Biosynthesis and release of beta-endorphin-, N-acetyl beta-endorphin-, beta-endorphin-(1-27)-, and N-acetyl beta-endorphin-(1-27)-like peptides by rat pituitary neurointermediate lobe: beta-endorphin is not further processed by anterior lobe

Abstract

Continuous labeling and pulse-chase techniques were employed to study the synthesis and secretion of multiple forms of immunoreactive beta-endorphin by cultured dispersed rat anterior lobe cells and intact neurointermediate pituitary lobe. Cell and medium extract immunoreactive beta-endorphin (specific immunoprecipitation and radioimmunoassay) exhibiting a Kav similar to authentic beta-endorphin upon gel filtration was characterized further by nonequilibrium isoelectric focusing, cation exchange chromatography, reverse phase high pressure liquid chromatography, and partial tryptic and chymotryptic mapping. Intact neurointermediate lobes incorporated radiolabeled amino acids into four to six forms of immunoreactive beta-endorphin. Four of these forms were physicochemically similar to authentic beta-endorphin, N-acetylated beta-endorphin, beta-endorphin-(1-27), and N-acetylated beta-endorphin-(1-27). Pulse-chase studies indicated that a beta-lipotropin-like molecule served as a metabolic intermediate for a beta-endorphin-like molecule. As beta-endorphin-like material accumulated in the cell, some of it was N-acetylated (approximately 18% at 2 hr chase and approximately 65% at 18 hr chase). At later chase times, beta-endorphin-(1-27)- and N-acetylated beta-endorphin-(1-27)-like peptides were the predominant molecular species detected. All endorphin forms were detected in unlabeled tissue maintained in culture or tissue continuously labeled for 72 hr and were released into the medium under basal, stimulatory (10(-8) M norepinephrine), or inhibitory (10(-7) M dopamine) incubation conditions. In all cases, beta-endorphin-(1-27)-like species were the predominant forms (more than 70% of total) present in the cells and released into the medium. In contrast, approximately 90% of radiolabeled immunoreactive beta-endorphin extracted from anterior lobe cells and medium similarly incubated appeared to represent the authentic beta-endorphin molecule. Continuous labeling (72 hr) revealed the beta-lipotropin/beta-endorphin molar ratio to be approximately 4. We conclude that, in anterior lobe, most of the beta-endorphin is not processed further and is released intact, while in neurointermediate lobe, it serves as a biosynthetic intermediate.

The hypothalamo‐hypophyseal rat explant in vitro: endocrinological studies of the pars intermedia dopaminergic neural input.

“…A number of reports showing that POMC peptides are simultaneously released together and that these secretions are inhibited by dopamine (Liotta, Yamaguchi &;Krieger, 1981;Randle et al 1983b;Lowry, 1983) suggest that all or most of these peptides might be regulated in the same manner as has been described here 388 H YPOTHALAMO-H YPOPH YSEAL EXPLANTS IN VITRO for MSH. In addition, the evidence demonstrates that the tubero-hypophyseal dopamine system has already reached a significant level of development in explants taken from the 2-week-old rats used in this study (see Davis et al 1984).…”

The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

“…For example, the C-terminal cleavage of endorphin as determined by pulse-chase studies in NILs incubated in vitro [15,16] is 18 to 24 hours. The present results suggest that the cleavage, occurring presumably after Arg20 , is a late event in the intragranular processing of pressophysin.…”

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

“…In the intermediate lobe of the pituitary the predominant forms are the acetylated, truncated forms N=-acetyl-fl-endorphin-(1-31), N~-acetyl-fl-endorphin-(1-27) and N=-acetyl-fl-endorphin-(1-26), due to the processing pathway which involves a more rapid acetylation than proteolytic cleavage (Eipper and Mains, 1981;Glembotski, 1982a;Liotta et al, 1981;Zakarian and Smyth, 1982a,b). In the anterior lobe acetylation and proteolytic trimming does not take place and/7-endorphin-(1-31) is the predominantly occurring form (Mains and Eipper, 1981;Zakarian and Smyth, 1982a,b;.…”

“…However, in the intermediate lobe of the pituitary, in brain and presumably also in placenta and pancreas, main products are fl-endorphin and v-lipotropin, c~-melanotropin, corticotropin-like intermediate lobe peptide (CLIP) and several shorter NH 2 terminal pro-opiomelanocortin fragments ( Fig. 1) Eipper, 1978a,b, 1979;Roberts et aL, 1978;Crine et al, 1978Crine et al, , 1979Eipper and Mains, 1981;Liotta et al, 1981;. These peptides all arise during a discrete proteolytic cleavage mechanism which is directed by paired basic amino acids.…”