4-aminopyridine decreases progesterone production by porcine granulosa cells.
4-aminopyridine decreases progesterone production by porcine granulosa cells.
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DOI：
10.1186/1477-7827-1-31
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发表时间：
2003-04-01
期刊：
影响因子：
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通讯作者：
Freeman, Lisa C
中科院分区：
文献类型：
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作者：
Li, Yan;Ganta, Suhasini;Freeman, Lisa C
背景：离子通道是具有细胞特异性表达模式的相关基因大家族。颗粒细胞表达来自多个家族的电压门控性钾通道。方法：分离猪卵泡颗粒细胞，用4-AP单独或与FSH、8-CPT-cAMP、17β雌二醇和DIDS联合培养。补充实验确定了4-AP对自发建立的猪颗粒细胞系PGC-2的影响。用放射免疫分析法检测颗粒细胞或PGC-2的功能。台盼蓝拒染法检测细胞存活率。用DiBAC4(3)分光光度法和PBFI荧光法分别记录药物诱导的细胞膜电位和细胞内钾离子浓度的变化。免疫印迹法检测增殖细胞核抗原(PCNA)和类固醇合成急性调节蛋白(STAR)表达。结果：4-AP(2 MM)可减少有血清和无血清颗粒细胞培养液中孕酮的积聚，但仅在无血清条件下抑制细胞增殖。4-AP可减少PGC-2诱导的STAR表达、cAMP生成和雌二醇合成。在含血清的原代培养细胞中加入8-CPT-cAMP或17β雌二醇均可降低4-AP的抑制作用。4-AP处理还与细胞大小增加、细胞内钾离子浓度增加和静息膜电位超极化有关。药物引起的静息膜电位超极化可通过减少细胞外氯离子或向培养液中加入DIDS来防止。结论：4-AP通过在激素合成途径中的多个相互作用步骤发挥药物作用，抑制基础和FSH刺激的猪颗粒细胞孕酮的产生。4-AP对类固醇合成的这些抑制作用可能反映了药物引起的细胞内K+、Cl-浓度以及颗粒细胞静息膜电位的变化。
BACKGROUND: Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.METHODS: Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17beta, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.RESULTS: 4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17beta to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.CONCLUSION: 4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.