A New in vitro Assay System for Prolactin‐Releasing Activity and its Application for Demonstration of Porcine Hypothalamic Prolactin‐Releasing Factor (PRF)

Abstract

A new sensitive and reproducible assay system for prolactinreleasing activity (PRA) using a rat pituitary cell culture system was established. After pretreatment with bromocriptine (1.3 x M) in culture medium for 24 h r on the 4th day in culture, the 6 hr addition of thyrotropin releasing hormone (TRH) to culture medium increased prolactin secretion in a dose-dependent manner at concentrations of TRH ranging from 3.2 x lo-" M t o 2.0 x M; maximal prolactin response was 270% of the control value.By using this new assay system for PRA, prolactin-releasing factor (PRF) in porcine hypothalami was isolated and identified. On Sephadex G-25 gel-chromatography of acid extracts of porcine hypothalami, PRA was found in the small peptide portion. These fractions of PRA were further subjected to CM-cellulose (CM-52) ion exchange chromatography, followed by reverse-phase HPLC. PRA obtained by HPLC chromatography was neutralized by anti-TRH antibody. It was concluded that TRH is the main PRF in porcine hypothalamic extracts.