Abstract
Background contextVitamin C (ascorbic acid [AA]) is essential for the synthesis of collagen and also acts as an antioxidant in the intervertebral disc (IVD). However, there is very little information currently available on the identity of the transporter that facilitates AA entry into IVD cells and the factors that mediate the transport process. PurposeTo investigate the expression of the two known isoforms of Na+-coupled vitamin C transporter, SVCT1 and SVCT2, in IVD cells and its regulation by insulin-like growth factor 1 (IGF-1) and the steroid hormone dexamethasone. Study designTo identify the expression and functional activity of the sodium-dependent vitamin C transporter (SVCT) in the IVD. MethodsUptake studies were carried out with rabbit annulus fibrosis and nucleus pulposus cells in 24-well plates using [14C]-AA. To characterize SVCT transporter, uptake was done in the presence and absence of Na+ in the uptake buffer. Time dependency, Na+ activation kinetics, saturation kinetics, and substrate selectivity studies were performed. Regulatory studies were performed in the presence of IGF-1 and the steroid hormone dexamethasone. Gene expression was analyzed by quantitative polymerase chain reaction. ResultsOur real-time polymerase chain reaction results showed the presence of SVCT2 but not SVCT1 in IVD cells. Uptake of vitamin C in IVD cells is Na+ dependent and saturable. The Michaelis constant for the process is 96±11 μM. The activation of vitamin C uptake by Na+ exhibits a sigmoidal relationship, indicating involvement of more than one Na+ in the activation process. The uptake system does not recognize any other water-soluble vitamin as a substrate. Immunocytochemical analysis shows robust expression of SVCT2 protein in IVD cells. The growth factors IGF-1 and the steroid hormone dexamethasone upregulate the expression of SVCT2 in IVD cells. ConclusionsOur studies demonstrate that the active SVCT2 is expressed in IVD cells and that the expression of this transporter is regulated by growth factors IGF-1 and dexamethasone.
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