High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake of l-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulated l-[¹⁴C]ascorbate during incubations of 1-30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparent K_m=30±2 μm and V_max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138 mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeled l-ascorbate was a more potent inhibitor (IC₅₀=30±5 μm) of l-[¹⁴C]ascorbate transport than was d-isoascorbate (IC₅₀=380±55 μm). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.