Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium

Nicholas A Delamere, Miguel Coca-Prados, Shelinder Aggarwal

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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/μg 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 μM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 μM). 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 (10-6M), 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.

Original languageEnglish (US)
Pages (from-to)102-108
Number of pages7
JournalBBA - Biomembranes
Volume1149
Issue number1
DOIs
StatePublished - Jun 18 1993
Externally publishedYes

Fingerprint

Phorbol 12,13-Dibutyrate
Ascorbic Acid
Epithelium
Cells
Rabbits
Protein Kinase C
Cell Line
Sodium
Phloretin
Staurosporine
Protein C Inhibitor
Aqueous Humor
Ouabain
Protein Kinase Inhibitors
Cultured Cells
Down-Regulation
Epithelial Cells
Chemical activation
Tissue
Fluids

Keywords

  • Ascorbic acid
  • ATPase, Na/K-
  • Ciliary epithelium, non-pigmented
  • cyclic AMP
  • Membrane transport
  • Protein kinase C

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium. / Delamere, Nicholas A; Coca-Prados, Miguel; Aggarwal, Shelinder.

In: BBA - Biomembranes, Vol. 1149, No. 1, 18.06.1993, p. 102-108.

Research output: Contribution to journalArticle

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abstract = "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/μg 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 μM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 μM). 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 (10-6M), 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.",
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