Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented 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 which secrete aqueous humor into the eye and concentrate ascorbic acid in the newly-formed fluid. The cultured non-pigmented epithelial cells accumulated ascorbic acid at a rate of 3-5 pmol/micrograms protein per h. As in freshly-isolated native tissue, the ascorbate uptake mechanism was sodium-dependent and could be inhibited by phloretin (apparent Ki = 2-10(-5) M). Phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, reduced the ascorbate uptake rate. The PDBu effect was concentration-dependent; at a concentration of 10(-6) M, PDBu reduced the ascorbate uptake rate to 65% of the control value. PDBu reduced the maximal rate of ascorbate uptake (determined at 200-500 microM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 microM). The PDBu-induced inhibition of ascorbate uptake persisted in the presence of ouabain and in low sodium (25 mM Na) medium, suggesting that the effect is not secondary to a change in the sodium gradient. Furthermore, no detectable elevation of cell sodium content was seen in cells equilibrated with 22Na prior to PDBu treatment. The PDBu-induced inhibition of ascorbate uptake was apparently mediated by protein kinase C because the effect was not observed in the presence of staurosporine (10(-6) M), a protein kinase C inhibitor, or in cells in which protein kinase C was downregulated. These observations suggest that activation of protein kinase C causes inhibition of the ascorbate transporter in this cultured cell line.
Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Research Institute, University of Louisville School of Medicine 40292.
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Delamere
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M
Coca-Prados
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Aggarwal
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002924:Ciliary Body / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D010693:Phloretin / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D019311:Staurosporine
D000470:Alkaloids; D002352:Carrier Proteins; D015240:Phorbol 12,13-Dibutyrate; D003994:Bucladesine; D012964:Sodium; D019311:Staurosporine; D001205:Ascorbic Acid; D010693:Phloretin