Proceedings: Control of prolactin biosynthesis in vitro by insulin.

Abstract

Prolactin synthesis and release can be investigated in vitro by incubation of the anterior pituitary gland. When isolated rat anterior pituitaries are incubated in vitro with labelled amino acids, radioactivity is incorporated into prolactin, growth hormone and other pituitary proteins. The anterior pituitary has been established as an insulin-sensitive tissue (Goodner & Freinkel, 1961) and Goodner & Krouse (1972) reported that insulin stimulates protein synthesis in the rat anterior pituitary gland. Insulin is required for a response of mammary-gland explants to prolactin and these two hormones act together in the regulation of alveolar cell differentiation during pregnancy. Betteridge &Wallis (1973) showed that insulin stimulates the biosynthesis of growth hormone in oitro. It was therefore decided to investigate the effect of insulin on prolactin synthesis in a system in vitro. Anterior pituitary glands were obtained from Sprague-Dawley rats and incubated in Krebs-Ringer bicarbonate medium containing 1 1.2m~-glucose and rH]leucine (5pCi/ ml). Paired hemi-pituitaries were used for experimental and control observations. Incubations lasted for 8h at 37°C in a humified C02+ air (5:95) atmosphere. At the end of this period the glands were removed from the medium, weighed and homogenized. Portions of the media and pituitary homogenates were precipitated with trichloroacetic acid and analysed for incorporation of radioactive leucine into total pituitary protein. Prolactin and growth hormone were separated as discrete bands by polyacrylamidegel electrophoresis of further portions of media and pituitary homogenates. The prolactin content was determined by densitometry of the stained polyacrylamide gels. Incorporation of radioactive leucine into prolactin was estimated by scintillation counting after excising the appropriate band and solubilizing with HzO2. Total prolactin synthesis was calculated by combining results from media and pituitary homogenates. In this system in oitro prolactin is actively synthesized and released. Over the 8 h incubation period, the rate of prolactin synthesis and release increases fairly linearly, as does the rate of total protein synthesis. Pituitary glands from mature female rats synthesized more prolactin than those from male rats. Total prolactin synthesis, expressed as a percentage of the total pituitary protein synthesis, was 20% in females and 3 % in males. Control of prolactin synthesis and release was further investigated by using female rats. The effect of bovine insulin on the biosynthesis of prolactin in the female anterior pituitary gland was investigated with this system in oitro. Over the range 1 m to 1~