The Expression and Role of Hyperpolarization-Activated and Cyclic Nucleotide-Gated Channels in Endocrine Anterior Pituitary Cells
The Expression and Role of Hyperpolarization-Activated and Cyclic Nucleotide-Gated Channels in Endocrine Anterior Pituitary Cells
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DOI：
10.1210/me.2011-1207
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发表时间：
2012-01-01
影响因子：
--
通讯作者：
Stojilkovic, Stanko S.
中科院分区：
文献类型：
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作者：
Kretschmannova, Karla;Kucka, Marek;Stojilkovic, Stanko S.
垂体细胞不依赖于外部刺激而产生动作电位，这种自发性电活动受到多种下丘脑和垂体内激动剂的调节。在这里，我们集中在超极化激活和环核苷酸门控（HCN）通道在培养的大鼠垂体前叶细胞的电活动的潜在作用。定量RT-PCR分析显示，HCN 2和HCN 3亚基的mRNA转录表达水平较高，HCN 1和HCN 4亚基的表达较低。Western免疫印迹分析正常和GH（3）永生化垂体细胞的裂解物显示具有适当分子量的HCN 2、HCN 3和HCN 4条带。电生理学实验表明，存在一个缓慢发展的超极化激活的内向电流，这是由Cs+和ZD 7288，在促性腺激素细胞，促甲状腺激素细胞，促生长激素细胞，和一小部分的催乳激素细胞，以及在其他身份不明的垂体细胞类型。腺苷酸环化酶的刺激和8-Br-cAMP的加入增强了这种电流和去极化的细胞膜，而8-Br-cGMP没有改变电流和超极化的细胞膜。基础腺苷酸环化酶活性的抑制和磷脂酶C信号通路的刺激抑制该电流。HCN通道的抑制会影响放电频率，但不会消除自发性电活动。这些实验表明，cAMP和cGMP对内分泌垂体细胞的兴奋性具有相反的影响，在培养的细胞中的基础cAMP生产足以整合电活动中的大部分HCN通道，并且磷脂酶C的激活引起的磷脂酰肌醇4，5-二磷酸的耗尽使它们沉默。（分子内分泌学26：153-164，2012）
Pituitary cells fire action potentials independently of external stimuli, and such spontaneous electrical activity is modulated by a large variety of hypothalamic and intrapituitary agonists. Here, we focused on the potential role of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in electrical activity of cultured rat anterior pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from normal and GH(3) immortalized pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3, and HCN4. Electrophysiological experiments showed the presence of a slowly developing hyperpolarization-activated inward current, which was blocked by Cs+ and ZD7288, in gonadotrophs, thyrotrophs, somatotrophs, and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. Inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity. These experiments indicate that cAMP and cGMP have opposite effects on the excitability of endocrine pituitary cells, that basal cAMP production in cultured cells is sufficient to integrate the majority of HCN channels in electrical activity, and that depletion of phosphatidylinositol 4,5-bisphosphate caused by activation of phospholipase C silences them. (Molecular Endocrinology 26: 153-164, 2012)