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The mas-like G protein-coupled receptors form a subfamily of G protein-coupled receptors that includes variable member numbers across different species and that have been shown to bind a wide variety of ligands from peptides to amino acid derivatives. While screening a library of peptides against different orphan G protein-coupled receptors, we found that human salusin beta activates the mouse mas-like G protein-coupled receptor, mMrgA1 with an EC(50) of about 300 nM. Salusin beta is a bioactive peptide recently discovered through bioinformatics analysis which stimulates arginine-vasopressin release from rat pituitary and causes rapid and profound hypotension and bradycardia. However, when we further analyzed the generality of the mMrgA1 activation, we found that human salusin beta does not activate corresponding human mas-like G protein-coupled receptors. Our results show that human salusin beta is a surrogate ligand of the mouse MrgA1 and raises a cautionary flag for experiments that analyze the pharmacological profiles of mas-like G protein-coupled receptors from different species.

Estrogen Is known to affect the regulation of all six of the established anterior pituitary gland (AP) hormones, but little is known of the specifics of its regulation of the AP hormones, their isoforms, and nonhormonal AP proteins. We used difference gel electrophoresis in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Two-month-old rats were ovariectomized and used at 6 months of age. They were injected subcutaneously with sesame oil vehicle or 50 μg estradiol valerate in vehicle and studied 48 hrs later, approximately 3 hrs before the time of the anticipated onset of the estrogen-induced surges of gonadotropins in blood. The APs were pooled, and the soluble protein fraction was examined in replicate analyses. After DeCyder software analysis, we identified 26 protein spots that had a 1.5-fold or greater average increase in the experimental group relative to the controls. Nineteen showed a 1.5-fold or greater decrease. Estrogen increased levels of the more acidic isoforms of growth hormone and prolactin and of proteins involved in protein synthesis, folding, and secretion (e.g., eukaryotic translation elongation factor 2, ERp57, ERp29, Hsc70-ps1, calreticulin, coatomer delta subunit, and secretogranin II) and of some metabolic enzymes (e.g., arginosuccinate synthetase, enolase 1, creatine kinase B, phosphoglycerate mutase, malate dehydrogenase, pyruvate kinase, and aldolase A). The majority of the downregulated proteins were involved in RNA or DNA interactions (e.g., five heterogeneous nuclear ribonucleoproteins, DEAD-box proteins 17 and 48, ssDNA binding protein PUR-alpha, PTB-associated splicing factor, and Pigpen protein), but isovaleryl coenzyme A dehydrogenase, mitochondrial aldehyde dehydrogenase, stathmin 1, vinculin, radixin, and secretogranin III were also reduced. Our results indicate that estrogen acts in vivo within 48 hrs to modulate levels of a significant number of AP proteins.

Brevican is the most abundant chondroitin sulfate proteoglycan in the extracellular matrix (ECM) of the adult rat brain. It has been found only in the central nervous system (CNS). In this study, we found that secreted brevican transcript was detectable in the pituitary of both male and female adult rats by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification. In posterior lobe of pituitary, pituicytes were heavily labelled. In anterior and intermediate lobes of pituitary, signals for brevican transcripts were observed in cells of various sizes. These data demonstrated that secreted brevican mRNA is expressed in the adult rat pituitary and brevican might not be a CNS-specific ECM.

Kampo medicine (Chinese herbal medicine) has been reported to be effective in the treatment of pituitary-ovarian dysfunction in young women and in the treatment of undefined symptoms in perimenopausal women. It has been considered that both the diagnosis and treatment of ovulatory failure in adolescent girls or young women are necessary for the sake of future fertile capacity. However, treatment for the above patients is not easy as steroid hormones and strong stimulators of ovulation should not be recommended because of the several side-effects it causes. Furthermore, young women, especially teenagers, seem to be reluctant to visit gynecology clinics. A number of herbal medicines have been used for many centuries in China and Japan for the treatment of menstrual disorders and infertility. In general, the traditional Chinese herbal prescriptions are rather inexpensive and safe with little side-effects, and have properties for normalizing biological balances. Unkei-to is known to stimulate the synthesis and release of gonadotropins in the rat pituitary. In humans, Unkei-to is known to be effective in various menstrual disorders, abnormal uterine bleeding, and infertility. In the treatment of ovulatory failure, recent dynamic studies have revealed the mechanism by which Unkei-to stimulates the diencephalon-pituitary-ovarian axis in humans. In this paper, we critically review the clinical data available (to date) in order to assess the efficacy of Unkei-to in inducing ovulation in several types of ovulatory disturbances. (Reprod Med Biol 2003; 2: 45-61).

There has been considerable interest in the relationship between hormone-secreting endocrine cells (HSEC) and their microvessels (MVN) in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). Therefore, this study was designed to visualize and to reveal the relationship between hormone secreting endocrine cells and their microvessel environment including vascular endothelial cells in 3D using rat pituitary glands under various experimental conditions by confocal laser scanning microscopy (CLSM). By CLSM, the 3D distributions of MVN were visualized and revealed a relationship between HSEC and MVN in experimental pituitary glands and human pituitary adenomas. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.

Bioceramic materials are used for the reconstruction or replacement of the damaged parts of the human body. In this study an improved procedure is described for producing ceramic scaffolds with controlled porosity. Bioinert alumina ceramic was used to make porous scaffolds by using indirect fused deposition modeling (FDM), a commercially available rapid prototyping (RP) technique. Porous alumina samples were coated with hydroxyapatite (HAp) to increase the biocompatibility of the scaffolds. Initial biological responses of the porous alumina scaffolds were assessed in vitro using rat pituitary tumor cells (PR1). Both porous alumina and HAp coated alumina ceramics provided favorable sites for cell attachments in a physiological solution at 37 °C, which suggests that these materials would promote good bonding while used as bone implants in vivo. Based on these preliminary studies, similar tests were performed with human osteosarcoma cells. Cell proliferation studies show that both the ceramic materials can potentially provide a non-toxic surface for bone bonding when implanted in vivo.

The expression of mRNA for voltage-dependent (Kv) and inward-rectifying K channels (Kir) was studied in clonal rat somato-mammotroph cells (GH3/B6 cells) and rat pituitary using reverse transcription-polymerase chain reaction (RT-PCR). In GH3/B6 cells transcripts for 16 different Kv channel alpha-subunits (seven Shaker-related: Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv3.2, Kv4.1, Kv5.1; six EAG: eag1, erg1, erg2, elk1-elk3; three KCNQ: KCNQ1-KCNQ3) and for five different Kir channel alpha-subunits (Kir1.1, Kir2.3, Kir3.2, Kir3.3, Kir6.2) were found. In addition, transcripts for a short isoform of Kvbeta2 and transcripts for Kvbeta3 subunits were present. In rat pituitary transcripts for 21 different Kv channel alpha-subunits (11 Shaker-related: Kv1.3, Kv1.4, Kv1.6, Kv2.1, Kv2.2, Kv3.2, Kv3.4, Kv4.1, Kv4.2, Kv4.3, Kv6.1; seven EAG: eag1, erg1-erg3, elk1-elk3; three KCNQ: KCNQ1-KCNQ3) and nine Kir channel alpha-subunits (Kir1.1, Kir2.2, Kir3.1-Kir3.4, Kir4.1, Kir6.1, Kir6. 2) were found. In addition, all tested auxiliary subunits (Kvbeta1-Kvbeta3, minK, SUR1, SUR2) are expressed in the pituitary. The results indicate that the macroscopic K currents in GH3/B6 and pituitary cells are presumably mediated by K channels constructed by a larger number of K channel alpha-subunits and auxiliary beta-subunits than previously distinguished electrophysiologically and pharmacologically.

In the joint experimental and computational efforts reported here to obtain novel chemical entities as growth hormone secretagogues (GHSs), a small database of peptides and non-peptides known to have GHS activity was used to generate and assess a 3D pharmacophore for this activity. This pharmacophore was obtained using a systematic and efficient procedure, "DistComp", developed in our laboratory. The 3D pharmacophore identified was then used to search 3D databases to explore chemical structures that could be novel GHSs. A number of these were chosen for synthesis and assessment of their ability to release growth hormone (GH) from rat pituitary cells. Among the compounds tested, those with a benzothiazepin scaffold were discovered with micromolar activity. To facilitate lead optimization, a second program, a site-dependent fragment QSAR procedure was developed. This program calculates a library of chemical and physical properties of "fragments" or chemical components in a known pharmacophore and determines which, if any, of these properties are important for the observed activity. The combined use of the 3D pharmacophore and the results of the site-dependent fragment QSAR analysis led to the discovery and synthesis of a novel series of potent GHSs, a number of which had nanomolar in vitro activity.

The effects of arachidonic acid (5,8,11,14-eicosatetraenoic acid, AA) and 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, were examined on the transient [IK(f)] and the delayed rectifier-like [IK(s)] voltage-gated potassium currents in rat pituitary melanotrophs. The main questions addressed were whether AA and ETYA blocked IK(f) and if any blocking action was specific. Macroscopic currents were measured using the patch clamp technique. Bath application of 20 µM AA reduced IK(f), however, the degree of the block varied between cells. In contrast, ETYA consistently inhibited IK(f). Fitting of the charge transfer or the peak current amplitude yielded KD estimates for ETYA of 1.2 µM and 3.3 µM, respectively. The reduction by ETYA of peak IK(f) was always associated with an increased rate of current decay, but there was no detectable change of the kinetics of activation. ETYA caused a small left shift of the IK(f) steady-state inactivation curve and significantly slowed recovery from inactivation. At 20 µM, ETYA also reduced IK(s), indicating that it is not specific. The possibility that ETYA acts as an open-channel blocker is discussed.Key words: transient potassium current, melanotroph, eicosatetraynoic acid, ETYA, arachidonic acid.

We studied the proliferation of pituitary corticotrophs quantitatively by labeling the proliferating cells with bromodeoxyuridine (BrdU) and carrying out immunocytochemistry for ACTH in rat fetuses at 19.5 days of gestation. In addition to labeling proliferating cells with a single injection of BrdU, we used double BrdU administrations at 9:00 and 19:00 for a more sensitive detection of proliferating cells. With this double administration, the number of cells labeled with either BrdU or both BrdU and ACTH increased by 1.75 and 2.3 times, respectively, compared with the single BrdU injection. The labeled cells further increased when the sections were stained for the proliferating cell nuclear antigen (PCNA) instead of BrdU. The number of cells labeled with PCNA or both PCNA and ACTH was 1.37 and 1.68 times that of the cells labeled with either BrdU or both BrdU and ACTH, respectively. The ratio of BrdU/ACTH-labeled cells or PCNA/ACTH-labeled cells to all corticotrophs was 13.6% and 24.3%, respectively, much higher than the ratios in fetuses having a single BrdU injection (6.6%). These results indicate that the mitosis of existing corticotrophs contributes greatly to their increase during the late fetal period.

Objective: The study investigates the effects of intravenously administered GnRH on plasma β-endorphin levels in female proestrous rats. Study design: Sixteen adult female proestrous Wistar rats, 220–250 g, were implanted with two indwelling catheters, one intracarotid and one intrajugular. Ten ng GnRH/100 μl distilled water or 100 μl saline (control) were infused in eight animals per group every 20 min from 12:30 to 14:30 h. Blood was withdrawn through the intracarotid catheter just before the initial infusion (12:30 h) and at 14:00, 15:30, 16:30 and 17:30 h for the determination of plasma β-endorphin levels. The Mann–Whitney test was used for comparison between GnRH-treated and control rats and the Wilcoxon test within each treatment group. Results: β-Endorphin levels of GnRH-treated rats were not significantly different at any sampling moment neither compared to preinfusion time nor to the corresponding controls. Conclusions: Intravenously administered GnRH was not sufficient for any possible effect on the secretion of β-endorphin from rat pituitary and further investigation is needed to demonstrate if a different experimental model would have any significant effect.

In the rat pituitary, activin stimulates whereas inhibin prevents FSH synthesis and secretion. Besides, the activin binding protein follistatin neutralizes the action of activin. The control of the FSH secondary surge at early estrus is not completely understood. To investigate the regulation of the inhibin/activin alpha-, betaA- and betaB-subunits and follistatin mRNA expression in the pituitary during the time of the FSH secondary surge, cyclic rats treated with LHRH antagonist (ANT) and ovine LH (oLH), progesterone (P), the anti-steroid RU486, adrenalectomy (ADX) or ADX plus corticosterone (B), were killed at early estrus. The serum concentrations of FSH were measured and the mRNA levels of the above mentioned transcripts were analysed and quantitated by using RNase protection assays. ANT abolished the FSH secondary surge and increased mRNA for alpha- and betaA-subunits and follistatin, but reduced that for betaB-subunit. Both oLH and P reversed these effects. RU486 blocked the effect of oLH on FSH levels and prevented the reduction in the mRNA for follistatin. ADX in ANT+oLH-treated rats reduced the serum FSH concentrations, enhanced mRNA for betaA- and betaB-subunits and, similar to RU486, blocked the drop in follistatin mRNA. Finally, replacement of B in ADX animals reversed these effects. These results demonstrate that, in the cyclic rat, the preovulatory secretion of LH and the surges of P and B on proestrus regulate the synthesis of inhibin/activin subunits and follistatin mRNA in the rat pituitary at early estrus, probably by reducing inhibin and follistatin and increasing activin. Moreover, these effects of LH, P and B at the pituitary level, together with the decrease in the amount of inhibin coming from the ovary, might be responsible for the occurrence of the FSH secondary surge.

SUMMARY There has been considerable interest in the relationship between hormonesecreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways. (J Histochem Cytochem 48:569‐577, 2000)

Sapecin B is an antibacterial protein isolated and purified from culture medium of the embryonic cell line derived from the flesh fly (Sarcophaga peregrina). It has structural simil

We have investigated the effect of melatonin on cell proliferation and modulation, in the GH3 experimental rat pituitary cell line; the expression of oncogenes c-myc, c-jun and the tumor suppressor gene p53 were also analyzed basally and after exposure to melatonin (10–6, 10–8 and 10–10 M). Melatonin exhibited an antiproliferative effect at all the doses tested, decreasing the proliferating index by 50%. After exposure to melatonin, a decrease in Ki67 and Proliferation cell nuclear antigen occurred acute- and transiently (at 2 h) after a single dose which recovered at 4 h, as well as chronically after repeated 12-hour doses which persisted at 48 h; a similar behavior was observed both acute- and chronically for c-myc and c-jun, while it was opposite for p53, rising acute- and transiently as well as after repeated exposure. These results demonstrate that melatonin modulates the proliferation mechanisms of the GH3 cells.

Both dehydration and adrenalectomy of rats caused a significant reduction of immunoreactive beta-endorphin in the pituitary gland. Beta-Endorphin in hypothalamus, however, was not altered in either dehydrated or adrenalectomized rats.

Systematic investigation of acyclic analogs of L-692,429, the prototype benzolactam growth hormone secretagogue, has helped to further define the structural requirements for the release of growth hormone from rat pituitary cells for this class of secretagogues.

Pituitary adenylate cyclase-activating polypetide (PACAP) exists in two amidated forms, PACAP38 and PACAP27, which are expressed in the magnocellular and parvocellular neurons of the paraventricular nucleus (PVN) and the magnocellular neurons of the supraoptic nucleus (SON) of the hypothalamus. The prohormone convertases PC1 and PC2, subtilisin-like PCs of the Kex2 family, are expressed in neuroendocrine cells. Immunocytochemistry and in situ hybridization of PC1 and PC2 in the hypothalamus have shown that PC1 and PC2 are also present in the PVN and SON. Therefore, it is possible that the precursor of PACAP is processed by PC1 and/or PC2 in the hypothalamic nuclei and then converted to its mature forms. To test this hypothesis, rat pituitary GH4C1 cells were supertransfected with human PACAP cDNA and either rat PC1 or PC2 cDNA. The acid extracts of these cells were analyzed by reversed-phase HPLC for proPACAP, PACAP38 and/or PACAP27 radioimmunoassays using three antibodies with different recognition sites, and then bioassayed for the ability to stimulate adenylate cyclase. The cells transfected with PACAP cDNA alone yielded PACAP-like immunoreactivity (PACAP-li) corresponding to molecular weights between 15 and 20 kDa without PACAP bioactivity. Cotransfection of these cells with PC1 or PC2 generated PACAP-li, which coeluted with synthetic PACAP38 and PACAP27, respectively. Western blot also revealed 4.5- and 3.0-kDa PACAP-li bands, which correspond to the molecular weights of PACAP38 and PACAP27, respectively. The HPLC fractions containing PACAP-li, which were coeluted with synthetic PACAP38 and PACAP27, showed marked bioactivities. These findings suggest that the precursor of PACAP expressed in the PVN and SON of the hypothalamus could be efficiently processed by PC1 and PC2, and then converted to mature, bioactive PACAP38 and PACAP27.

Summary A series of thioureas containing two aromatic groups and one amino group was synthesized as potential growth hormone secretagogues. The growth hormone (GH) releasing properties of these compounds ( 10–24 ) were evaluated using primary rat pituitary cells. The most potent compound 20 showed potency with an EC 50 of 1.8 μTwo constrained analogs 23 and 24 showed similar activity.

La presente etude a eu pour but d'examiner le taux de glycosylation basale de la hormone luteinisante (LH), en faisant la distinction entre la glycosylation post-translationnelle (distale) et la glycosylation co-translationnelle (proximale). Les experiences decrivent la relation temporelle entre la biosynthese des chaines peptidiques (par l'incorporation de [ 14 C]leucine: [ 14 C]Leu-LH) et la glycosylation distale (par l'incorporation de [ 3 H]galactose: [ 3 H]Gal-H) et proximale (par l'incorporation de [ 3 H]mannose: [ 3 H]Man-LH) de la LH par les cellules hypophysaires de rat en culture primaire. Les effets du cycloheximide (inhibiteur de translation) et de la tunicamycine (inhibiteur de glycosylation) sur les taux de synthese et de liberation de [ 3 H]Man-LH et de [ 3 H]Gal-LH ont aussi ete examines. Les taux de synthese et de liberation (entre 0 et 2 h, et entre 2 et 5 h) ont ete calcules par une analyse de regression, et la signification statistique des differences a ete determinee par le test de pente. Le taux de synthese de [ 3 H]Man-LH (pente = 45,59) a ete semblable a celui de [ 14 C]Leu-LH (pente = 41,39), ce qui etait en accord avec l'hypothese que l'addition du noyau riche en mannose est un processus de co-translation. La liberation de [ 3 H]Man-LH (pente = 4,32) et celle de [ 14 C]Leu-LH (pente = 2,53) ont montre une periode de latence d'approximativement 2 h. La dynamique de la secretion de [ 3 H]Gal-LH pendant la periode d'incubation, illustree par un taux de synthese plus faible (pente = 26,40) et un taux de liberation plus rapide (pente = 6,34), a differe de celle de [ 3 H]Man-LH. La LH marquee au [ 3 H]Gal a ete liberee au debut de l'incubation, ce qui montre que le galactose est incorpore a la fin du processus de secretion aux molecules LH qui sont immediatement liberees. Le blocage de la translation de LH par le cycloheximide a ete associe a une diminution correspondante de l'incorporation de [ 3 H]Man. En revanche, l'addition de tunicamycine aux cellules hypophysaires a inhibe les taux de synthese et de liberation de [ 3 H]Man-LH, sans affecter ceux de [ 14 C]Leu-LH. Ces resultats montrent que la glycosylation proximale depend de la synthese des chaines peptidiques, alors que la translation ne depend pas de l'addition du noyau poly-mannose. Les taux de synthese et de liberation de [ 3 H]Gal-LH ont ete moins influences par les antibiotiques, et l'inhibition n'a ete significative qu'aux doses plus elevees et lors de traitements plus longs. Les presents resultats demontrent l'independance des deux etapes de glycosylation de la LH, taux de synthese de [ 3 H]Gal-LH etant d'un facteur 1,7 fois plus lent et celui de la liberation d'un facteur 1,5 plus rapide que ceux de [ 3 H]Man-LH, respectivement. Les resultats suggerent aussi que la glycosylation n'est pas une etape essentielle dans le processus de secretion de la LH, etant donne que l'hormone, qui est normalement secretee sous une forme glycosylee, a ete synthetisee, transportee et liberee sans les chaines laterales hydrocarbonees.

We examined the effects of nitric oxide (NO) modulators on rat pituitary LH content in vivo and studied their response to LHRH-stimulated LH secretion in vitro in ovariectomized adult female Sprague Dawley rats. Alzet mini pumps (flow rate 10 microl/h) delivering either normal saline (Group I, 1.2 mg nitroglycerin, a donor of NO (Group II) or 50 mg of nitro-L-Arginine methyl ester, a NO synthase (NOS) inhibitor (Group III), were subcutaneously implanted into experimental animals. Following 36 h infusion, pituitaries were removed and either frozen for LH quantitation, or fragmented and challenged in the superfusion system with 10 min pulses of LHRH (1 ng/ml) at 90 min intervals for 10 hours. LH was assayed by radio-immunoassay (RIA) in the homogenates of pituitaries and in aliquots of the superfusate collected every 10 mins. Significantly lower pituitary LH levels were noted in Group III (150.3 +/- 18.6 ng) in comparison to Groups I (215.6 +/- 5.5 ng; p0.04) or II (221.2 +/- 14.9 ng; p0.01), suggesting that low levels of NO stimulate LH secretion in vivo. The pituitary LH contents were not significantly different in Groups I and II. In vitro studies reveal that exogenous LHRH stimulated response, measured as average pulse response (90 minute period after LHRH), and total LH released during the 10 hour perfusion, was 290 +/- 23.6 ng and 1646.7 +/- 270.8 ng, respectively, in Group III; 57.9 +/- 3.1, and 344.7 +/- 24.3 ng in Group I, and 105.3 +/- 6.3, and 633.7 +/- 77.1 mg in Group II. Thus, our in vitro studies demonstrate significantly enhanced (p0.05) LHRH- stimulated LH secretion in Group III in comparison to Groups I and II, while Group II shows higher responsiveness than Group I (p0.05). The results of the current studies provide evidence that NOS inhibition facilitates pituitary LH secretion. The differential responses to LHRH-stimulated LH secretion in vitro in the 3 groups suggest a possible role of NO in modulating pituitary LHRH receptor concentrations. However, this will have to be tested by further studies.

Whole cell patch-clamp techniques were used on clonal pituitary cells (GH3) and primary cultures of somatotrophs and lactotrophs to study currents that would be active at or below voltages for the threshold for action potential generation. When GH3 cells were held at -60 mV and pulsed to -120 mV, a slow-activating sustained inward current was observed (-16.5 +/- 1.5 pA in physiological baths, n = 72; approximately 1 s to half-maximal activation, voltage for 50% activation - 101 mV). The current was insensitive to bath application of 10 mM tetraethylammonium, 10 mM 4-aminopyridine, and 1 mM barium but was completely blocked by 3 mM cesium. The current was found to be a mixed cation current with a sodium permeability of 0.29 relative to potassium. These properties indicate that the current belongs to the hyperpolarization-activated cation current (Ih), or I(f), family of currents. However, the current was not altered by the addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the pipette or forskolin to the bath. A similar but smaller current was observed in 15 of 16 somatotrophs but in only 1 of 9 lactotrophs. Application of cesium to spontaneously spiking GH3 cells or somatotrophs had no effect. However, cesium did block an inward holding current observed at -80 mV. These results demonstrate that the I(h) in pituitary cells does not serve as a pacemaking current but suggest that it may influence membrane potential responses when somatotrophs become hyperpolarized.

The present study was designed to characterize bihormonal cells in rat pituitary cells which secrete PRL and gonadotropins. This was done by using sandwich cell immunoblot assay (CIBA) and reverse hemolytic plaque assay (RHPA) in combination with immunocytochemistry (ICC) and by measuring the intracellular free calcium concentration ([Ca2+]i). The result of the experiment with sandwich CIBA revealed that the populations of LH- and FSH-secreting cells in the PRL-secreting cells were 6.23% and 5.91%, respectively, and the populations of the PRL secreting cells in the LH- and FSH-secreting cells were 18.4% and 15.5%, respectively. Additional studies by the combined techniques of RHPA with ICC revealed that the populations of LH- and FSH-containing cells in the PRL secreting cells were 4.43% and 2.40%, respectively, which were consistent with the results of Sandwich CIBA. Some of the PRL-secreting cells determined by RHPA showed responsiveness to TRH and GnRH in [Ca2+]i. These results suggest that bihormonal cells which secrete both PRL and LH, or both PRL and FSH are present in the normal rat pituitaries.

Endothelins (ETs) and their receptor subtypes A and B (ETA and ETB) are expressed in the various components of the mammalian hypothalamo-pituitary-adrenal (HPA) axis, but their involvement in the functional regulation of HPA is controversial. To gain insight into this topic, we have investigated the effects of ET-1 and/or the specific antagonists of ETA and ETB receptors (BQ-123 and BQ-788, respectively) on the plasma concentrations of ACTH, corticosterone and aldosterone of non-stressed (control) and ether- or cold-stressed rats. The study of the effects of the administration of the two ET-receptor antagonists alone could provide informations about the possible action of endogenous ETs on the HPA axis. Exogenous ET-1 increased ACTH, corticosterone and aldosterone blood levels in control rats, as well as evoked a sizable enhancement of the HPA axis response to ether stress and a marked depression of the response to cold stress. BQ-123 and BQ-788 did not prevent the stimulatory effect of exogenous ET-1 in control rats, but when administered alone, raised the plasma concentrations of ACTH, corticosterone and aldosterone. Both ET-receptor antagonists magnified the HPA axis response to ether and cold stresses, but their effect was not counteracted by exogenous ET-1. Although very difficult to interpret, our present findings allow us to conclude that endogenous ETs play a role in the maintenance of the basal activity of rat HPA axis acting through ETA and ETB receptor subtypes, which are partially insensitive to BQ-123 and BQ-788. Conversely, the involvement of ETs in the modulation of the HPA axis responses to various stresses is very doubtful.

Due to a typesetting error, there was an error in the name of the sixth author. The correct author name is: Ettore C. degli Uberti

Numerous studies have established a link between estrogen levels and activity of the hypothalamic pituitary adrenal (HPA) system. Considering the ``weak estrogenic{''} effect of soy isoflavones, this study was designed to evaluate the influence of soy extract treatment on some morphofunctional parameters of rat pituitary corticotropes in vivo. Adult male orchidectomized Wistar rats received estradiol-dipropionate or soy extract in oil/ethanol solvent subcutaneously for 3 weeks. Both controls, sham-operated (So) and orchidectomized (Orx) rats, were injected with solvent, in the same regime. Changes in pituitary volume, total volume, total number and volume of individual corticotropes were evaluated stereologically, while ACTH levels were determined biochemically. In comparison with So rats, estradiol treatment provoked increases (p < 0.05) of ACTH level (166\%), pituitary weight (167\%) and volume (102\%), total volume (20\%) and total number of corticotropes (18\%). In comparison to Orx, following estradiol treatment elevation (p < 0.05) of ADE level (69\%), pituitary weight (131\%) and volume (82\%), total (30\%) and individual volume (29\%) of corticotropes was observed. Soy extract treatment led to enhancement (p < 0.05) of: ACTH level (28\%), total (25\%) and individual volume (20\%) of corticotropes. It can be concluded that soy extract acts in a similar way to estradiol, but the increased activity of corticotropes was weaker. (C) 2013 Elsevier GmbH. All rights reserved. Ministry of Education and Science of Serbia {[}173009]

A series of spiroindane growth hormone secretagogues that vary in their amino side chains is reported. Variations in these side chains markedly affect growth hormone release in vitro and in vivo . The best side chain in this series of secretagogues is α-methylalanine. L-162,752 not only stimulated GH release from rat pituitary cells, but also induced GH release in dogs upon i.v. and p.o. administration.

A series of analogs of the non-peptidic benzolactam growth hormone secretagogue L-692,429 was prepared with heteroatom substitutions at the 5-position of the benzolactam ring. Replacement of the 5-position with sulfur or oxygen resulted in secretagogues of approximate equal potency to L-692,429 in the rat pituitary cell assay. Substitution of the 5-position with a carbonyl group or hydroxyl group and replacement of the benzolactam with the 1,4-benzodiazapin-2-one ring system gave analogs of greatly diminished in vitro activity.

To further investigate how sex steroids regulate galanin (GAL) in the rat pituitary and hypothalamus, we examined the effects of prepubertal gonadectomy (Gx) and long-term (9 weeks) replacement with estradiol (E2) or testosterone (T) on pituitary and hypothalamic GAL concentrations in Wistar rats (5-6/group). Sham-operated animals served as controls (CTR). Pituitary GAL concentration was markedly higher in random-cycling CTR-females than in CTR-males (1391 +/- 247 vs 39 +/- 5 pg/mg protein, P < 0.01) and decreased after Gx only in females (20 +/- 3 pg/mg protein, P < 0.01). E2 strongly increased pituitary GAL in Gx-females and Gx-males (4470 +/- 365 and 3853 +/- 347 pg/mg protein, P < 0.01), whereas T had no effect. Inversely, hypothalamic GAL was higher in CTR males than in CTR females (5.4 +/- 0.3 vs 4.0 +/- 0.5 ng/mg protein, P < 0.05), and decreased significantly after gonadectomy in males (3.7 +/- 0.2 ng/mg protein, P < 0.01). The only steroid treatment that significantly modified hypothalamic GAL in Gx animals was administration of E2 to females (5.7 +/- 0.4 ng/mg protein, P < 0.01 vs non-treated Gx). We also studied in hypophysectomized (Hx) rats (8/group) the effects of sex steroids on hypothalamic GAL concentration and distribution. The low hypothalamic GAL concentration observed in male and female Hx rats (1.0 +/- 0.1 ng/mg protein) was significantly increased by T in males and in females (respectively, by 40% and by 50%, P < 0.02) and by E2 in males (by 60%, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Long term incubation of pituitary tumor GH3 cultures with epidermal growth factor (EGF) induces reciprocal changes in PRL and GH production. However, it is not known whether EGF alters the cellular composition of these cultures. Another unanswered question is whether chronic treatment with EGF stimulates PRL secretion from nonneoplastic pituitary cells. In this study, GH3 cells and pituitary cells from neonatal (10-day-old) rats were cultured for 6 and 2 days, respectively, in the absence or presence of 5 nM EGF. Cells containing PRL and/or GH were then enumerated using light microscopic immunocytochemistry. In addition, neonatal pituitary cells were subjected to reverse hemolytic plaque assays for PRL. EGF treatment drastically increased the proportion of classical lactotropes (cells that secrete only PRL) in the GH3 cultures, from about 0.5% to 8% of all cells, without modifying the percentage of GH-positive cells. A similar action of EGF was observed in the primary cultures. Moreover, EGF enhanced by 240% the amount of PRL secreted from the neonatal lactotrope population during 1-h incubations under basal conditions. This effect was mediated by a selective increase in the relative number of PRL secretors forming large plaques. The results suggest that EGF promotes the differentiation of classical lactotropes in both GH3 cultures and pituitary cultures from neonatal rats, and that these cells are characterized by a high basal rate of PRL secretion.

We have examined statistically the actions of thyrotropin-releasing hormone (TRH) and Bay K 8644, an L-type Ca(2+)-channel agonist, on the frequency and shape of cytosolic Ca2+ spikes in individual GH4C1 rat pituitary cells. TRH induced a brief (0-40 s) suppression of Ca2+ spikes followed by a period (40-200 s) of increased spike frequency. TRH treatment reduced the rate of rise and amplitude of Ca2+ spikes, and increased the rate of fall, relative to spontaneous spikes before treatment. TRH had no significant effect on the correlation between spike amplitude and the spike decay time constant tau, suggesting that the increased rate of fall was due to enhanced Ca2+ extrusion and not to decreased Ca(2+)-induced Ca2+ release. Bay K rapidly (t1/2 = 9-13 s) induced a 2-fold increase in the rate of rise of spikes with no change in the total rise time, leading to an increase in spike amplitude. It increased by 2-fold the fall time of spikes, as predicted solely by the previously observed relationship between spike amplitude and fall time. Bay K therefore appeared to increase the number of Ca2+ channels participating in each spike event without altering the kinetics of channel activation or deactivation, and without influencing Ca2+ extrusion. After addition of Bay K, the interval between spikes gradually (t1/2 approximately 100 s) increased, whereas the rate of rise remained constant and maximal. To explain these actions of TRH and Bay K, we postulate that a fraction of L-type Ca2+ channels are inactivated during each spike and must be re-activated in order to participate in a subsequent spike. We conclude further that the changes in spike frequency and profiles induced by these secretagogues are most consistent with a model in which TRH induces increases in both Ca2+ influx and efflux while Bay K induces a large increase in Ca2+ influx but has little effect on efflux.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel 38-residue neuropeptide which stimulates adenylate cyclase activity in rat pituitary cells as well as in other neuronal and non-neuronal tissues. In this study we have investigated whether PACAP27 and PACAP38 may stimulate either cyclic AMP accumulation or phosphoinositide formation in cultured cerebellar granule cells. In cultures at 8 days of maturation in vitro (DIV), a 15-min exposure to PACAP27 or PACAP38 equally promoted a concentration-dependent increase in intracellular cAMP content: the effect was significant at 1-5 nM and maximal between 10 and 100 nM, while VIP was 1,000-fold less potent in elevating cAMP levels. In the presence of 3-isobutyl-1-methylxanthine (200 microM), stimulation by PACAP was present already at 0.1 nM and was maximal (6-fold increase) at 1 nM. A rapid elevation in intracellular cAMP (about 80%) was observed within a 30-second exposure to 10 microM PACAP38 or PACAP27; the maximal activity of PACAP was present between 15 and 30 min and progressively declined at 60 min without reaching basal values. PACAP27 and PACAP38, but not VIP, were also able to stimulate inositol phospholipid hydrolysis: PACAP38 (EC50: 0.16 nM) was 10-fold more potent than PACAP27 (EC50: 2.1 nM) in stimulating [3H]inositol phosphate formation. The effect of PACAP was rapid: fractionation of [3H]inositol phosphates revealed that inositol trisphosphate and inositol bisphosphate increased earlier (within 20 s) than inositol monophosphate (within 60 s).(ABSTRACT TRUNCATED AT 250 WORDS)

Activin A-, AB- and B-responding cells were characterized by their responsiveness in cytosolic free calcium ([Ca 2+ ] i ) to four hypothalamic releasing hormones, CRH, GHRH, TRH and GnRH. First, rat pituitary cells responding to activin A, AB and B in [Ca 2+ ] i were determined in a mixed population of pituitary cells. The populations of the activin A-, AB-, and B-responding cells were 13.9%, 9.3% and 13.2%, respectively. Overlapping of response among each population of activin- responding cells were present in some of the cells. The cells responding of activin A, AB, and B were then characterized by their responses to CRH, GHRH, TRH and GnRH. Most of the cells responding to activin A, AB, and B also responded to GHRH or TRH. These results reveal that there are distinct differences among each population of activin A-, AB- and B-responding cells and that there is still functional overlapping of responsiveness among these populations. The characterization of activin-responding cells suggests involvement of somatotropes and lactotropes in activin-induced biological events in the pituitary.

The activity of GnSI/AF was measured in pig follicular fluid (pFF) from 58 individual follicles of various sizes, by bioassay using rat pituitary cells, to investigate the relationship between gonadotrophin surge inhibiting/attenuating factor (GnSI/AF) activity and follicular development. In addition, the correlation between GnSI/AF and inhibin activities and the content of sex steroids (oestradiol, progesterone and testosterone) of follicles was examined. The activity of GnSI/AF in pFF varied significantly (0.155-1.69 U microliter-1) with size of the follicle. The activities (mean +/- SEM) were intermediate and constant in follicles with diameters from 3 to 5 mm (0.583 +/- 0.080 U microliter-1, n = 24), were higher and reached the highest value in follicles with diameters between 6 and 8 mm (0.863 +/- 0.068 U microliter-1, n = 21), and were lower, reaching the lowest value in follicles with diameters of 9 and 10 mm (0.401 +/- 0.089 U microliter-1, n = 13). In contrast, inhibin activity was almost constant during the development of follicles, although individual values varied from 0.9 to 2.5 U microliters-1. For follicles with diameters of 4-8 mm, inhibin activity was 1.754 +/- 0.042 U microliters-1 (n = 39); activity was higher in the smallest follicles with diameters of 3 mm (2.063 +/- 0.015 U microliters-1, n = 6) and was lower in follicles with diameter of 9 mm, reaching the lowest value in follicles with diameter of 10 mm (1.176 +/- 0.068 U microliters-1, n = 7); inhibin activity was not significantly correlated with GnSI/AF activity.(ABSTRACT TRUNCATED AT 250 WORDS)

We determined the most suitable conditions for measuring the somatostatin (SRIF) level by brain microdialysis and investigated its release from the hypothalamus. The relative recovery rate of SRIF was 8.4 ± 0.5%(mean ± SE) using a polycarbonate (PC) membrane with the push-pull method at a flow rate of 2 μl/min. Using tubes with an internal diameter of 0.28 mm and lengths of 5, 25, 50 and 100 cm, the relative recovery rates using a PC membrane with the push method were 8.2 ± 0.5%, 7.3 ± 0.6%, 6.2 ± 0.5% and 4.1 ± 0.6%, respectively. When using tubes with an internal diameter of 0.1 mm and lengths of 5, 25, 50 and 100 cm, the relative recovery rates were 7.3 ± 0.7%, 5.6 ± 1.0%, 3.5 ± 1.1% and 1.4 ± 0.7%, respectively. The relative recovery rate was 5.2 ± 0.5% with a polysulfone (PS-F, Fresenius) membrane, 4.5 ± 0.4% with a PS-H (Hospal) membrane, 2.6 ± 0.2% with an ethylenevinyl alcohol membrane (EVAL), 5.1 ± 0.8% with a polyvinyl alcohol (PVA) membrane and 10.4 ± 0.8% with a PS-K (Kaneka) membrane. With the push method, the extracellular SRIF level in rat pituitary was 42.8 ± 1.8 pg/ml with a PC membrane, 23.1 ± 2.9 pg/ml with an EVAL membrane at a flow rate of 2 μl/min. With the push-pull method, it was 52.7 ± 5.2 pg/ml using a PC membrane, 33.5 ± 2.8 pg/ml using a PVA membrane and 54.4 ± 3.2 pg/ml using a PS-K membrane. Intraperitoneal injection of urethane significantly increased SRIF from a basal level of 36.8 ± 10.6 pg/ml to 66.6 ± 12.5 pg/ml (P

The hypothesis that testosterone and inhibin interact in the control of FSH secretion in rams was tested. Adult rams were castrated and were simultaneously given testosterone implants and 3-times daily sc injections of 0, 0.4, 0.8 or 1.6 ml charcoal-treated bovine follicular fluid (bFF). After 1 wk, the implants were removed, and the bFF injections continued as before. Blood samples were taken daily for mean LH, FSH and testosterone concentrations, and every 10 min for 12 h in the presence and in the absence of testosterone for assessment of pulsatile LH release. The bFF specifically inhibited FSH secretion from rat pituitary cells in culture. In the presence of testosterone, there were no main effects of bFF on mean plasma FSH or LH concentrations, nor were these values different from their pre-treatment means (P>0.05). Treatment with bFF did not affect LH pulse frequency or amplitude, but the number of rams showing LH pulses was reduced in the 0.8 and 1.6-ml dose groups (P

The effect of a s.c. bolus injection of 2 micrograms galanin on the hypothalamo-pituitary-adrenocortical (HPA) axis were investigated in both normal and ether-stressed (2 min ether-vapor inhalation) or cold-stressed (20 min at 4 degrees C) rats. Blood concentrations of ACTH, aldosterone (ALDO) and corticosterone (B) were measured by specific RIA, 1, 2 or 4 h after galanin injection. Galanin administration to normal rats resulted in a marked rise in the blood levels of ACTH, ALDO and B at 1h and 2 h, the values returned to the baseline after 4 h. Ether and cold stresses notably raised the blood levels of ACTH, ALDO and B, and these rises lasted unchanged until 4 h. Galanin markedly potentiated ACTH and ALDO responses to ether stress at 1 and 2 h, but B response remained unchanged. ACTH response to cold stress was not affected by galanin; however, galanin magnified ALDO response to cold stress at 4 h, and enhanced at 1 h and depressed at 2 h that of B. In light of these findings the following conclusions can be drawn: (i) galanin exerts a stimulatory effect on HPA axis of rats under basal conditions; (ii) under our experimental conditions, ether stress exerts a stronger stimulation of HPA axis than cold stress; (iii) the galaninergic mechanisms involved in the stimulation of ACTH release do not interfere with ether stress-activated ones controlling ACTH secretion, and are probably similar to those underlying the effect of cold stress; (iv) steroidogenic capacity of adrenal cortex, at least in term of glucocorticoid hormones, is a rate-limiting step in the response of rat HPA axis to severe stresses; and (v) galanin exerts a direct secretory action of the rat adrenal gland, that can manifest itself only in the case of submaximally cold stress-stimulated HPA axis.