Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

The effect of thyrotrophin-releasing hormone (TRH, 10(-7) M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone-releasing hormone (LH-RH, 10(-7) M). Actinomycin D (2 X 10(-5) M) and cycloheximide (10(-4) M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).     

ACTH radioimmunocytochemistry (RICH) on rat anterior pituitary cells

Summary Radioimmunocytochemistry (RICH) was applied to detect corticotrophs in adult rat pituitaries and 8-day-old anterior pituitary monolayers by incubating sections and cultures with ¹²⁵I-ACTH-anti ACTH immune complexes. After incubations autoradiography was made. In comparison, conventional immunostaining was carried out on adjacent sections and parallel cultures. It has been esteblished that RICH is suitable for detection of corticotrophs.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

Effects of TRH on cell proliferation of rat pituitary cells (GH3)

Chronic treatment (more than 3 d) of GH3 cells, cloned rat pituitary cells producing prolactin, with 100 nM TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e. insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity (MSA) instead of serum. TRH stimulated prolactin production by GH3 cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 nM, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 nM, it inhibited the growth significantly. In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on the growth of GH3 cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five factors other than MSA. In conclusion, TRH could inhibit cell growth of GH3 in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium.     

Effects of TRH on cell proliferation of rat pituitary cells (GH3)

Summary Chronic treatment (more than 3 d) of GH₃ cells, cloned rat pituitary cells producing prolactin, with 100 nM TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e. insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity (MSA) instead of serum. TRH stimulated prolactin production by GH₃ cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 nM, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 nM, it inhibited the growth significantly. In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on the growth of GH₃ cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five factors other than MSA. In conclusion, TRH could inhibit cell growth of GH₃ in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium.     

Calcium-independent phosphatidylinositol response in gonadotropin-releasing-hormone-stimulated pituitary cells.

This paper describes the effect of gonadotropin-releasing hormone (GnRH, gonadoliberin) on phospholipid metabolism in cultured rat pituitary cells. The cells were incubated with [32P]Pi to label endogenous phospholipids (10-60 min) and then stimulated with GnRH for up to 60 min. Cellular phospholipids were separated by two-dimensional t.l.c. and the radioactivity was determined. Phosphatidylinositol (PI), a minor constituent of cellular phospholipids (7.7%), was the major labelled phospholipid, accounting for 45% of the total radioactivity, at early periods after pulse labelling. On the other hand, phosphatidylcholine, the major cellular phospholipid (37%), was labelled only to 32% of the total radioactivity. The remaining label was distributed among phosphatidylethanolamine (4.2%), cardiolipin (3.4%), phosphatidic acid (PA, 2.5%), and phosphatidylserine (1.8%). GnRH doubled 32P labelling of PA and PI significantly at 1 and 5 min of incubation respectively in the presence or absence of extracellular Ca2+. Labelling of other phospholipids was not affected by GnRH treatment. The half-maximal stimulating dose (ED50) for PI labelling and lutropin release was 0.75 nM and 0.5 nM respectively, and the stimulatory effect was blocked by the potent GnRH antagonist [D-Glp1,pClPhe2,D-Trp3,6]GnRH. GnRH-stimulated PA and PI labelling could not be demonstrated after 1 and 45 min of incubation respectively, or when the prelabelling was conducted for 60 min rather than 10 min. These results suggest heterogeneous compartmentalization of gonadotroph PA and PI pools and that increased PI turnover might be a transducing signal for Ca2+ gating that follows gonadotroph GnRH-receptor activation.     

Autohistoradiographic localization of thyrotropin-releasing hormone (TRH)-binding sites in cultured rat pituitary cells

Using an autohistoradiographic technique, it has been possible to localize specific binding sites for [³H]thyrotropin-releasing hormone (TRH) in rat anterior pituitary cells in primary culture. A low percentage (3 %) of the cells were highly labelled while 10–20% of the remaining cells showed a lower level of accumulation of radioactivity. These data provide morphological evidence for the presence of TRH-binding sites in pituitary cells and suggest a large variation of their density.     

Brain cortex phosphatidylserine inhibits phosphatidylinositol turnover in rat anterior pituitary glands

The in vitro effect of bovine brain cortex phosphatidylserine on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine of rat anterior pituitary glands was studied. Phosphatidylserine (0.1 to 66.6 microM) decreased the incorporation of 32Pi into phosphatidylinositol, but not phosphatidylcholine or phosphatidylethanolamine, in a concentration-related manner. The inhibitory effect of phosphatidylinositol was similar to that of dopamine in the same experimental conditions. The combined effects of submaximal concentrations of dopamine and phosphatidylserine elicited an apparently additive inhibitory effect on phosphatidylinositol synthesis. The inhibitory effect of phosphatidylserine was completely reversed by haloperidol and sulpiride and only partially by pimozide, antidopaminergic agents which per se do not affect phosphatidylinositol synthesis. The stimulatory effect of TRH to increase 32Pi incorporation into phosphatidylinositol was decreased by phosphatidylserine. These observations suggest that the decrease in prolactin release in the presence of phosphatidylserine may be evoked through a dopaminergic mechanism.     

Mechanism involved in synergistic adrenocorticotropin response to activating protein kinase-A and -C in rat anterior pituitary cells

Activation of protein kinase C (PKC) stimulates adrenocorticotropin (ACTH) release synergistically in the presence of corticotropin releasing factor (CRF). We examined the effect of a cyclic nucleotide-specific phosphodiesterase inhibitor, 1-isoamyl-3-isobutylxanthine (IIX), on arginine vasopressin (AVP)-induced ACTH release and intracellular cAMP accumulation in normal rat anterior pituitary cells. IIX alone elevated intracellular cAMP accumulation. IIX potentiated AVP-induced ACTH release synergistically without further increase in cAMP accumulation, suggesting that synergistic ACTH release has an alternative mechanism other than the synergistic elevation of intracellular cAMP accumulation which has been reported. Phorbol 12-myristate-13-acetate (PMA) also induced synergistic ACTH release when incubated with IIX. IIX had no additional effect on ACTH response when incubated with maximal dose of CRF, forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Moreover, the combination of PMA and 8-Br-cAMP produced synergistic ACTH response. In conclusion, the synergistic ACTH release from rat pituitary corticotrophs occurs at least in the presence of directly activating events of PKC and PKA as well as PKC-induced inhibition of phosphodiesterase activity.     

ACTH radioimmunocytochemistry (RICH) on rat anterior pituitary cells.

Radioimmunocytochemistry (RICH) was applied to detect corticotrophs in adult rat pituitaries and 8-day-old anterior pituitary monolayers by incubating sections and cultures with 125I-ACTH-anti ACTH immune complexes. After incubations autoradiography was made. In comparison, "conventional" immunostaining was carried out on adjacent sections and parallel cultures. It has been established that RICH is suitable for detection of corticotrophs.     

Thyroidectomy cells and their response to thyrotrophin releasing hormone (TRH) in the rat

Summary Thyroidectomy cells of the rat pituitary gland were studied by the peroxidase-antibody labeling procedure and by electron microscopy. Secretory granules accumulated in these cells in response to a short-term treatment with thyroxine, and the cells were then reactive to the peroxidase-antibody labeling procedure. An intravenous injection of synthetic thyrotrophin releasing hormone (TRH) to thyroxine-treated, thyroidectomized rats provoked an acute and active extrusion of secretory granules from the thyroidectomy cells. The secretory granules in these cells were mostly haloed after primary fixation in osmium tetroxide. It is concluded that TRH causes thyroidectomy cells to release their secretory granules, and presumably TSH, by the usual process of exocytosis or granule extrusion.This study was supported by USPHS Grant AM 12583.     

Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect.

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoreactive thyroliberin (TRH) precursor forms in human hypothalamus and anterior pituitary tissues.

Immunoreactive (IR) proTRH forms were characterized in human hypothalamic tissue with two antisera raised against a hepta- and a decapeptide containing the TRH progenitor sequence (-Gln-His-Pro-Gly-). A similar study was performed in human normal and adenomatous anterior pituitaries, tissues in which TRH synthesis has been previously suggested. IR-proTRH was found in all the samples ranging from 42-775 fmol/mg proteins. Size exclusion chromatography identified a major 25-35 kDa form and a minor 4-8 kDa form. The existence of the major form was confirmed by immunoblotting. The results suggest that both human hypothalamic and normal or adenomatous anterior pituitary tissues synthesize similar IR-proTRH forms.     

Effects of VIP, TRH, GABA and dopamine on prolactin release from superfused rat anterior pituitary cells

Effects of VIP, TRH, dopamine and GABA on the secretion of prolactin (PRL) from rat pituitary cells were studied in vitro with a sensitive superfusion method. Dispersed anterior pituitary cells were placed on a Sephadex G-25 column and continuously eluted with KRBG buffer. Infusion of TRH (10(-11) - 10(-8)M) and VIP (10(-9) - 10(-6)M) resulted in a dose-related increase in PRL release. LHRH (10(-8) - 10(-5)M) had no effect on PRL release. On the other hand, infusion of dopamine (10(-9) - 10(-6)M) and GABA (10(-8) - 10(-4)M) suppressed not only the basal PRL release from dispersed pituitary cells but also the PRL response to TRH and VIP. The potency of TRH to stimulate PRL release is greater than that of VIP, and the potency of dopamine to inhibit PRL secretion is stronger than that of GABA on a molar basis. These results indicate that TRH and VIP have a stimulating role whereas dopamine and GABA have an inhibitory role in the regulation of PRL secretion at the pituitary level in the rat.     

Mechanism of action of benzo(a)pyrene and nicotine on hormone production by rat pituitary tumor cells

The effects of the cyclic aromatic hydrocarbon, benzo(a)pyrene (BaP) and that of the tobacco alkaloid, nicotine, on prolactin (PRL) and growth hormone (GH) synthesis by rat pituitary tumor cells in culture (GH cells) have been studied. Treatment of GH cells with nicotine (0.1–300 μg/ml) neither affected the growth, nor significantly altered the general pattern of hormone production in these cells. BaP at concentrations greater than 5 μg/ml irreversively inhibited the growth of these cells. The sublethal concentrations of BaP, which did not affect either 1) cell growth, or 2) amino acid transport or 3) total protein synthesis or degradation, did however inhibit specifically, hormone synthesis by these cells. More interestingly concentrations of nicotine which did not affect either cell growth or hormone synthesis, modulated both of these cellular processes in the presence of BaP. A concentration dependent stimulation of microsomal BaP monooxygenase activity was observed in nicotine or BaP treated cells. The effects of these drugs on stimulation of BaP monooxygenase activity seems to be additive. Nicotine also enhanced the association of radioactivity (presumably [³H] BaP metabolites) with DNA in [³H] BaP treated cells. It is concluded that nicotine by itself did not demonstrate any cytotoxic effect nor influence hormone synthesis in GH cells. However, this constituent of tobacco smoke stimulated BaP monooxygenase activity and the interaction of [³H] BaP metabolites with cellular DNA and also modulated BaP induced inhibition of hormone synthesis in GH cells.     

Functional ATP receptors in rat anterior pituitary cells

The effects ofATP and other nucleotides on the cytosolicCa²⁺ concentration([Ca²⁺]_i)of single immunocytochemically typed anterior pituitary (AP) cells havebeen studied. ATP increased[Ca²⁺]_iin a large percentage (60-88%) of all five AP cell types:lactotropes, somatotropes, corticotropes, gonadotropes, andthyrotropes. Additivity experiments suggest the presence of at leasttwo different receptors, one accepting both ATP and UTP (U receptor),producing Ca²⁺ release from theintracellular stores, and the other preferring ATP (A receptor),producing Ca²⁺ (andMn²⁺) entry. The characteristicsof the U and A receptors were consistent with those ofP2Y₂ andP2X₂, respectively, and theirdistribution in the different AP cell types was not homogeneous. Thepresence of other ATP receptors suchP2Y₁ orP2X₂/P2X₃heteropolymers in a small fraction of the cells cannot be excluded.Thus functional ionophoric P2X receptors, which are typical of neuraltissue, are also present in the pituitary gland and could contribute to regulation of the gland's function.