Relaxin stimulates prolactin secretion from anterior pituitary cells

The anterior pituitary has recently been implicated as a relaxin target issue because of the cAMP elevation after relaxin treatment. We attempted to correlate this finding with an endocrine response to relaxin in rats. Anterior pituitary cells were enzymatically dispersed and subjected to the reverse hemolytic plaque assay. PRL secretion was significantly stimulated 1.31-fold by human relaxin at the lowest concentration studied (30 pM) and maximally stimulated 1.65-fold at 0.3 nM relaxin. Antibodies directed against relaxin inhibited this effect, as did the PRL inhibitory hormone, dopamine. In contrast to the response of PRL cells, there was no effect or a slight inhibition of LH release after incubation with relaxin. In conclusion, we propose that one of the pituitary cell types responsive to relaxin in culture is the PRL-secreting mammotroph.     

Kallidin stimulates prolactin secretion from individual rat anterior pituitary cells

Kinins are localized within the adenohypophysis where they have been shown to stimulate the release of pituitary hormones. In the present study we have investigated the effect of [Lys]-bradykinin (kallidin) on prolactin secretion at the single cell level from cultured male rat anterior pituitary cells. This was assessed by use of a reverse haemolytic plaque assay which permits quantitative evaluation of the proportion of all pituitary cells which are secreting prolactin, and the amount of prolactin secreted per lactotroph (plaque area). The rate of plaque development was used as an index of the rate of hormone secretion in time-course studies. Kallidin induced a dose-dependent increase in both the percentage of plaque-forming cells and the median plaque area during the first 2 and 3 h of incubation respectively. The threshold concentration of kallidin was 10 nmol/l. After 4 h of kallidin stimulation there was no difference between treated and control monolayers with respect to median plaque area and the total secretion index. Although recruitment of additional cells into the secretory pool cannot be excluded, this seems unlikely since at 3 and 4 h little or no difference was observed in the number of plaque-forming cells. The data suggest that initially kallidin accelerated the rate of prolactin secretion primarily by inducing an increase in the number of cells secreting prolactin, and subsequently by increasing the amount of hormone secreted per lactotroph. The results presented here are consistent with the proposed role of the kallikrein-kinin system in the paracrine or autocrine control of prolactin release from the pituitary gland.     

5-Hydroxyeicosatetraenoic acid increases prolactin release from rat anterior pituitary cells

The enzymatic breakdown of phospholipids to form arachidonic acid and its subsequent conversion to metabolites produced via the lipoxygenase pathway in anterior pituitary cells may contribute to the process of PRL release. The incubation of primary cultures of pituitary cells from female rats with the lipoxygenase product 5-hydroxyeicosatetraenoic acid (5-HETE; 5-100 microM) significantly increased PRL release in a concentration-dependent manner. The release of PRL induced by 45 microM 5-HETE was completely blocked by 1 microM dopamine. Penfluridol, an agent that binds to and inactivates several Ca+2-binding proteins, including calmodulin, decreased (P less than 0.01) basal and 5-HETE-stimulated PRL release. Similarly, 50 microM D-600, a Ca+2 channel antagonist, significantly (P less than 0.01) reduced basal and 5-HETE-induced PRL release. BW755c or RHC 80267, both of which reduce the production of arachidonic acid metabolites, including 5-HETE, significantly reduced basal PRL release. The inhibitory effects of BW755c and RHC 80267 on PRL release, however, could be overcome by the addition of 5-HETE. In conclusion, 5-HETE or similar lipoxygenase metabolites may be important cellular components in the process of PRL release, and the inhibitory action of dopamine on PRL would seem to be mediated at some step after stimulation by these metabolites.     

Isolation of prolactin from salmon pituitary

Prolactin(s) was isolated from salmon pituitary by chromatography on concanavalin A, gel filtration, and ion-exchange chromatography. The prolactin not adsorbed on DEAE Bio-Gel A (DEAE 1) had the same molecular weight by get filtration before and after ion-exchange chromatography. This salmon prolactin had a molecular weight of 24,300 by gel filtration and 20,500 by SDS gel electrophoresis. The isoelectric point was 6.0 when determined by isoelectric focusing. This prolactin was homogeneous by electrophoresis under acidic and basic conditions and essentially homogeneous by isoelectric focusing. The protein consistently maintained plasma sodium levels in hypophysectomized Poecilia latipinna at very low dosage (50 ng/fish). Antibodies against this prolactin localized specifically in the prolactin cells of rainbow trout pituitary. A protein fraction more strongly adsorbed on DEAE Bio-Gel A (DEAE 4) also had some prolactin activity in the Poecilia assay. Prior to DEAE chromatography this protein occurred in the same molecular weight fraction as the nonadsorbed protein but after DEAE chromatography the fraction contained three proteins with molecular weights of 23,000 (64%), 46,000 (21%), and 66,000 (15%). The results suggest that the higher molecular weight proteins are aggregates formed during isolation but the 23,000 molecular weight protein may be a modified form of the unadsorbed prolactin. The growth hormone fraction (DEAE 2) had no prolactin activity when tested at 10 times the minimum effective dose of salmon prolactin. The remaining DEAE fractions, 3 and 5, were also inactive in the Poecilia assay.     

Bradykinin parallels thyrotropin-releasing hormone actions on prolactin release from rat anterior pituitary cells

Bradykinin (BK), a nonapeptide, originally discovered in blood, is also present in neurons and fibers of the hypothalamus. We tested the putative releasing factor properties of BK on prolactin (PRL) release from anterior pituitary cells in vitro. BK stimulated the release of PRL in a dose-dependent manner, the threshold concentration being in the range. 0.1-1.0 nM. The release of PRL induced by BK at 1 nM concentration was about 2-fold, delayed and sustained over many minutes. Higher concentrations of BK stimulated PRL release in two phases. The shape of the BK-induced PRL release was superficially similar to that induced by thyrotropin-releasing hormone (TRH). 10 nM BK and 10 nM TRH induced about a 4-fold increase in PRL release within 5 min, followed by a gradual recovery to basal secretion. These results indicate that this peptide can act directly at the anterior pituitary gland to release PRL. Phorbol ester also promoted PRL release over the range of 1-10 nM, but the time course of the release was somewhat different.     

Platelet-activating factor stimulates prolactin release from dispersed rat anterior pituitary cells in vitro

The biologically active phospholipid (platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated PRL release from dispersed rat anterior pituitary cells in culture. PAF-induced PRL release was dose dependent, with threshold stimulation at 1 nM and maximal stimulation at 100 nM. Stimulation occurred as early as 1 min of incubation and persisted for 2 h. The action of PAF on PRL release is consistent with a receptor-mediated mechanism based on the observations that the action of PAF is blocked by dopamine agonists and the PAF receptor antagonists L 652731 and SRI 63072. The structural analogs 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, which lack the biological activity of PAF, are not able to stimulate PRL release over the dose range 0.2-2 microM. In addition, the PAF precursor lyso PAF and diacyl-sn-glycero-3-phosphocholine (phosphatidylcholine) were ineffective in stimulating PRL release. PAF induced the secretion of PRL and GH but not that of LH or TSH from hemipituitaries in short term incubations. PAF did not effect PRL release from GH3 cells. In conclusion, these data indicate that PAF stimulates PRL release from primary cultures of rat anterior pituitary cells in a dose-related, rapid, and specific manner.     

Mammary gland prolactin receptor and pituitary prolactin secretion in lactating mice with different lactational performance

SHN female mice, a high mammary tumour strain, are superior to SLN, a low mammary tumour strain, in lactational performance. Mammary gland prolactin receptor and pituitary prolactin secretion during lactation were compared between these strains. The binding activity, the number of receptor sites per mg tissue and the association constant were measured by the in vitro incubation of mammary gland slices with 125I-labelled bovine prolactin, and the pituitary and plasma levels of prolactin were assayed by homologous radioimmunoassay. There was only a slight difference between strains in any of the parameters for prolactin receptor and for prolactin secretion on either day 4 or day 9 of the first lactation. Almost all the correlation coefficients between each parameter for prolactin receptor and the pituitary or plasma level of prolactin were not statistically significant. These findings suggest that any parameter for prolactin examined in this study is not always directly indicative of lactational performance and further show that the individual variation in the pituitary prolactin secretion during lactation is not so great as to alter the prolactin receptor.     

Involvement of calcium ions in dopamine inhibition of prolactin secretion from sheep pituitary cells

The involvement of calcium in the regulation of prolactin secretion and a possible inhibitory mechanism of action for dopamine have been investigated. Basal prolactin secretion from cultured ovine pituitary cells was dependent on the concentration of calcium ions (Ca²⁺) in the medium and was inhibited by the presence of verapamil (10 μM). The divalent cation ionophore A23187 (1 μM) caused a rapid stimulation of prolactin release from the cells. The effect was essentially complete within 10 min and subsequently secretion of prolactin occurred at close to the basal rate. A23187 had no effect on cell cyclic AMP levels. Dopamine (0.1 μM) but not verapamil (10 μM) inhibited the A23187 (10 μM) induced release of prolactin. Inhibition of basal and A23187 (1 μM) stimulated prolactin secretion occurred over a similar range of dopamine concentrations. The dopamine receptor antagonist haloperidol (1 μM) reversed the inhibitory effect of dopamine (0.1 μM) on A23187-stimulated prolactin release. These results provide evidence to support the concept that control of Ca²⁺ handling by lactotrophs may be of fundamental importance in the regulation of prolactin secretion.     

Antisera to vasoactive intestinal polypeptide inhibit basal prolactin release from dispersed anterior pituitary cells

Vasoactive intestinal polypeptide (VIP) has been identified in hypothalamic tissue, is secreted into hypophysial portal blood, and stimulates prolactin (PRL) release in vivo and in vitro. It has been proposed, therefore, that VIP is a physiologic PRL-releasing factor. In this study, we confirm that VIP stimulates PRL release from rat pituitary cells in vitro, and demonstrate that an anti-VIP antiserum blocks VIP-induced PRL secretion. Surprisingly, the anti-VIP antiserum inhibited basal PRL secretion from rat pituitary cells in 3 separate experiments. Data from these experiments were pooled, as the responses were similar, revealing basal PRL release of 10.7 +/- 1.3 ng rPRL/10(5) cells (X +/- SE), while anti-VIP antisera significantly inhibited release to 4.4 +/- 0.6 ng rPRL/10(5) cells (p less than 0.001). PRL release in incubates containing control non-immune sera did not differ from basal release, 8.1 ng rPRL/10(5) cells. A further control experiment was conducted wherein cells were incubated with an anti-ACTH antiserum, representing another hyperimmune serum, which had no effect on PRL secretion. These data suggest that VIP, in addition to its possible role as a hypothalamic-derived PRL-releasing factor, may play a role within the pituitary as a regulator of basal PRL secretion.     

Cell-density dependence of the rate of prolactin secretion from perifused rat anterior pituitary cells

Anterior pituitary glands from female rats were dispersed enzymically in the absence of dopamine. Dispersed cells (10(6)-10(7)) were layered onto columns containing Bio-Gel P-2 and were then perifused for 3 h with Dulbecco's Modified Eagle's Medium. The prolactin content of the perifusate and cell homogenates was determined by radioimmunoassay. Prolactin secretion during the third hour of perifusion increased as the loading of cells increased. However, the increase was not linear, and when secretion rate per 10(6) cells was calculated it was found that increased loading decreased the rate, which fell to a plateau of 1.3 +/- 0.1 (S.E.M.) ng/min per 10(6) cells at a loading of about 8 x 10(6) cells from 3.8 +/- 0.1 ng/min per 10(6) cells for a loading of 10(6) cells. This cell-density dependence of the rate of prolactin secretion in the perifusion system may be due to intercellular contact since the isolation of the tissue removes the influence of hypothalamic factors, while localized build up of prolactin (potentially causing direct autoregulation on the lactotroph) seems unlikely because of the continuous flow of medium.     

15-Lipoxygenase products stimulate prolactin secretion from a cloned strain of rat pituitary cells

Arachidonic acid and its lipoxygenase products may contribute to the process of prolactin (PRL) release. In the present study we investigate the role of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on PRL secretion from GH3 cells. The incubation of GH3 cells with the lipoxygenase product 15-HETE significantly increased PRL release in a concentration-dependent manner. Nordihydroguaiaretic acid (NDGA), which reduces the production of arachidonate metabolites via the lipoxygenase pathway, also reduced basal and TRH or arachidonic-acid-stimulated-PRL release. The inhibitory effect of NDGA on PRL release could be overcome by the addition of 15-HETE. The time course curve of PRL release from cells challenged by 15-HETE had the same profile as that of cells stimulated by TRH. The stimulating effect of 15-HPETE (ED50 = 0.7 x 10(-9) M), which is the direct precursor of 15-HETE, on PRL release was greater than TRH or 15-HETE (ED50 = 6.5 x 10(-9) M). Furthermore 15-HPETE and 15-HETE seemed to affect the release of newly synthesized PRL. These data indicate that 15-HETE and 15-HPETE could be important intracellular components in the control of PRL secretion and may account for at least a part of arachidonate-induced PRL release from GH3.     

Tumor necrosis factor-alpha increases release of arachidonate and prolactin from rat anterior pituitary cells

We investigated the effect of tumor necrosis factor-alpha (TNF alpha), a product of activated macrophages, on the release of arachidonate from dispersed anterior pituitary cells. Primary cultures of anterior pituitary cells from rats were preincubated with [3H]arachidonate to label their phospholipid-containing components. The cells were then washed and incubated with vehicle or test agents, and PRL release into the medium and [3H]arachidonate cleaved from phospholipid were measured. TNF alpha significantly increased the release of both PRL and [3H] arachidonate release in a time- and dose-dependent manner. Other cytokines, such as interleukin-1 alpha, interleukin-1 beta, and gamma-interferon, had no effect on [3H]arachidonate release. To define the role of calcium in TNF alpha-induced arachidonate release, dispersed pituitary cells were incubated with low calcium medium, which decreased arachidonate release in response to TNF alpha. TNF alpha potentiated the release of [3H]arachidonate and PRL promoted by phospholipase-A2 and melittin, and markedly shifted the dose-response curve to the left. Inhibitors of phospholipase-A2, such as p-bromophenacyl bromide and quinacrine, had no effect on TNF alpha-induced [3H]arachidonate and PRL release. BW755C, an inhibitor of the conversion of arachidonate to its metabolites, decreased TNF alpha-induced PRL release, while indomethacin, a prostaglandin synthesis inhibitor, had no effect on TNF alpha-induced PRL release. These data indicate that arachidonate metabolites may be involved in the process of TNF alpha-induced PRL release.     

A possible role for lipoxygenase and epoxygenase arachidonate metabolites in prolactin release from pituitary cells

We studied the effects of selected leukotrienes and hydroxyeicosatetraenoic acids (HETEs) on prolactin release from primary cultures of female rats anterior pituitary cells. Leukotrienes B4, C4, and D4 had no effect on basal prolactin release; however, they did enhance prolactin release that was stimulated by 1 or 5 nM thyrotropin-releasing hormone (TRH). Leukotriene C4 also enhanced prolactin release that was induced by phorbol myristate acetate (a protein kinase C activator) by maitotoxin (a calcium uptake stimulator), and by angiotensin II. 5-HETE, 12-HETE, and 15-HETE stimulated basal prolactin release at high concentrations (1 microM and greater), and 5-HETE and 12-HETE enhanced TRH- and angiotensin II-induced prolactin release at lower (nanomolar) concentrations as well. In order to determine the role of endogenous arachidonate metabolites in prolactin release, pituitary cell cultures were exposed to selected inhibitors of the 5-lipoxygenase enzyme, which metabolizes arachidonate to leukotrienes and 5-HETE, and to those of the epoxygenase enzyme, which metabolizes arachidonate to epoxyeicosatrienoic acids. These inhibitors decreased basal and secretagogue-induced prolactin release. In additional experiments, it was determined that TRH enhances the liberation from pituitary cells of arachidonate metabolites with high-performance liquid chromatography elution profiles similar to those of leukotriene C4 and omega-OH-leukotriene B4 (a metabolite of leukotriene B4) and the HETEs. Therefore, the production of leukotrienes, HETEs, and epoxyeicosatrienoic acids may be necessary for the normal release of prolactin.     

Cortistatin inhibits growth hormone release from human fetal and adenoma pituitary cells and prolactin secretion from cultured prolactinomas

CONTEXT: Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin. OBJECTIVE/DESIGN: The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion. SETTING: This study was performed in the endocrine laboratory of a tertiary academic medical center. MATERIALS: Primary cell cultures of human fetal (21-25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study. INTERVENTIONS: Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed. OUTCOME MEASURES: GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR. RESULTS: CST-14 (10 nm) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nm) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nm) suppressed the GH response to GHRH and ghrelin analog (10 nm each) by 30-50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nm) showed a 20-40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent. CONCLUSIONS: This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.