Functional Lactotroph Heterogeneity in Lactating Rats and Invitro Modification by 17beta-Estradiol

Lactotrophs from lactating rats were separated by unit gravity sedimentation on a continuous density gradient of bovine serum albumin and were identified in two populations located in the light fractions (fractions 3-5) and in the heavy fractions (fractions 7-9) of the gradient. After 7 days in vitro, the effects on prolactin release of thyrotropin-releasing hormone (TRH) and dopamine before and after pretreatment with 17-beta-estradiol were studied by a continuous perifusion system and reverse hemolytic plaque assay. Light fraction lactotrophs spontaneously released large quantities of prolactin (22 ng/ml/2 min/10(6) cells) and this basal release was markedly elevated (51 ng/ml/2 min/10(6) cells) by pretreatment with 17-beta-estradiol (10(-8) M, 48 h), while the amount of intracellular prolactin remained stable. Mean hemolytic plaque area was increased in the same manner by 17-beta-estradiol pretreatment but the number of cells and the percentage of plaque-forming cells were not changed. Perifusion of dopamine-containing medium (10(-7) M) almost completely blocked basal prolactin release from light fraction cells and this inhibition was markedly reduced by 17-beta-estradiol pretreatment. TRH-containing medium (10(-7) M) weakly stimulated basal prolactin release (about 190% from basal) and this response was significantly enhanced (to about 300% of basal release) by 17-beta-estradiol pretreatment. Both dopaminergic inhibition and TRH-stimulatory effects were dose-dependent and their half maximal effect values were not changed evaluated at the single cell level by the reverse hemolytic plaque assay corroborated the results obtained from perifusion experiments. Lactotrophs from heavy fractions released small amounts of prolactin (12 ng/ml/2 min/10(5) cells) and neither this basal release nor the amount of intracellular prolactin were markedly modified by 17-beta-estradiol pretreatment. As opposed to the light fraction cells, lactotrophs found in heavy fractions were very sensitive to TRH (10(-7) M) stimulation with maximal stimulation reaching ten times basal release, but were less sensitive to dopamine (10(-7) M), with an inhibition of only 40% basal prolactin liberation. Pretreatment of heavy fraction lactotrophs with 17-beta-estradiol induced similar effects to those observed after pretreatment of light fraction cells: the stimulation by TRH was increased (from 11 times to 16 times) whereas the inhibition by dopamine was diminished (from 35% to 60%), but cell number and the percentage of prolactinsecreting cells remained unchanged. From the above results, we suggest that: 1) lactotrophs in the lactating rat purity can be divided into two major subpopulations with regard to cellular size and density, prolactin production and responsiveness to TRH and dopamine; 2) 17-beta-estradiol pretreatment increases basal prolactin release from light fraction cells but does not affect basal prolactin release from heavy fraction cells in this way; 3) pretreatment with 17-beta-estradiol enhances TRH stimulation and reduces dopaminergic inhibition of prolactin release from lactotrophs