Yoshida T, Mio M, Tasaka K
Department of Pharmacology, Faculty of Pharmaceutical Science, Okayama University, Japan.
Biochem Pharmacol. 1992 Oct 6;44(7):1357-65. doi: 10.1016/0006-2952(92)90537-s.
In order to clarify the mechanism of substance P (SP)-induced cortisol secretion from bovine adrenocortical (BAC) cells, protein synthesis at the early stage of SP-stimulation in BAC cells was investigated. Both SP and adrenocorticotropic hormone (ACTH) increased [3H]leucine uptake into BAC cells in a dose-dependent fashion. Although the SP-induced [3H]leucine uptake precedes the cortisol secretion, ACTH was slower in inducing [3H]leucine uptake and cortisol secretion. Protein synthesis inhibitors, actinomycin D and cycloheximide, were potent in inhibiting the SP-induced cortisol secretion. SDS-PAGE analysis, revealed that a 240 kDa protein is newly synthesized in BAC cells in response to SP but not ACTH. It was also indicated that the production of this 240 kDa protein was elicited about 30 min after stimulation by SP. Moreover, A23187 and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also caused a rapid [3H]leucine uptake and production of 240 kDa protein. In contrast, dibutyryl cAMP did not induce the synthesis of this 240 kDa protein. Calmidazolium, a calmodulin inhibitor, effectively inhibited not only [3H]leucine uptake but also 240 kDa protein production due to SP. On the other hand, KT-5720, an inhibitor of protein kinase A, had no effect on [3H]leucine uptake or 240 kDa production. Using the [125I]calmodulin-membrane overlay method, it was found that the 240 kDa protein was a newly synthesized calmodulin binding protein. From the present study, it was concluded that the de novo synthesis of this 240 kDa protein may be intimately related to the cortisol secretion in SP-stimulated BAC cells associated with an activation of the Ca-calmodulin pathway.
为了阐明P物质（SP）诱导牛肾上腺皮质（BAC）细胞分泌皮质醇的机制，研究了BAC细胞在SP刺激早期的蛋白质合成情况。SP和促肾上腺皮质激素（ACTH）均以剂量依赖的方式增加了[3H]亮氨酸进入BAC细胞的摄取量。虽然SP诱导的[3H]亮氨酸摄取先于皮质醇分泌，但ACTH诱导[3H]亮氨酸摄取和皮质醇分泌的速度较慢。蛋白质合成抑制剂放线菌素D和环己酰亚胺能有效抑制SP诱导的皮质醇分泌。SDS-PAGE分析显示，BAC细胞中响应SP而非ACTH新合成了一种240 kDa的蛋白质。还表明，这种240 kDa蛋白质的产生在SP刺激后约30分钟开始。此外，A23187和12-O-十四烷酰佛波醇-13-乙酸酯（TPA）也引起了快速的[3H]亮氨酸摄取和240 kDa蛋白质的产生。相比之下，二丁酰cAMP并未诱导这种240 kDa蛋白质的合成。钙调蛋白抑制剂卡米达唑不仅有效抑制了[3H]亮氨酸摄取，还抑制了SP诱导的240 kDa蛋白质的产生。另一方面，蛋白激酶A抑制剂KT-5720对[3H]亮氨酸摄取或240 kDa蛋白质的产生没有影响。使用[125I]钙调蛋白-膜覆盖法发现，这种240 kDa蛋白质是一种新合成的钙调蛋白结合蛋白。从本研究得出结论，这种240 kDa蛋白质的从头合成可能与SP刺激的BAC细胞中与钙-钙调蛋白途径激活相关的皮质醇分泌密切相关。
