Pituitary localization of 3H-spiroperidol by an Uptake/Storage Mechanism?

The lack of a pituitary imaging agent combined with the considerable clinical value for such an agent prompted an examination of ³H-spiroperidol (³HSp). Spiroperidol was selected for initial evaluation based on its high affinity for D₂ receptors which are known to be present in the pituitary. A time course study of ³HSp concentration in rat pituitary and other tissues was conducted. Pituitary activity levels were found to be constant from 5 min to 4 h and were about 8 times levels in corpus striatum at 1 h. Blocking studies with (+)-butaclamol and with unlabelled spiroperidol suggested the existence of both a D₂ receptor mediated binding localization and a second uptake which is postulated to be an internalization process. Further studies involving ultracentrifugation of pituitary homogenates resulted in evidence for association of ³HSp with dense subcellular particles. ³HSp thus appears to be internalized by pituitary cells.     

雌激素诱发大鼠原位与移植垂体催乳素瘤中催乳素基因与TGFα和TGF …

利用本实验室建立的１７β－雌二醇诱致Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ（ＳＤ）大鼠原位垂体和异体移植于肾囊的垂体同时形成催乳素瘤的动物模型,采用Ｎｏｒｔｈｅｍ印迹杂交方法,我们观察了Ｅ２长期作用（１２０ｄ）后诱发的原位与移植垂体ＰＲＬ瘤中ＰＲＬ基因和两种转化生长因子ＴＧＦα和ＴＧＦβ１基因表达水平的改变。结果表明：在Ｅ２长期作用后,原位垂体与异体移植于肾囊,从而远离下丘脑的垂体均可形成垂体ＰＲＬ瘤;原位     

The influence of vasoactive intestinal polypeptide and cholecystokinin of prolactin release in rat and human monolayer cultures

We have evaluated the effects of the gut-brain peptides, VIP and CCK, on pituitary PRL secretion in monolayer cultures of normal and tumor bearing rodent and human pituitary tissue. In cultures prepared with normal human pituitary tissue obtained from three patients with metastatic breast cancer, VIP at 10^?7M and 10^?9M (but not 10^?11M) significantly (p<.05) increased PRL secretion in the wells by 6 hrs. Similar concentrations of VIP also significantly (p<.05) promoted PRL release from pituitary tissue obtained by transphenoidal hypophysectomy from one of two prolactinoma patients. Dopamine (10^?5M) inhibition of PRL secretion was not affected by 10^?11 to 10^?7M VIP. In contrast to these findings VIP did not significantly influence 6 hr rat PRL release in monolayer cultures of normal or transformed cells (GH₃) with or without the addition of bacitracin (10^?5M).CCK₃₃ significantly (p<.01) increased rat PRL release in human pituitary monolayer cultures at 10^?5M. The more biologically potent CCK₈ significantly (p<.02) increased rat PRL release at a 10-fold lower concentration, 10^?6M. In contrast, CCK₈ 10^?8 to 10^?6M, did not significantly influence PRL release from normal human pituitary cultures or from tumor bearing human (prolactinoma) and rat (GH₃) cultures. We conclude that 1) the gut-brain peptides, VIP and CCK, can directly stimulate pituitary PRL release and 2) VIP may be a physiologic prolactin releasing factor in man.     

The effects of ACTH 1-17 on GH secretion in vitro

Recently we demonstrated that ACTH 1-17 infusion in normal subjects is able to stimulate growth hormone (GH) secretion. In order to study the mechanism by which ACTH 1-17 induces this hormonal secretory pattern, we examined the effects of ACTH 1-17 addition to primary cultures of rat anterior pituitary cells and of two human pituitary adenomas (a mixed GH- and PRL-secreting adenoma and a prolactinoma) on GH and PRL secretion. Normal rat pituitary cells responded to rGRF with a dose-dependent increase of rGH: ACTH 1-17 induced a slight not significant increase of rGH secretion even at micromolar concentrations. Furthermore no additive effect of ACTH 1-17 on rGRF-stimulated GH release was observed. No significant stimulatory effect was also documented in the human tumors studied. These results suggest that the GH releasing activity of ACTH 1-17 observed in vivo is mediated via a direct action on CNS.     

Dopamine receptors in rat pituitary and estradiol-induced pituitary tumor: effect of chronic treatment with bromocriptine

We have investigated dopamine (DA) receptors in estradiol-induced PRL-secreting pituitary tumors and intact pituitary tissue. Female rats were injected at 3-week intervals with 2 mg estradiol valerate (EV) or with diluent. After 21 weeks, adenomatous changes in the pituitary gland of EV-treated rats were seen and plasma PRL concentrations reached 2 micrograms/ml. Bromocriptine (2.5 mg/kg) was then administered for 1 month to half of the control rats and half of the rats bearing tumors. Anterior pituitary weight was increased in EV-treated rats compared to controls while the affinity and the density of DA receptors as assessed by [3H]spiperone binding remained unchanged. Bromocriptine (CB-154) induced a 70% decrease in the density of DA receptors without any change in affinity both in normal pituitaries and in tumors. Concurrently, the elevated plasma concentrations of PRL in the tumor bearing rats were decreased to control values following the CB-154 treatment. Our data suggest that rats with primary estrogen-induced PRL secreting tumors have normal pituitary DA receptors.     

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D₂ receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [³H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [³H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [³H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [³H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D₂ receptor and should prove useful in studies on functional reconstitution of the receptor.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

Prolactin and growth hormone synthesis and thymidine incorporation in dissociated rat pituitary tumor cells

The effect of in vivo diethylstilbestrol (DES) treatment on the MtT/W15 transplantable pituitary tumor was examined in dissociated pituitary cells by measuring the rate of incorporation of [3H]thymidine into DNA and the synthesis of prolactin (PRL) and growth hormone (GH) as assessed by the rate of incorporation of [3H]leucine. MtT/W15 transplantable pituitary tumors from rats treated for 3 weeks with DES showed significant reduction in the extent of [3H]thymidine incorporation compared with tumor cells from untreated rats (2231 +/- 182 vs 172 +/- 17 dpm/10(5) cells; n = 3). In addition, tumor cells from DES-treated rats showed a significant increase in GH synthesis compared with tumor cells from untreated rats. In contrast to these findings, dissociated pituitary cells from non-tumor-bearing rats given 10 mg DES in Silastic tubing for 3 weeks showed a three-fold increase in PRL synthesis compared to cells from untreated control rats (29.3 +/- 1.5 vs 10.0 +/- 0.9% of total radioactivity in gel; n = 3. There was also a four-fold increase in the rate of [3H]thymidine incorporation after DES-treatment in non-tumor-bearing rats (695 +/- 114 vs 178 +/- 13.9 dpm/10(5) cells; n = 3). These results indicate that DES inhibits MtT/W15 pituitary tumor cell proliferation, while stimulating synthesis of GH.     

HER2/ErbB2 receptor signaling in rat and human prolactinoma cells: strategy for targeted prolactinoma therapy

Dopamine agonist resistance or intolerance is encountered in approximately 20% of prolactinoma patients. Because human epidermal growth factor receptor 2 (HER2)/ErbB2 is overexpressed in prolactinomas and ErbB receptor ligands regulate prolactin (PRL) gene expression, we tested the role of HER2/ErbB2 in prolactinoma hormone regulation and adenoma cell proliferation to assess the rationale for targeting this receptor for prolactinoma therapy. As we showed prolactinoma HER2 overexpression, we generated constitutively active HER2-stable GH3 cell transfectants (HER2CA). PRL mRNA levels were induced approximately 250-fold and PRL secretion was enhanced 100-fold in HER2CA cells, which also exhibited increased proliferation. Lapatinib, a dual tyrosine kinase inhibitor (TKI) of both epidermal growth factor receptor (EGFR)/ErbB1 and HER2, blocked receptor signaling, and suppressed PRL expression more than gefitinib, a TKI of EGFR/ErbB1. Lapatinib also suppressed colony formation in soft agar more than gefitinib. Oral lapatinib treatment caused tumor shrinkage and serum PRL suppression both in HER2CA transfectant-inoculated Wistar-Furth rats and in estrogen-induced Fischer344 rat prolactinomas. In cultured human cells derived from resected prolactinoma tissue, lapatinib suppressed both PRL mRNA expression and secretion. These results demonstrate that prolactinoma HER2 potently induces PRL and regulates experimental prolactinoma cell proliferation. Because pituitary HER2 signaling is abrogated by TKIs, this receptor could be an effective target for prolactinoma therapy.     

Interactions of steroids with prolactin secretion in vitro

Estrogens prevent or diminish the sensitivity to dopamine of prolactin (PRL) secretion by cultured rat pituitary cells. Cultured tumor cells prepared from a transplantable rat PRL-secreting tumor were insensitive to dopamine and bromocriptine, while the anti-estrogen tamoxifen restored this sensitivity. Cultured normal human pituitary cells were shown to be more sensitive to dopamine, if they were preincubated with estradiol, while cultured human prolactinoma cells became insensitive to bromocriptine after they were exposed to estrogens. This sensitivity was restored, however, by tamoxifen. These results point to an important species difference between primates and rodents with regard to the normal regulation of PRL secretion.     

Prolactin Induces Apoptosis of Lactotropes in Female Rodents

Anterior pituitary cell turnover occurring during female sexual cycle is a poorly understood process that involves complex regulation of cell proliferation and apoptosis by multiple hormones. In rats, the prolactin (PRL) surge that occurs at proestrus coincides with the highest apoptotic rate. Since anterior pituitary cells express the prolactin receptor (PRLR), we aimed to address the actual role of PRL in the regulation of pituitary cell turnover in cycling females. We showed that acute hyperprolactinemia induced in ovariectomized rats using PRL injection or dopamine antagonist treatment rapidly increased apoptosis and decreased proliferation specifically of PRL producing cells (lactotropes), suggesting a direct regulation of these cell responses by PRL. To demonstrate that apoptosis naturally occurring at proestrus was regulated by transient elevation of endogenous PRL levels, we used PRLR-deficient female mice (PRLRKO) in which PRL signaling is totally abolished. According to our hypothesis, no increase in lactotrope apoptotic rate was observed at proestrus, which likely contributes to pituitary tumorigenesis observed in these animals. To decipher the molecular mechanisms underlying PRL effects, we explored the isoform-specific pattern of PRLR expression in cycling wild type females. This analysis revealed dramatic changes of long versus short PRLR ratio during the estrous cycle, which is particularly relevant since these isoforms exhibit distinct signaling properties. This pattern was markedly altered in a model of chronic PRLR signaling blockade involving transgenic mice expressing a pure PRLR antagonist (TG^{Δ1–9-G129R-hPRL}), providing evidence that PRL regulates the expression of its own receptor in an isoform-specific manner. Taken together, these results demonstrate that i) the PRL surge occurring during proestrus is a major proapoptotic signal for lactotropes, and ii) partial or total deficiencies in PRLR signaling in the anterior pituitary may result in pituitary hyperplasia and eventual prolactinoma development, as observed in TG^{Δ1–9-G129R-hPRL} and PRLRKO mice, respectively.     

Uptake and retention of3H-estradiol by gonadotrophs and lactotrophs in the pituitary glands of the guinea pig,hamster and gerbil

Summary Nuclear uptake and retention of³H-estradiol by luteinizing hormone (LH) and prolactin (PRL) cells was examined in three species of rodents (guinea pigs, hamsters and gerbils) using the combined techniques of immunocyto-chemistry and autoradiography. Castrated animals were injected with³H-estradiol and decapitated 1.5 h later. The pituitary glands were processed for thaw-mount autoradiography followed by conventional immunocytochemical staining for LH and PRL.³H-estradiol accumulated in more than 80% of the anterior pituitary cells in the gerbils, while only 33 and 22% of the cells accumulated³H-estradiol in the hamsters and guinea pigs, respectively. A varying percentage of immunoreactive LH and PRL cells in all three species were found also to contain binding sites for estradiol. Some LH and PRL cells in hamsters and guinea pigs and only some in PRL cells of gerbils were found to be devoid of grains. Quantitative analysis revealed that the number of grains per nucleus differed considerably from cell to cell. LH cells of guinea pigs accumulated much larger amounts of³H-estradiol than did the PRL cells, while the LH cells in the hamsters and gerbils accumulated only slightly more³H-estradiol than the PRL cells.These results confirm the previous observations in rats and baboons that demonstrated tremendous species differences in percentage of cells in the anterior pituitary gland that accumulated³H-estradiol. Also, these data suggest that there are functionally heterogeneous cell types among the LH and PRL cells in hamsters, guinea pigs and gerbils as has been previously demonstrated in rats and baboons.     

Effect of zinc administration on thyrotropin releasing hormone-stimulated prolactinemia in healthy men

Previous in vitro studies have demonstrated zinc (Zn⁺⁺) inhibition of basal and of potassium (K⁺) or thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion, in a selective, reversible, and dose-dependent manner. Thus, Zn⁺⁺ may regulate physiologically pituitary PRL secretion. Furthermore, studies with patients with uremia, cirrhosis or prolactinoma, have shown the coexistence of hypozincemia and hyperprolactinemia and zinc supplementation did not correct hyperprolactinemia in these patients. In normal individuals Zn⁺⁺ administration produced controversial results on PRL secretion. Here, we investigated whether zinc administration affects TRH-stimulated PRL in healthy men. We found that Zn⁺⁺ administration does not change the TRH-stimulated PRL. Therefore, in normal conditions, Zn⁺⁺ does not inhibit TRH-stimulated prolactinemia. In addition, we found that acute increases of blood PRL and TRH do not alter blood Zn⁺⁺ levels.     

Culture requirements for optimal expression of 1α,25-dihydroxyvitamin D3-enhanced thyrotropin secretion

Summary An effect of the hormone, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on hormone secretion by normal rat pituitary cells was investigated in vitro. Based on previous findings using GH₄C₁ cells, dispersed anterior pituitary cell cultures were prepared and maintained in serum-free conditions for up to 6 d. Under these circumstances, there was no effect of 1,25(OH)₂D₃ to alter medium or cell-associated levels of thyrotropin (TSH), prolactin (PRL), or growth hormone (GH). Cultures maintained under these conditions had lower medium and cell-associated hormone levels and lesser responses to agonists than cultures maintained in serum-supplemented medium. In the presence of 10% charcoal-treated fetal bovine serum, treatment with 10⁻⁸ M 1,25(OH)₂D₃ for 24 h selectively increased TRH (10⁻¹⁰ to 10⁻⁷ M)-induced TSH secretion (P<0.001), with maximal enhancement observed at 10⁻⁹ M TSH-releasing hormone (TRH). Enhancement of TSH secretion by 1,25(OH)₂D₃ was detected after 15 min exposure to TRH. There was no effect on agonist-induced PRL or GH secretion or on cell-associated hormone levels. The effect was evident after 24 h treatment with 1,25(OH)₂D₃, and decreased thereafter. Several other steroid hormones had no effect on 10⁻⁹ M TRH-induced TSH secretion. These data contrast with the effect of 1,25(OH)₂D₃ in GH cells. They suggest that 1,25(OH)₂D₃ may act selectively in the normal pituitary to modulate TSH secretion.     

垂体催乳素瘤的病因及发病机制的实验研究

应用在同一只雄性ＳＤ大鼠中,保留原位垂体并在肾囊内植入一异体垂体,同时背部埋植装有１７－β－雌二醇（１７－β－ｅｓｔｒａｄｉｏｌ,Ｅ２）的药泵,经Ｅ２在体作用６０￣１２０天后,其原位垂体和异体移植入肾囊的垂体同时形成２垂体催乳素（ｐｒｏｌａｃｔｉｎ,ＰＲＬ）瘤的动物模型,研究ＰＲＬ瘤的发病机制。结果表明,Ｅ２长期作用可诱发原位垂体和远离下丘脑的移植垂体同时形成ＰＲＬ瘤,并伴高ＰＲＬ血症和ＰＲＬ基因     

Effects of chronic bromocriptine treatment of an estrone-induced, prolactin-secreting rat pituitary adenoma

Bromocriptine (BROM), a dopamine (DA) agonist, is commonly and successfully used for long-term treatment of human prolactinomas. We have studied the effects of chronic BROM administration to female 344 Fisher/Lis rats bearing an estrone-induced, prolactin (PRL)-secreting pituitary tumor recently characterized as a model for human prolactinoma. The animals were injected twice daily with BROM (2.5 mg/kg) or with diluent. After 1 month of treatment, the animals were sacrificed, and plasma collected and stored at -20 degrees C for PRL radioimmunoassay. The pituitary tumors were removed and tumoral mammotrophs dispersed enzymatically for studies of DA receptor binding and PRL release in vitro. BROM treatment significantly reduced tumor weight, cell size, rough endoplasmic reticulum, Golgi complexes and plasma PRL levels. [3H]-spiroperidol binding to tumoral mammotrophs was also evaluated. BROM induced a significant decrease in the number of DA binding sites without any changes in affinity. These results indicate that chronic BROM treatment of an animal model of prolactinoma induces tumor involution, reduction of PRL release and probably synthesis, and down regulation of dopaminergic binding sites.     

Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression

Pituitary tumor-transforming gene (PTTG) originally isolated from GH-secreting pituitary adenoma cells causes in vitro cell transformation, in vivo tumorigenesis, and induces basic fibroblast growth factor. These functions require an intact C-terminal proline-proline-serine-proline motif. PTTG1 is abundantly expressed in human pituitary tumors and plays a role in the early stages of experimental prolactinoma formation. We now determined direct effects of PTTG1 on hormonal phenotypes of functional pituitary tumor cells. Overexpression of PTTG1 C terminus (amino acids 147-202) containing intact proline-proline-serine-proline motifs in rat prolactin (PRL)- and GH-secreting GH3 cells markedly abrogates PRL mRNA expression by more than 90% (P < 0.001) and hormone levels (P < 0.001) and PRL promoter activity (P < 0.01) compared with control vector cells or to a PTTG1 C terminus mutant (P163A, S165Q, P166L, P170L, P172A, and P173L). Wild-type PTTG1 C-terminal transfectants formed smaller (P < 0.05) sc tumors in rats compared with control or mutated PTTG1 C-terminal transfectants. Estrogen (10 nm) treatment for 48 h partially restored PRL expression in stable wild-type PTTG1 C-terminal transfectants. These results indicate that targeting PTTG1-mediated signaling alters the hormonal phenotype in pituitary cells and disrupted PTTG1 action may be a potential subcellular therapeutic tool for repressing PRL hypersecretion.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

Does neonatal androgen modulate uptake and retention of 3H-estradiol in gonadotrophs and lactotrophs?

Summary Castrated adult male and female and androgenized female rats (AS rats) were injected i.v. with ³H-estradiol (E₂). Nuclear uptake and retention of the ³H-steroid was examined in pituitary luteinizing hormone (LH) and prolactin (PRL) cells by the combined techniques of autoradiography and immunocytochemistry. About 80% of PRL cells were found to concentrate the radioactive steroid compound in all experimental groups, while 89%, 82% and 68% of LH cells were found to be labeled in AS rats, normal female and male rats, respectively. This suggests that there are subpopulations of LH or PRL cells that contain no or, if any, small numbers of E₂ receptor. Statistical analysis revealed that PRL cells take up more radioactivity than LH cells in male rats, while there is no significant difference between female and AS rats. Variations in E₂ uptake (coefficient of variation) was higher in LH cells than in PRL cells in male rats and in AS rats. In females, on the contrary, coefficient of variation was larger in PRL cells. Thus the characteristics of nuclear uptake and retention of estradiol in LH and PRL cells appear to be modulated in part by neonatal androgen since the pattern found in AS rats is different than that found in normal male and female rats.