Tsujii T, Ishizaka K, Winters S J
Department of Medicine, University of Pittsburgh, Pennsylvania 15213-2582.
Endocrinology. 1994 Sep;135(3):826-33. doi: 10.1210/endo.135.3.7915230.
Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a neuropeptide related to vasoactive intestinal peptide-secretin-glucagon which stimulates adenylate cyclase in cultured rat pituitary cells and stimulates LH and FSH release in vitro and in vivo. Because the cAMP-protein kinase-A pathway regulates the gonadotropin subunit messenger RNAs (mRNAs) and modulates GnRH-stimulated gonadotropin secretion in vitro, we examined the effects of PACAP38 on gonadotropin secretion and subunit mRNA levels. Anterior pituitary cells were prepared from 7-week-old male rats castrated at 5 weeks of age. In monolayer cultures stimulated with GnRH, 0.1-10 nM PACAP38 decreased (P < 0.05) the EC50 for GnRH dose-dependently without affecting the maximum LH secretory response. Cells were next stimulated with 1-min pulses of 2.5 nM GnRH every hour for 9 h in the absence or presence of 10 nM PACAP38, which was perifused continuously. The amplitude of GnRH-induced LH, FSH, and alpha-subunit secretory episodes from PACAP38-treated cells rose (P < 0.01) gradually to 233 +/- 54%, 197 +/- 44%, and 378 +/- 104%, respectively (mean +/- SEM; n = 5 experiments), of the value for control cells lacking PACAP38. This enhancement was sustained for at least 3 h after PACAP38 was removed from the perifusion medium. With PACAP treatment, interpulse secretion of LH and alpha-subunit increased gradually (P < 0.01) to 174 +/- 21% and 212 +/- 64% of the value for chambers stimulated with GnRH alone (control), respectively, whereas interpulse secretion of FSH declined (P < 0.001) to 75 +/- 7% of the control value. In contrast to the gradual effect of PACAP38 to enhance GnRH-induced hormone secretion, PACAP38 alone produced a transient burst of gonadotropin secretion. At the completion of the perifusions, total RNA was extracted and gonadotropin subunit mRNA levels were determined by Northern analysis. GnRH increased (P < 0.01) FSH beta mRNA to 438 +/- 52% of the level in cells stimulated with medium alone (control). Adding PACAP38 to the perifusion medium partially blocked (P < 0.01) the effect of GnRH (178 +/- 20% of the control value), and PACAP38 alone reduced (P < 0.01) FSH beta mRNA levels to 31 +/- 3% of the control value. By contrast, alpha-subunit mRNA levels were increased by both PACAP38 (143 +/- 4% of the control value; P < 0.01) and GnRH (121 +/- 2% of the control value; P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)
垂体腺苷酸环化酶激活多肽-38（PACAP38）是一种与血管活性肠肽-促胰液素-胰高血糖素相关的神经肽，它能刺激培养的大鼠垂体细胞中的腺苷酸环化酶，并在体内外刺激促黄体生成素（LH）和促卵泡生成素（FSH）的释放。由于环磷酸腺苷-蛋白激酶-A途径调节促性腺激素亚基信使核糖核酸（mRNA），并在体外调节促性腺激素释放激素（GnRH）刺激的促性腺激素分泌，我们研究了PACAP38对促性腺激素分泌和亚基mRNA水平的影响。垂体前叶细胞取自5周龄时阉割的7周龄雄性大鼠。在GnRH刺激的单层培养中，0.1 - 10 nM的PACAP38剂量依赖性地降低（P < 0.05）GnRH的半数有效浓度（EC50），而不影响最大LH分泌反应。接下来，在不存在或存在10 nM PACAP38（持续灌流）的情况下，每小时用2.5 nM GnRH刺激细胞1分钟，共刺激9小时。来自PACAP38处理细胞的GnRH诱导的LH、FSH和α亚基分泌脉冲的幅度分别逐渐上升（P < 0.01）至缺乏PACAP38的对照细胞值的233±54%、197±44%和378±104%（平均值±标准误；n = 5次实验）。在从灌流培养基中去除PACAP38后，这种增强作用至少持续3小时。用PACAP处理时，LH和α亚基的脉冲间期分泌分别逐渐增加（P < 0.01）至单独用GnRH刺激的培养室（对照）值的174±21%和212±64%，而FSH的脉冲间期分泌下降（P < 0.001）至对照值的75±7%。与PACAP38增强GnRH诱导的激素分泌的渐进性作用相反，单独的PACAP38会产生促性腺激素分泌的短暂爆发。在灌流结束时，提取总RNA并通过Northern分析测定促性腺激素亚基mRNA水平。GnRH将FSHβ mRNA增加（P < 0.01）至单独用培养基刺激的细胞（对照）水平的438±52%。向灌流培养基中添加PACAP38部分阻断（P < 0.01）GnRH的作用（为对照值的178±20%），而单独的PACAP38将FSHβ mRNA水平降低（P < 0.01）至对照值的31±3%。相比之下，α亚基mRNA水平在PACAP38（为对照值的143±4%；P < 0.01）和GnRH（为对照值的121±2%；P < 0.05）作用下均升高。（摘要截短至4百字）
