Prolactin-deficient variants of GH3 rat pituitary tumor cells: linked expression of prolactin and another hormonally responsive protein in GH3 cells.

GH3 cells normally synthesize and secrete two pituitary polypeptide hormones, prolactin and growth hormone. From an ethyl methane sulfonate-mutagenized population, prolactin low-producing variants have been isolated at a frequency near 20%. Intracellular prolactin synthesis in the variants was reduced 40- to 100-fold compared to wild-type cells while growth hormone synthesis varied less than 2-fold. This decrease was paralleled by a decrease in intracellular preprolactin mRNA. Although reduced, prolactin synthesis was still repressible by glucocorticoids. There was a coordinate loss of expression of p21, a thyroid and glucocorticoid hormone-regulated protein, in GH3 cells, whereas the synthesis and regulation of other hormonally responsive proteins were unimpaired in the variants. Since p21 expression was coordinately regained in a high-producing prolactin revertant cell, expression of the two proteins is tightly coupled in GH3 cells. The stability of the low-producing phenotype differed among variants. One (B2) gave rise to revertants at about 20% frequency even after two rounds of subcloning, whereas another (B3) was more stable in that only 1 weak revertant was found in 47 subclones. The reversion frequency of B3 cells was also measured at less than 0.5%. Unmutagenized GH3 cells were phenotypically stable in that no prolactin-deficient variant was found among 57 subclones. Since variants were ony found after ethyl methane sulfonate mutagenesis, the DNA alkylating agent appears to have promoted an epigenetic change in pituitary gene expression.     

Regulated expression of the prolactin gene in rat pituitary tumor cells

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.     

Platelet-activating factor affects cytosolic free calcium concentration and prolactin secretion in GH4C1 rat pituitary cells.

Platelet-activating factor (PAF) is a naturally occurring pleiotropic mediator which acts via specific membrane receptors. In certain target cells, PAF causes elevations in cytosolic free Ca2+ concentration ([Ca2+]i); however, little is known of the effects of PAF on endocrine cells. Therefore, we have investigated the actions of PAF on [Ca2+]i in prolactin-secreting GH4C1 cells and have compared the effects with the well documented actions on these cells of thyrotropin-releasing hormone (TRH). GH4C1 cells were loaded with quin2/AM and fluorescence was measured in suspended populations. PAF induced a dose-dependent (10-100 microM) rise in [Ca2+]i which was slower in onset than that caused by TRH, peaking (200 to 400% above basal [Ca2+]i) at about 12 sec, and decaying over about 3 min to basal [Ca2+]i. Unlike TRH, PAF did not cause a secondary plateau phase of rise in [Ca2+]i. The terpene PAF receptor antagonist BN52021 inhibited the action of PAF on [Ca2+]i. Voltage-dependent Ca2+ channel blocker, verapamil (200 microM), antagonized the action of PAF on [Ca2+]i as did chelation of extracellular Ca2+. PAF also stimulated the secretion of prolactin in a dose-dependent manner (10 to 50 microM). The concentrations of PAF required to evoke responses in GH4C1 cells were considerably higher than those required in several other known PAF target cell types. The high concentration requirement in GH4C1 cells may be due to rapid degradation of PAF or the presence of low affinity receptors. We conclude that PAF can act, via cell surface receptors, on pituitary GH4C1 cells to alter [Ca2+]i by a pathway that enhances influx of extracellular Ca2+ through voltage-gated channels and then to enhance the secretion of prolactin.     

The intracellular calcium antagonist, TMB-8, inhibits prolactin gene expression in GH3 cells

We examined the effects of the drug, TMB-8, which promotes sequestration of intracellular Ca2+, on the ability of extracellular Ca2+ to stimulate prolactin gene expression in GH3 cells. TMB-8 inhibited prolactin mRNA levels in a dose-dependent manner in the concentration range of 2.5-10 microM. Prolactin mRNA levels were increased about 18-fold by the addition of 0.1 mM CaCl2, and about 25-fold by the addition of 0.4 mM CaCl2. Addition of 10 microM TMB-8 reduced these levels to about 4-fold and 7-fold, respectively. At 10 microM TMB-8 did not effect total protein synthesis or the Ca2+-induced aggregation of the cells, indicating a selective inhibition by the drug of prolactin gene expression. Both TMB-8 and the calmodulin inhibitor, calmidazolium, reversed the effects of Ca2+ on prolactin mRNA levels in cells that had been pretreated for 2 days with 0.4 mM CaCl2.     

Effects of RHC 80267, a diglyceride lipase inhibitor, on prolactin secretion and calcium uptake in GH3 pituitary cells

The effect of the diglyceride lipase inhibitor RHC 80267 on the prolactin secretory process was examined in clonal anterior pituitary GH3 cells. This compound reduced basal prolactin secretion as well as secretion induced by TRH and phospholipase C but not that induced by phorbol myristate acetate. Although exogenous phospholipase C increased diglyceride, no increase in the products of diglyceride lipase was detected. Moreover, low doses of RHC 80267 were observed to effectively block potassium-stimulated 45calcium influx. It is unlikely that RHC 80267 inhibits prolactin release solely by inhibiting diglyceride lipase. These data suggest blockade of plasma membrane calcium channels as an alternate mechanism for the inhibitory actions of RHC 80267 on intact GH3 cells. These observations may have implications for RHC 80267 action in other cell types.     

Stimulation of single L-type calcium channels in rat pituitary GH3 cells by thyrotropin-releasing hormone.

Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels.     

Laminin induces formation of neurite-like processes and potentiates prolactin secretion by GH3 rat pituitary cells

Tumor-derived GH3 rat pituitary cell lines are widely utilized to study mechanisms of prolactin secretion and responsiveness to secretagogues. These cells served here as a model with which to study relationships between shape and function. When GH3 cells were routinely grown in serum-supplemented medium, they exhibited the polygonal phenotype of epithelial cells, with scarce secretory granules. In contrast, when seeded in a serum-free medium, they attached loosely and contained more secretory granules. In both cases, they released prolactin in a nonpolarized manner. We show in the present work that laminin extracted from Englebreth-Holm-Swarm (EHS) tumors was a potent attachment and spreading factor for GH3/B6 cells seeded in serum-free medium. Moreover, it induced the formation of neurite-like processes, which were increased in number and length by chronic treatment with a specific secretagogue, thyroliberin (TRH). These changes in cell shape were correlated with a potentiation of prolactin secretion, both basal and TRH-stimulated. Furthermore, using immunocytochemistry and electron microscopy, we revealed--at the dilated tip of processes--an accumulation not only of prolactin, but also of synaptophysin, a vesicle membrane marker, and of several organelles, such as secretory granules, smooth vesicles, dense bodies and mitochondria. The cytoplasmic processes contained long parallel bundles of microtubules and showed a strong immunoreactivity for beta 2-tubulin. In addition, we found immunocyto-chemical evidence for the presence of 200-k Da neurofilament protein in GH3/B6 cell processes as well as in neurites of cultured hypothalamic neurons. We conclude that, in GH3/B6 cells, laminin induced the differentiation of neurite-like processes, which were the site of polarized organelle transport and exhibited some neuronal markers.     

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.     

Induction of prolactin synthesis in rat pituitary tumor cells by 5-bromodeoxyuridine.

The clonal strain of pituitary tumor cells GH₁2C₁ does not produce detectable amounts of prolactin (<5 ng/mg cell protein per 24 hr), although it does synthesize growth hormone. When GH₁2C₁ cells were grown in the presence of 5-bromodeoxyuridine (BrdU, 3 μg/ml), the cells did produce prolactin as determined by quantitative microcomplement fixation and incorporation of ³H-leucine into ³H-prolactin. BGH₁2C₁ and F₁BGH₁2C₁, two BrdU-resistant (r) substrains derived from GH₁2C₁ which grow in the presence of 30 μg/ml BrdU, also synthesized prolactin (100–500 ng/mg cell protein per 24 hr). Growth of BrdU^r strains was not dependent upon on the presence of the drug in the medium; however, the continued production of prolactin by F₁BGH₁2C₁ cells was dependent upon the presence of BrdU. Growth hormone production in both BrdU^s and BrdU^r strains was not affected by BrdU. Resistance of F₁BGH₁2C₁ cells to BrdU was not due to a defect in BrdU uptake. Thymidine inhibited the incorporation of ³H-BrdU into DNA in both sensitive and resistant strains, and also reduced BrdU-induced prolactin synthesis in F₁BGH₁2C₁. We postulate that induction of prolactin synthesis by BrdU in GH₁2C₁ and F₁BGH₁2C₁ cells is mediated by the incorporation of the drug into cellular DNA. Furthermore, the lack of measurable prolactin synthesis by the parent strain GH₁2C₁ is not due to deletion of the gene for prolactin, but is probably the result of regulatory mechanisms which do not permit expression of this gene.     

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.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour 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 caffeine on the influx of extracellular calcium in GH4C1 pituitary cells

Caffeine increases intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular Ca²⁺ stores. Caffeine also can change [Ca²⁺]_i by affecting Ca²⁺ influx through voltage-operated Ca²⁺ channels (VOCCs). In the present study, we investigated the effects of caffeine on Ca²⁺ entry in GH₄C₁ pituitary cells. Pretreatment of the cells with caffeine attenuated the high K⁺-evoked influx of ⁴⁵Ca²⁺ in a dose-dependent manner. This inhibition was not secondary to the caffeine-evoked elevation of [Ca²⁺]_i because caffeine was able to inhibit VOCCs also in the presence of the intracellular Ca²⁺ chelator BAPTA. However, the inhibitory effect of caffeine on ⁴⁵Ca²⁺ entry appeared to be dependent on the degree of depolarization of the plasma membrane. Only in cells depolarized with relatively high concentrations of K⁺ (20, 35, and 50 mM) was the caffeine-induced inhibition observed. A similar inhibitory effect of caffeine on the high K⁺-evoked calcium and barium entry was observed in experiments using Fura 2. Neither IBMX, forskolin nor dibutyryl cAMP reduced the enhanced [Ca²⁺]_i induced by 50 mM K⁺, suggesting that the effect of caffeine was not due to increased intracellular cAMP. Furthermore, high doses of caffeine inhibited the plateau level of the TRH-induced increase in [Ca²⁺]_i, which is caused partly by influx of Ca²⁺ through VOCCs. The inhibitory effect of caffeine was, in part, due to an hyperpolarization of the plasma membrane observed at high doses of caffeine. On the other hand, low doses of caffeine enhanced depolarization-evoked Ba²⁺ entry as well as the TRH-evoked plateau level of [Ca²⁺]_i. We conclude that caffeine has a dual effect on Ca²⁺ entry through activated VOCCs in GH₄C₁ cells: at low concentrations caffeine enhances Ca²⁺ entry, whereas high concentrations of caffeine block Ca²⁺ entry. J. Cell. Physiol. 171:52–60, 1997. © 1997 Wiley-Liss, Inc.     

Sphingosine kinase regulates voltage operated calcium channels in GH4C1 rat pituitary cells

We have previously shown that sphingosine inhibits depolarisation-induced calcium influx through voltage-operated calcium channels (VOCCs) in GH(4)C(1) cells, whereas sphingosine-1-phosphate (S1P) does not. In the present study we investigated whether sphingosine kinase modulates VOCC activity in GH(4)C(1) cells by removing inhibitory sphingosine. Sphingosine and the structurally similar sphingosine kinase inhibitor dimethylsphingosine (DMS) both rapidly attenuated the calcium influx evoked by depolarisation. The inhibitory effect declined over time to a greater extent in cells treated with sphingosine than in cells treated with DMS, indicating that sphingosine is being metabolised more rapidly. When the specific sphingosine kinase inhibitor 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKi) was added to the cells after depolarisation there was likewise a reduction of the calcium response. This inhibitory effect was slow and reached a plateau about 3 min after application. In contrast, the sphingosine-mediated inhibition was immediate, suggesting that the SKi-induced inhibition was due to build-up of cellular sphingosine. In experiments on cells overexpressing sphingosine kinase, the inhibitory effect of sphingosine was reversed faster than in control cells. The effect was not due to the produced S1P, since S1P did not have any effect on VOCCs even at concentrations as high as 50 microM. In patch-clamp experiments the calcium entry through VOCCs was attenuated in GH(4)C(1) cells overexpressing a kinase-dead sphingosine kinase, compared with cells overexpressing the wild type sphingosine kinase. In addition, in cells treated with SKi the calcium entry through VOCCs was attenuated compared with control cells. Our results provide compelling evidence that sphingosine kinase regulates the function of voltage-operated calcium channels in GH(4)C(1) cells, not through its catalytic product, but by removal of the substrate sphingosine.     

Nuclear calcium signaling by inositol trisphosphate in GH3 pituitary cells

It has been proposed that nuclear and cytosolic Ca(2+) ([Ca(2+)](N) and [Ca(2+)](C)) may be regulated independently. We address here the issue of whether inositol trisphosphate (IP(3)) can, bypassing changes of [Ca(2+)](C), produce direct release of Ca(2+) into the nucleoplasm. We have used targeted aequorins to selectively measure and compare the changes in [Ca(2+)](C) and [Ca(2+)](N) induced by IP(3) in GH(3) pituitary cells. Heparin, an IP(3) inhibitor that does not permeate the nuclear pores, abolished the [Ca(2+)](C) peaks but inhibited only partly the [Ca(2+)](N) peaks. The permeant inhibitor 2-aminoethoxy-diphenyl-borate (2-APB) blocked both responses. Removal of ATP also inhibited more strongly the [Ca(2+)](C) than [Ca(2+)](N) peak. The [Ca(2+)](N) and [Ca(2+)](C) responses differed also in their sensitivity to IP(3), the nuclear response showing higher affinity. Among IP(3) receptors, type 2 (IP(3)R2) has a higher affinity for IP(3) and is not inactivated by ATP removal. We find that IP(3)R2 immunoreactivity is present inside the nucleus whereas the other IP(3)R subtypes are detected only in the cytoplasm. The nuclear envelope (NE) of GH(3) cells showed deep invaginations into the nucleoplasm, with cytosol and cytoplasmic organella inside. These results indicate that GH(3) pituitary cells possess mechanisms able to produce selective increases of [Ca(2+)](N).     

Co-regulation of pituitary tumor cell adhesion and prolactin gene expression by glucocorticoid

Rat 235-1 pituitary tumor cells are lactotrophs producing high levels of prolactin (PRL). Dexamethasone (Dex, 100 nM) inhibits PRL gene expression in 235-1 cells by 50%, while simultaneously decreasing cell replication and cell-cell aggregation. To determine the time course of Dex action, we used a quantitative assay for cell-cell interaction, based on the number of single cells present before and after re-aggregation of dispersed cells. 235-1 cells were cultured in growth medium or medium plus 100 nM Dex for 1–4 days before assay. Control cells had 90% re-aggregation on all days of assay. Aggregation of Dex-treated cells decreased to 55% by day 4. Dex treatment also reduced cell numbers by 40%, but this decrease did not contribute to reduced aggregation. To determine the mechanism of Dex-inhibited cell-cell adhesion, we examined the expression of cadherins and catenins. Cadherin-related mRNAs (P- and N-cadherin probes) were detectable in 235-1 cells, but their levels were unchanged by Dex. A pan-cadherin antibody was unable to detect classical cadherins in these cells. Both α- and β-catenins were detected by Western blotting and their levels were decreased by Dex. Unlike control aggregates, aggregates of Dex-treated cells were able to inhibit expression of PRL mRNA when added to monolayers of 235-1 cells. These data suggest that Dex influences cadherin function by inhibiting catenin expression and that this has the functional consequence of altering 235-1 cell-cell interactions. Overall the data show that Dex affects important aspects of lactotroph function other than PRL gene expression. These changes may include physical alterations in pituitary cell contacts that further support a change in functional state. J. Cell. Physiol. 174:115–124, 1998. © 1998 Wiley-Liss, Inc.