Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: substrate specificity and sensitivity to transport inhibitors

Abstract

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na(+)-dependent uptake system for L-ascorbate (J Membr Biol 111:83-91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37 degrees C. Uptake of [L-14C]ascorbate (5 microM) was inhibited 98 +/- 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 +/- 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (less than 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) approximately equal to sulfinpyrazone greater than furosemide approximately equal to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.