Dopamine inhibits prolactin liberation acting via the D2 type receptor. Two different electrophysiological responses to dopamine have been shown to characterise two types of lactotroph isolated from the lactating female rat. It is now known that differential splicing of the pre-messenger RNA coding for the D2 receptor leads to the production of two D2 subtypes, D2(415) and D2(444). These subtypes differ in the region which is believed to be responsible for the binding of G proteins, and could thus lead to the activation of different intracellular second messenger systems. Here we show that the pre-messenger RNA for the D2 receptor is differentially spliced in such a way that the ratio D2(415)/D2(444) is significantly different (2.91 +/- 0.6 vs 1.29 +/- 0.14) between two populations of lactotrophs, each enriched in cells showing one type of response to DA. We further show that the ratio D2(415)/D2(444) can be changed by treatment of prolactin cells in primary culture with progesterone or testosterone. Estrogen did not change the ratio, but diminished the total amount of D2 cDNA. Regulation of differential splicing by sex steroids could provide a mechanism for modifying lactotroph responsiveness to DA in different physiological situations.

Enrichment of dispersed pituitary cells from normal lactating rats on a continuous BSA gradient permitted the isolation of two prolactin cell populations – light and heavy. Hormone release studies indicated that spontaneous prolactin (PRL) release of the heavy fraction cells was particularly sensitive to TRH stimulation (mean of 450%) and that this effect of TRH was totally inhibited by Ca²⁺ channel blockers (Co²⁺, Cd²⁺, Ni²⁺ and Mn²⁺). For this reason, the electrophysiological response to TRHwas investigated on heavy fraction cells. Experiments performed on 264 cells after 4–12 days in culture showed that these cells could be divided into two groups. The first group, called high resting potential (HRP) cells, constituted 73% of the total and was characterized by a mean resting potential of –60 mV and a mean input membrane resistance of 700 MΩ, and these cells displayed plateau potentials, which were triggered by application of brief (2 s), large (1 nA) depolarizing or hyperpolarizing current steps. The plateaux were characterized by a sustained depolarization at a potential near –20 mV and they were concomitant with an increase of the membrane conductance. The repolarization consisted of a slight, gradual hyperpolarization followed by a rapid return to the resting potential. The second group of cells (the remaining 27%; called low resting potential or LRP cells) was characterized by a mean resting potential of –45 mV and a mean input membrane resistance of 250 MΩ. These cells were excitable, 30% of them displaying spontaneous activity but never showing plateaux. Electrophysiological experiments showed that the majority of the HRP cells (99%) responded to TRH but were insensitive to dopamine (DA) ejections (up to 10^–6M) at resting potential. Ejection of TRH onto HRP cells induced a slow depolarization (10–15 mV) concomitant with a decrease of the membrane resistance. This led the cell membrane potential to a critical value (≈–50 mV), at which the plateau response was triggered. The plateau lasted for about 10 s from potentials greater than –100 mV, and could reach 10 min from holding potentials close to the resting potential. The amplitude of the plateau varied according to the holding potential and the reversal potential was found to be -20 mV. Local application of tetraethylammonium chloride (TEA, 30 mM) only slightly affected the amplitude of the plateaux but they were shortened or totally blocked by Co²⁺, Cd²⁺ and Ni²⁺. Contrary to is lack of effect on these cells at their resting potential, the ejection of DA onto cells showing plateau activity shortened or totally abolished the plateau. This effect was mimicked by the dopaminergic agonist bromocriptine. On the other hand, 60% of the LRP cells were insensitive to TRH (10^–6M) but showed a hyperpolarization after DA application (40% were insensitive either to DA or to TRH). We conclude that two subpopulations of PRL cells may exist, one which, at resti...

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 vifro, the effects on prolactin release of thyrotropin-releasing hormone (TRH) and dopamine before and after pretreatment with 17B-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 rnin/lO6 cells) and this basal release was markedly elevated (51 ng/ml/2 min/106 cells) by pretreatment with 17/l-estradiol (10. M, 48 h), while the amount of intracellular prolactin remained stable. Mean hemolytic plaque area was increased in the same manner by 17j-estradiol pretreatment but the number of cells and the percentage of plaque-forming cells were not changed. Perifusion of dopamine-containing medium (10 M) almost completely blocked basal prolactin release from light fraction cells and this inhibition was markedly reduced by 17LGestradiol pretreatment. TRH-containing medium (10 -' M) weakly stimulated basal prolactin release (about 190% from basal) and this response was significantly enhanced (to about 300% of basal release) by 17P-estradiol pretreatment. Both dopaminergic inhibition and TRH-stimulatory effects were dose-dependent and their half maximal effect values were not changed by 17b-estradiol Pretreatment. Secretion of prolactin 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/106 cells) and neither this basal release nor the amount of intracellular prolactin were markedly modified by 17p-estradiol pretreatment. As opposed to the light fraction cells, lactotrophs found in heavy fractions were very sensitive to TRH M) stimulation with maximal stimulation reaching ten times basal release, but were less sensitive to dopamine (10 ' M), with an inhibition of only 40% basal prolactin liberation. Pretreatment of heavy fraction lactotrophs with l7D-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 6O%), but cell number and the percentage of prolactinsecreting cells remained unchanged. From the above results, we suggest that: 1) lactotrophs in the lactating rat pituitary can be divided into two major subpopulations with regard to cellular size and density, prolactin production and responsiveness to TRH and dopamine; 2) 17b-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 17b-estradiol enhances TRH stimulation and reduc...

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...