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 language | English (US) |
|---|---|
| Pages (from-to) | 102-108 |
| Number of pages | 7 |
| Journal | BBA - Biomembranes |
| Volume | 1149 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jun 18 1993 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium
AU - Delamere, Nicholas A
AU - Coca-Prados, Miguel
AU - Aggarwal, Shelinder
PY - 1993/6/18
Y1 - 1993/6/18
N2 - 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.
AB - 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.
KW - Ascorbic acid
KW - ATPase, Na/K-
KW - Ciliary epithelium, non-pigmented
KW - cyclic AMP
KW - Membrane transport
KW - Protein kinase C
UR - http://www.scopus.com/inward/record.url?scp=0027156570&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027156570&partnerID=8YFLogxK
U2 - 10.1016/0005-2736(93)90030-4
DO - 10.1016/0005-2736(93)90030-4
M3 - Article
C2 - 8391316
AN - SCOPUS:0027156570
VL - 1149
SP - 102
EP - 108
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
SN - 0005-2736
IS - 1
ER -