de novo synthesis of calmodulin binding protein in substance P-induced steroidogenesis in bovine adrenocortical cells.
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.
Yoshida
T
T
Department of Pharmacology, Faculty of Pharmaceutical Science, Okayama University, Japan.
Mio
M
M
Tasaka
K
K
D000302:Adrenal Cortex / Q000187:drug effects* / Q000378:metabolism; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis*; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006854:Hydrocortisone / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D013373:Substance P / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002148:Calmodulin-Binding Proteins; D013373:Substance P; D000001:Calcimycin; D000324:Adrenocorticotropic Hormone; D007930:Leucine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D006854:Hydrocortisone