Pituitary Cell Cultures Research Articles

English

This study examined the potential of catfish pituitary cells to proliferate in culture media and that of hormones produced in their primary culture to induce spawning in African catfish. The trypsinized pituitary cells were cultured in three culture media RPMI 1640, McCoy 5a and M2 media with addition of 10% fetal bovine serum. Female catfish (800±200 g) were induced using the culture medium that gave the shortest population doubling time and highest cell count (RPMI 1640). The eggs were fertilized with sperm cells in vitro. The fertilization and hatchability rates were determined. The blood samples of the induced spawners were collected at O (before injection), 3, 6, 9 and 12 h, respectively after injection. The collected samples of blood plasma were analyzed using enzyme-linked immunosorbent assay for quantitative determinations of Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH). A significant increase in the cell counts over the initial seeding density of 4.1 × 106/ml after 12 h of culture was established in each of the three culture media used. The induced spawning bio-assay which served as a biochemical marker for gonadotrophin specific function showed that 97.5 g/kg of mature oocytes were spawned from cultured pituitary cells (CPC), 127.5 g/kg from induction with fresh pituitary gland (FPG) and 157 g/Kg from gonadotrophin releasing hormone analogue (Ovaprim) induced catfishes (800±200 g). The fertilization rate of cultured pituitary cells (CPC) differed significantly (P 0.05). The results of the plasma gonadotrophin revealed that the highest levels of plasma LH and FSH were reached between 9 and 12 h of ovulation in all the inducers used. The two pituitary based hormones (FSH and LH) played complementary roles during ovulation and spawning of catfish eggs. This study provides an insight to the possibility of using hormones from the primary culture of pituitary cells to induce spawning in African catfish. Key words: Pituitary cells, primary culture, induced spawning, ovulation.

In Vivo and In Vitro Arsenic Exposition Induces Oxidative Stress in Anterior Pituitary Gland.

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereas luteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 µmol/L iAs significantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin release both in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.

Central effect of vasotocin 4 receptor (VT4R/V1aR) antagonists on the stress response and food intake in chicks given neuropeptide Y (NPY)

Previous studies identified SR-49059 as a most effective antagonist of the avian vasotocin 4 receptor (VT4R) compared to other candidate blockers including the Manning compound using in silico 3 dimensional (3D) modeling/docking analysis of the chicken VT4R and an in vitro anterior pituitary cell culture study. The present experiments were designed to validate whether SR-49059 and the Manning compound would likewise be effective in vivo in blocking the VT4R when applied intracerebroventricularly (ICV) to chicks. Two treatments were tested, a stressor (immobilization) and administration of neuropeptide Y (NPY), a potent orexigenic compound. In the first experiment, birds were given the Manning compound, SR-49059 or physiological saline ICV followed by immobilization stress. Blood samples were taken and corticosterone (CORT) was determined by radioimmunoassay. It was hypothesized that both antagonists would reduce the stress response. A second experiment examined the role of the VT4R in food intake regulation. The Manning compound, SR-49059 or physiological saline was administered prior to NPY and food intake was monitored for 1h. It was hypothesized that each of the two antagonists coupled with NPY would augment food intake above the intake resulting from saline plus NPY administration. Related to the second experiment was a third that examined the difference between the effect of central administration of NPY versus SR-49059 in releasing CORT. Results of the first study showed that the Manning compound or SR-49059 prior to stress decreased CORT levels compared to controls while the second experiment showed that SR-49059 or the Manning compound plus NPY, enhanced food intake above that of the experimental group given saline and NPY. The last study showed that NPY increased plasma CORT above birds given SR-49059 centrally or saline administered controls. Taken together, results suggest that the avian VT4R is involved in the central neuroendocrine stress response as well as functions in appetite regulation by mediating an anorexigenic effect similar to what has been reported in mammals for the V1aR. In conclusion, similar to the past in silico and in vitro tests, the current in vivo experiments showed SR-49059 to be a most efficacious avian vasotocin receptor antagonist. Therefore based upon results of functional tests utilizing a highly specific mammalian antagonist, SR-49059, to the mammalian V1aR that likewise was most effective in blocking the avian VT4R and past reported high sequence homology between the mammalian V1aR and the VT4R, it is recommended that the chicken VT4R be renamed the avian V1aR to facilitate better communication among scientists involved in comparative studies.

Efficacy of 5-aminolevulinic acid based photodynamic therapy in pituitary adenomas—experimental study on rat and human cell cultures

BackgroundIncomplete resection of pituitary adenomas may result in recurrence. As adjuvant irradiation is not riskless, alternative treatment options should be investigated. 5-aminolevulinic acid based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. The present study examined the efficacy of 5-ALA PDT in vitro on benign pituitary adenoma cell cultures. MethodsIn group I experiments were performed on immortalized rat pituitary adenoma cells (GH3). The cultured cells were treated with different 5-ALA concentrations ranging from 7.5–16.5μg/ml. In Group II human pituitary adenoma cell cultures were obtained from surgically resected adenoma tissue (n=15). These were incubated with 5-ALA concentrations from 12.5–100μg/ml. The concentration ranges had been determined in preliminary dose-finding tests. For both groups incubation time was four hours and PDT was performed by exposition to laser light (635nm, 625s, 18.75J/cm2). Cell viability was examined by WST-1 assay. ResultsIn both groups PDT showed a 5-ALA concentration-dependent effect on cell death. In group I lower 5-ALA concentrations were necessary to destroy all cells as compared to group II. Moreover, in group II, the different subtypes of human adenomas showed different sensitivities to 5-ALA-based PDT (secreting vs. non-secreting). Especially corticotroph adenomas were highly sensitive to 5-ALA PDT. ConclusionsThe GH3 cell line was an useful in vitro model to optimize different PDT parameters. Human pituitary adenoma cells could also be killed by 5-ALA PDT, however this required higher 5-ALA concentrations. Furthermore, the results suggested different 5-ALA sensitivities between different human adenoma cell types. More experiments are necessary to confirm these preliminary results.

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Retinoic acid and retinaldehyde dehydrogenase are not involved in the specific induction of the follicle-stimulating hormone β subunit by trichostatin A, a selective inhibitor of histone deacetylase

The selective histone deacetylase inhibitor, trichostatin A (TSA), increases follicle-stimulating hormone β subunit (FSHβ) mRNA expression but not α- and luteinizing hormone β (LHβ)-subunits in both the pituitary gonadotrophic cell line LβT2 and primary cultures of rat anterior pituitary cells. TSA increased histone acetylation in whole cell lysates in both cells. In addition, retinaldehyde dehydrogenases (RALDHs), which are retinoic acid (RA)-synthesizing enzymes, were induced by TSA in these cells. Anacardic acid, a histone acetyltransferase inhibitor that prevents histone acetylation, significantly inhibited TSA-induced FSHβ mRNA expression as well as TSA-induced RALDH2 and RALDH3 mRNA expression. Similar to the effect of TSA, gonadotropin-releasing hormone stimulated RALDH expression in LβT2 cells. RA directly applied to the pituitary cells stimulated the transcriptional activity of the FSHβ promoter. In addition, α- and LHβ-subunit promoters were also activated by RA. Our results suggest that TSA specifically increases FSHβ expression with a concomitant increase in RALDHs; however, RALDH and RA are not directly involved in the specific regulation of FSHβ by TSA.

CXCL10/CXCR3 signaling mediates inhibitory action by interferon-gamma on CRF-stimulated adrenocorticotropic hormone (ACTH) release.

Secretion of hormones by the anterior pituitary gland can be stimulated or inhibited by paracrine factors that are produced during inflammatory reactions. The inflammation cytokine interferon-gamma (IFN-γ) is known to inhibit corticotropin-releasing factor (CRF)-stimulated adrenocorticotropin (ACTH) release but its signaling mechanism is not yet known. Using rat anterior pituitary, we previously demonstrated that the CXC chemokine ligand 10 (CXCL10), known as interferon-γ (IFN-γ) inducible protein 10kDa, is expressed in dendritic cell-like S100β protein-positive (DC-like S100β-positive) cells and that its receptor CXCR3 is expressed in ACTH-producing cells. DC-like S100β-positive cells are a subpopulation of folliculo-stellate cells in the anterior pituitary. In the present study, we examine whether CXCL10/CXCR3 signaling between DC-like S100β-positive cells and ACTH-producing cells mediates inhibition of CRF-activated ACTH-release by IFN-γ, using a CXCR3 antagonist in the primary pituitary cell culture. We found that IFN-γ up-regulated Cxcl10 expression via JAK/STAT signaling and proopiomelanocortin (Pomc) expression, while we reconfirmed that IFN-γ inhibits CRF-stimulated ACTH-release. Next, we used a CXCR3 agonist in primary culture to analyze whether CXCL10 induces Pomc-expression and ACTH-release using a CXCR3 agonist in the primary culture. The CXCR3 agonist significantly stimulated Pomc-expression and inhibited CRF-induced ACTH-release, while ACTH-release in the absence of CRF did not change. Thus, the present study leads us to an assumption that CXCL10/CXCR3 signaling mediates inhibition of the CRF-stimulated ACTH-release by IFN-γ. Our findings bring us to an assumption that CXCL10 from DC-like S100β-positive cells acts as a local modulator of ACTH-release during inflammation.

In vitro effects of somatostatin on the growth hormone-insulin-like growth factor axis in orange-spotted grouper (Epinephelus coioides)

Growth in vertebrates is mainly mediated by the growth hormone (GH)-insulin-like growth factor (IGF) axis, and somatostatin (SRIF) inhibits growth by decreasing GH release at the pituitary level and antagonizing the release and action of GHRH in the hypothalamus. However, the effects of SRIF on the regulation of growth at levels other than GH release from the pituitary gland are less well known. In the present study, we comprehensively examined the pituitary and peripheral actions of SRIF on the GH-IGF axis in grouper using a primary pituitary and hepatocyte cell culture system. Our results showed that SRIF inhibited GH release at the pituitary level, but had no influence on GH mRNA expression. Basal hepatic GH receptor 1 (GHR1), IGF-I and IGF-II mRNA levels declined over time, whereas GHR2 mRNA levels remained stable throughout the culture period. GH stimulated the hepatic expression of GHR and IGF mRNAs in a dose-dependent manner, while SRIF suppressed both basal and GH-stimulated expression of GHR and IGF mRNAs in primary cultured hepatocytes. The inhibition of GHR and IGF mRNA levels by SRIF was not attributed to the rate of mRNA degradation. To the best of our knowledge, we demonstrated the effects of SRIF on basal and GH-stimulated IGF-II mRNA levels in teleosts for the first time. These results indicate that SRIF regulates growth at the level of the pituitary and peripheral liver.

Role of stress proteins and hormones in bioregulation of ontogeny

In this short review article we tried to overview diffusely spread data on the role of stress proteins and hormones in ontogeny. The work presented here is a product of our long-term studies beginning from the middle of eighties of the last century and performed both in Russia and Brazil. It involves the results obtained with the use of experimental models on laboratory animals in vivo and in vitro, as well as later theoretical research in world literature databases. In experimental studies we used laboratory rats of different age groups and primary cultures of pituitary and liver cells for evaluating respectively body and organ growth and production of immunoreactive growth hormone (GH) and serum albumin (SA), as well as biosynthesis of DNA, total RNA and protein. The results obtained, showing important role of glucocorticoids (GC) in regulation of perinatal pituitary and liver functions and postnatal growth, were reinterpreted by us recently in the frame of DOHaD concept and as related to perinatal imprinting/programming phenomena. It is concluded that the present moment is quite appropriate for the widening of our studies both to the side of early embryonal development and in direction to aging, thus completing the whole cycle of life history / course research, as referred to stress proteins and hormones.

Kisspeptin Antagonists Reveal Kisspeptin 1 and Kisspeptin 2 Differential Regulation of Reproduction in the Teleost, Morone saxatilis.

The importance of kisspeptin in regulating vertebrate reproduction has been well established, but the exact mechanism continues to unfold. Unlike mammals, many lower vertebrates possess a dual kisspeptin system, Kiss1 and Kiss2. To decipher the roles of the kisspeptins in fish, we identified two potential kisspeptin antagonists, pep 234 and pep 359, by screening analogs for their ability to inactivate striped bass Kiss1 and Kiss2 receptors expressed in COS7 cells. Pep 234 (a mammalian KISS1 antagonist) antagonizes Kiss1r signaling activated by Kiss1 and Kiss2, and pep 359 (a novel analog) antagonizes Kiss2 activation of both receptors. In vitro studies using brain slices demonstrated that only Kiss2 can upregulate the expression of the hypophysiotropic gnrh1, which was subsequently diminished by pep 234 and pep 359. In primary pituitary cell cultures, the two antagonists revealed a complex network of putative endogenous and exogenous regulation by kisspeptin. While both kisspeptins stimulate Fsh expression and secretion, Kiss2 predominately induces Lh secretion. Pep 234 and 359 treatment of spawning males hindered sperm production. This effect was accompanied with decreased brain gnrh1 and gnrh2 mRNA levels and peptide content in the pituitary, and increased levels of pituitary Lh, probably due to attenuation of Lh release. Strikingly, the mRNA levels of arginine-vasotocin, the neurons of which in the preoptic area coexpress kiss2r, were dramatically reduced by the antagonists. Our results demonstrate differential actions of Kiss1 and Kiss2 systems along the hypothalamic-pituitary axis and interactions with other neuropeptides, and further reinforce the importance of kisspeptin in the execution of spawning.

Inhibin Biosynthesis and Activity Are Limited by a Prodomain-Derived Peptide.

Gonadal-derived inhibin A and B are essential factors in mammalian reproduction, negatively regulating pituitary production of FSH. Inhibins are synthesized as heterodimers of α- and β-subunits, each comprising an N-terminal pro- and C-terminal mature domain. After dimerization, the inhibin α- and β-subunit prodomains are enzymatically cleaved from the mature domains at consensus RXXR sites (site1). Interestingly, the inhibin α-subunit is a unique TGF-β ligand, comprising a second cleavage site (site2) within its prodomain. Cleavage at site2 in the inhibin α-subunit prodomain releases a 43-amino acid proα-peptide. We aimed to determine the influence of the proα-peptide on inhibin synthesis and bioactivity. Blocking proα-peptide release by silencing cleavage site2 (Arg56-Arg61) inhibited both inhibin A and B synthesis. Ligand blot analysis and solid-phase binding assays indicated that the proα-peptide binds specifically to a mature 30-kDa inhibin (mean Kd 86 nM) but was unable to bind related activins. The proα-peptide suppressed inhibin A and B bioactivity in primary rat pituitary cell cultures. Mechanistically, the proα-peptide blocked inhibin A binding to its coreceptor, betaglycan (IC50 131 nM), and the subsequent sequestration of the activin type II receptor (IC50 156 nM), which underscores inhibin's biological activity. Based on the sequential mutations across the inhibin α-subunit, the proα-peptide binding site was localized to residues Arg341-Thr354, corresponding directly to the betaglycan binding region. Together our findings indicate that the proα-peptide limits the synthesis and bioactivity of inhibins.

Lactotrophs: the new and major source for VEGF secretion and the influence of ECM on rat pituitary function in vitro.

Vascular endothelial growth factor (VEGF) plays a pivotal role in pituitary endocrine function by influencing fenestration and blood vessel growth. Folliculostellate (FS) cells, which represent only a small number of pituitary cells, are recognized to produce VEGF. Tissue sections and primary pituitary cell cultures from rat pituitary glands were performed to co-localize VEGF and pituitary lactotrophs, which represents nearly 50% of all pituitary cells, by immunofluorescence. VEGF is co-localized with prolactin-producing cells in vivo and in vitro. FS cells are present infrequently in vivo (1.6%) and in vitro (2.4%). Culture supernatants were analyzed for the presence of VEGF by ELISA. VEGF levels are always significantly lower in supernatants from the cells that are seeded on Matrigel extracellular matrix (ECM) compared to the cells grown on plastic. Lower VEGF concentrations in supernatants from the pituitary cells cultured on ECM may reflect a more adequate cell environment compared to culture on plastic. These results demonstrate for the first time, that VEGF is expressed by lactotrophs, which outnumber FS cells. These results are of potential clinical relevance especially in oncology for the interpretation of studies investigating anti-angiogenic treatment of pituitary tumors.

Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110β, p110γ, and p110δ in pituitary gonadotropes and somatotropes.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110β, p110γ, and p110δ). Importantly, p110β and p110γ, but not p110α or p110δ, can be directly activated by the Gβγ heterodimer of Gαβγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110β, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110β only regulate basal LH secretion. However, p110β and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110β and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gβγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

Melatonin regulates somatotrope and lactotrope function through common and distinct signaling pathways in cultured primary pituitary cells from female primates.

Melatonin (MT) is secreted by the pineal gland and exhibits a striking circadian rhythm in its release. Depending on the species studied, some pituitary hormones also display marked circadian/seasonal patterns and rhythms of secretion. However, the precise relationship between MT and pituitary function remains controversial, and studies focusing on the direct role of MT in normal pituitary cells are limited to nonprimate species. Here, adult normal primate (baboons) primary pituitary cell cultures were used to determine the direct impact of MT on the functioning of all pituitary cell types from the pars distalis. MT increased GH and prolactin (PRL) expression/release in a dose- and time-dependent fashion, a response that was blocked by somatostatin. However, MT did not significantly affect ACTH, FSH, LH, or TSH expression/release. MT did not alter GHRH- or ghrelin-induced GH and/or PRL secretions, suggesting that MT may activate similar signaling pathways as ghrelin/GHRH. The effects of MT on GH/PRL release, which are likely mediated through MT1 receptor, involve both common (adenylyl cyclase/protein kinase A/extracellular calcium-channels) and distinct (phospholipase C/intracellular calcium-channels) signaling pathways. Actions of MT on pituitary cells also included regulation of the expression of other key components for the control of somatotrope/lactotrope function (GHRH, ghrelin, and somatostatin receptors). These results show, for the first time in a primate model, that MT directly regulates somatotrope/lactotrope function, thereby lending support to the notion that the actions of MT on these cells might substantially contribute to the define daily patterns of GH and PRL observed in primates and perhaps in humans.

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells

Nitric oxide (NO) and Ca2+ are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca2+ signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca2+-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca2+ availability and voltage-sensitive Ca2+ channels, as well as intracellular Ca2+ store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca2+-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca2+-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca2+-dependent transduction pathways.

Urotensin II inhibits arginine vasotocin and stimulates isotocin release from nerve endings in the pituitary of gilthead sea bream (Sparus aurata).

The aim of this in vitro study was to determine whether arginine vasotocin (AVT) and isotocin (IT) release from nerve endings is affected by urotensin II (UII) in gilthead sea bream pituitary. Primary cultures of pituitary cells were exposed to 10(-12), 10(-10), and 10(-8) M UII for 6, 24, and 48 hr. AVT and IT contents were determined in the culture media by high performance liquid chromatography (HPLC). UII at all doses decreased AVT release after 6, 24, and 48 hr of incubation. IT release was increased by UII only after 24 hr of incubation. This study, for the first time, indicates that UII affects AVT and IT release from nerve endings in the pituitary of Sparus aurata. It is presumed that UII together with AVT and IT may control response to different salinities in fishes.

Dopamine-2 receptor activation suppresses PACAP expression in gonadotrophs.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed at a high level in the fetal pituitary and decreases profoundly between embryonic day 19 and postnatal day 1 (PN1), with a further decrease from PN1 to PN4. In this series of experiments, we investigated the hypothesis that dopamine 2 receptor (Drd2) activation interrupts a cAMP-dependent feed-forward loop that maintains PACAP expression at a high level in the fetal pituitary. Using single-cell RT-PCR of pituitary cell cultures from newborn rats, Drd2 mRNA was identified in gonadotrophs that were also positive for PACAP mRNA. PACAP expression in pituitary cultures from embryonic day 19 rats was suppressed by the PACAP6-38 antagonist and by the Drd2 agonist bromocriptine. Increasing concentrations of bromocriptine inhibited cAMP production as well as cAMP signaling based on cAMP response element-luciferase activity, decreased PACAP promoter activity, and decreased PACAP mRNA levels in αT3-1 gonadotroph cells. Furthermore, blockade of dopamine receptors by injecting haloperidol into newborn rat pups partially reversed the developmental decline in pituitary PACAP mRNA that occurs between PN1 and PN4. These results provide evidence that dopamine receptor signaling regulates PACAP expression under physiological conditions and lend support to the hypothesis that a rise in hypothalamic dopamine at birth abrogates cAMP signaling in fetal gonadotrophs to interrupt a feed-forward mechanism that maintains PACAP expression at a high level in the fetal pituitary. We propose that this perinatal decline in pituitary PACAP reduces pituitary follistatin which permits GnRH receptors and FSH-β to increase to facilitate activation of the neonatal gonad.

Regulation of gene expression of vasotocin and corticotropin-releasing hormone receptors in the avian anterior pituitary by corticosterone

The effect of chronic stress (CS) on gene expression of the chicken arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) receptors [VT2R, VT4R, CRH-R1, and CRH-R2] was examined by measuring receptor mRNA levels in the anterior pituitary gland of the chicken after chronic immobilization stress compared to acute stress (AS). Radioimmunoassay results showed that blood circulating corticosterone (CORT) levels in the CS group were significantly decreased compared to that of birds in the AS group (P<0.05). The VT2R and CRH-R2 mRNA in CS birds were significantly decreased to that of controls. The VT4R mRNA was significantly decreased compared to controls in AC birds and was further decreased in the CS group compared to controls (P<0.05). The CRH-R1 mRNA was significantly decreased in the AS birds compared to controls. However, there was no significant difference of CRH-R1 mRNA between acute stress and chronic stress birds. Using primary anterior pituitary cell cultures, the effect of exogenous CORT on VT/CRH receptor gene expression was examined. Receptor mRNA levels were measured after treatment of CORT followed by AVT/CRH administration. The CORT pretreatment resulted in a dose-dependent decrease of proopiomelanocortin heteronuclear RNA, a molecular marker of a stress-induced anterior pituitary. Without CORT pretreatment of anterior pituitary cell cultures, the VT2R, VT4R and CRH-R1mRNA levels were significantly increased within 15min and then decreased at 1h and 6h by AVT/CRH administration (P<0.05). Pretreatment of CORT in anterior pituitary cells induced a dose-dependent increase of VT2R, VT4R and CRH-R2 mRNA levels, and a significant decrease of CRH-R1 mRNA levels at only the high dose (10ng/ml) of CORT (P<0.05).Taken together, results suggest a modulatory role of CORT on the regulation of VT/CRH receptor gene expression in the avian anterior pituitary gland dependent upon CORT levels.