title: Phosphorylation of rat and human adrenocorticotropin-related peptides: physiological regulation and studies of secretion.
abstract: Possible physiological factors that might control the extent of phosphorylation of ACTH-related peptides in the rat pituitary were investigated both in vivo and with cultured anterior or neurointermediate pituitary cells. Phosphorylated and nonphosphorylated forms of ACTH and corticotropin-like intermediate lobe peptide [CLIP or ACTH(18-39)] were separated by isoelectric focusing or high performance liquid chromatography and quantified by immunoassay. Radiolabeled ACTH- or CLIP-related peptides from cultured pituitary cells incubated in [3H]tyrosine were isolated by immunoprecipitation or immunoadsorption, fractionated as above, and detected by liquid scintillation counting. Both in the animal and in culture, the extent of phosphorylation of anterior pituitary cellular ACTH did not vary when the release of ACTH was stimulated (by metyrapone treatment, corticotropin-releasing factor, or cAMP) or inhibited (by dexamethasone). Long term cultures of rat anterior pituitary cells continued to phosphorylate ACTH in both serum-containing and serum-free medium; the extent of phosphorylation of ACTH rose from about 55% in vivo to 80% within 1 week in culture. By comparison, the extent of phosphorylation of intermediate pituitary CLIP decreased from about 75% in vivo to about 40-50% in culture. The extent of phosphorylation of the ACTH and CLIP secreted by primary pituitary cultures simply reflected the extent of phosphorylation of ACTH and CLIP in the cells, in both basal and stimulated states. Human ACTH(1-39) contains the same amino acid sequence that is phosphorylated in rat and mouse ACTH(1-39). Based on isoelectric focusing and high performance liquid chromatography of the tryptic peptides of human ACTH (with and without alkaline phosphatase treatment), about one third of the ACTH in the human pituitary is phosphorylated.
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D053497:Corticotropin-Like Intermediate Lobe Peptide; D003470:Culture Media; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D010902:Pituitary Gland / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D010904:Pituitary Gland, Posterior / Q000378:metabolism; D051381:Rats; D014357:Trypsin
chemical_list: D003470:Culture Media; D010446:Peptide Fragments; D010455:Peptides; D053497:Corticotropin-Like Intermediate Lobe Peptide; D000324:Adrenocorticotropic Hormone; D014357:Trypsin