Requirement for sodium-dependent ascorbic acid transport in osteoblast
function.
Franceschi, Renny T., John X. Wilson, and S. Jeffrey Dixon.
Department of Periodontics, Prevention, and Geriatrics, School of
Dentistry and Department of Biological Chemistry, School of Medicine,
University of Michigan, Ann Arbor, Michigan 48109-1078 and the Department of
Physiology and Division of Oral Biology, Faculty of Dentistry, The University
of Western Ontario, London, Ontario, Canada N6A5C1
APStracts 2:0051C, 1995.
Ascorbic acid is necessary for expression of the osteoblast phenotype. We
examined whether sodium-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 of
ascorbate. MC3T3-E1 cells were found to possess a saturable, stereoselective,
sodium-dependent ascorbic acid transport activity that is sensitive to the
transport inhibitors, sulfinpyrazone, 4,4'-diisothiocyanostilbene-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 transporters. Upon addition of 100[mu]M
ascorbic acid to the extracellular medium, intracellular concentrations of 10
mM were reached within 5-10 h and remained constant for up to 24 h. A good
correlation was observed between intracellular ascorbic acid concentration
and rate of hydroxyproline synthesis. Although ascorbic acid was transported
preferentially compared with D-isoascorbic acid, both isomers had equivalent
activity 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, ascorbic acid
transport inhibitors which prevented intracellular accumulation of vitamin
blocked the synthesis of hydroxyproline. Thus, sodium-dependent ascorbic acid
transport is required for MC3T3-E1 cells to achieve the millimolar
intracellular vitamin C concentrations necessary for maximal prolyl
hydroxylase activity and expression of the osteoblast phenotype.
Received 25 August 1994; accepted in final form 20 December 1994
APS Manuscript Number C0410-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1994 The American Physiological Society.
Published in APStracts on 27 February 1995.