Ahmad Aamir, Khundmiri Syed J, Pribble Francesca, Merchant Michael L, Ameen Mohammed, Klein Jon B, Levi Moshe, Lederer Eleanor D
Department of Medicine, University of Louisville, Louisville, KY 40202, USA.
Cell Physiol Biochem. 2011;27(6):703-14. doi: 10.1159/000330079. Epub 2011 Jun 17.
BACKGROUND/AIMS: Total body phosphate homeostasis is regulated by expression of type IIa sodium phosphate cotransporter (NpT2a) in the apical membrane (BBM) of renal proximal tubule cells. NpT2a expression is regulated by dietary phosphate and PTH but the mechanisms for trafficking of the protein are unknown. Based on 2D gel electrophoresis and mass spectroscopy data that changes in dietary phosphate stimulated changes in BBM expression of vacuolar H(+)-ATPase, we hypothesized that vacuolar H(+)-ATPase plays a significant role in regulation of NpT2a in opossum kidney (OK) cells, a model for renal proximal tubule transport.
Role of vacuolar H(+)-ATPase was studied in opossum kidney (OK) cells by examining the effect of inhibition of vacuolar H(+)-ATPase on Pi uptake and NpT2a expression.
Pretreatment of OK cells with bafilomycin A(1) and concanamycin A, inhibitors of vacuolar H(+)-ATPases, blocked high phosphate- and PTH-induced degradation of NpT2a, but had no effect on high phosphate or PTH induced inhibition of sodium-dependent phosphate transport. Exposure of the cells to bafilomycin A(1) significantly decreased phosphate transport and apical membrane expression of NpT2a. Treatment with brefeldin A, an inhibitor of Golgi transport, decreased phosphate transport and apical membrane expression of NpT2a while treatment of cells with both brefeldin A and bafilomycin A(1) had no additive effect.
We conclude that vacuolar H(+)-ATPase plays a significant role in exocytosis of NpT2a into the apical membrane and in degradation of NpT2a but has no role in endocytosis.
背景/目的：机体总磷稳态由肾近端小管细胞顶膜（BBM）中IIa型钠磷共转运体（NpT2a）的表达调节。NpT2a的表达受饮食磷和甲状旁腺激素（PTH）调控，但其蛋白转运机制尚不清楚。基于二维凝胶电泳和质谱数据，即饮食磷的变化刺激了液泡H(+)-ATP酶的BBM表达变化，我们推测液泡H(+)-ATP酶在负鼠肾（OK）细胞（一种肾近端小管转运模型）中NpT2a的调节中起重要作用。
通过检测液泡H(+)-ATP酶抑制对磷摄取和NpT2a表达的影响，研究其在负鼠肾（OK）细胞中的作用。
用液泡H(+)-ATP酶抑制剂巴弗洛霉素A(1)和 concanamycin A预处理OK细胞，可阻断高磷和PTH诱导的NpT2a降解，但对高磷或PTH诱导的钠依赖性磷转运抑制无影响。细胞暴露于巴弗洛霉素A(1)显著降低了磷转运和NpT2a的顶膜表达。用高尔基体转运抑制剂布雷菲德菌素A处理可降低磷转运和NpT2a的顶膜表达，而用布雷菲德菌素A和巴弗洛霉素A(1)同时处理细胞则无叠加效应。
我们得出结论，液泡H(+)-ATP酶在NpT2a向顶膜的胞吐作用和NpT2a的降解中起重要作用，但在胞吞作用中不起作用。
