REQUIREMENT FOR NA-DEPENDENT ASCORBIC-ACID TRANSPORT IN OSTEOBLAST FUNCTION()

Ascorbic acid is necessary for expression of the osteoblast phenotype. We examined whether Na+-dependent transport is required for MC3T3-E1 preosteoblast cells to respond to vitamin C and investigated the role of membrane transport in the intracellular accumulation and function o f ascorbate. MC3T3-E1 cells were found to possess a saturable, stereos elective, Na+-dependent ascorbic acid transport activity that is sensi tive to the transport inhibitors sulfinpyrazone, 4,4'-diisothiocyanost ilbene-2,2'-disulfonic acid, and phloretin. Transport activity showed no competition with glucose or 2-deoxyglucose and was not inhibited by cytochalasin B, indicating that it is distinct from known hexose tran sporters. On addition of 100 mu M ascorbic acid to the extracellular m edium, intracellular concentrations of 10 mM were reached within 5-10 h and remained constant for up to 24 h. A good correlation was observe d between intracellular ascorbic acid concentration and rate of hydrox yproline synthesis. Although ascorbic acid was transported preferentia lly compared with D-isoascorbic acid, both isomers had equivalent acti vity in stimulating hydroxyproline formation once they entered cells. Marked stereoselectivity for extracellular L-ascorbic acid relative to D-isoascorbic acid was also seen when alkaline phosphatase and total hydroxyproline were measured after 6 days in culture. Moreover, ascorb ic acid transport inhibitors that prevented intracellular accumulation of vitamin blocked the synthesis of hydroxyproline. Thus Na+-dependen t ascorbic acid transport is required for MC3T3-E1 cells to achieve th e millimolar intracellular vitamin C concentrations necessary for maxi mal prolyl hydroxylase activity and expression of the osteoblast pheno type.