STUDIES ON REGULATION OF THE ASCORBIC-ACID TRANSPORTER IN A CELL-LINEDERIVED FROM RABBIT NONPIGMENTED CILIARY EPITHELIUM

A cell line was derived from rabbit non-pigmented ciliary epithelium. The non-pigmented ciliary epithelium is one of the two cell layers whi ch secrete aqueous humor into the eye and concentrate ascorbic acid in the newly-formed fluid. The cultured non-pigmented epithelial cells a ccumulated ascorbic acid at a rate of 3-5 pmol/mug protein per h. As i n freshly-isolated native tissue, the ascorbate uptake mechanism was s odium-dependent and could be inhibited by phloretin (apparent K(i) = 2 -10(-5) M). Phorbol 12,13-dibutyrate (PDBu), a protein kinase C activa tor, reduced the ascorbate uptake rate. The PDBu effect was concentrat ion-dependent; at a concentration of 10(-6) M, PDBu reduced the ascorb ate uptake rate to 65% of the control value. PDBu reduced the maximal rate of ascorbate uptake (determined at 200-500 muM external ascorbate ) but caused no detectable change in the K(m) for ascorbic acid (appro x. 80 muM). The PDBu-induced inhibition of ascorbate uptake persisted in the presence of ouabain and in low sodium (25 mM Na) medium, sugges ting that the effect is not secondary to a change in the sodium gradie nt. Furthermore, no detectable elevation of cell sodium content was se en in cells equilibrated with Na-22 prior to PDBu treatment. The PDBu- induced inhibition of ascorbate uptake was apparently mediated by prot ein kinase C because the effect was not observed in the presence of st aurosporine (10(-6) M), a protein kinase C inhibitor, or in cells in w hich protein kinase C was downregulated. These observations suggest th at activation of protein kinase C causes inhibition of the ascorbate t ransporter in this cultured cell line.