Characterization of gamma-melanotropin-like immunoreactivity and its secretion in an adrenocorticotropin-producing mouse pituitary tumor cell line.

Abstract

gamma MSH, a putative hormone in the N-terminal region of the ACTH/beta-endorphin (beta-EP) precursor protein, was studied by RIA with an antiserum against gamma 3MSH in ACTH-producing mouse pituitary tumor cells, AtT-20/D16v. Serial dilution of the culture medium or the cell extract gave parallel lines to the standard curve in the RIA for gamma MSH. Rat median eminence extracts enhanced the release of gamma MSH-like immunoreactivity (gamma MSH-LI) concomitant with ACTH-like immunoreactivity (ACTH-LI) and beta-EP-like immunoreactivity (beta-EP-LI). Dexamethasone suppressed the release of gamma MSH-LI as well as ACTH-LI and beta-EP-LI. Gel exclusion chromatography of the culture medium and the cell extract has revealed that gamma MSH-LI consists of two peaks; one eluted near the elution position of beta-lipotropin and the other near the elution position of beta-EP. There was no peak corresponding to the elution position of synthetic gamma 3MSH. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has demonstrated that gamma MSH-LI migrated at five positions with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, respectively. The 31K gamma MSH coincided with the migration position of 31K ACTH of 31K beta-EP, and 21-23K gamma MSH coincided with the position of 21-23K ACTH on SDS-PAGE. The 16-17K gamma MSH coincided with the mouse 16K fragment (reported by Eipper and Mains) of ACTH-beta-lipotropin precursor protein in the migration in SDS-PAGE and in immunoreactivity to anti-gamma MSH antiserum. [3H]Glucosamine was incorporated into 16K, 13K, and 3.8K gamma MSH. These results suggest that AtT-20/D16v cells produce gamma MSH-LIs with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, and they are secreted concomitantly with ACTH-LI and beta-EP-LI.