van de Pavert S A, Clarke I J, Rao A, Vrana K E, Schwartz J
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157, USA.
J Endocrinol. 1997 Jul;154(1):139-47. doi: 10.1677/joe.0.1540139.
Although arginine-vasopressin (AVP) is reported to produce greater ACTH biosynthetic and secretary responses than does corticotropin-releasing hormone (CRH) in sheep anterior pituitary cells, neither factor appears to increase pro-opiomelanocortin (POMC) mRNA levels, as does CRH in the cells of some other species. Since only a fraction of cells that express POMC mRNA may be able to respond to AVP, the aim of this study was to further delineate the regulation of POMC mRNA in ovine anterior pituitary corticotrophs, as a whole and in functional subpopulations of corticotrophs. We measured the effects of AVP, CRH or activation of protein kinase C by phorbol myristate acetate (PMA) in cultured cells. We compared responses in intact populations with those of cultures from which CRH-target cells were pharmacologically eliminated. Dissociated adult ovine anterior pituitary cells were cultured overnight, treated with either vehicle (intact) or a CRH-toxin conjugate that specifically eliminates CRH-target cells (CRH-target-depleted), washed, returned to culture and subsequently challenged with vehicle, AVP (100 nM), CRH (10 nM) or PMA (1 microM) for 5 h. The media were assayed for ACTH by RIA and the cells for POMC mRNA by Northern blot analysis. In intact populations, AVP and CRH increased ACTH secretion from 6.5 +/- 1.2 to 216 +/- 22 and 81 +/- 14 ng/well respectively, but only AVP caused an increase in steady-state POMC mRNA levels (+48 +/- 10%). Direct activation of protein kinase C with PMA mimicked the effect of AVP on ACTH secretion (318 +/- 16 ng/well), but did not alter POMC mRNA levels. In CRH-target-depleted populations, control ACTH secretion (11 +/- 3 ng/well) and POMC mRNA (+69 +/- 7%) were elevated, compared with intact populations. AVP (55 +/- 8 ng/well) and PMA (120 +/- 17 ng/ well), but not CRH, increased ACTH secretion; POMC mRNA was not significantly elevated by any of the treatments. Taken together, these data provide further support for the notion of dissociation between secretion of ACTH and expression of POMC mRNA, and demonstrate that AVP increases steady-state POMC mRNA levels in ovine anterior pituitary cells. The data are also consistent with the concept that complex interactions, possibly including those between cells, influence ACTH secretion and steady-state POMC mRNA levels.
尽管据报道，在绵羊垂体前叶细胞中，精氨酸加压素（AVP）比促肾上腺皮质激素释放激素（CRH）能产生更大的促肾上腺皮质激素（ACTH）生物合成和分泌反应，但与其他一些物种细胞中的CRH不同，这两种因子似乎都不会增加阿黑皮素原（POMC）mRNA水平。由于只有一小部分表达POMC mRNA的细胞可能对AVP有反应，本研究的目的是进一步阐明绵羊垂体前叶促肾上腺皮质细胞整体以及促肾上腺皮质细胞功能亚群中POMC mRNA的调控机制。我们在培养细胞中测量了AVP、CRH或佛波酯（PMA）激活蛋白激酶C的作用。我们比较了完整细胞群体与经药理学方法消除CRH靶细胞的培养物的反应。将成年绵羊垂体前叶细胞解离后培养过夜，用溶剂（完整细胞）或特异性消除CRH靶细胞的CRH毒素偶联物（CRH靶细胞缺失）处理，洗涤后放回培养，随后用溶剂、AVP（100 nM）、CRH（10 nM）或PMA（1 μM）刺激5小时。通过放射免疫分析法（RIA）检测培养基中的ACTH，通过Northern印迹分析检测细胞中的POMC mRNA。在完整细胞群体中，AVP和CRH分别使ACTH分泌从6.5±1.2增加到216±22和81±14 ng/孔，但只有AVP使POMC mRNA稳态水平增加（+48±10%）。用PMA直接激活蛋白激酶C模拟了AVP对ACTH分泌的作用（318±16 ng/孔），但未改变POMC mRNA水平。在CRH靶细胞缺失的群体中，与完整细胞群体相比，对照ACTH分泌（11±3 ng/孔）和POMC mRNA（+69±7%）升高。AVP（55±8 ng/孔）和PMA（120±17 ng/孔）增加了ACTH分泌，但CRH没有；任何处理均未使POMC mRNA显著升高。综上所述，这些数据进一步支持了ACTH分泌与POMC mRNA表达之间解离的观点，并证明AVP可增加绵羊垂体前叶细胞中POMC mRNA的稳态水平。这些数据也与复杂相互作用（可能包括细胞间相互作用）影响ACTH分泌和POMC mRNA稳态水平的概念一致。
