Pituitary immunoreactive calcitonin-like material: Lack of evidence for cross-reactivity with pro-opiomelanocortin

Abstract

In mammals, calcitonin (CT) is synthesized, stored, and secreted by intrathyroidal C cells. Several reports have suggested the presence of immunoreactive CT in the pituitary gland. We have studied the rat pituitary gland using a radioimmunoassay for CT and have also found immunoreactive CT-like material. Assay of extracts of whole rat pituitary glands was performed using a radioimmunoassay for human CT, which gave identical dilution curves with synthetic human CT (hCT), synthetic rat CT (rCT), and mouse and rat thyroid extracts, but not with a variety of other pituitary and hypothalamic peptides. Immunoreactive CT (iCT) content of extracts of whole pituitary glands ranged from 6 to 72 pg/mg wet weight of tissue (60–840 pg/whole pituitary gland), whereas iCT was not measureable (less than 5 pg/mg tissue) in similar extracts of hypothalamus and cerebral cortex. Gel filtration studies of pituitary extracts showed a peak of iCT, which eluted with 125I-rCT and diluted in parallel with rCT. To investigate whether the pituitary iCT was related to pro-opiomelanocortin, extracts of ACTH-producing AtT20 D16 cells from mice, which contain the ACTH precursor in large quantities, were examined and no iCT was found. Immunohistochemical studies of rat pituitary glands with peroxidase-antiperoxidase and immunofluorescent techniques showed positive staining for CT in cells in the pars anterior, but not in the pars intermedia or pars nervosa; this staining was not eliminated when the antiserum was absorbed with CT under conditions that completely obliterated staining of rat thyroid glands. Double staining demonstrated essentially two distinct populations of cells, one positive for CT and another positive for ACTH, with less than 1% of the cells positive for both ACTH and CT. Immunoreactive CT-like material was present in the pituitary glands of rats thyroparathyroidectomized 18 days before they were killed, but was diminished. Biosynthetic labeling in vitro of rat pituitary glands with 3H-leucine showed incorporation into prolactin; there was no incorporation into CT. No in vitro secretion of iCT by whole rat pituitary glands either basally or after high K + stimulation was observed. We conclude that: (1) a substance that has certain immunologic and size characteristics of CT is present in minute amounts in the pituitary gland of rats; (2) this material is not a part of the ACTH precursor; and (3) positive immunohistochemical staining in pituitary glands may not be specific for authentic CT.