Prolactin-like Biological Activity in the Pituitary Glands of the Marsupial Monodelphis domestica and of the Amphibian Rana pipiens Detected by a Colorimetric Nb2 Lymphoma Cell Proliferation Assay

Abstract

Abstract

An inexpensive and reliable colorimetric microplate version of the Nb2 lymphoma cell proliferation bioassay for prolactin (PRL) was developed and optimized. The useful range of the assay is between 0.1 and 12.8 ng/ml in terms of rat pituitary PRL. The assay can accommodate up to 20 μl sample/well. The physiological relevance of the assay was verified by measuring thyrotropin-releasing hormone (TRH)-induced secretion of PRL in pituitary cultures and in serum samples of neonatal rats. Through the use of the colorimetric Nb2 assay, PRL-like bioactivities were demonstrated in pituitary extracts of the marsupial, Monodelphis domestica (1.47 ng PRL/μg protein) and of the amphibian, Rana pipiens (1.86 ng PRL/μg protein). Marsupial and amphibian PRLs are predicted to have low specific activities in the Nb2 assay. Since the PRL values were calculated in terms of a rat PRL standard, they probably underestimate the amounts of PRL present. Parallel dose-response curves were obtained with these pituitary extracts and standard rat PRL over a wide range of dilutions. The Nb2 bioassay may serve as a tool for the purification of PRL from these species. The colorimetric version of the Nb2 bioassay may be a useful alternative to traditional Nb2 assays that rely on direct cell count or [³H]thymidine uptake.