ascorbic-acid and Pituitary-Neoplasms

Ascorbate is an important cofactor in the biosynthesis of alpha-amidated endocrine and neural peptides. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme responsible for the generation of mature COOH-terminal alpha-amidated 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 alpha-amidation of alpha MSH. 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 alpha-amidated peptides. The AtT20 corticotropic pituitary tumor cell line also contains PAM and a potential site for COOH-terminal alpha-amidation of the pro-ACTH/endorphin-derived hinge peptide and was, thus, used for the study of cellular ascorbate transport. Radiolabeled L-[1-14C]ascorbate ([1-14C]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 intra- and extracellular, during the course of the experiments. The uptake of [1-14C]ascorbate was saturable (Km = 31.5 microM), sodium and temperature dependent, and stereoselective. The products of ascorbate autooxidation, dehydroascorbate and 2,3-diketogulonic acid, did not inhibit [1-14C]ascorbate uptake. To study the presence of ascorbate in the secretory granules, cells were incubated with [1-14C]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 [1-14C]ascorbate did not change significantly with stimulation, suggesting that very little of the cellular ascorbate was contained within secretory granules.

Topics: 2,3-Diketogulonic Acid; 8-Bromo Cyclic Adenosine Monophosphate; Adrenocorticotropic Hormone; Animals; Ascorbic Acid; Biological Transport; Cell Line; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Dose-Response Relationship, Drug; Isoproterenol; Kinetics; Mice; Mixed Function Oxygenases; Multienzyme Complexes; Osmolar Concentration; Oxidoreductases Acting on CH-NH Group Donors; Pituitary Neoplasms; Pro-Opiomelanocortin; Sodium; Temperature

Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.

Topics: Amides; Amino Acids; Animals; Ascorbic Acid; Cell Line; Chromatography, Gel; Male; Mice; Molecular Weight; Peptide Fragments; Pituitary Gland; Pituitary Gland, Anterior; Pituitary Neoplasms; Pro-Opiomelanocortin; Protein Processing, Post-Translational; Rats

Ascorbic acid uptake in AtT-20 tumor cells and primary cultures of rat anterior and intermediate pituitary was sodium-dependent and showed half-maximal saturation between 9 and 18 microM ascorbate. When incubated in [14C]ascorbic acid at concentrations similar to those in serum (50 microM), all of the cells concentrated ascorbate 20- to 40-fold, producing intracellular ascorbate concentrations of 1-2 mM. HPLC analyses showed that over 90% of the intracellular label comigrated with authentic ascorbic acid. Although ascorbate was rapidly oxidized in culture medium in the absence of cells, incubation of ascorbate in culture medium in the presence of cells stabilized the ascorbate substantially. Unlike systems that transport dehydroascorbic acid, the ascorbate transport systems in all three preparations were not inhibited by glucose. Thus all three systems possess similar saturable, high-affinity, sodium-dependent active transport systems for ascorbic acid.

Topics: Animals; Ascorbic Acid; Biological Transport; Cell Line; Glucose; Kinetics; Pituitary Gland; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Sodium

Secretion of peptidyl-glycine alpha-amidating monooxygenase (PAM) activity and enkephalin convertase (a carboxypeptidase B-like enzyme) activity from AtT-20 mouse corticotrope tumor cells was compared with secretion of pro-ACTH/endorphin-derived peptides. Upon stimulation of secretion by corticotropin-releasing factor, a beta-adrenergic agonist, or barium ions, secretion of PAM activity, enkephalin convertase activity, and immunoactive hormone rose in parallel. During inhibition of stimulated secretion with glucocorticoids or cobalt ions, secretion of PAM activity, enkephalin convertase activity, and immunoactive hormone fell in parallel. No measurable secretion of cytosolic, mitochondrial, or lysosomal marker enzymes was detected, indicating that the secretion of PAM activity and enkephalin convertase activity was specific and not a result of cell damage or lysis. The kinetic characteristics, apparent mol wt, and enzymatic properties determined for the secreted enzymes were similar to those for the enzymes from secretory granules. In AtT-20 cells treated with glucocorticoids for up to 8 days, cellular levels of immunoactive ACTH/endorphin-related peptides decreased to one third of control levels. Levels of PAM activity in cell extracts declined in parallel with levels of hormone. In contrast, levels of enkephalin convertase activity, cell protein, lactate dehydrogenase, fumarase, and lysosomal carboxypeptidase did not decline in response to chronic glucocorticoid exposure.

Topics: Animals; Ascorbic Acid; Carboxypeptidase H; Carboxypeptidases; Cell Line; Copper; Copper Sulfate; Corticosterone; Corticotropin-Releasing Hormone; Dexamethasone; Ditiocarb; Kinetics; Mice; Mixed Function Oxygenases; Molecular Weight; Multienzyme Complexes; Oxidoreductases Acting on CH-NH Group Donors; Pituitary Neoplasms

Mouse anterior pituitary corticotropic tumor cells (AtT-20/D-16v) were homogenized and subjected to subcellular fractionation. A secretory granule associated enzyme activity was detected which could convert D-Tyr-Val-Gly into D-Tyr-Val-NH2. The enzyme activity was dependent on the presence of molecular oxygen, copper ions, and reduced ascorbate. Potent endogenous inhibitors of the enzyme obscured the activity unless appropriate levels of copper ions were added. The production of radiolabeled D-Tyr-Val-NH2 from labeled D-Tyr-Val-Gly was inhibited by a wide variety of peptides possessing COOH-terminal glycine residues but not by a number of other peptides, suggesting that many peptides with COOH-terminal glycine residues can function as substrates in the alpha-amidation reaction. Kinetic studies with varied concentrations of D-Tyr-Val-Gly demonstrated Michaelis-Menten kinetics; both the Km for D-Tyr-Val-Gly and maximum velocity (Vmax) increased upon addition of ascorbate to the reaction. Under optimized assay conditions, the secretory granule pool contains enough alpha-amidation activity to alpha-amidate all the relevant peptides in granules in a small fraction of the total time required for complete biosynthetic processing. Secretion of alpha-amidation activity was stimulated along with secretion of pro-ACTH/endorphin-derived peptides upon addition of synthetic corticotropin releasing factor or 8-bromo-cAMP.

Topics: Amides; Animals; Ascorbic Acid; Cytoplasmic Granules; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Kinetics; Mice; Oligopeptides; Pituitary Gland, Anterior; Pituitary Neoplasms; Protein Processing, Post-Translational

Topics: Adenoma, Chromophobe; Ascorbic Acid; Breast Neoplasms; Choriocarcinoma; Chorionic Gonadotropin; Female; Hemagglutination Inhibition Tests; Humans; Immune Sera; Luteinizing Hormone; Menopause; Middle Aged; Pituitary Neoplasms; Pregnancy; Uterine Neoplasms

Topics: Adrenal Glands; Adrenalectomy; Adrenocorticotropic Hormone; Aniline Compounds; Animals; Ascorbic Acid; Body Weight; Corticosterone; Fats; Growth Hormone; Liver; Liver Glycogen; Neoplasm Transplantation; Neoplasms, Experimental; Organ Size; Pituitary Neoplasms; Prolactin; Rats

Topics: Adenine Nucleotides; Adrenal Gland Neoplasms; Adrenocorticotropic Hormone; Animals; Ascorbic Acid; Endocrine Gland Neoplasms; Glycogen; Metabolism; Mice; NADP; Phosphotransferases; Pituitary Neoplasms; Research; Steroids; Tissue Culture Techniques