In vitro Effects of 17Beta‐Estradiol on Thyrotropin‐Releasing Hormone‐Induced and Dopamine‐lnhibited Prolactin Release from Adult Male Rat Lactotrophs in Primary Culture

Abstract

Abstract Continuous cell perifusion and reverse hemolytic plaque assay have been used to show a regulatory action of 17 beta-estradiol on lactotroph responsiveness to thyrotropin-releasing hormone (TRH) or dopamine (DA) in vitro. Lactotroph-enriched cell cultures were obtained from adult male rats after trypsinization and mechanical dissociation followed by separation on a continuous bovine serum albumin gradient at unit gravity. After 7 days in culture, perifusion experiments showed that prolactin was continuously released and this release was increased by TRH and decreased by DA. Both TRH-induced secretion and DA-induced inhibition of prolactin release were dose-dependent with a half maximal effect obtained at 7 x 10(-9) M for TRH and at 10(-9) M for DA. It was shown by reverse hemolytic plaque assay that about 55% of the cells were plaque-forming (lysis of red blood cells) and were thus identified as prolactin-secreting cells. This was similar to a previous result obtained by immunofluorescent staining. Heterogeneity among lactotrophs with regard to the quantity of prolactin released was clearly shown by the varying plaque areas in all preparations. In order to make a quantitative analysis of the effect of 17 beta-estradiol on TRH-stimulation and DAergic inhibition in these heterogeneous prolactin cells, they were divided into two groups: large plaques (>/= 3 x 10(3)mu m(2)) constituted about 35% of all plaque-forming cells, and small plaques (< 3 x 10(3)mu m(2)), about 65%. Pretreatment with 17beta-estradiol (10(-8) M) either for 10 h or 48 h markedly increased TRH-stimulated prolactin release and decreased the inhibitory effect of DA both in perifusion and reverse hemolytic plaque assay experiments. However, these pretreatments did not change the values of half maximum dose for TRH and DA. TRH transformed about 7% of the small plaques into large plaques and this proportion was increased to 25% after 17beta-estradiol treatment. On the contrary, DA and its more stable analogue bromocriptine increased the percentage of small plaques by 10% to 15% but this effect was decreased after 17beta-estradiol treatment. We conclude that: 1) Normal rat pituitary lactotrophs show heterogeneity with respect to their spontaneous release and responsiveness to TRH and DA; 2) pretreatment with 17beta-estradiol increases the response to TRH and decreases the response to DA without altering the doses at which they have half maximal effect; 3) there is no significant difference between the effect of 17beta-estradiol obtained after 10 h and after 48 h pretreatment.