PROTRH PEPTIDES ARE SYNTHESIZED AND SECRETED BY ANTERIOR PITUITARY CELLS IN LONG-TERM CULTURE

Abstract

The expression of two ProTRH derived peptides, thyrotropin--releasing hormone (TRH) and PrePro-TRH25-50 (PYE27) was studied in anterior pituitary (AP) cells cultured in monolayer for up to 21 days. TRH levels in extracted cells rose from undetectable at 3 days to 267 +/- 22.5 fmol/well (p less than 0.01) at 21 days in culture. When AP tissue was extracted without dissociation or culture TRH was undetectable. The molar ratio of TRH/PYE27 was approximately 5:1 as predicted by the structure of PreProTRH. Extracts of cultured AP cells coeluted with TRH and PYE27 standards when subjected to HPLC analysis. Basal TRH secretion was 13.2 +/- 1.8 fmol/well/30 min at 18 days in culture; depolarizing concentrations of K+ (55 mM) caused a 2.2 fold (p less than 0.01) Ca++ dependent increase in TRH release. Immunostaining for PYE27 was found in approximately 10% of the cell population. Our results suggest that authentic ProTRH peptides are synthesized by AP cells in long term culture but not in situ. While the mechanism of activation of the PreProTRH gene needs to be elucidated we propose that TRH and/or other ProTRH derived peptides may exert paracrine effects on AP function.