Adaptive regulation of ascorbate transport in osteoblastic cells

Osteoblasts possess a concentrative L‐ascorbate (vitamin C) uptake mechanism involving a Na⁺‐dependent ascorbate transporter located in the plasma membrane. The transporter is specific for ascorbate and stereoselective for L‐ascorbate over D‐isoascorbate. The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system. L‐ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 μM L‐[¹⁴C]ascorbate. The initial rate of L‐[¹⁴C]ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L‐ascorbate‐replete medium was 89 + 8 nmol/g protein per minute. Following removal of L‐ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 + 13 nmol/g protein per minute. Conversely, the initial rate of uptake by cells grown in ascorbate‐free medium decreased following the addition of L‐ascorbate, but not D‐isoascorbate, to the medium. The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L‐ascorbate did not affect uptake of 2‐deoxy‐D‐glucose. Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (V_max) without changes in the affinity of the transport system for L‐ascorbate. These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation. Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.