Ascorbate transport by atT20 mouse pituitary corticotropic tumor cells: Uptake and secretion studies

Paul P. Shields, Thomas R. Gibson, Christopher C. Glembotski

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Ascorbate is an important cofactor in the biosynthesis of αamidated endocrine and neural peptides. Peptidylglycine αamidating monooxygenase (PAM) is the enzyme responsible for the generation of mature COOH-terminal aamidated peptides from COOH-terminal glycine-extended peptides, and this enzyme requires ascorbate for full activity in vitro. Also, cultured intermediate pituitary lobe cells contain PAM and require ascorbate for the COOH-terminal αamidation of aMSH. Since pituitary cells are not capable of synthesizing ascorbate, the ability of the cells to accumulate the cofactor must play an important role in the biosynthesis of αamidated peptides. The AtT20 corticotropic pituitary tumor cell line also contains PAM and a potential site for COOH-terminal αamidation of the pro-ACTH/endorphin-derived hinge peptide and was, thus, used for the study of cellular ascorbate transport. Radiolabeled L-[l-14C]ascorbate ([l-14C]ascorbate) was incubated with the cells under various conditions, and the accumulation of radioactivity by the cells was followed. Reverse phase HPLC was used to identify the integrity of the labeled ascorbate, both intrα and extracellular, during the course of the experiments. The uptake of [1-14C]ascorbate was saturable (Km = 31.5 μM), sodium and temperature dependent, and stereoselective. The products of ascorbate autooxidation, dehydroascorbate and 2,3-diketogulonic acid, did not inhibit [l-14C]ascorbate uptake. To study the presence of ascorbate in the secretory granules, cells were incubated with [l14C]ascorbate and then induced to secrete with isoproterenol or 8-bromo-cAMP. A 2- to 6-fold stimulation of ACTH secretion over the basal secretion rate was observed; however, the secretion of intracellular [l-14C]ascorbate did not change significantly with stimulation, suggesting that very little of the cellular ascorbate was contained within secretory granules.

Original languageEnglish (US)
Pages (from-to)1452-1460
Number of pages9
JournalEndocrinology
Volume118
Issue number4
DOIs
StatePublished - 1986
Externally publishedYes

ASJC Scopus subject areas

  • Endocrinology

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