Multi time-resolved spectroscopic analysis on molecular mechanisms of steroid hormone synthesis in adrenal cortex.

用促肾上腺皮质激素、三磷酸腺苷或血管紧张素Ⅱ刺激类固醇激素的合成。在单个肾上腺皮质束状细胞中，所有受检者的肾上腺皮质束细胞均观察到钙信号。在所有的情况下，我们发现钙信号细胞和非钙信号细胞共存。钙信号和酶在单个细胞内分布的多重成像显示，钙信号细胞不含有3β-羟基类固醇脱氢酶和细胞色素P450scc和P450c21，这意味着钙信号细胞不合成类固醇激素。另一方面，非钙信号转导细胞含有所有这些酶，表明非钙信号转导细胞是类固醇合成细胞。在共聚焦显微镜下，也观察到细胞核内钙离子浓度的增加。我们成功地用磷光探针标记了肾上腺素(ADX)和肾上腺素还原酶(ADR)，而不降低电子转移活性。通过测量时间分辨磷光各向异性，研究了ADX与P450scc和ADR与P450scc之间的蛋白质-蛋白质相互作用。各向异性测量表明，P450scc、ADX和ADR形成了三者之间的缔合。

ACTH,ATP or angiotensin II was used to stimulate the steroidogenesis. In a single adrenocortical fasciculata cell, calcium signal was observed for all stmuli examined. In all cases, we found the co-exsitence of Ca-signaling cells and non-Ca-signaling cells. Multiple imaging of calcium signal and enzyme distribution within a single cell revealed that a Ca signaling cell did not contain 3beta-hydroxysteroid dehydrogenase and cytochrome P450scc and P450c21, implying that a Ca-signaling cell does not synthesize steroid hormones. On the other hand, non-Ca-signaling cell contained all these enzymes, indicating that the non-Ca-signaling cell is a steroidogenic cell. With a confocal microscope, the increase of calcium concentration was also observed in nucleus.We succeeded to label adrenodoxin (ADX) and adrenodoxin reductase (ADR) with phosphorescent probes without decreasing the electron transfer activity. Protein-protein interactions were investigated between ADX and P450scc and between ADR and P450scc by measuring time-resolved phosphorescence anisotropy. The anistropy measurements revealed the formation of ternery association consisting of P450scc, ADX and ADR.