Childs G V, Unabia G
Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77550.
Mol Endocrinol. 1989 Jan;3(1):117-26. doi: 10.1210/mend-3-1-117.
Arginine vasopressin (AVP) potentiates corticotrope responses to CRH by increasing the percentage of target cells that secrete in a reverse hemolytic plaque assay for ACTH. The present studies were designed to test more specific effects of AVP and its second messengers on CRH binding to individual corticotropes. Spectrophotometric analyses of 560 corticotropes from fractions enriched to 90% by counterflow centrifugation showed a 30% increase in the average area of the dark blue label for biotinylated CRH after a 1-h exposure to 10 nM AVP or after activation of protein kinase-C [by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or L calcium channels (by Bay K 8644). In addition, computer analysis of the color of the label (wavelength 476-483) showed a 13% increase in saturation (intensity of the blue) and a 23% decrease in brightness (amount of white) after stimulation. The gray level readings of the blue color were also 18% lower after stimulation, which indicates an increase in density (less light transmitted). Taken together, the increases in label area and intensity indicated that activation of L calcium channels or protein kinase-C enhanced CRH binding by individual corticotropes. When mixed pituitary cell populations were analyzed for percentages of labeled cells, exposure to Bay K 8644, TPA, angiotensin II, or AVP resulted in 30-40% increases in the percentage of CRH-bound cells. Dual reactions for biotinylated CRH and ACTH showed that most of the added CRH-bound cells stored ACTH. The effect of exposure to two of the activators was not additive, however. If L calcium channels were blocked with nimodipine, the protein kinase-C-mediated enhancement in CRH binding and ACTH release was blocked, indicating that these actions are dependent on extracellular calcium. In contrast, nimodipine did not block the TPA-mediated enhancement of ACTH storage. These studies show that the potentiation of CRH-mediated ACTH release by AVP or angiotensin II may occur by the enhancement of CRH binding to individual corticotropes. This appears to promote the cytochemical detection of additional CRH-bound corticotropes which may stem from a reserve cell population that normally has levels of CRH receptors or ACTH stores below thresholds needed for detection. The source of these cells (from stem cells or multipotential cells) remains to be determined.
精氨酸加压素（AVP）通过增加在促肾上腺皮质激素（ACTH）反向溶血空斑试验中分泌的靶细胞百分比，增强促肾上腺皮质激素释放激素（CRH）对促肾上腺皮质激素细胞的反应。本研究旨在测试AVP及其第二信使对CRH与单个促肾上腺皮质激素细胞结合的更具体影响。对通过逆流离心富集至90%的级分中的560个促肾上腺皮质激素细胞进行分光光度分析，结果显示，在暴露于10 nM AVP 1小时后，或在激活蛋白激酶C[通过12-O-十四烷酰佛波醇-13-乙酸酯（TPA）]或L型钙通道（通过Bay K 8644）后，生物素化CRH的深蓝色标记平均面积增加了30%。此外，对标记颜色（波长476 - 483）的计算机分析显示，刺激后饱和度增加了13%（蓝色强度），亮度降低了23%（白色量）。刺激后蓝色的灰度读数也降低了18%，这表明密度增加（透射光减少）。综合来看，标记面积和强度的增加表明L型钙通道或蛋白激酶C的激活增强了单个促肾上腺皮质激素细胞对CRH的结合。当分析混合垂体细胞群体中标记细胞的百分比时，暴露于Bay K 8644、TPA、血管紧张素II或AVP导致结合CRH的细胞百分比增加了30 - 40%。对生物素化CRH和ACTH的双重反应表明，大多数添加的结合CRH的细胞储存ACTH。然而，暴露于两种激活剂的效果并非相加。如果用尼莫地平阻断L型钙通道，蛋白激酶C介导的CRH结合增强和ACTH释放被阻断，表明这些作用依赖于细胞外钙。相反，尼莫地平并未阻断TPA介导的ACTH储存增强。这些研究表明，AVP或血管紧张素II对CRH介导的ACTH释放的增强作用可能是通过增强CRH与单个促肾上腺皮质激素细胞的结合来实现的。这似乎促进了额外结合CRH的促肾上腺皮质激素细胞的细胞化学检测，这些细胞可能来自一个储备细胞群体，该群体通常具有低于检测所需阈值的CRH受体水平或ACTH储存量。这些细胞的来源（来自干细胞或多能细胞）仍有待确定。
