Lederer E D, Sohi S S, Mathiesen J M, Klein J B
Department of Internal Medicine, Veterans Affairs Medical Center, Louisville 40206; and Department of Internal Medicine, University of Louisville, Louisville, Kentucky 40202-1718, USA.
Am J Physiol. 1998 Aug;275(2):F270-7. doi: 10.1152/ajprenal.1998.275.2.F270.
The purpose of the present study was to determine the effect of protein kinase A and protein kinase C activation on the membrane expression of NaPi-4, the type II sodium-phosphate cotransporter in OK cells. NaPi-4 expression was measured using polyclonal antisera produced in rabbits against a peptide identical to the carboxy-terminal 12-amino acid sequence of NaPi-4. The antisera identified an apically localized protein by confocal imaging of intact OK cells and a broad band of 110-140 kDa by immunoblot analysis of OK cell membranes. Treatment of OK cells with parathyroid hormone (PTH) decreased the intensity of the 110- to 140-kDa band, which was detectable by 2 h, maximal by 4 h at 62%, and sustained for 24 h. 8-Bromo-cAMP (8-BrcAMP) inhibited NaPi-4 expression for up to 24 h by over 90%. However, phorbol 12-myristate 13-acetate inhibited NaPi-4 expression by less than 10%. PTH-(3-34), a fragment which stimulates only protein kinase C, inhibited phosphate transport but also had no effect on NaPi-4 expression. We conclude that protein kinase A but not protein kinase C inhibits sodium-phosphate uptake in OK cells by downregulation of NaPi-4 expression.
本研究的目的是确定蛋白激酶A和蛋白激酶C激活对OK细胞中II型钠-磷酸共转运体NaPi-4膜表达的影响。使用针对与NaPi-4羧基末端12个氨基酸序列相同的肽段在兔体内产生的多克隆抗血清来检测NaPi-4的表达。通过对完整OK细胞进行共聚焦成像，该抗血清鉴定出一种位于顶端的蛋白，并且通过对OK细胞膜进行免疫印迹分析鉴定出一条110 - 140 kDa的宽带。用甲状旁腺激素（PTH）处理OK细胞可降低110至140 kDa条带的强度，2小时时可检测到，4小时时达到最大值，为62%，并持续24小时。8-溴-环磷酸腺苷（8-BrcAMP）在长达24小时内抑制NaPi-4表达超过90%。然而，佛波醇12-肉豆蔻酸酯13-乙酸酯抑制NaPi-4表达不到10%。仅刺激蛋白激酶C的片段PTH-(3 - 34)抑制磷酸盐转运，但对NaPi-4表达也没有影响。我们得出结论，蛋白激酶A而非蛋白激酶C通过下调NaPi-4表达来抑制OK细胞中的钠-磷酸盐摄取。
