Chicken Pituitary Cells Research Articles

Molecular Cloning and Functional Characterization of Three 5-HT Receptor Genes (HTR1B, HTR1E, and HTR1F) in Chickens.

The serotonin (5-hydroxytryptamine, 5-HT) signaling system is involved in a variety of physiological functions, including the control of cognition, reward, learning, memory, and vasoconstriction in vertebrates. Contrary to the extensive studies in the mammalian system, little is known about the molecular characteristics of the avian serotonin signaling network. In this study, we cloned and characterized the full-length cDNA of three serotonin receptor genes (HTR1B, HTR1E and HTR1F) in chicken pituitaries. Synteny analyses indicated that HTR1B, HTR1E and HTR1F were highly conserved across vertebrates. Cell-based luciferase reporter assays showed that the three chicken HTRs were functional, capable of binding their natural ligands (5-HT) or selective agonists (CP94253, BRL54443, and LY344864) and inhibiting intracellular cAMP production in a dose-dependent manner. Moreover, activation of these receptors could stimulate the MAPK/ERK signaling cascade. Quantitative real-time PCR analyses revealed that HTR1B, HTR1E and HTR1F were primarily expressed in various brain regions and the pituitary. In cultured chicken pituitary cells, we found that LY344864 could significantly inhibit the secretion of PRL stimulated by vasoactive intestinal peptide (VIP) or forskolin, revealing that HTR1F might be involved in the release of prolactin in chicken. Our findings provide insights into the molecular mechanism and facilitate a better understanding of the serotonergic modulation via HTR1B, HTR1E and HTR1F in avian species.

Identification of the receptors for somatostatin (SST) and cortistatin (CST) in chickens and investigation of the roles of cSST28, cSST14, and cCST14 in inhibiting cGHRH1–27NH2-induced growth hormone secretion in cultured chicken pituitary cells

Somatostatin receptors (SSTRs) are proposed to mediate the actions of somatostatin (SST) and its related peptide, cortistatin (CST), in vertebrates. However, the identity, functionality, and tissue expression of these receptors remain largely unknown in most non-mammalian vertebrates including birds. In this study, five SSTRs (named cSSTR1, cSSTR2, cSSTR3, cSSTR4, cSSTR5) were cloned from chicken brain by RT-PCR. Using a pGL3-CRE-luciferase reporter system, we demonstrated that activation of each cSSTR expressed in CHO cells by cSST28, cSST14 and cCST14 treatment could inhibit forskolin-induced luciferase activity of CHO cells, indicating the functional coupling of all cSSTRs to Gi protein(s). Interestingly, cSSTR1-4 expressed in CHO cells could be activated by cSST28, cSST14 and cCST14 with high potencies, suggesting that they may function as the receptors common for these peptides. In contrast, cSSTR5 could be potently activated by cSST28 only, indicating that it is a cSST28-specific receptor. Using RT-PCR, wide expression of cSSTRs was detected in chicken tissues including pituitary. In accordance with their expression in pituitary, cSST28, cSST14, and cCST14 were demonstrated to inhibit basal and novel cGHRH1–27NH2-induced GH secretion in cultured chicken pituitary cells dose-dependently (0–10nM) by Western blot analysis, suggesting the involvement of cSSTR(s) common for these peptides in mediating their inhibitory actions. Collectively, our study establishes a molecular basis to elucidate the roles of SST/CST in birds and provide insights into the roles of SST/CST in vertebrates, such as their conserved actions on pituitary.

Prostaglandin-D synthetase induces transcription of the LH beta subunit in the primary culture of chicken anterior pituitary cells via the PPAR signaling pathway

Downstream effects of prostaglandin-D synthetase (PGDS) in a primary culture of chicken ( Gallus gallus) anterior pituitary cells were investigated to study how PGDS regulated laying in hens. Either PGDS downstream metabolite, PGD 2 or PGJ 2, elevated LHB mRNA and LHB protein levels in dose- and time-dependent manners, and treatment with arachidonic acid (1 μM) alone upregulated 15-deoxy-Δ 12,14-PGJ 2 (15-d-PGJ 2; derived from PGJ 2)/PGJ 2, LHB mRNA, and LHB protein levels (P < 0.05) in the primary culture of chicken pituitary anterior cells. Transfection of the plasmid Enhanced Green Fluorescent Protein-PGDS plasmid into these cells in medium containing 1 μM arachidonic acid additionally increased 15-d-PGJ 2/PGJ 2, LHB mRNA, and LHB protein levels (P < 0.05). In the hypothalamus/pituitary gland of laying hens, there was a high correlation (r = 0.64; P < 0.05) between PGDS and the peroxisome proliferator-activated receptor A ( PPARA) mRNA level in a high egg production strain, the L2 Taiwan country chickens. In commercial Single-Comb White Leghorn layers, there were high correlation coefficients between PGDS and PPARA (r = 0.65; P < 0.05) and between PGDS and PPARG (r = 0.67; P < 0.01) mRNA levels. A broad-range PPARs agonist, GW9578 (5 to 500 nM), enhanced LHB mRNA and LHB protein levels (P < 0.05). The PPARA-specific (GW6471) and PPARG-specific (T0070907) antagonists suppressed endogenous LHB mRNA and LHB protein levels (P < 0.05); in addition, both antagonists attenuated arachidonic acid–induced LHB mRNA levels (P < 0.05) and PGDS-induced (in the presence of 1 μM arachidonic acid) LHB mRNA and LHB protein (P < 0.05) levels in the primary culture of chicken anterior pituitary cells. Higher LHB mRNA/LHB protein ratios in PGD 2-, PGJ 2-, arachidonic acid-, PGDS-, and GW9578-induced as well as GW6471- and T0070907-suppressed anterior pituitary cells suggested that LHB transcription occurred before translation. In conclusion, PGDS induced LHB transcription and subsequent translation via the PPAR signaling pathway.

Immunohistochemical characterization of chicken pituitary cells containing the vasotocin VT2 receptor

Research in mammals has demonstrated a variety of regulatory effects of vasopressin and oxytocin on endocrine functions of the anterior pituitary gland. Less evidence is available regarding the hypophysiotropic action of arginine vasotocin (AVT) comprising vasopressic and oxytocic activities in birds. Some hypophysiotropic effects of AVT may result from its interactions with brain circuits controlling pituitary functions, whereas others are caused by its direct affect on pituitary cells. Use of an antiserum to the vasotocin receptor VT2 (VT2R) has revealed numerous immunoreactive cells in the anterior pituitary gland of the chicken. The objective of the present study has been to identify endocrine phenotypes of chicken pituitary cells containing VT2R by means of immunohistochemical labeling. VT2R immunoreactivity has been found in all cells immunoreactive for adrenocorticotropin and alpha-melanotropin. Approximately 10% of labeled lactotropes are also immunoreactive for VT2R and lie around the anatomical boundary dividing the cephalic and caudal lobes. In both corticotropes/melanotropes and lactotropes, immunoreactive VT2R is present in a narrow layer outlining cell bodies. Immunoreactive VT2R is not found in gonadotropes, thyrotropes, or somatotropes. These results provide evidence for the important role of VT2Rs in mediating effects of AVT on endocrine secretion from corticotropes and, partially, from lactotropes.

The chicken pituitary-specific transcription factor pit-1 is involved in the hypothalamic regulation of pituitary hormones

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.

PPAR expression and activity in murine skin tumors

Downstream effects of prostaglandin-D synthetase (PGDS) in a primary culture of chicken (Gallus gallus) anterior pituitary cells were investigated to study how PGDS regulated laying in hens. Either PGDS downstream metabolite, PGD2 or PGJ2, elevated LHB mRNA and LHB protein levels in dose- and time-dependent manners, and treatment with arachidonic acid (1 μM) alone upregulated 15-deoxy-Δ12,14-PGJ2 (15-d-PGJ2; derived from PGJ2)/PGJ2, LHB mRNA, and LHB protein levels (P < 0.05) in the primary culture of chicken pituitary anterior cells. Transfection of the plasmid Enhanced Green Fluorescent Protein-PGDS plasmid into these cells in medium containing 1 μM arachidonic acid additionally increased 15-d-PGJ2/PGJ2, LHB mRNA, and LHB protein levels (P < 0.05). In the hypothalamus/pituitary gland of laying hens, there was a high correlation (r = 0.64; P < 0.05) between PGDS and the peroxisome proliferator-activated receptor A (PPARA) mRNA level in a high egg production strain, the L2 Taiwan country chickens. In commercial Single-Comb White Leghorn layers, there were high correlation coefficients between PGDS and PPARA (r = 0.65; P < 0.05) and between PGDS and PPARG (r = 0.67; P < 0.01) mRNA levels. A broad-range PPARs agonist, GW9578 (5 to 500 nM), enhanced LHB mRNA and LHB protein levels (P < 0.05). The PPARA-specific (GW6471) and PPARG-specific (T0070907) antagonists suppressed endogenous LHB mRNA and LHB protein levels (P < 0.05); in addition, both antagonists attenuated arachidonic acid–induced LHB mRNA levels (P < 0.05) and PGDS-induced (in the presence of 1 μM arachidonic acid) LHB mRNA and LHB protein (P < 0.05) levels in the primary culture of chicken anterior pituitary cells. Higher LHB mRNA/LHB protein ratios in PGD2-, PGJ2-, arachidonic acid-, PGDS-, and GW9578-induced as well as GW6471- and T0070907-suppressed anterior pituitary cells suggested that LHB transcription occurred before translation. In conclusion, PGDS induced LHB transcription and subsequent translation via the PPAR signaling pathway.

Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors

The pharmacology of mammalian and avian gonadotropin-releasing (GnRH) receptors differs for agonist analogues. We have therefore compared the activities of mammalian-based GnRH antagonists in sheep and chicken gonadotropes to further elucidate the different structural requirements of the receptors. The antagonist activities of ten GnRH analogues were compared in cultured sheep and chicken pituitary cells by determining the dose required to cause a 50% inhibition of luteinizing hormone secretion (IC 50) induced by GnRH at its half-maximal concentration (EC 50). Nine analogues showed high antagonist activity in the sheep bioassay. Analogue IC 50s varied between half and twice ((1.22 – 6.06) × 10 −10 M) the GnRH EC 50 (3 × 10 −10 M). One of these peptides exhibited partial agonist activity. In contrast, eight of the analogues showed low antagonist activity in chicken pituitary cells, with IC 50s varying from 46 to 1490 times ((1.4–44.7) × 10 −7 M) the GnRH EC 50 (3 × 10 −9 M) and had a different order of potencies compared with that in the sheep. Furthermore, two analogues did not display antagonist activity at all in the chicken bioassay, but acted as pure agonists, stimulating LH secretion. These findings demonstrate marked differences in pharmacology between the avian and mammalian pituitary GnRH receptors and emphasize that GnRH antagonists, selected for their efficacy in mammals, cannot necessarily be used for physiological studies in non-mammalian vertebrates. The distinctly different pharmacology of the receptors and structural requirements of analogues for agonist/antagonist activity establish a basis for identifying receptor features involved in ligand-induced signal propagation using chimaeras of cloned sheep and chicken receptors.

Estrogen Reduces Sensitivity of Chicken Pituitary Cells to GnRH.

Primary monolayer culture cells prepared from the anterior pituitary of 1-day-old male chicks were used to examine the effect of estrogen on GnRH-induced LH release. The half-maximal response (ED50) values of several kinds of GnRH determined by dose-response incubations with day-4 culture cells were in the following ascending order (in nM) : (D-Lys6)GnRH (a synthetic analogue; 0.07), chicken GnRH-II (0.24), chicken GnRH-I (0.51), and mammalian GnRH (0.82). By the use of the most potent and stable GnRH, (D-Lys6) GnRH, at a dose (1 nM) less than that inducing a maximal LH release, the GnRH-induced LH release was found to decrease in day-4 culture cells which had been pre-incubated with estradiol-17β (E2) for 12, 24 or 48 h. Whereas basal LH secretion was not changed by pre-incubation with E2. Following pre-incubation with E2 or diethylstilbestrol for 24 h the dose-response curves for the GnRH-induced LH release were shifted to a higher concentration with the ED50 enhanced. The results suggest that estrogen reduces the sensitivity of the chicken anterior pituitary cells to GnRH in vitro.

Phosphorylation of prolactin and growth hormone.

To determine whether GH and prolactin could be phosphorylated, turkey GH, chicken GH, chicken prolactin and turkey prolactin were incubated in vitro with the catalytic subunit of protein kinase A and [gamma-32P]ATP. Phosphorylation was assessed after sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and autoradiography. Polyacrylamide electrophoresis showed that both purified native chicken GH and turkey GH were phosphorylated under the conditions employed. However, the glycosylated variant of chicken GH did not appear to be labelled. Chicken prolactin, turkey prolactin and the glycosylated variant of turkey prolactin were all intensely phosphorylated by protein kinase A. Ovine and rat prolactins could also be phosphorylated by protein kinase A. The phosphate content of different native prolactin (turkey, ovine and rat) and GH (ovine and chicken) preparations was also determined and found to be significant. Chicken pituitary cells in primary culture incorporated 32P in GH- and prolactin-like bands isolated by non-denaturing polyacrylamide gel electrophoresis, and this was stimulated by phorbol myristate acetate. Phosphorylation of GH and prolactin may thus explain some of the charge heterogeneity of these hormones.

Triiodothyronine (T 3) inhibition of growth hormone secretion by chicken pituitary cells in vitro

These studies examined the cellular basis for the inhibitory effects of triiodothyronine (T 3) on growth hormone-releasing factor (GRF)-evoked growth hormone (GH) release from chicken anterior pituitary cells in vitro. A primary monolayer culture of anterior pituitaries from 4- to 8-week-old White Leghorn cockerels was performed as previously described by this laboratory. Following a 72-hr preincubation period, cells were washed and incubated (2 hr) with either secretagogues or media alone (control). T 3 (20 ng/ml) or vehicle was added to cells during both the preincubation (48–72 hr) and incubation (2 hr) period. Triiodothyronine reduced ( P < 0.05) GH release (ng/ml) in response to (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) the cAMP analog and protein kinase A activator, 8-bromo cAMP; and (4) the phorbol ester and protein kinase C activator, phorbol 12-myristate 13-acetate. Triiodothyronine reduced ( P < 0.05) the intracellular content of GH and total GH (released GH and intracellular GH) irrespectively of whether secretagogues were also present. When GH release was expressed as a percentage of total GH [released GH/(intracellular GH + released GH)], percentage GH released in response to GRF, or the protein kinase A, protein kinase C, or calcium pathway activators was not as great in T 3-treated versus non-T 3-treated cells. These data indicate that T 3 inhibits GRF-evoked GH release by reducing the availability of intracellular stores of GH and by also inhibiting second messenger-stimulated GH release pathways.

Second Messenger Pathways Mediating Chicken Luteinizing Hormone Secretion from Dispersed Pituitary Cells1

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible involvement of adenylyl cyclase-cAMP-protein kinase A pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.

A sex difference in responsiveness to gonadotrophin-releasing hormone in chicken pituitary cells: A study on intracellular calcium

A sex difference in responsiveness to gonadotrophin-releasing hormone in chicken pituitary cells: A study on intracellular calcium

Influence of catecholamines, prostaglandins and thyroid hormones on growth hormone secretion by chicken pituitary cells in vitro

In young chickens plasma concentrations of growth hormone (GH) are depressed by prostaglandins (PG) E 1 and E 2, epinephrine, norepinephrine, α 2 and β agonists or thyroid hormones. A primary culture of chicken adenohypopyseal cells was used to examine the direct effects of these agents at the level of the pituitary as evaluated by GH release in the presence and absence of growth hormone releasing factor (GRF). Following collagenase dispersion and culture (preincubation, 48 hr) cells were exposed (incubation, 2 hr) to test agents, except for thyroid hormones which were added during the preincubation, and incubation period. Growth hormone release was increased (P<.05) in the presence of PGE 1 (10 −8M by 34%; 10 −7M by 54%), PGE 2 (10 −8M by 29%; 10 −7M by 29%), PGF 2α (10 −8M by 28%), and the β agonist isoproterenol (10 −7M by 46%). Basal GH release from chicken pituitary cells was not affected by dopamine, norepinephrine, epinephrine, thyroxine (T 4), triiodothyronine (T 3), or α adrenergic agonists. Growth hormone releasing factor stimulated GH release was not affected by the presence of prostaglandins E 1, E 2 or F 2α in the incubation media. However, GRF stimulated GH release was reduced by high doses of catecholamines: dopamine (10 −6M by 34%), norepinephrine (10 −6M by 74%), epinephrine (10 −8M by 47%; 10 −7M by 41%; 10 −6M by 89%), and by the α 1 adrenergic agonist, phenylephrine (10 −7M by 52%), the α 2 agonist, clonidine (10 −8M by 34%; 10 −7M by 83%) and the β agonist, isoproterenol (10 −7M by 64%). Similarly, GRF-induced GH release was decreased by coincubation with 10 or 100 ng/ml of T 3 by 75% and 89%, respectively. In contrast, T 4 did not affect GH release evoked by GRF although T 3 depressed GH release in the presence of both thyrotropin releasing hormone (TRH) and GRF. To determine the temporal relationship for T 3 inhibition of GRF stimulated GH release, T 3 (1.3 × 10 −8M) was added during either the preincubation, incubation period or both. T 3 did not affect basal GH release during any time period, however, T 3 supplementation during the preincubation (48 hr) or both the preincubation (48 hr) and incubation (2 hr) period did suppress GRF stimulated GH release. These data suggest that the decrease in plasma concentration of GH observed in young chickens injected with catecholamines or T 3 may involve direct effects on the pituitary gland. However, the inhibitory effects of PGE 1, PGE 2 and PGF 2α on in vivo GH secretion appears to be mediated at another site, presumably the hypothalamus.

Possible participation of calcium in growth hormone release and in thyrotropin-releasing hormone and human pancreatic growth hormone-releasing factor synergy in a primary culture of chicken pituitary cells

We previously reported that thyrotropin-releasing hormone (TRH) and human pancreatic growth hormone-releasing factor (hpGRF) exert synergistic (greater than additive) effects on growth hormone (GH) release from chicken pituitary cells in primary culture. In the present studies the possible participation of calcium in GH release and in TRH and hpGRF synergy was investigated. Following dispersion with collagenase, cells were cultured for 48 hr prior to exposure (2 hr) to test agents. Cultured cells were exposed to a range of calcium concentrations (0, 0.02, 0.2, and 2.0 m M) in the presence and absence of secretagogues. These results demonstrated that basal GH release was not altered by the concentration of calcium in the medium: however, secretagogue-induced GH release required calcium. Thus, TRH, hpGRF, 8 Br-cAMP, or forskolin stimulated GH release in the absence of calcium. Furthermore, synergistic GH release evoked by TRH and hpGRF, 8 Br-cAMP, or forskolin was observed only at the highest calcium concentration (2.0 m M). In other studies, ionomycin (10 −5 M), a calcium ionophore, stimulated GH release to a value about 125% over the basal (absence of test agent) value. Ionomycin-induced GH release was not affected by TRH (5.0 ng/ml); the combined effects of ionomycin (10 −7–10 −5 M) and hpGRF (5.0 ng/ml) on GH release were less than additive. However, ionomycin (10 −5 M) further increased GH release over that resulting from the synergistic action of TRH and hpGRF (5.0 ng/ml each). Verapamil (a calcium channel blocker) did not affect GH release induced by either TRH or hpGRF (5.0 ng/ml each). However, this agent did inhibit synergistic GH release evoked by TRH and hpGRF, 8 Br-cAMP, forskolin, or isobutylmethylxanthine. These results suggest that calcium participates in secretagogue-induced GH release from chicken somatotrophs in vitro.

Scanning Electron Microscopic study on chicken pituitary cells

The cytology and histology of the vertebrate pituitary glands have been studied extensively under light microscopy and transmission electron microscopy. These informations form the basis for the generally accepted concept that the specific cell types synthesize, store and release their particular hormones. These hormonal products are stored in membrane-bound secretion granules in the cytoplasm.Tanaka developed the 0 smium-DMS 0-0 smium (ODO) method which can remove the excess cytoplasmic matrices from the frozen-fractured surface of pre-fixed cells and revealed intracellular structures of many kind s of cells from liver, retina, etc. In this work, we have used the ODO method and Field Emission SEM to obtain 3-dimensional images of the fine structure of pituitary cells, as a new approach to have a better idea about the intracellular organization of the hormone secretory cells .Chicken pituitary glands were fixed in 0.5% glutaraldehyde in 0.2M phosphate buffer(pH=7.2) for ½ h; rinsed with the same buffer; postfixed in 0.5% OsO4 for another ½h. After been rinsed throughly, specimens were passed through 25% and 50% dimethyl sulfoxide(DMSO) solution for 30 minutes each.

Gonadal Steroid Modulation of Signal Transduction and Luteinizing Hormone Release in Cultured Chicken Pituitary Cells*

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.

Dual Pathways of Calcium Entry in Spike and Plateau Phases of Luteinizing Hormone Release from Chicken Pituitary Cells: Sequential Activation of Receptor-Operated and Voltage-Sensitive Calcium Channels by Gonadotropin-Releasing Hormone

It has previously been shown that, in pituitary gonadotrope cells, the initial rise in cytosolic Ca2+ induced by GnRH is due to a Ca2+ mobilization from intracellular stores. This raises the possibility that the initial transient spike phase of LH release might be fully or partially independent of extracellular Ca2+. We have therefore characterized the extracellular Ca2+ requirements, and the sensitivity to Ca2+ channel blockers, of the spike and plateau phases of secretion separately. In the absence of extracellular Ca2+ the spike and plateau phases were inhibited by 65 +/- 4% and 106 +/- 3%, respectively. Both phases exhibited a similar dependence on concentration of extracellular Ca2+. However, voltage-sensitive Ca2+ channel blockers D600 and nifedipine had a negligible effect on the spike phase, while inhibiting the plateau phase by approximately 50%. In contrast, ruthenium red, Gd3+ ions, and Co2+ ions inhibited both spike and plateau phases to a similar extent as removal of extracellular Ca2+. A fraction (35 +/- 4%) of spike phase release was resistant to removal of extracellular Ca2+. This fraction was abolished after calcium depletion of the cells by preincubation with EGTA in the presence of calcium ionophore A23187, indicating that it depends on intracellular Ca2+ stores. Neither absence of extracellular Ca2+, nor the presence of ruthenium red or Gd3+ prevented mobilization of 45Ca2+ from intracellular stores by GnRH. We conclude that mobilization of intracellular stored Ca2+ is insufficient by itself to account for full spike phase LH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of endogenous chicken gonadotrophin-releasing hormone-I and -II on chicken pituitary cells.

The presence of two endogenous forms of gonadotrophin-releasing hormone (GnRH) in the chicken hypothalamus (chicken GnRH-I ([Gln8]GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]GnRH)), and the stimulation of gonadotrophins by both forms, suggests the possible existence of GnRH receptor subtypes and gonadotroph subtypes in the chicken pituitary. This question was investigated by assessing the effects of various combinations of the two known forms of chicken hypothalamic GnRH and antagonist analogues of GnRH on LH release from dispersed chicken anterior pituitary cells in both static and perifused systems. The relative inhibition of chicken GnRH-I-stimulated and chicken GnRH-II-stimulated LH release by 12 GnRH antagonists did not differ significantly, suggesting a single GnRH receptor type. Chicken GnRH-II was approximately sixfold more potent than chicken GnRH-I in releasing LH. Release of LH in response to maximal doses of chicken GnRH-I and chicken GnRH-II and to a mixture of both was similar and the two peptides were not additive in their effects, consistent with the presence of a single type of LH gonadotroph and a GnRH receptor which binds both forms of GnRH. Each form of GnRH desensitized cells to subsequent stimulation with the other form, providing additional evidence for a single type of LH gonadotroph. These findings suggest that chicken GnRH-I and -II stimulate gonadotrophin release through a single GnRH receptor type on a single class of LH gonadotroph in the chicken pituitary.

Barium-induced LH release from chicken pituitary cells: synergism with phorbol ester.

Barium is known to elicit secretion in a number of cell systems. The mechanism of Ba2+ stimulation of LH release in cultured chicken pituitary cells was investigated in the present study. Barium-stimulated LH release was inhibited by extracellular Ca2+, indicating that Ba2+ does not act by stimulating Ca2+ entry. Simultaneous stimulation of the cells with Ba2+ and phorbol ester produced a synergistic response, similar to the synergism obtained with phorbol ester and treatments which increase cytosolic Ca2+. Both Ba2+-stimulated LH release and the synergism of Ba2+ with phorbol ester were inhibited by calcium channel blockers (Co2+, methoxyverapamil and nifedipine) and by calmodulin antagonists (trifluoperazine and chlorpromazine). These results indicate that the actions of Ba2+ are dependent on its entry through Ca2+ channels, and suggest that calmodulin activation is necessary for the synergism between Ba2+ and phorbol ester. Thus, synergism does not result from a direct effect of divalent cations on C-kinase.