Oki Y, Nicholson W E, Orth D N
Department of Medicine, Vanderbilt University Medical Center, Nashville 37232.
Endocrinology. 1990 Jul;127(1):350-7. doi: 10.1210/endo-127-1-350.
Arginine vasopressin (AVP) stimulates biphasic release of ACTH from anterior pituitary corticotrophs. The response consists of an initial transient spike phase lasting less than 3 min and a subsequent sustained plateau phase that persists for as long as AVP is present. AVP also acts synergistically with CRF on ACTH release. We have previously shown that the initial spike phase of the response mainly requires release of intracellular Ca2+ and is independent of calmodulin, whereas the sustained plateau phase, like the monophasic sustained response elicited by CRF, involves the influx of extracellular Ca2+ via L-type voltage-sensitive Ca2+ channels and activation of calmodulin. We have also shown that the synergism between AVP and CRF does not require extracellular Ca2+ influx. In this study we examined the role of Ca2+/phospholipid-dependent protein kinase-C (PKC) in the two phases of the response to AVP and in the synergism between AVP and CRF. We exploited the observation that prolonged exposure to phorbol esters down-regulates PKC. Dispersed adult male rat anterior pituitary cells were incubated in static suspension culture for 4-5 days, 0.5 microM phorbol 12-myristate 13-acetate (PMA) or 0.0005% dimethylsulfoxide vehicle alone was added, and the incubation was continued for 24 h. The cells were preperifused with PMA-free perifusion medium for 3 h and then perifused with various agents for 10-20 min. Effluent fractions were collected every 30 sec or 1 min and subjected to ACTH RIA. Pretreatment with PMA inhibited the subsequent response to 100 nM PMA and 100 microM dioctanoylglycerol, but not to 5 microM forskolin or to depolarization with 56 mM KCl, demonstrating specific down-regulation of PKC. PMA pretreatment had no effect on the initial spike phase of the response to AVP, but inhibited the sustained plateau phase by 57% (P less than 0.005) and, consequently, the integrated total response by 33% (P less than 0.05). Pretreatment had no effect on the response to CRF. However, pretreatment with PMA completely blocked both phases of the synergistic response to the combination of AVP and CRF. These results indicate that activation of PKC is required for the sustained phase of the response to AVP and both phases of its synergistic interaction with the protein kinase-A pathway, but is not involved in the initial spike phase of the response to AVP, which presumably is mediated by inositol 1,4,5-trisphosphate-stimulated mobilization of intracellular Ca2+, or in the independent activation of the protein kinase-A pathway by CRF.
精氨酸加压素（AVP）刺激垂体前叶促肾上腺皮质激素细胞双相释放促肾上腺皮质激素（ACTH）。该反应包括一个持续时间不到3分钟的初始瞬时峰值阶段和随后只要AVP存在就持续的持续平台期。AVP在ACTH释放方面也与促肾上腺皮质激素释放因子（CRF）协同作用。我们之前已经表明，反应的初始峰值阶段主要需要细胞内Ca2+的释放，且不依赖于钙调蛋白，而持续平台期，就像CRF引发的单相持续反应一样，涉及细胞外Ca2+通过L型电压敏感性Ca2+通道的内流以及钙调蛋白的激活。我们还表明，AVP与CRF之间的协同作用不需要细胞外Ca2+内流。在本研究中，我们研究了Ca2+/磷脂依赖性蛋白激酶C（PKC）在对AVP反应的两个阶段以及AVP与CRF之间协同作用中的作用。我们利用了长时间暴露于佛波酯会下调PKC这一观察结果。将成年雄性大鼠分散的垂体前叶细胞在静态悬浮培养中孵育4 - 5天，单独添加0.5微摩尔佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）或0.0005%二甲基亚砜载体，然后继续孵育24小时。细胞先用不含PMA的灌流培养基预灌流3小时，然后用各种试剂灌流10 - 20分钟。每30秒或1分钟收集流出部分，并进行ACTH放射免疫分析。用PMA预处理可抑制随后对100纳摩尔PMA和100微摩尔二辛酰甘油的反应，但不影响对5微摩尔福斯高林或56毫摩尔氯化钾去极化的反应，这证明了PKC的特异性下调。PMA预处理对AVP反应的初始峰值阶段没有影响，但使持续平台期降低了57%（P小于0.005），因此，综合总反应降低了33%（P小于0.05）。预处理对CRF反应没有影响。然而，用PMA预处理完全阻断了对AVP和CRF组合的协同反应的两个阶段。这些结果表明，PKC的激活是对AVP反应的持续阶段及其与蛋白激酶A途径协同相互作用的两个阶段所必需的，但不参与对AVP反应的初始峰值阶段，该阶段大概是由肌醇1,4,5 - 三磷酸刺激的细胞内Ca2+动员介导的，也不参与CRF对蛋白激酶A途径的独立激活。
