Primary Cultures Of Rat Pituitary Cells Research Articles

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.

Curcumin Inhibits the Growth, Induces Apoptosis and Modulates the Function of Pituitary Folliculostellate Cells

The polyphenol curcumin (diferuloylmethane) is the active componenet of the spice plant Curcuma longa and has been shown to exert multiple actions on mammalian cells. We have studied its effect on folliculostellate (FS) TtT/GF mouse pituitary cells, representative of a multifunctional, endocrine inactive cell type of the anterior pituitary. Proliferation of TtT/GF cells was inhibited by curcumin in a monolayer cell culture and in the colony formation assay in soft agar. Fluorescence-activated cell-sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G₂/M accompanied by inhibition of cyclin D₁ protein expression. Curcumin had a small effect on necrosis of TtT/GF cells, but it mainly stimulated apoptosis as demonstrated by FACS analysis (Annexin V-fluorescein isothiocyannate/7-aminoactinomycin D staining). Curcumin-induced apoptosis involved suppression of Bcl-2, stimulation of cleaved caspase-3 and induction of DNA fragmentation. Functional studies on FS cell-derived compounds showed that curcumin inhibited mRNA synthesis and release of angiogenic vascular endothelial growth factor-A (VEGF-A). Immune-like functions of FS cells were impaired since curcumin downregulated Toll-like receptor 4, reduced nuclear factor-ĸB expression and suppressed bacterial endotoxin-induced interleukin-6 (IL-6) secretion. The inhibitory action of curcumin on VEGF-A and IL-6 production was also found in primary rat pituitary cell cultures, in which FS cells are the only source of these proteins. The observed effects of curcumin on FS cell growth, apoptosis and functions may have therapeutic consequences for the intrapituitary regulation of hormone production and release as well as for pituitary tumor pathogenesis.

Role of Glucocorticoids in the Regulation of Pituitary Somatostatin Receptor Subtype (sst1–sst5) mRNA Levels: Evidence for Direct and Somatostatin-Mediated Effects

Glucocorticoids can differentially regulate somatostatin (SRIH) receptor subtype expression depending on the duration of treatment, dose used and tissue type examined. In order to determine if glucocorticoids are critical regulators of pituitary SRIH receptor synthesis in vivo, we examined the effect of adrenalectomy (ADX), with and without dexamethasone (DEX; 200 µg/day for 8 days) treatment, on the relative expression levels of the SRIH receptor subtypes, sst1–sst5, by multiplex RT-PCR. ADX increased pituitary sst2 mRNA levels, but did not significantly alter mRNA levels of the other SRIH receptor subtypes. These findings indicate that pituitary sst2 synthesis is normally under inhibitory control of endogenous glucocorticoids. High-dose DEX resulted in a decrease in sst1–sst4 mRNA and an increase in sst5 mRNA, independent of adrenal status. DEX also decreased sst2, sst3 and sst4 mRNA levels and increased sst5 mRNA levels by short-term in vitro application (10 nM, 4 h) in primary rat pituitary cell cultures, indicating DEX regulation of sst2–sst5 in vivo is at least in part due to a direct action at the level of the pituitary. However, the inhibitory actions of DEX on sst1 mRNA levels observed in vivo were not consistently replicated in vitro. In order to determine if the somatotrope population of the pituitary would display a similar response to DEX, fluorescent-activated cell sorting was used to obtain somatotrope-enriched cultures (>95% growth hormone immunopositive cells). DEX treatment (10 nM, 4 h) of somatotropes decreased sst2 and sst3, but did not alter sst5 mRNA levels. These results indicate that the effects of DEX on sst5 mRNA levels observed in unsorted pituitary cell cultures might be due to changes in sst5 expression in pituitary cell types other than somatotropes. Since excess glucocorticoids are thought to enhance SRIH tone, we also tested if ligand activation of SRIH receptor subtypes in vitro could mimic any of the actions of DEX on SRIH receptor mRNA levels observed in vivo. To this end, unsorted pituitary cell cultures and somatotrope-enriched cultures were treated with SRIH (1 and 100 nM) for 4 h. SRIH increased sst3 and sst5 mRNA levels, in both culture systems. These results suggest that the DEX-induced increase in endogenous SRIH tone may contribute to enhanced sst5 mRNA levels observed in vivo. However, the stimulatory actions of SRIH on sst3 mRNA levels observed in vitro might be overridden by direct inhibitory actions of DEX.

Transforming growth factor-beta stimulates vascular endothelial growth factor production by folliculostellate pituitary cells.

TGF-beta isoforms are expressed in the anterior pituitary and modulate the growth and function of endocrine pituitary cells. Recently, TGF-beta has been shown to stimulate growth and basic fibroblast growth factor secretion in nonendocrine folliculostellate (FS) pituitary cells. We therefore studied whether the production of FS cell-derived vascular endothelial growth factor (VEGF), the most important regulator of vascular permeability and angiogenesis, is affected by TGF-beta. We observed by RT-PCR that TtT/GF cells, which are FS mouse pituitary tumor cells, synthesize TGF-beta1, -beta2, and -beta3. They also express TGF-beta receptors types 1 and 2, as well as Smad2, Smad3, and Smad4 proteins, which are essential for TGF-betabinding and signaling. Stimulation of TtT/GF cells with either TGF-beta1 or TGF-beta3 induced a rapid translocation of Smad2 into the cell nuclei. Both TGF-beta isoforms dose dependently stimulated VEGF production in TtT/GF cells, but not in lactosomatotroph GH3 cells. Time-course studies and suppression of TGF-beta-induced VEGF production by cycloheximide suggest that TGF-beta induces de novo synthesis of VEGF in folliculostellate cells, which is completely blocked by dexamethasone. In primary rat pituitary cell cultures, TGF-beta1 and -beta3 stimulated VEGF production. TGF-beta stimulation of VEGF production by folliculostellate cells could modulate intrapituitary vascular permeability and integrity as well as angiogenesis in an auto-/paracrine manner.

Modulation of pituitary somatostatin receptor subtype (sst1-5) messenger ribonucleic acid levels by changes in the growth hormone axis.

The role of individual components of the hypothalamic-pituitary-GH axis in the modulation of pituitary somatostatin (SRIF) receptor subtype (sst1-5) synthesis was assessed using multiplex RT-PCR to measure receptor messenger RNA (mRNA) levels in normal rats and spontaneous dwarf rats (SDRs). In SDRs, a strain with no immunodetectable GH, pituitary sst1 and sst2 mRNA levels were elevated, sst5 mRNA levels were reduced, and sst3 and sst4 mRNA levels did not significantly differ from those in normal controls. Treatment of SDRs with GH (72 h), but not insulin-like growth factor I, significantly decreased sst2 mRNA levels and increased sst4 and sst5 mRNA levels above vehicle-treated control levels. To test whether more rapid changes in circulating GH levels could alter SRIF receptor subtype expression, normal rats were infused (iv) with GH-releasing hormone (GHRH) for 4 h in the presence or absence of SRIF antiserum. GHRH infusion increased pituitary sst1 and sst2 and decreased sst5, but had no effect on sst3 and sst4 mRNA levels. Immunoneutralization of SRIF, which produced a rise in circulating GH levels, did not alter basal or GHRH-mediated SRIF receptor subtype expression. These observations indicate that acute suppression of SRIF tone does not regulate pituitary SRIF receptor subtype mRNA levels in vivo. The possibility that elevated circulating GH concentrations induced by GHRH infusion were responsible for the observed changes in SRIF receptor subtype mRNA levels was examined by infusing SDRs with GHRH for 4 h. GHRH did not increase sst1 mRNA levels in SDRs above their already elevated value. However, GHRH infusion produced an increase in sst2 and a decrease in sst5 mRNA levels similar to those observed in normal rats, indicating that the acute effects of GHRH on SRIF receptor subtype expression are independent of circulating GH levels. Primary rat pituitary cell cultures were incubated with GHRH (10 nM) or forskolin (10 microM) for 4 h to determine whether GHRH could directly mediate SRIF receptor subtype mRNA. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst5 mRNA levels, but had no effect on sst3 and sst4, similar to the results in vivo. The effect of forskolin mimicked that of GHRH on sst1, sst2, and sst5 mRNA, suggesting that GHRH acts through cAMP to directly mediate gene transcription or mRNA stability of these SRIF receptor subtypes. In addition, forskolin reduced sst3 and sst4 expression. These results strongly suggest that rat pituitary sst1, sst2, and sst5 mRNA levels are regulated both in vivo and in vitro by GHRH. The stimulatory action of GHRH on sst1 and sst2 and the inhibitory action on sst5 indicate that these receptor subtypes have independent and unique roles in the modulation of pituitary GH release.

Glucocorticoids regulate pituitary growth hormone secretagogue receptor gene expression.

Glucocorticoids regulate growth hormone (GH) secretion by modulating both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids increase GH and GH-releasing hormone receptor (GHRH-R) gene expression. To test if glucocorticoids might also regulate the pituitary expression of the recently identified GH secretagogue (GHS) receptor, GHS-R; adult male rats were adrenalectomized or sham operated, and treated with the synthetic glucocorticoid (dexamethasone, 200 microg/day) or vehicle for 8 days. Pituitary GHS-R mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Adrenalectomy decreased pituitary GHS-R mRNA to 45% of vehicle-treated, sham-operated rats (P < 0.05). Administration of dexamethasone increased GHS-R mRNA levels in sham-operated as well as in adrenalectomized rats (199 +/- 24% (P < 0.05) and 369 +/- 48% (P < 0.01) of vehicle-treated controls). Addition of dexamethasone to primary rat pituitary cell cultures increased GHS-R mRNA levels in a dose- and time-dependent manner while the transcriptional inhibitor, actinomycin D, completely blocked the stimulatory action of dexamethasone. Taken together, these results suggest glucocorticoids directly increase pituitary GHS-R mRNA levels by stimulating GHS-R gene transcription.

Estrogen regulation of peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid levels by a nuclear posttranscriptional event.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyze the conversion of glycine-extended peptides into COOH-terminal amidated peptides. We have previously shown that PAM messenger RNA (mRNA) levels in the anterior pituitary of intact cycling adult female rats showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17beta-estradiol was accompanied by a 4.5 +/- 0.5-fold decrease in total PAM mRNA and a 2-fold decrease in PAM activity in the anterior pituitary gland. To investigate the cellular site at which 17beta-estradiol acts to affect the PAM mRNA, we made parallel measurements of the relative levels of PAM mRNA and nuclear precursor RNA and the relative rate of gene transcription after treatments designed to alter the estrogen status. The transcription rate experiments indicated that these 17beta-estradiol effects were not due to reduced PAM gene activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. Primary rat pituitary cell cultures from OVX and OVX-17beta-estradiol-treated rats in the presence of actinomycin D showed that 17beta-estradiol treatment decreased the half-life of PAM mRNA from 15-16 h to 8-9 h. There was no effect of 17beta-estradiol on PAM mRNA poly(A) tail length or site of polyadenylation. However, in this study the down-regulation of PAM was identified as a nuclear event. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that decreased PAM expression after 17beta-estradiol treatment was largely due to intranuclear destabilization of the primary transcript. The levels of nuclear precursor RNA were decreased roughly 5- to 6-fold in OVX + 17beta-estradiol compared with OVX rats. The decrease in PAM mRNA is blocked by cycloheximide, indicating that its requires new protein synthesis. Mechanisms that would generate such an effect include altered stability of unprocessed message in the nucleus. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for PAM mRNA.

A 361 base pair region of the rat FSH- β promoter contains multiple progesterone receptor-binding sequences and confers progesterone responsiveness

The rat is frequently used as a model to study the role of progesterone (P) in regulating FSH secretion and synthesis. The ability of P to modulate rat FSH- β mRNA levels suggests the presence of a functional hormone response element. We have found three PRE-like sequences upstream of the transcription start site in the rat FSH- β gene. These sequences are herein referred to as PRE-like sequence #1, #2 and #3 with #1 being most distal from the start site. The current studies determined whether these PRE-like sequences bound P receptor (PR) and were functional in regulating the induction of expression by P. Electrophoretic mobility shift assays (EMSA) demonstrated that a single 289 base pair (bp) DNA fragment encompassing all three PRE-like sequences specifically bound PR. Further, PR bound with high affinity to double-stranded oligonucleotides representing individual PRE-like sequences #1, #2 and, with lower affinity to a double-stranded oligonucleotide representing PRE-like sequence, #3. We have cloned a 361 bp sequence from the promoter region of the rat FSH- β gene encompassing all three PRE-like sequences into a luciferase reporter vector (pGL3-promoter) yielding pFSH β361-luc+ which when transiently transfected into primary rat pituitary cell cultures, conferred P-responsiveness to a heterologous promoter. P-responsiveness was dependent upon the presence of PR and was blocked by the PR antagonist RU-486. These data strongly suggest the presence of functional PRE’s in the rat FSH- β gene promoter.

Differential regulation in the release of bioactive versus immunoactive gonadotropins from cultured rat pituitary cells by inhibin and androgens.

Using in vitro bioassays and radioimmunoassays, the authors examined the interactions between purified 32kD ovine inhibin, testosterone, and dihydrotestosterone on basal and GnRH-stimulated secretion of gonadotropins in primary rat pituitary cell cultures. This study demonstrates that inhibin and androgens: 1) differentially regulate gonadotropin levels, 2) cause changes in the amounts of bioactive and immunoactive FSH and LH released. In conclusion, differences in bio- and immuno-FSH and LH secretion suggest that the endocrine milieu results in not only quantitative but qualitative changes in the secreted gonadotropin isoforms.

Vitamin D-enhanced thyrotrophin release from rat pituitary cells: effects of Ca2+, dihydropyridines and ionomycin

Vitamin D may regulate pituitary function, as there are selective effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on gene expression in clonal pituitary tumour cells, and on TRH-induced TSH release in normal rat pituitary cells in vitro. The role of Ca2+ in 1,25-(OH)2D3-enhanced TSH release from primary rat pituitary cell cultures was investigated. Pretreatment with 10 nmol 1,25-(OH)2D3/l for 24 h augmented KCl (3-60 mmol/l)-induced TSH release over 1 h at all KCl concentrations greater than 7.5 mmol/l (P less than 0.001), with a 76% enhancement of TSH release induced by 30 mmol KCl/l (P less than 0.001). The Ca2+ channel antagonist nifedipine (10 nmol/l-10 mumol/l) caused a concentration-dependent inhibition of KCl (60 mmol/l)-induced TSH secretion. Pretreatment with 1,25-(OH)2D3 enhanced KCl-induced release at all concentrations of nifedipine (P less than 0.001). The Ca2+ selective divalent cation ionophore ionomycin (1 nmol/l-1 mumol/l), and the Ca2+ channel agonist BAY K 8644 (10 nmol/l-1 mumol/l) increased prolactin secretion but did not increase TSH release, and 1,25-(OH)2D3 had no effect. At an extracellular Ca2+ concentration of less than 500 nmol/l, TRH-induced TSH release was observed only after treatment with 1,25-(OH)2D3 (P less than 0.01). As the extracellular Ca2+ concentration was increased, greater increments of TRH-induced TSH release were observed following pretreatment with 1,25-(OH)2D3 (P less than 0.01). However, the effect of 1,25-(OH)2D3 in the thyrotroph was independent of the pretreatment extracellular Ca2+ concentration. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in-vitro responsiveness to TRH and KCl. These data suggest that the action of 1,25-(OH)2D3 in the thyrotroph is to enhance intracellular signal transduction. They further support a permissive or regulatory role of vitamin D in the normal pituitary gland.

Pituitary Vasoactive Intestinal Peptide Regulates Prolactin Secretion in the Hypothyroid Rat

In this study, we demonstrated that the cell content and basal secretion of vasoactive intestinal peptide (VIP) in primary rat pituitary cell cultures were increased in hypothyroidism. VIP release from hypothyroid pituitary cells in vitro was stimulated by thyrotropin releasing hormone (TRH 10(-8) to 10(-6) M) and growth hormone (GH)-releasing hormone (GHRH 10(-9) to 10(-8) M) but not by corticotropin-releasing hormone or luteinizing hormone-releasing hormone in concentrations up to 10(-6) M. In the presence of anti-VIP antisera, there was a significant decrease in basal prolactin secretion from cultured hypothyroid pituitary cells (p less than 0.005) indicating that VIP exerts a tonic stimulatory effect on prolactin (PRL) secretion. The increment in PRL secretion following TRH was not affected by exposure to anti-VIP indicating that PRL release after TRH is not mediated by VIP at the pituitary level. In contrast to changes in PRL, exposure to anti-VIP had no effect on basal GH secretion, indicating that the PRL changes are hormone specific. Similarly, GHRH-induced GH release was unaffected by VIP immunoneutralization.

Endocrine effects of degradation-stabilized thyrotropin-releasing hormone (TRH) analogues on primary rat pituitary cell cultures

Journal Article Endocrine effects of degradation-stabilized thyrotropin-releasing hormone (TRH) analogues on primary rat pituitary cell cultures Get access V Kirschner, V Kirschner Universitäts-Frauenklinik, Abt. für Gyndkologische Endokrinologie, Heidelberg Search for other works by this author on: Oxford Academic Google Scholar L Kiesel, L Kiesel Universitäts-Frauenklinik, Abt. für Gyndkologische Endokrinologie, Heidelberg Search for other works by this author on: Oxford Academic Google Scholar T Rabe, T Rabe Universitäts-Frauenklinik, Abt. für Gyndkologische Endokrinologie, Heidelberg Search for other works by this author on: Oxford Academic Google Scholar B Runnebaum B Runnebaum Universitäts-Frauenklinik, Abt. für Gyndkologische Endokrinologie, Heidelberg Search for other works by this author on: Oxford Academic Google Scholar Acta Endocrinologica (Norway), Volume 117, Issue 4_Supplement, Apr 1988, Pages S94–S95, https://doi.org/10.1530/acta.0.117S094 Published: 01 April 1988

12-O-tetradecanoyl phorbol-13-acetate stimulates rat growth hormone (GH) release through different pathways from that of human pancreatic GH-releasing factor.

The mechanism(s) of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on rat (r) GH release was studied in primary rat pituitary cell cultures. TPA stimulated rGH release (3.2- to 4.1-fold above control value) and rTSH and rLH release (1.4- and 1.7-fold above control values, respectively), but not rPRL release. The ED50 of TPA on rGH secretion was 1.3 X 10(-9) M compared to 4.5 X 10(-11) M for human pancreatic GH-releasing factor [hpGRF-(1-44)]. If maximally effective doses of TPA or hpGRF-(1-44) were added to the cells, the magnitudes of the increase in rGH release were quite similar for both agents when the incubation period was less than 12 h. When (Bu)2cAMP was added simultaneously with various doses of TPA, (Bu)2cAMP increased rGH release beyond the maximal effect of TPA. There was an additive effect when hpGRF-(1-44) and TPA were used to stimulate rGH release. These results indicate that TPA enhances rGH release through a different pathway than hpGRF-(1-44). TPA failed to increase the formation of intra- and extracellular cAMP, whereas hpGRF-(1-44) increased both, suggesting that TPA stimulates rGH release through an cAMP-independent pathway(s). Protein kinase C has been postulated to be a receptor for TPA in human platelets. When phospholipase C, which activates protein kinase C via the formation of diacylglycerol, was added to the cells, rGH release was stimulated in a dose-dependent manner. This effect was not blocked by indomethacin. These results may suggest that activation of protein kinase C leads to rGH release. The observations are consistent with the hypothesis that TPA activates protein kinase C and causes the release of rGH in normal pituitary cells in culture. These findings indicate that the mechanism(s) of action of TPA on rGH release is different from that of hpGRF-(1-44).

GONADOTROPIN RELEASE FROM PITUITARY CULTURES FOLLOWING ACTIVATION OF ENDOGENOUS ION CHANNELS

In primary rat pituitary cell cultures, activation of Na+ channels resulted in Na+ and Ca2+ dependent gonadotropin release. This occurred with the same efficacy and over the same time course as that stimulated by gonadotropin releasing hormone (GnRH). In contrast, GnRH required extracellular Ca2+, but not Na+ for its action. These results indicate that the gonadotrope has ion channels which resemble those found in neural tissues.