Liu J P, Robinson P J, Funder J W, Engler D
Medical Research Centre, Prince Henry's Hospital, Melbourne, Victoria, Australia.
J Biol Chem. 1990 Aug 25;265(24):14136-42.
This study was undertaken to define the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) release and biosynthesis in cultured ovine anterior pituitary cells and to define the intracellular mechanisms responsible for their action. At 4 h, CRF and AVP increased both ACTH release and total ACTH content, with AVP clearly the more potent agonist (maximal ACTH release: AVP, 22.8-fold; CRF, 7.6-fold; maximal increment in total ACTH content: AVP, 1.9-fold; CRF, 1.1-fold; EC50 for ACTH release: AVP, 2.3 +/- 0.5 nM; CRF, 9.2 +/- 5.0 nM). The increase in total ACTH content was interpreted to reflect an augmentation of ACTH biosynthesis since it was abolished by 10 microM cycloheximide. Exposure of the anterior pituitary cells to increasing concentrations of forskolin or 8-bromo-cAMP elicited increases in ACTH release and total ACTH content that were similar to those caused by CRF. A 30-min incubation with phorbol 12-myristate 13-acetate (PMA) caused a dose-related translocation of protein kinase C from the cytosol to the cell membrane; after 4 h, the increases in ACTH release and total ACTH content in response to increasing concentrations of PMA were similar to those caused by AVP. Chronic (24 h) exposure to 150 nM PMA caused an almost total depletion of both cytosolic and membrane-bound protein kinase C activities. When protein kinase C-depleted cells were subsequently exposed to AVP, the increases in ACTH release and total ACTH content were markedly attenuated, but the responses to CRF were preserved. Finally, the combination of CRF and AVP, CRF and PMA, or AVP and 8-bromo-cAMP increased ACTH release and total ACTH content in a synergistic manner. We conclude that: 1) in ovine anterior pituitary cells, AVP is the predominant regulator of ACTH secretion and biosynthesis; 2) the action of AVP is predominantly mediated by activation of protein kinase C, whereas the action of CRF is likely to be mediated by activation of the cAMP-dependent protein kinase (protein kinase A); and 3) the ability of CRF and AVP to increase total ACTH content and secretion in a synergistic manner provides a demonstration in normal pituitary cells that protein kinases C and A may interact in a unidirectional manner to regulate ACTH biosynthesis in addition to ACTH release. This interaction may take place within, or between, individual corticotropes.
本研究旨在确定促肾上腺皮质激素释放因子（CRF）和精氨酸加压素（AVP）在培养的绵羊垂体前叶细胞中对促肾上腺皮质激素（ACTH）释放和生物合成的调节作用，并确定其作用的细胞内机制。在4小时时，CRF和AVP均增加了ACTH的释放和ACTH的总含量，其中AVP显然是更有效的激动剂（ACTH最大释放量：AVP为22.8倍；CRF为7.6倍；ACTH总含量最大增加量：AVP为1.9倍；CRF为1.1倍；ACTH释放的半数有效浓度：AVP为2.3±0.5 nM；CRF为9.2±5.0 nM）。ACTH总含量的增加被解释为反映了ACTH生物合成的增加，因为它被10μM的环己酰亚胺所消除。垂体前叶细胞暴露于浓度不断增加的福斯可林或8-溴-cAMP中，会引起ACTH释放和ACTH总含量的增加，这与CRF引起的情况相似。用佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）孵育30分钟会导致蛋白激酶C从细胞质向细胞膜发生剂量相关的转位；4小时后，随着PMA浓度增加，ACTH释放和ACTH总含量的增加与AVP引起的情况相似。长期（24小时）暴露于150 nM PMA会导致细胞质和膜结合的蛋白激酶C活性几乎完全耗尽。当蛋白激酶C耗尽的细胞随后暴露于AVP时，ACTH释放和ACTH总含量的增加明显减弱，但对CRF的反应得以保留。最后，CRF与AVP、CRF与PMA或AVP与8-溴-cAMP的组合以协同方式增加了ACTH释放和ACTH总含量。我们得出以下结论：1）在绵羊垂体前叶细胞中，AVP是ACTH分泌和生物合成的主要调节因子；2）AVP的作用主要通过蛋白激酶C的激活介导，而CRF的作用可能通过cAMP依赖性蛋白激酶（蛋白激酶A）的激活介导；3）CRF和AVP以协同方式增加ACTH总含量和分泌的能力在正常垂体细胞中证明，蛋白激酶C和A可能以单向方式相互作用，除了调节ACTH释放外，还调节ACTH生物合成。这种相互作用可能发生在单个促肾上腺皮质激素细胞内或细胞之间。
