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.     

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 thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: a comparison between serum-free and serum-supplemented media

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.     

Polyphosphoinositide hydrolysis by phospholipase C is accelerated by thyrotropin releasing hormone (TRH) in clonal rat pituitary cells (GH3 cells)

Thyrotropin releasing hormone (TRH) accelerates the turnover of phosphatidylinositol in GH3 cells ('phospholipid response'). From the analysis of inositol phosphates in the presence of Li+ which inhibits their dephosphorylation, it can be concluded that the hydrolysis of phosphatidylinositol 4,5-biphosphate, and possibly of phosphatidylinositol 4-phosphate by phospholipase C is markedly accelerated by TRH. It appears that this reaction initiates the acceleration of phosphatidylinositol turnover. The specificity of hormonally regulated phospholipase C reaction for polyphosphoinositides has important implications for the potential role of the phospholipid response as a mechanism of membrane signal transduction.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

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)     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).     

Potassium channel inhibition reduces cell proliferation in the GH3 pituitary cell line

Potassium (K⁺) conductances are known to be involved in cell proliferation of a number of nonexcitable cell types. The nature of the mechanism by which K⁺ channel inhibition reduces cell proliferation has remained elusive despite intensive search. We investigated whether such a phenomenon could be demonstrated in excitable cells, using the GH3 pituitary cell line as a cell model. Our aims were: (1) to study the effect of K⁺ channel inhibition on the proliferation of GH3 cells; and (2) to investigate the putative intracellular signals involved in this inhibition. Tetraethylammonium chloride (TEA), a blocker of the calcium (Ca²⁺)-dependent K⁺ conductances of GH3, was found to reversibly inhibit cell proliferation, as measured by ³H-thymidine incorporation. Cell cycle block specifically occurred at the G1/S phase of the cell cycle. This inhibition of proliferation was observed for 1–4 mM TEA, which suppressed most of the Ca²⁺-activated K⁺ current and part of the inward rectifying K⁺ current, as shown by electrophysiological experiments. Increasing extracellular K⁺ concentrations with KCl also inhibited cell proliferation in a dose-dependent manner. Both TEA and KCl depolarized the cells and increased intracellular Ca²⁺ levels ([Ca²⁺]i), showing that, in this type of excitable cell, inhibition of cell proliferation can be associated with elevated Ca²⁺ levels. Ca²⁺ and membrane resting potential (MRP) were considered as possible messengers of this inhibition. Our results suggest that cell cycle arrest of GH3 cells by K⁺ channel block probably involves an additional pathway, distinct from those of Ca²⁺ and MRP. J. Cell. Physiol. 177:402–410, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells)

Changes in the cytosolic free Ca2+ concentration, [Ca2+]i, have been proposed to mediate the regulation of the secretion of pituitary hormones by hypothalamic peptides. Using an intracellularly trapped fluorescent Ca2+ probe, quin2, [Ca2+]i was monitored in GH3 cells. Somatostatin lowers [Ca2+]i in a dose dependent manner from a prestimulatory level of 120 +/- 4 nM (SEM, n = 13) to 78 +/- 9 nM (n = 5) at 10(-7)M; the effect is half maximal at 2 X 10(-9) M somatostatin. The decrease in [Ca2+]i occurs rapidly after somatostatin addition and a lowered steady state [Ca2+]i is maintained for several minutes. Somatostatin does not inhibit the rapid rise in [Ca2+]i elicited by thyrotropin releasing hormone (TRH) and can still cause a decrease in [Ca2+]i in the presence of TRH (10(-7)M). Concomitantly with its action on [Ca2+]i somatostatin causes hyperpolarization of GH3 cells assessed with the fluorescent probe bis-oxonol. The lowering of [Ca2+]i by somatostatin is however not only due to reduced Ca2+ influx through voltage dependent Ca2+ channels, since it persists in the presence of the channel blocker verapamil. These results suggest that somatostatin may exert its inhibitory action on pituitary hormone secretion by decreasing [Ca2+]i.     

d-Aspartate in a prolactin-secreting clonal strain of rat pituitary tumor cells (GH(3))

d-Aspartate (d-Asp) is found in prolactin (PRL)-containing cells of the rat anterior pituitary gland [Lee et al., Brain Res. 838, 193-199, 1999]. In order to determine whether d-Asp is actually produced by the anterior pituitary gland and whether it plays a physiological role in PRL function, a PRL-secreting clonal strain of rat pituitary tumor cells (GH(3)) was employed in this study. HPLC analysis and immunocytochemical staining detected the presence and synthesis of d-Asp in the cytoplasm of these cells. In addition, thyrotropin-releasing hormone-stimulated PRL secretion was increased in a dose-dependent fashion by d-Asp from these cells. These results suggest that the anterior pituitary gland synthesizes d-Asp and that d-Asp acts as a messenger in this gland.     

The mechanism of [3H]dexamethasone uptake into prolactin producing rat pituitary cells (GH3 cells) in culture

Glucocorticosteroids stimulate growth hormone (GH) synthesis and inhibit prolactin (PRL) synthesis and cell growth in cultured GH3 cells, a clonal cell strain derived from a rat pituitary tumour. This model system was used to study the mechanism by which glucocorticosteroids enter target cells. The cellular uptake of [3H]dexamethasone was temperature dependent and was further inhibited by addition of an excess amount of cold dexamethasone. Half maximal uptake was obtained after about 5 min at 37 degrees C. The initial rates of [3H]dexamethasone uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]dexamethasone in intact cells studied at different temperatures resulted in linear Arrhenius plots, with a calculated energy of activation of 91.0 kJ x mole-1 x degree-1. Scatchard analysis of specifically cell bound [3H]dexamethasone at equilibrium (0 degrees C) showed a straight line with a calculated dissociation constant (Kd) of 1.6 x 10(-9) M and a maximal uptake of 180 x 10(-15) mole/mg cell protein. Specific binding of [3H]dexamethasone to cytosol proteins could only be demonstrated at 0 degrees C. These results indicate that [3H]dexamethasone diffuses passively into the cell, and binds to specific receptors in an energy dependent way.     

Hydrocortisone binding receptor in the rat pituitary GH3 cell line.

A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH₃ cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [³H]HC with the receptor has an apparent dissociation constant (K_d) of about 6.0 × 10^?9M and a concentration of binding sites of approx. 1 × 10^?13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10⁶ M^?1 min^?1. The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [³H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.     

Ketoconazole inhibits the conductance K+ induced by thyreoliberin (TRH) in GH3 cells

Ketoconazole an imidazole derivative used in clinical practice as an antifungal medicine interferes with the phospholipid dependent "second messenger" pathway. In this work we show that in the GH3 clonal cell line, ketoconazole (10 microM) inhibits part of the complex pattern of electrical activity induced by Thyrotropin-Releasing Hormone (TRH, 10 nM). The phase blocked by ketoconazole represents the Ca2+ -activated K+ conductance.     

Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis

Background  

Prolactinoma is the most frequent pituitary tumor in humans. The dopamine D₂ receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma.     

Electroporation of rat pituitary (GH) cell lines: optimal parameters and effects on endogenous hormone production

An efficient electroporation procedure was established for the genetic transformation of two clonal strains of hormone producing rat pituitary cells (GH12C1 and GH3). We used the bacterial chloramphenicol acetyltransferase (CAT) gene as reporter gene to determine optimal conditions for electroporation. The conditions found to be optimal, measured as expression of the highest CAT activity, were 240-300 V and a DNA concentration of 30-60 micrograms/ml in sucrose buffer. Cell viability was then about 50 per cent. Maximum CAT activity was seen 24 hours after electroporation. The electroporation procedure, in the presence or absence of DNA, caused a transient decrease in endogenous growth hormone (GH) and prolactin (PRL) production.     

Effects of fatty acids on BK channels in GH(3) cells

Ca²⁺-activated K⁺ (BK) channels inGH₃ cells are activated by arachidonic acid (AA). Becausecytosolic phospholipase A₂ can produce other unsaturatedfree fatty acids (FFA), we examined the effects of FFA on BK channelsin excised patches. Control recordings were made at several holdingpotentials. The desired FFA was added to the bath solution, and thevoltage paradigm was repeated. AA increased the activity of BK channelsby 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic,linoleic, linolenic, eicosapentaenoic, and the triple bond analog ofAA, eicosatetraynoic acid, all increased BK channel activity, whereasstearic (saturated) or the trans isomers elaidic,linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationshipsto the left without a change in slope, suggesting no change in thesensitivity of the voltage sensor. Measurements of membrane fluidityshowed no correlation between the change of membrane fluidity and thechange in BK channel activation. In addition, AA effects on BK channelswere unaffected in the presence of N-acetylcysteine.Arachidonyl-CoA, a membrane impermeable analog of AA, activateschannels when applied to the cytosolic surface of excised patches,suggesting an effect of FFAs from the cytosolic surface of BK channels.Our data imply a direct interaction between cis FFA and theBK channel protein.

     

Potassium channel expression level is dependent on the proliferation state in the GH3 pituitary cell line

Previously, we showed that the peakdensity of the transient outward K⁺ current(I_to) expressed in GH3 cells was different inthe S phase than in other phases of the cell cycle. Using cellsynchronization, we show here that I_to dropsprecisely at the quiescent (G₀ phase)/proliferating transition. This change is not due to a modification in the voltage dependence of I_to, but rather to a modificationin its inactivation kinetics. Molecular determination of K⁺channel subunits showed that I_to required theexpression of Kv1.4, Kv4.1, and Kv4.3. We found that the increase inI_to density during the quiescent state wasaccompanied by an increase in Kv1.4 protein expression, whereas Kv4.3expression remained unchanged. We further demonstrate that the linkbetween I_to expression and cell proliferation isnot mediated by variations in cell excitability. These results providenew evidence for the cell cycle dependence ofI_to expression, which could be relevant inunderstanding the mechanisms leading to pituitary adenomas.

     

Long-term induced effects by only one treatment with ketoconazole on rat tumoral pituitary cells (GH3/B6 strains)

Ketoconazole, an imidazole is a powerful antimycotic that has recently been used in the treatment of endocrinological and lipid metabolism disorders as well as in anti-cancer chemotherapy. When rat mammosomatotropic cells are treated for 30 hrs., this drug produces different effects depending on whether cells are normal or tumoral (GH3/B6). A dose of 10 microM had no effect in normal cells, however, in tumoral cells it had dramatic effects: (i) 50% of the cells are killed and those which survive no longer proliferate and they secrete GH but not PRL; (ii) they respond to GHRH in a dose-dependent manner, while normal cells do not under similar culture conditions; (iii) the lipid composition of the membrane is modified as indicated by the increase in arachidonic acid turn-over and the dramatic change in the distribution of its metabolites. For the moment we cannot explain these data.