH neurons in the female rat hypothalamus. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze HCN channel gene expression in GT1-7 cells.
Double-label fluorescence immunohistochemistry showed that 43% of hypothalamic GnRH neurons immunostained for HCN2 and 90% of GnRH neurons immunostained for HCN3. RT-PCR and Western blot showed expression of all four HCN channel subunits in GT1-7 cells. Double-label immunocytochemistry showed cytoplasmic immunostaining of HCN2 and HCN3 in GT1-7 cells.
This study demonstrates for the first time that HCN channels are expressed in GnRH neurons in the rat hypothalamus and GT1-7 cells. Our research supports the hypothesis that HCN channels may be involved in electrical bursting activity and pulsatile GnRH secretion in endogenous GnRH neurons and GT1-7 cells.
进行当前研究以确定超极化激活的环核苷酸门控（HCN1 - 4）通道是否在雌性大鼠下丘脑促性腺激素释放激素（GnRH）神经元以及永生化GnRH神经元（GT1 - 7细胞）中表达。
采用双标荧光免疫组织化学法使HCN1 - 4通道与GnRH在雌性大鼠下丘脑的GnRH神经元中共定位。运用逆转录聚合酶链反应（RT - PCR）、蛋白质印迹法和免疫细胞化学法分析GT1 - 7细胞中HCN通道基因的表达情况。
双标荧光免疫组织化学显示，43%的下丘脑GnRH神经元HCN2免疫染色阳性，90%的GnRH神经元HCN3免疫染色阳性。RT - PCR和蛋白质印迹显示GT1 - 7细胞中所有四种HCN通道亚基均有表达。双标免疫细胞化学显示GT1 - 7细胞中HCN2和HCN3呈胞质免疫染色阳性。
本研究首次证明HCN通道在大鼠下丘脑GnRH神经元和GT1 - 7细胞中表达。我们的研究支持以下假说：HCN通道可能参与内源性GnRH神经元和GT1 - 7细胞的电爆发活动及GnRH的脉冲式分泌。
