Angiotensinogen secretion by single rat pituitary cells: detection by a reverse haemolytic plaque assay and cell identification by immunocytochemistry.

A reverse haemolytic plaque assay (RHPA) for angiotensinogen was developed in rat hepatoma H4 cells and applied to investigate the possible secretion of angiotensinogen from rat pituitary cells in primary culture. Over a 24-hour incubation period in Cunningham chambers plaques with a mean area of 2,800 +/- 430 and 590 +/- 220 microns2/plaque (SD, n = 6) formed around all viable H4 cells and 2.8 +/- 0.59% of viable pituitary cells respectively. As a positive control PRL secretion from lactotrophs was routinely checked by the RHPA and shown to form plaques with a mean area of 4,050 +/- 1,850 microns2/plaque after a 4-hour incubation. By comparing plaque size in H4 cells with angiotensinogen release in cell culture, as quantified by radioimmunoassay, the secretion rate of angiotensinogen from pituitary cells was calculated as 22 +/- 8 ng/10(6) cells/24 h. Plaque-forming cells consisted of two morphologically distinct populations; 78% being small cells (less than 6 microns diameter) containing little cytoplasm and 22% were large (greater than 9 microns diameter) cells with an abundant cytoplasm. Immunocytochemical staining of pituitary cells after formation of plaques with anti-angiotensinogen, anti-LH and anti-PRL antiserum showed that the large plaque-forming cells were gonadotrophs and none were lactotrophs. All plaque-forming cells stained for angiotensinogen but only 44% of the viable cells which stained for angiotensinogen actually formed plaques. The possibility that cellular angiotensinogen was imported from extracellular sources was investigated by incubation of pituitary cells with pure 125I-angiotensinogen for periods up to 24 h. No uptake of the radiolabelled protein was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of endothelins on the secretion of prolactin, luteinizing hormone, and follicle-stimulating hormone are mediated by different guanine nucleotide-binding proteins

Different bacterial toxins capable of modifying specific alpha-subunits of G-proteins were used to characterize the guanine nucleotide-binding protein (G-protein) dependency of the effects of endothelins (ETs) on PRL, LH, and FSH secretion. Primary cultures of anterior pituitary cells obtained from female rats were preincubated for 24 h with 20 ng/ml pertussis toxin (PTX) or 2 micrograms/ml cholera toxin (CTX) before challenge with ETs. Both ET-1 and ET-3 elicited a concentration-dependent inhibition of PRL secretion and stimulated the release of LH and FSH secretion on pituitary cells not treated with toxins. Based on the calculated ration of the half-maximal effective concentrations (EC50) of ET-1 and ET-3, ET-1 showed 7800, 20, and 14 times greater potency than ET-3 on PRL, LH, and FSH secretion, respectively. PTX, a selective inhibitor of Gi and several other G proteins, increased the basal secretion of PRL and completely eliminated the responsiveness of lactotroph cells to ET-1 and ET-3. Pretreatment with PTX caused a markedly different effect on LH and FSH secretion: while basal LH release was slightly increased, FSH secretion was markedly depressed by PTX. Moreover, while ET-induced LH secretion was enhanced by PTX, the effectiveness of ETs on FSH release was completely abolished. CTX, known as an activator of Gs proteins, decreased the basal secretory activity of lactotrophs but did not influence the ET-induced decrease of PRL release. CTX pretreatment (like PTX before) elicited a strikingly different effect on LH and FSH: while basal LH secretion was enhanced, basal FSH secretion was markedly inhibited by CTX. Moreover, while the effectiveness of ETs on LH secretion was not changed significantly, the stimulatory effect of ETs on FSH secretion was diminished after CTX pretreatment. Thus, the inhibition of PRL secretion by ETs requires a PTX-sensitive G protein while the ET-induced stimulation of FSH secretion involves both PTX- and CTX-sensitive elements. The fact that pretreatments with PTX or CTX influenced basal secretion of PRL, LH, and FSH suggests that PTX- and/or CTX-sensitive G proteins are directly involved in the process of exocytosis. Additionally, these findings might indicate an active paracrine/autocrine regulation of pituitary cells in culture that are impaired or enhanced by the bacterial toxins employed. Though the broad substrate specificity of PTX and CTX and the multiplicity of G protein families did not allow us to identify the specific G protein(s) involved, these data reveal the diversity of ET-induced intracellular signaling mechanisms in lactotrophs and gonadotrophs.     

Inhibition of protein phosphatase 2A (PP2A) mimics suckling-induced sensitization of mammotropes: involvement of a pertussis toxin (PTX) sensitive G-protein and the adenylate cyclase (AC).

In lactating rats, suckling renders mammotropes more responsive to prolactin (PRL)-releasing stimuli and less responsive to PRL-inhibiting secretagogues. We have previously shown that a decrease in the activity of protein phosphatase 2A (PP2A) may be responsible for the decrease in responsiveness to the inhibitory secretagogue dopamine (DA). In our present experiments, we have studied the involvement of the adenylate cyclase (AC), stimulatory and inhibitory GTP-binding proteins and also the role of PP2A in the sensitization phenomenon. Pituitary cells obtained from mother rats separated from their pups for 4 h prior to dispersion (non-suckled), suckled for 10 or 30 min after the separation period (suckled) and without separation (continual suckling) were incubated in the presence of different doses of forskolin to activate AC and DA. In a further study, pituitary cells of non-suckled rats were pretreated with cholera toxin (CTX) or pertussis toxin (PTX) and tested for the stimulatory action of forskolin or TRH on PRL release. Ocadaic acid (OA) pretreatment has been used to investigate the involvement of PP2A. Hormone secretion was measured by the reverse hemolytic plaque assay (RHPA). Our results have shown that cells from non-suckled rats were unresponsive to forskolin. A 10-min suckling stimulus sensitizes pituitary mammotropes to respond with a PRL release to a dose-dependent activation of AC by forskolin. This sensitization of AC becomes a permanent feature of the cells when suckling continues for an additional 20 min. We have also found that pituitary mammotropes from non-suckled dams respond to forskolin or TRH with PRL release when they were preincubated with either PTX or the PP2A inhibitor OA. It clearly indicates that the non-responsive pituitary can be shifted to the responsive stage by uncoupling of inhibitory G-protein from its receptor as well as by inhibition of PP2A. This latter finding, consonant with our previous results, suggests that suckling may cause selective changes in the function of G(i) of mammotropes due to a rapid phosphorylation which can remove tonic, GTP-dependent inhibitory function.     

Acute effect of basic fibroblast growth factor on secretion of prolactin as assessed by the reverse hemolytic plaque assay

The effect of basic fibroblast growth factor (bFGF) on acute secretion of PRL by pituitary lactotrophs was examined under basal conditions and after treatment with TRH or dopamine. We used the reverse hemolytic plaque assay (RHPA) to determine the amount of PRL secreted per lactotroph and the percentage of pituitary cells secreting PRL. Young (2- to 3-month-old) female Sprague-Dawley rats were ovariectomized and 1 week later implanted with a Silastic capsule containing 180 micrograms/ml estradiol in sesame oil. Three days later, rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. In Exp I, time and dose responses to bFGF were determined using the RHPA. Basic FGF reduced (P less than 0.0001) the mean basal secretion of prolactin per lactotroph. The effect was similar at 30, 60, 120, and 240 min of incubation. The reduction in PRL was greatest at 3.36 x 10(-6) M, while lesser reductions were observed at 1.68 x 10(-6) and 5.60 x 10(-7) M. A dose of 3.36 x 10(-6) M (60 ng/ml) and an incubation time of 60 min were subsequently used in Exp II. In Exp II, we examined the effects of bFGF on TRH stimulation and dopamine inhibition of PRL secretion. PRL secretion was maximally stimulated (P less than 0.01) by 10(-7) M TRH. Basic FGF blocked the TRH-induced increase in PRL secretion. PRL secretion was maximally reduced (P less than 0.001) by 10(-5) M dopamine. Coincubation of bFGF with dopamine reduced (P less than 0.01) the mean plaque area to the same extent as dopamine alone. In each experimental situation changes in mean plaque area reflected a shift in the frequency distribution of the plaque area. Neither bFGF, TRH, dopamine, nor the combined treatments influenced the percentage of pituitary cells secreting PRL compared to basal conditions. We have demonstrated that 1) bFGF reduces the basal secretion of PRL in an acute manner; 2) bFGF blocks the TRH-induced increase in PRL; and 3) bFGF does not potentiate the inhibitory effect of dopamine on PRL secretion and, therefore, may act in part through the same inhibitory pathway as dopamine. We conclude from these data that bFGF, or related factors, could play a role in the regulation of PRL secretion.     

Structure-function correlations of growth hormone or/and prolactin-producing pituitary adenomas: an in vitro study with the reverse hemolytic plaque assay

The purpose of this study was to detect in vitro growth hormone (GH) and prolactin (PRL) secretion from adenomas clinically associated with GH or PRL hypersecretion. The reverse hemolytic plaque assay (RHPA) was applied in order to reveal possible differences among various morphologic adenoma types, and to examine the inhibitory effects of octreotide on GH release as well. The 20 surgically resected pituitary adenomas studied included 15 from acromegalic patients and 5 from patients with hyperprolactinemia. All adenomas were diagnosed by histology, immunocytochemistry and electron microscopy. Among tumors associated with acromegaly, 5 were densely granulated (DG), 5 were sparsely granulated (SG) somatotroph (SM) adenomas, 2 were mammosomatotroph (MSM) and 3 mixed somatotroph-lactotroph cell (mixed SM-LT) adenomas; tumors causing hyperprolactinemia included 4 lactotroph (LT) adenomas and 1 mixed SM-LT adenoma. GH release assessed by the RHPA corresponded to in vivo hormone secretion and to tissue immunoreactivity. Statistical analysis showed significant differences among all morphologic types of SM adenomas, exclusive of SG-SM adenomas compared to mixed SM-LT adenomas. The mean plaque size in DG-SM and MSM adenomas was significantly greater than that of SG-SM and mixed SM-LT adenomas, indicating higher GH secretion by the former two types during the same incubation time. PRL secretion was documented in 2 mixed SM-LT adenomas. Plaques for PRL, but not for GH were formed in all LT adenomas. In all SM and LT adenomas, cells producing large plaques represented a minority of the plaque-forming cell population, however, they accounted for the largest part of the total plaque area, thus the largest part of hormone secretion. Octreotide effects on GH release were studied in 6 adenomas by the RHPA. Octreotide treatment induced a rapid and significant reduction in GH secretion by SM cells in vitro, with a selective effect on high-secreting cells.     

Pharmacologic evidence that a D2 receptor subtype mediates dopaminergic stimulation of prolactin secretion from the anterior pituitary gland.

The ability of low concentrations of dopamine (DA) to stimulate the secretion of prolactin (PRL) was examined in perifused or monolayer cultures of anterior pituitary cells. In cultures perifused with media containing 100 nM DA, changing the DA concentration to either 1 or 100 pM caused a significant dose-dependent stimulatory PRL secretory response within 6 min when compared to the PRL secretory response to removal of DA altogether. Picomolar concentrations of DA caused a biphasic PRL secretory response. This response is characterized by an immediate increase in the rate of PRL secretion similar to that seen when the cells were treated with 100 nM thyrotropin-releasing hormone followed by a decrease in the rate of PRL secretion to levels comparable to cells receiving media alone. In a monolayer culture system DA, at concentrations between 10 nM and 1.0 pM, caused significant stimulation of PRL secretion relative to media alone. Maximal stimulation occurred at nanomolar concentrations of DA (approximately 60% greater than control). Although the D2 agonists, bromocriptine and 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin hydrochloride (PPHT) caused significant (p less than 0.05) inhibition of PRL secretion at nanomolar concentrations and above, neither had stimulatory activity. The D1 agonists, SKF 38393 and SKF 82958, had no effect on PRL secretion when tested at 0.1 pM to 1 microM. These data suggest that DA not only inhibits PRL secretion in vitro, but also stimulates PRL secretion at relatively low concentrations. Stimulation is mediated by a DA receptor which is neither recognized by D2 nor D1 agonists, suggesting a possible third DA receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes of prolactin response to dopamine during the rat estrous cycle

The effects of dopamine (DA) on prolactin (PRL) secretion in anterior pituitary tissue from rats selected during various stages of the estrous cycle were analyzed under in vitro conditions. The results were examined in relation to plasma PRL, estradiol and progesterone levels. During the estrous cycle there was a marked variation in the responsiveness of the lactotrophs to the inhibitory action of DA. Rapid changes occurred during proestrus: pituitary lactotrophs were not sensitive to the inhibitory action of 10 nM DA at 15.00 and 17.00 h and were less sensitive to 1 microM DA compared to 09.00 h (p less than 0.01), 12.00 h (p less than 0.05) and 19.00 h (p less than 0.05). This lowest PRL response to DA was associated with the preovulatory PRL surge. The recovery of a higher PRL response at 19.00 h coincided with the decrease of plasma PRL levels. During the remainder of the cycle, plasma PRL levels remained low in estrus, diestrus 1 and diestrus 2. Lactotrophs were sensitive to 1 microM and 10 nM DA during estrus and diestrus 2 but a significant lower PRL response to 1 microM DA (p less than 0.05) and no PRL response to 10 nM DA was observed in diestrus 1. These data show that alterations in the sensitivity of the lactotrophs' responsiveness to DA occur in the anterior pituitary gland during the rat estrous cycle and can lead to the removal of DA inhibition in the presence of physiological DA concentrations. This phenomenon is consistent with the fact that DA could be involved in the preovulatory PRL surge during the estrous cycle.     

Effects of gonadal steroids on somatotroph function in the rat: analysis by the reverse hemolytic plaque assay

The mechanism by which gonadal steroids modulate GH secretion is not known. We have used the reverse hemolytic plaque assay to examine whether gonadal steroid-induced modulation of GH secretion is effected by changes in the population of somatotrophs and/or alterations in their secretory properties. Two groups of Sprague-Dawley rats were studied: group 1 (n = 6) comprised male (M), castrate (Cx), and testosterone-replaced castrate male (Cx + T) rats and group 2 (n = 5) consisted of male (M), female (F), and 17 beta-estradiol-replaced castrate male (Cx + E) rats. The number of plaque-forming cells (expressed as both absolute number and a percentage of all cells) was determined, and secretory status was assessed by measuring plaque areas in response to 0, 0.01, 0.1, 1, 10, and 100 nM GHRH. While mean basal GH plaque areas were similar among the treatment groups of group 1, the maximal GH plaque area was significantly decreased in Cx [16.8 +/- 2.4 vs. 26.4 +/- 3.9 X 10(6) microns2 (mean +/- SEM); P less than 0.05], but not in Cx + T (27.5 +/- 4.1 microns2) rats. The GHRH EC50 was unaffected by castration or T replacement. The percentage and absolute population of somatotrophs were reduced in Cx, but not in Cx + T, rats, while the numbers of lactotrophs remained unchanged in these treatment groups. For group 2, the mean peak GH plaque area was reduced in Cx + E (16.5 +/- 2.9 microns2; P less than 0.001) compared to that in M rats (36.2 +/- 2.3 microns2), but was not significantly different from that in F (13.0 +/- 1.5 microns2) rats. The EC50 was significantly (P less than 0.025) greater in Cx + E (10.9 +/- 2.3 nM) and F (7.9 +/- 1.6 nM) compared to M rats (2.8 +/- 0.7 nM). The absolute somatotroph and lactotroph populations were increased in Cx + E compared to M and F rats, as were the populations of other pituitary cell types. Testosterone enhances GH secretion by increasing the secretory capacity, but not the sensitivity, of somatotrophs to GHRH and by recruiting the function of a subpopulation of somatotrophs. Estradiol reduces the secretory capacity and sensitivity of somatotrophs to GHRH, but increases the population of somatotrophs, lactotrophs, and non-GH- and non-PRL-secreting cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Postnatal development of pituitary lactotropes in the rat measured by reverse hemolytic plaque assay

Developmental changes and sexual differences in the lactotrope population were studied with a reverse hemolytic plaque assay for detection and measurement of PRL secretion from individual cells in conjunction with PRL immunocytochemistry (ICC). Pituitary glands from both sexes at different ages were monodispersed with 0.1% trypsin. Freshly dispersed cells were incubated in Cunningham chambers for measurement of the fraction of plaque-forming cells and the size of plaques formed, or attached to glass slides for measurement of the fraction of cells staining for PRL by ICC. The percentage of plaque-forming cells in both sexes gradually increased with age from about 5% of the total anterior pituitary cell population at 5 days of age to the adult levels of about 54% in the females at proestrus and about 37% in the males. Sexual differences in the percentage of plaque-forming cells were consistently seen at 40 days of age and thereafter. The mean size of plaques in both sexes also increased with age from about 1,100 microns 2 at 5 days of age to a peak level of about 15,600 microns 2 in the females at proestrus and about 6,700 microns 2 in the males at 60 days of age; then fell to the adult levels of about 5,500 microns 2 in the females at proestrus and about 3,200 microns 2 in males. Sexual differences first appeared at 40 days of age and were estrogen-dependent. The results from ICC closely matched those from the plaque assay, except at 5 days of age and in the adult males. At 5 days, the fraction of cells staining for PRL was twice that of cells forming plaques. In adult male rats there were about 47% immunostained PRL cells but only about 39% plaque-forming cells. This difference, however, disappeared after estrogen treatment. These results demonstrate that sexual differences in the lactotrope population develop around puberty in rats and appear to be estrogen dependent.     

TRH raises cytosolic Ca2+ in human adenomatous lactotrophs

The effect of TRH on cytosolic free calcium concentrations, [Ca2+]i, was evaluated on cell suspensions obtained from 6 human PRL secreting pituitary adenomas. In these cells resting [Ca2+]i levels were variable (mean +/- SE; 103.8 +/- 6.5, n = 25); the addition of 100 nM TRH caused a marked [Ca2+]i rise within 20 sec., the peak values ranging from 200 to 437 nM (285 +/- 10.8 nM, n = 10). The transients induced by TRH were composed by a rapid increase, due to mobilization of calcium from intracellular stores, followed within a few seconds by a lower plateau which was due to stimulated influx from the extracellular space. In fact, when EGTA and verapamil were applied after TRH they caused the Ca2+ plateau to dissipate rapidly. The addition of 1 microM dopamine (DA) caused a substantial decrease of resting [Ca2+]i (about 10-30%) as well as an inhibition of the plateau phase induced by TRH. The effect of DA completely depended on extracellular Ca2+. The TRH-induced transients observed in adenomatous cells were quite similar in size and time course to those recorded in normal rat lactotrophs. As previously observed in rat lactotrophs, in adenomatous cells treatment with pertussis toxin (PTx, 1 microgram/ml for 4 h) was unable to affect the [Ca2+]i transients induced by TRH while completely abolished the effect of DA. The effects of TRH on in vivo and in vitro PRL secretion were also evaluated. Before surgery, no patient showed a positive response to the iv administration of 200 micrograms TRH (serum PRL levels: 95 +/- 62 ng/ml in basal conditions vs 124 +/- 92 after TRH, P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional expression of the dopamine-activated K(+) current in lactotrophs during the estrous cycle in female rats: correlation with prolactin secretory responses

It is well established that hypothalamic dopamine (DA) is the major physiologic regulator of prolactin (PRL) secretion, exerting a tonic inhibition throughout most of the estrous cycle. A dramatic drop in the amount of DA perfusing the anterior pituitary occurs in the afternoon of proestrus and is critical for the production of the surge of PRL that occurs at that time. In my laboratory, we have identified and characterized a DA-activated K(+) channel (K(DA)) in lactotrophs derived from proestrous rats that underlies DA-induced membrane hyperpolarization of lactotrophs. We have also demonstrated that this hyperpolarization plays a critical role in both the inhibition of PRL release from proestrous cells and the PRL secretory rebound that occurs following DA withdrawal. We now report that the ability of DA to activate the K(DA) channel and elicit hyperpolarization in primary lactotrophs changes dramatically during the estrous cycle. Lactotrophs isolated from cycling female rats were studied using whole-cell voltage clamp. DA (1 microM) elicited a robust membrane K(+) current in the majority of proestrous lactotrophs (86%; 24.0 +/- 2.9 pA). By contrast, DA activated a considerably smaller membrane current (3.3 pA) in very few lactotrophs isolated from rats on either diestrus or estrus (8 and 0%, respectively). Using a perifusion system to examine temporal patterns of PRL release, we found that following application and withdrawal of DA, proestrous cells produced a robust secretory rebound, but diestrous and estrous cells did not. However, DA inhibited PRL release to the same extent regardless the stage of the cycle from which the cells were derived. These data are consistent with the presence of multiple DA effectors in lactotrophs and demonstrate that their relative importance shifts dramatically with changes in the endocrine status of the animal. We propose that the DA-activated K(+) channel (K(DA)) is a critical effector governing the unique secretory profile of PRL observed in proestrous animals.     

Suckling unmasks the stimulatory effect of dopamine on prolactin release: possible role for alpha-melanocyte-stimulating hormone as a mammotrope responsiveness factor.

Mounting evidence indicates that dopamine (DA) can stimulate as well as inhibit PRL release when given in appropriately low doses. In the present study, we investigated whether the suckling stimulus could influence this response. Pituitary cultures from suckled or nonsuckled rats were exposed to DA (10(-16) - 10(-6) M) during a reverse hemolytic plaque assay for PRL. Pituitary cells from nonsuckled rats exhibited only the inhibitory response to DA; exposure to high-dose DA (10(-6) M) reduced plaque area to 42.3 +/- 7.2% (mean +/- SEM) of control. A low dose of DA (10(-12) M) had no effect on PRL secretion (79.3 +/- 13.3% of control). In striking contrast, a brief suckling stimulus (10 min) rendered the mammotropes responsive to stimulation by low-dose DA (to 152.7 +/- 12.5% of control). Thus, suckling appears to be a requirement for expression of the stimulatory effect of DA in lactators. In a subsequent series of experiments we explored the possibility that a hypophysial factor, released during nursing, might mimic the effects of suckling on mammotrope responsiveness. Accordingly, we tested the effects of alpha-melanocyte-stimulating hormone (10(-7) M) and low-dose DA, alone or in combination, on pituitary cells from nonsuckled rats. Although neither agent alone had a dramatic effect on PRL secretion, concurrent administration of both of these significantly stimulated PRL release to 130.0 +/- 4.2% of control. Taken together, these results demonstrate that suckling renders mammotropes responsive to the stimulatory effects of DA. Moreover, our data indicate that alpha-melanocyte-stimulating hormone could function as a responsiveness factor in this phenomenon.     

Full oestrogenic activity of C19-delta 5 adrenal steroids in rat pituitary lactotrophs and somatotrophs

Oestrogens are known to exert specific stimulatory effects on basal and dopamine (DA)-inhibited prolactin (PRL) release as well as on PRL cell content in rat adenohypophysial cells in primary culture. Recently, we have demonstrated that classical oestrogens increase growth hormone (GH) release and cellular GH content in rat pituitary somatotrophs. Since there is evidence that 5-androstene-3 beta, 17 beta-diol (delta 5-diol), a metabolite of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) can induce typical oestrogenic responses in target tissues, we have investigated the effect of C19 adrenal steroids and compared them to that of 17 beta-oestradiol (E2) on the above-indicated parameters. Following a 72-h incubation, maximal concentrations of E2, delta 5-diol and DHEA caused a similar increase in PRL cell content at respective EC50 values of 0.020, 14 and 115 nM. The sensitivity of lactotrophs to DA action was decreased by approximately 4-fold after a 48-h exposure to maximal concentrations of delta 5-diol or DHEA. The time course of the antidopaminergic effect of delta 5-diol and DHEA was almost superimposable to that of E2. A 72-h incubation with 5 microM DHEA-S, a concentration within the range of blood levels found in women, doubled (P less than or equal to 0.001) cellular PRL accumulation. On the other hand, androstenedione (delta 4-dione) was without effect on any of the parameters measured. All stimulatory effects induced by maximal effective concentrations of delta 5-diol, DHEA or DHEA-S were competitively inhibited by simultaneous incubation with the antioestrogen LY156758. The amplitude of the stimulatory effects of delta 5-diol and DHEA on GH cell content as well as on spontaneous GH-releasing factor (GRF)-induced GH release was superimposable to that observed with E2. The effect of the steroids on GH cell content was exerted at 0.016 nM (E2), 12 nM (delta 5-diol) and 250 nM (DHEA). Simultaneous incubation with LY156758 completely blocked the effect of maximally effective concentrations of E2, delta 5-diol and DHEA in somatotrophs. Furthermore, a physiological concentration of DHEA-S (5 microM) enhanced spontaneous as well as GRF-induced GH release. On the other hand, delta 4-dione as well as testosterone and dihydrotestosterone did not alter GH release. The present data demonstrate that delta 5-diol, DHEA and DHEA-S can exert full oestrogenic effects in lactotrophs and somatotrophs, thus supporting their potential oestrogenic role under both physiological and pathological conditions.     

Muscarinic regulation of basal versus thyrotropin-releasing hormone-induced prolactin secretion in rat anterior pituitary cells. differential roles of nitric oxide and intracellular calcium mobilization

Acetylcholine (ACh), synthesized in the pituitary, can act locally to modulate pituitary function. We used rat primary anterior pituitary (AP) cells to investigate how ACh affects pituitary prolactin (PRL) secretion in the presence or absence of known PRL regulators: thyrotropin-releasing hormone (TRH), 17beta-estradiol (E(2)) and triiodothyronine (T(3)). Cultured AP cells were prepared from ovariectomized rats and pretreated with diluent, 0.6 nM E(2), 10 nM T(3), or E(2) plus T(3) for 5 days, then challenged with various doses of ACh or muscarinic receptor agonists (oxotremorine or carbachol) and TRH (100 nM) for 20 min. Significant ACh (10(-5) M) suppression of both basal and TRH-induced PRL secretion was not evident in diluent-, E(2)- or T(3)-pretreated cells, but observed only in cells pretreated with both E(2) and T(3). Moreover, in E(2) plus T(3)-pretreated cells, oxotremorine and carbachol, like ACh (10(-7)-10(-5) M), suppressed both responses in a dose- related manner. Pertussis toxin (PTX; 100 ng/ml) as well as atropine (a muscarinic receptor antagonist; 1 mM) blocked these effects of cholinomimetics. ACh also inhibited both PRL responses elicited by drugs elevating intracellular cAMP (10 microM forskolin) or Ca(2+) (1 microM Bay K-8644) in a PTX-sensitive manner. ACh inhibition of basal PRL secretion was unaltered by intracellular Ca(2+) mobilization blockers, TMB-8 (100 microM) and thapsigargin (1 microM), but abrogated by the nitric oxide synthase inhibitor (300 microM L-NAME). ACh inhibition of TRH-induced PRL secretion was accentuated by TMB-8 and alleviated by thapsigargin or L-NAME. In summary, muscarinic inhibition of either basal or TRH-induced PRL secretion was augmented by E(2) and T(3), and involved the PTX-sensitive cAMP/Ca(2+) pathways. Furthermore, nitric oxide mediated the basal rather than TRH-induced PRL response to ACh, whereas the intracellular Ca(2+) mobilization concerned the TRH-induced rather than the basal PRL response to ACh. Thus, ACh synthesized in the AP appears to inhibit basal vs. TRH-induced PRL secretion via different mechanisms.     

Influence of dopamine on the altered release of prolactin, luteinizing hormone, and follicle-stimulating hormone induced by interleukin-2 in vitro.

Interleukin-2 (IL-2) alters the release of anterior pituitary hormones at femtomolar concentrations from hemipituitaries incubated in vitro. This cytokine significantly lowered luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and stimulated prolactin (PRL) release, thus demonstrating a reciprocal action of the lymphokine on lactotrophs and gonadotrophs. Since dopamine (DA) is a powerful inhibitor of PRL release, in the present experiments were evaluated possible dose dependent effects of DA on IL-2-induced alterations of the release of PRL, LH, and FSH. Hemipituitaries were incubated with varying concentrations of DA, a combination of IL-2 plus DA, or a combination of haloperidol (1 x 10(-5) M) with DA for 1 h, followed subsequently by incubation with medium containing only high potassium (K+) to study the effects on depolarization-induced hormone release. DA induced a dose-related, significant lowering of the basal PRL release with a minimal effective dose (MED) of less than 19 nM. The depolarization-induced PRL release was also inhibited, but the MED was 100-fold higher than the MED to inhibit basal PRL release. DA at much higher concentrations (30, 60, and 90 microM) significantly reduced pituitary PRL content. The addition of 0.187, 3.75, 15, or 60 microM DA to IL-2-induced PRL release. IL-2 (10(-15) M) produced a significant decrease in LH and FSH release. The combination of 3.75 or 15 microM DA plus IL-2 failed to alter the IL-2 suppressed LH release, whereas the addition of 0.187 microM DA to IL-2 blocked its suppressive influence, and 60 microM DA added to Il-2 produced an additive inhibitory effect. Thus, the interaction of IL-2 and DA is biphasic on LH release. The significant reduction of FSH release induced by IL-2 was blocked in the presence of 0.187, 3.75, 15, or 60 microM DA. DA alone at relatively high concentrations of 30, 60, and 90 microM suppressed basal LH and FSH release. The effects of DA on PRL, LH, and FSH at all doses tested were blocked by the DA receptor blocker, haloperidol which by itself at the concentration tested (1 x 10(-5) M) had no effect. Thus, the actions of DA at all concentrations tested appear to be mediated via DA receptors. In conclusion, DA was capable of blocking the stimulatory action of IL-2 on PRL release and its inhibitory action on FSH release by a DA receptor mediated action.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of age and long term ovariectomy on prolactin secretion, as assessed by the reverse hemolytic plaque assay

PRL secretion from pituitary lactotrophs was assessed using the reverse hemolytic plaque assay in young (2- to 3-month-old), middle-aged (10- to 12-month-old), and middle-aged long term ovariectomized (LT-OVX) rats to investigate whether 1) a change in the percentage of pituitary cells secreting PRL is detectable in middle-aged animals, 2) the amount of PRL secreted per cell changes with age, 3) aging involves a change in responsiveness to TRH and/or dopamine (DA), and 4) LT-OVX can prevent any of these changes. Young and middle-aged rats were OVX for 1 week. LT-OVX rats were OVX at 2-3 months of age and used when they were 10-12 months old. All animals were implanted with Silastic capsules containing estradiol (E2) in sesame oil and killed 3 or 8 days later. Anterior pituitaries were collected, and cells were dispersed and prepared for the reverse hemolytic plaque assay. Three days after E2 was implanted, the percentage of anterior pituitary cells that secrete PRL was higher in middle-aged compared to young rats. LT-OVX prevented this increase; the percentage of cells secreting PRL was significantly lower in LT-OVX than in both young and middle-aged rats. Basal secretion of PRL per cell was not different in young compared to middle-aged rats and was significantly lower in LT-OVX than in either young or middle-aged rats. TRH induced similar increases in plaque size in young and middle-aged rats, but had no effect in LT-OVX rats. DA (10(-7) M) inhibited plaque size only in LT-OVX rats; however, higher concentrations of DA were equally effective in the three experimental groups. Eight days after E2 was implanted, the percentage of cells that secrete PRL increased in LT-OVX rats, but was still significantly lower than that in middle-aged animals. After 8 days of E2 treatment, PRL release was similar in the three experimental groups under basal conditions. In LT-OVX rats TRH produced a small increase in PRL secretion (30-40%); DA suppressed PRL release in a similar manner in the three groups. These data demonstrate that middle-aged rats exhibit an increase in the percentage of cells secreting PRL without a concomitant detectable change in the amount of PRL released by single cells and/or a change in responsiveness to TRH or DA. Long term estrogen deprivation prevents this age-related change, suppresses responsiveness to TRH, and enhances sensitivity to DA.     

Dopamine receptors on intact anterior pituitary cells in culture: functional association with the inhibition of prolactin and thyrotropin

Dopamine (DA) and the dopaminergic agonists bromocriptine and apomorphine inhibit the secretion of TSH as well as that of PRL by rat anterior pituitary (AP) cells in monolayer culture. The order of potency of the drugs is the same for the inhibition of both hormones: bromocriptine ED50 = 0.006 nM against PRL and 0.017 nM against TSH; apomorphine ED50 = 2.9 and 4.8 nM, respectively, and DA, ED50 = 30 and 370 nM, respectively. The dopaminergic antagonists domperidone (DOM) and metoclopramide prevent the inhibition of TSH and PRL by 10(-6) M DA (IC50 = 0.012 and 0.32 nM for metoclopramide against PRL and TSH, respectively; similarly, IC50 = 0.01 and 0.61 nM for DOM). The action of butaclamol is shown to be stereospecific, in that the (+) isomer is 1000-fold more potent in reversing the inhibition of both TSH and PRL by 10(-6) M DA than the (-) isomer [IC50 = 1.1 and 7200 nM for the (+) and (-) isomers against PRL; similarly, 6.3 and 2600 nM against TSH]. The use of radioligand-binding techniques with tritiated DOM ([3H]DOM) and dihydroergocriptine ([3H]DHE) has demonstrated a high affinity dopaminergic binding site upon rat AP cells under the same conditions as the cell cultures used in the hormone secretion studies. Both ligands have been shown to label a site with high affinity (Kd = 1-2 nM) and low capacity (2-3 fmol/10(5) cells). At this site, dopaminergic agonists and antagonists compete with both radioligands and display a rank order of potency which is the same as that shown against TSH and PRL secretion and which is typically dopaminergic. For [3H]DHE: bromocriptine Ki (0.04 nM) greater than metoclopramide = DOM (0.07 nM) greater than (+)butaclamol (0.7 nM) greater than apomorphine (20 nM) greater than DA (700 nM) greater than (-)butaclamol (2000 nM). Similar data were derived using [3H]DOM. The high affinity site is saturable, has rapid association and dissociation rates, as determined for both radioligands used, and is temperature dependent. In contrast, both radioligands bind to a second binding site on the cells that is of lower affinity (Kd = 244 nM for [3H]DOM and 678 nM for [3H]DHE) and larger capacity (100 fmol/10(5) cells for both ligands). This second site is neither stereospecific nor, using the methodology presented here, does it discriminate between other dopaminergic compounds. It is thus not considered to represent specific DA receptor binding. It is concluded that the dopaminergic stimulus causing the inhibition of TSH and PRL secretion from rat AP cells in culture is mediated via a high affinity DA receptor present upon lactotrophs and thyrotrophs and that this receptor has similar characteristics on the two cell types.     

Nongenomic actions of testosterone on a subset of lactotrophs in the male rat pituitary

Rapid, nongenomic effects of testosterone on PRL release in vitro were investigated. Anterior pituitary tissue from adult male rats was stimulated in vitro for 5 or 20 min with testosterone (T; 1 or 100 nM) or testosterone-BSA (T-BSA; 1 or 100 nM) with or without 1.2 mM tannic acid, which enables visualization of secretory granule exocytosis. Within 5 min, both concentrations of T and T-BSA stimulated exocytosis from type 2 lactotrophs (characterized by small spherical granules), but not from type 1 lactotrophs (characterized by large polymorphic granules). The effects of T on type 2 lactotrophs could be blocked by preincubation with dopamine (500 nM), but were not time or concentration dependent, and could not be inhibited by 1) removal of extracellular Ca2+, 2) the L-type Ca2+ channel blocker nifedipine (100 nM), 3) the Ca2+-adenosine triphosphatase inhibitor thapsigargin (150 nM), 4) the PKC inhibitor retinal (10 microM), or 5) the gamma-aminobutyric acidA chloride channel blocker picrotoxin (100 microM). T-BSA (0.1 nM to 1 microM) for 5 or 20 min also caused an increased release of immunoreactive PRL into the medium compared with control incubations. T and T-BSA did not stimulate exocytosis from gonadotrophs or cause LH release. In conclusion, we report for the first time a rapid, nongenomic effect of T on PRL secretion.     

Transient dopamine withdrawal differentially potentiates thyrotropin releasing hormone-induced release of prolactin of various ages

Transient removal of dopamine (DA) potentiates the prolactin (PRL) releasing action of thyrotropin releasing hormone (TRH) in cultured lactotrophs. We have determined if this potentiating action on PRL release is related to the intracellular processing of PRL by studying this phenomenon in cells in which PRL was pulse-labeled at varying times. Anterior pituitaries from ovariectomized estradiol-treated rats were dispersed and cultured on plastic coverslips. Cells were incubated for 24 h in media containing 500 nM DA. Cells were exposed to three consecutive 20-min incubation periods. During the first period all cells were maintained in 500 nM DA, during the second period half of the cells were removed from DA inhibition and during the third period DA inhibition was restored and the cells challenged with 100 nM TRH. In experiments involving labeling of PRL, cells were incubated in leucine-free media containing 100 microCi/ml [3H] leucine for 1 h prior to treatment, for 1 h starting 5 h prior to treatment or for 16 h starting 24 h prior to treatment. Total PRL (by radioimmunoassay) and radiolabeled PRL (by liquid scintillation after electrophoretic isolation) were determined both in media and cells. The withdrawal of DA alone resulted in a 2-fold stimulation of total PRL release. The efficacy of TRH to release total PRL was also increased 2- to 4-fold after transient DA withdrawal. Whereas release of 1-hour-old labeled PRL was not affected by DA withdrawal, 5-hour-old and 8- to 24-hour-old [3H] PRLs were stimulated 2.6- and 2-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine inhibits prolactin release when cyclic adenosine 3',5'-monophosphate levels are elevated

We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase.