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

Continuous cell perifusion and reverse hemolytic plaque assay have been used to show a regulatory action of 178-estradiol on lactotroph responsiveness to thyrotropin-releasing hormone (TRH) or dopamine (DA) in vifro. 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 lo-' M for TRH and at lo-' 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 178-estradiol on TRH-stimulation and DAergic inhibition in these heterogeneous prolactin cells, they were divided into two groups: large plaques ( 2 3 x lo3 pm') constituted about 35% of all plaque-forming cells, and small plaques ( < 3 x l o 3 pm'), about 65%. Pretreatment with 178-estradiol (lo-' 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 178-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 178-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 178-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 17fl-estradiol obtained after 10 h and after 48 h pretreatment.It has long been shown that estrogens play a modulatory role in the control of prolactin (PRL) secretion although its mode of action remains unclear. Pretreatment with 17B-estradiol has been shown to increase the number of thyrotropin-releasing hormone (TRH) binding sites in pituitary homogenates from female rats, whilst concomitantly enhancing the amount of PRL released in response to iv injection of TRH (1, 2). Similarly, in tumoral PRL e l l s in culture, the number of TRH binding sites and TRHinduced PRL...

Voltage-Dependent Potassium Currents in Ovine Somatotrophs and Their Function in Growth Hormone Secretion

“…Cells in culture were resuspended after a brief trypsinization as described elsewhere [ 13], washed in M 199 and then loaded with lura 2/AM (1 \xM) for 30 min at 37 °C with gentle agitation. The cell sus pension was centrifuged for 3 min (1.500 rpm) and resuspended in HEPES buffer solution (HBS; 135 mM NaCl, 5 mM KC1, 1.2 mM CaCL, 1 mM MgCL.…”

Absence of direct regulation of prolactin cells by estradiol-17β in rainbow trout (Oncorhynchus mykiss)

“…17&Estradiol (E,) stimulates PRL secretion by a direct action on pituitary cells in which it both enhances PRL mRNA production dramatically and increases the mitotic activity of PRL cells (Lieberman et al, 1982;Takahashi and Kawashima, 1987). Moreover, in vitro studies suggest that the direct effect of E, on PRL cells could be exerted by two other mechanisms: an increase in their sensitivity to thyrotropin-releasing hormone (TRH), one of the most potent PRL-releasing factors (PRF), and a decrease in their sensitivity to dopamine, considered as the most likely PRL-inhibiting factor (De Lean et al, 1977;Pasqualini et al, 1986;Zhang et al, 1990). Further in vivo studies also indicate that estrogens not only act directly on the lactotrophs but also modulate activity of the central system which controls PRL pituitary release (Fink, 1988).…”

Functional Lactotroph Heterogeneity in Lactating Rats and in vitro Modification by 17Beta‐Estradiol

“…During lactation, the circulating PRL level is increased, which could indicate an important role in lactation for the cells with high sensitivity to TRH in the heavy fractions. In addition, heavy fraction PRL cells are present only in very small numbers in male (8) and young female rats (12). These findings provoke inquiries into the factor responsible for the appearance of the heavy fraction lactotrophs in the lactating female.…”

“…In addition, estrogens modulate the action of hypothalamic releasing factors which control PRL release: the stirnulatory effect of thyrotropin-releasing hormone (TRH) (4) is increased in G H 3 tumoral cells (5) while the inhibitory effect of dopamine (DA) (6) is decreased in pituitary cells of ovariectomized rats (7). We have previously reported that 178-estradiol pretreatment modifies responsiveness to DA and TRH of lactotrophs from adult male rats in primary culture (8). These findings can be linked with the isolation by gradient sedimentation of two populations of lactotrophs from lactating female rat pituitaries identified by their PRL secretion manner measured by RIA and electrophysiological responses to T R H and DA using microelectrode intracellular recording (9); one (light fraction cells), spontaneously releases large amounts of PRL in culture, is highly sensitive to dopaminergic inhibition but is only slightly stimulated by TRH, and the second (heavy fraction cells), secretes lower amounts of PRL, is poorly inhibited by DA but is very sensitive to TRH stimulation.…”