Ishizaka K, Tsujii T, Winters S J
Department of Medicine, Montefiore University Hospital, University of Pittsburgh, Pennsylvania 15213.
Endocrinology. 1993 Nov;133(5):2040-8. doi: 10.1210/endo.133.5.8404651.
cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.
在与促性腺激素释放激素（GnRH）脉冲共灌流的垂体细胞中，研究了环磷酸腺苷（cAMP）对促性腺激素分泌及亚基mRNA水平的调节作用。对5周龄时阉割的7周龄雄性大鼠的垂体细胞，每小时用1分钟的GnRH脉冲、腺苷酸环化酶激活剂福斯高林、细胞可渗透的cAMP类似物8-溴-cAMP（8Br-cAMP）或对照培养基刺激9至24小时。细胞还用非甾体抗炎药氟芬那酸处理，该药物可降低垂体cAMP水平。在共灌流期间，每10分钟收集流出液用于促卵泡激素（FSH）和促黄体生成素（LH）测定。共灌流结束时，提取总RNA，并用Northern分析法定量促性腺激素亚基mRNA水平。持续给予氟芬那酸逐渐将GnRH刺激的FSH和LH脉冲幅度分别降至对照值的40±4.7%和62±12%的最低点。氟芬那酸降低了（P<0.05）FSHβ和α亚基mRNA水平，并阻断了GnRH延长LHβmRNA的作用。福斯高林或8Br-cAMP脉冲释放LH和FSH，持续给予福斯高林或8Br-cAMP增强了GnRH对促性腺激素的刺激作用。福斯高林或8Br-cAMP脉冲给药时增加了（P<0.05）FSHβmRNA和α亚基mRNA水平，但持续给药时未增加，并延长了LHβmRNA。Nal-Glu GnRH拮抗剂阻断了GnRH脉冲的作用，但未阻断8Br-cAMP或福斯高林的作用。总之，用氟芬那酸降低细胞内cAMP水平减弱了GnRH刺激的促性腺激素分泌，降低了α亚基和FSHβmRNA水平，并阻断了GnRH延长LHβmRNA的作用，而8Br-cAMP或福斯高林则产生相反的作用。这些数据扩展了先前的结果，即cAMP调节促性腺激素分泌，并表明cAMP/A激酶途径调节每个促性腺激素亚基mRNA。
