Cadmium Mimics Estrogen-Driven Cell Proliferation and Prolactin Secretion from Anterior Pituitary Cells

Cadmium (Cd) is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2) involving E2 receptor (ER) activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health.     

Presence of Two Benzodiazepine Binding Sites in the Rat Hippocampus

Abstract: Characteristics of receptor binding of diazepam and flunitrazepam in three brain areas were compared. It was found that in the cerebral cortex and cerebellum the number of sites was similar for both ligands and that the affinity of diazepam was four times lower than the affinity of flunitrazepam. In contrast, when binding in the hippocampus was analyzed (assuming the presence of homogenous binding sites), it was found that the number of binding sites was higher and that the affinity was 17 times lower for diazepam than for flunitrazepam. This difference is due to the presence of two diazepam binding sites in this brain area, as demonstrated by a Scatchard analysis.     

牛泌乳素mRNA的分离及鉴定

本文报道了从牛脑垂体提取总RNA,经寡聚脱氧胸苷纤维素亲合层析分离获得牛脑垂体Poly(A)~+RNA。牛泌乳素mRNA经含羟甲基汞琼脂糖凝胶电泳分析,估计其长度约为1200个核苷酸。根据牛泌乳素的部分氨基酸序列推断并合成寡聚核苷酸探针,经Northern印迹杂交及放射自显影分析,证实了该mRNA中含有牛泌乳素mRNA的序列。在兔网织红细胞体外翻译体系中,牛泌乳素mRNA促进了~35S-甲硫氨酸参入,翻译合成的初级翻译产物能与兔抗羊PRL抗血清发生特异性免疫沉淀反应,SDS聚丙烯酰胺凝胶电泳及放射自显影分析结果表明,牛泌乳素前体的分子量约25000。     

Second Messengers Associated with the Action of AII and Dopamine D2 Receptors in Anterior Pituitary. Relationship with Prolactin Secretion

Abstract

We have shown that DA receptors of the D2 subtype inhibit prolactin release by several mechanisms. DA receptors inhibit cyclic AMP production through a GTP binding protein sensitive to the Bordetella pertussis toxin. However, this mechanism cannot be involved in the blockade of the AII stimulated prolactin secretion by DA. This blockade is probably partly due to the inhibition of the AII-stimulated inositol phosphate production by DA. This inhibition is also sensitive to the Bordella pertussis toxin. The toxin is able to ADP-ribosylate three substrates in anterior pituitary cells (39, 40 and 41 kDa). In addition, we show here that AII receptors inhibit adenylate cyclase of anterior pituitary cell homogenates, but not in intact cells.     

Prolactin and Somatotropin Binding by Granulosa Cells of Cows at Their Different Reproductive States

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3–5 to 6–10 mm, specific binding of ¹²⁵I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data indicate that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1–2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.     

Induction of Cytosolic Progestin Binding Sites by Catecholestrogens in Rat Pituitary Gland and Uterus: Different Potencies of 2- and 4-Hydroxyestradiol

The ability of catecholestrogens to induce cytosolic progestin binding sites in the hypothalamus, pituitary gland, and uterus of ovariectomised-adrenalectomised rats was demonstrated by the increase in high-affinity [3H]promegestone binding sites (KD 1.39, 0.50, and 0.54 nM, respectively) following a single subcutaneous injection (26.4 micrograms/animal) of the 3.4-dibenzoate ester of 4-hydroxyestradiol. The affinity and the time course of induction of these binding sites were very similar to those after a single injection of an equivalent dose (20 micrograms/animal) of estradiol 3-benzoate, exhibiting maximal receptor levels after 44 h. Widely differing efficacies in the induction of progestin binding sites were observed between the dibenzoate esters of 2- and 4-hydroxyestradiol. 2-Hydroxyestradiol 2,3-dibenzoate was ineffective in the pituitary gland up to a dose of 132 micrograms/animal, whereas 4-hydroxyestradiol dibenzoate was equipotent to estradiol benzoate, showing a maximal induction of progestin binding sites at single doses in the range of 13.2-26.4 micrograms/animal (equivalent to 10-20 micrograms of estradiol benzoate). As compared to the pituitary gland, the uterus was much more sensitive to the systemic administration of estrogen benzoates. At single doses in the range of 1.32-6.6 micrograms/animal (equivalent to 1-5 micrograms of estradiol benzoate), 4-hydroxyestradiol dibenzoate induced maximal levels of progestin receptors, and even 2-hydroxyestradiol dibenzoate, when given at a high dose (132.4 micrograms/animal, equivalent to 100 micrograms of estradiol benzoate), produced a slight increase in progestin binding sites.     

Solubilization of Kainic Acid Binding Sites from Rat Brain

Kainic acid binding sites were solubilized from rat brain using a combination of Triton X-100 and digitonin. The highest percentage of solubilized binding sites (45%) was obtained by treating brain membranes with 1% Triton-X-100 and 0.2% digitonin in 0.5 M potassium phosphate containing 20% glycerol. The solubilized binding sites were stable and amenable to analysis by gel filtration and lectin affinity chromatography. Computer assisted analyses demonstrated that the solubilized sites displayed high- and low-affinity binding constants similar to the membrane-bound sites. Competition experiments further supported the pharmacological similarities of the solubilized and membrane-bound sites. Gel filtration chromatography of the solubilized binding site indicated that the detergent-bound complex had a Stokes radius of 82.7 A. The [3H]kainic acid binding site appears to be glycosylated based on its capability to bind to lectins. The lectin, wheatgerm agglutinin, proved to be a potentially useful tool for characterization because the solubilized binding sites were bound and eluted in relatively high yield.     

Augmentation of Prolactin Release by alpha-Melanocyte Stimulating Hormone Is Possibly Mediated by Melanocortin 3-Receptors in the Mouse Anterior Pituitary Cells

Suckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by alpha-melanocyte stimulating hormone (alpha-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the alpha-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not alpha-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that alpha-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that alpha-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice.     

Forskolin Stimulates Adenylate Cyclase Activity, Cyclic AMP Accumulation, and Adrenocorticotropin Secretion from Mouse Anterior Pituitary Tumor Cells

Abstract: The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/D16-16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nu-cleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

Molecular Interactions of Etazolate with Benzodiazepine and Picrotoxinin Binding Sites

Pyrazolopyridines, such as etazolate (SQ 20009), enhance [3H]diazepam binding to a Lubrol-solubilized fraction that has specific binding sites for 3H benzodiazepines, [alpha-3H]dihydropicrotoxinin (DHP) and [3H]muscimol. Etazolate enhancement of [3H]diazepam binding was inhibited by picrotoxinin. Furthermore, etazolate inhibited the [3H]DHP binding in a Lubrol-solubilized fraction with an IC50 value of 6-8 microM. These results provide evidence that etazolate, like pentobarbital, modulates benzodiazepine binding via the DHP-sensitive site of the benzodiazepine-GABA receptor-ionophore complex.     

Antisense Strategy Unravels a Dopamine Receptor Distinct from the D2 Subtype, Uncoupled with Adenylyl Cyclase, Inhibiting Prolactin Release from Rat Pituitary Cells

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [³H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.     

Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary

Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 +/- 11 fmol/mg protein and a KD value of 1.4 +/- 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 microM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and NT lost its capacity to release PRL in the absence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

不同移植部位对成年大鼠睾丸间质细胞雄激素分泌功能的影响

目的:探索不同移植部位对移植的成年SD大鼠睾丸中睾丸间质细胞存活及雄激素分泌功能的影响。方法:将健康成年雄性SD大鼠随机分为对照组、假手术组、皮下组和肾包膜组。对照组大鼠不去势,其余大鼠于睾丸移植前1周行去势手术。对照组和假手术组去势后仅行背部皮肤切开,不进行睾丸移植;皮下组背部两侧各移植1/3个成年SD大鼠睾丸组织;肾包膜组每侧肾包膜下移植1/3个成年SD大鼠睾丸组织。4周后取材行HE和免疫组化染色,分析移植睾丸组织中睾丸间质细胞存活情况,ELISA法检测受体大鼠血清睾酮水平。结果:皮下组和肾包膜组移植物中难于见到完整的睾丸间质组织,但免疫组化染色发现大量HSD-17β1阳性细胞,对照组、皮下组和肾包膜组的HSD-17β1阳性细胞数分别为(24.33±4.30)、(9.83±4.05)和(12.67±2.81)个,对照组与皮下组相比差异具有统计学意义(p0.05);ELISA分析发现对照组、假手术组、皮下组和肾包膜组的血清睾酮浓度分别为(3.81±1.32)、(0.28±0.08)、(0.44±0.13)和(0.90±0.31)ng/m L,肾包膜组血清睾酮浓度高于假手术组(p0.01)和皮下组(p0.05),而皮下组血清睾酮水平高于假手术组,但两者差异无统计学意义(p0.05)。结论:移植的成年大鼠睾丸组织中的睾丸间质细胞可在受体肾包膜下或皮下存活,但肾包膜下移植可能更加有利于睾丸间质细胞存活和雄激素分泌。     

An Inactivating Inward-rectifying K Current Present in Prolactin Cells from the Pituitary of Lactating Rats

Primary cultures containing a high percentage of lactotrophs were obtained by dissociating the pituitary of rats following 14–18 days of lactation. Lactotrophs with a distinctive appearance were recorded after 1–35 days in vitro and identified by immunocytochemical staining for prolactin. Whole-cell voltage clamp measurements in isotonic KCl solution from a holding potential of −40 mV revealed the presence of inward-rectifying K currents with a time-dependent, Na⁺-independent inactivation at potentials negative to −60 mV. The time for complete inactivation was strikingly different between lactotrophs, varying between 1 sec and more than 5 sec at −120 mV, and was not related to time in culture. The reversal potential shifted 59 mV (25°C) for a tenfold change in external K⁺ concentration, demonstrating the selectivity of the channel for K⁺ over Na⁺. The inward-rectifying K current was blocked by 5 mm Ba²⁺ and partially blocked by 10 mm TEA. Chloramine-T (1 and 2 mm) produced a total block of the inward-rectifying K current in lactotrophs. Thyrotropin-releasing hormone (500 nm) significantly reduced the inward-rectifying K current in about half of the lactotrophs. This current is similar to the inward-rectifying K current previously characterized in clonal somatomammotrophic pituitary cells (GH₃B₆). The variability of the rate of inactivation of this current in lactotrophs and its responsiveness to TRH is discussed. Received: 28 September 1995/Revised: 11 December 1995     

Polarized Secretion of Human Corticosteroid Binding Globulin by MDCK and BeWo Cells

Polarized epithelial cells are able to faithfully direct certain secretory protein components to either their apical or basolateral environments. The mechanism by which these cells accomplish this is still not entirely understood. It is hypothesized that a membrane-associated "sorting receptor" recognizes an intrinsic signal contained within the sorted protein. This interaction directs the secretory protein into the appropriate domain-specific vesicle for transport to either the apical or basolateral face. The nature of this sorting signal and the recognition receptor have not been established. In an effort to understand this phenomenon, a study was undertaken to ascertain whether human corticosteroid binding globulin (hCBG) contains intrinsic signals capable of directing its secretion to a particular side of polarized epithelial cells. The results of these studies have revealed that hCBG is selectively secreted into the apical environment by both MDCK and BeWo cells. Furthermore, this polarized secretion is unaffected by either (1) agents that inhibit N-linked oligosaccharide processing or (2) lysomotrophic drugs, which alter the intravesicular pH. It is concluded that hCBG possesses an intrinsic signal for apical secretion, which can be recognized by two polarized cell types of differing origins. This signal does not appear to be present in the N-linked oligosaccharide moieties of hCBG nor is it affected hy an elevation of the intravesicular pH within the trans-Golgi network. The use of hCBG-transfected MDCK and BeWo cells constitute a useful model system for the investigation of the signals involved in the sorting of secreted proteins.     

Brainstem 5-Hydroxytrytamine1A Binding Sites are not Down-Regulated by Agonists which Induce Tolerance in the Rat: Myoclonus and other Serotonergic Behaviors

Abstract

To study the regulation of 5-HT_1A receptors in the brainstem, the region most relevant to the serotonin syndrome and to serotonin-responsive human myoclonic disorders, we chronically treated rats with various 5-HT_1A agonists and labeled 5-HT_1A sites with [³H]8-OH-DPAT. Daily injection for 30 consecutive days of 10 mg/kg ip 8-OH-DPAT (pre- and post-synaptic 5-HT_1A agonist) significantly decreased 8-OH-DPAT-evoked flat body posture, forelimb myoclonus, and hypothermia compared to chronic vehicle injection. There was no cross tolerance to 8-OH-DPAT in rats chronically injected with ipsapirone or buspirone (presynaptic 5-HT_1A agonists). However, none of the 5HT_1A agonists significantly altered B_max of brainstem 5-HT_1A binding sites. Chronic injection with other drugs such as 1-propranolol, (±) pindolol and spiperone (5-HT_1A and 5-HT₂ antagonists), methysergide (5-HT₁ and 5-HT₂ antagonist), and agonists and antagonists at various other 5-HT receptors also had no effect on binding parameters. These data demonstrate lack of cross-tolerance between pre- and post-synaptically acting 5-HT_1A agonists and absence of down-regulation of presynaptic 5-HT_1A sites at doses which induced tolerance of 5-HT_1A-mediated behaviors of the serotonin syndrome. They suggest changes in the post-synaptic cell rather than the receptor recognition site as the mechanism of tolerance.     

Regulation of Prolactin and Growth Hormone Production by Clonal Strains of Functional Pituitary Cells in Culture

This report describes the use of clonal strains of rat pituitarytumor cells to study the regulation of prolactin and growthhormone production. Emphasis is placed on the effects on prolactinproduction of the hypothalamic tripeptide pGlu-His-ProNH₂, alsocalled thyrotropin releasing hormone (TRH). TRH binds to specificcellular receptors and stimulates initially the release of previouslysynthesized prolactin; several hours later it stimulates thesynthesis of prolactin. Several kinds of experimental resultsare consistent with the hypothesis that cyclic AMP mediatesthe TRH-stimuIated release of prolactin. However, all of theeffects of TRH on these cells in culture are not mimicked bycyclic AMP analogs, for TRH decreases growth hormone productionwhile it increases the synthesis of prolactin, and the analogdibutyryl cyclic AMP increases the production of both hormones.