Saccharin induces morphological changes and enhances prolactin production in GH4C1 cells

Summary The artificial sweetener saccharin inhibits binding of epidermal growth factor (EGF) to cultured rat pituitary tumor cells (GH₄C₁ cells). Saccharin also causes morphological alterations in these cells, resulting in pronounced elongation, stretching, and firmer attachment of cells to the culture dishes. These alterations in cell shape are similar to those observed after treatment of GH₄C₁ cells with EGF and with thyrotropin-releasing hormone (TRH), both of which enhance prolactin (PRL) production in these cells. After assaying for PRL in saccharin-treated cultures, it was observed that this sweetener is also capable of stimulating PRL production two-to sixfold in a dose-dependent manner. Enhancement of PRL production can be observed at 0.5 mM saccharin, yet this is 10 times less than the saccharin concentration required to alter cell shape. These effects of saccharin on cell morphology and on PRL production are reversible in GH₄C₁ cell cultures. When added to cultures along with maximal concentrations of EGF or TRH, the effects of saccharin on PRL production are additive, suggesting that the actions of saccharin are mediated by a somewhat different pathway from that of the peptide hormones. Pulse labeling studies indicate that the enhancement of PRL production is highly specific inasmuch as saccharin was found to decrease the overall rate of protein synthesis in these cells. Saccharin also causes a decrease in the rate of DNA synthesis under these treatment conditions. Mitomycin C, which similarly inhibited DNA synthesis, had no effect on cell morphology or PRL production. This investigation was supported by a Faculty Research Grant from Wheaton College     

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.     

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.     

n-Butyrate effects thyroid hormone stimulation of prolactin production and mRNA levels in GH1 cells

Using cultured GH1 cells, a growth hormone and prolactin-producing rat pituitary cell line, we have shown that n-butyrate and other short chain carboxylic acids stimulate histone acetylation and elicit a reduction of thyroid hormone nuclear receptor which is inversely related to the extent of acetylation (Samuels, H. H., Stanley, F., Casanova, J., and Shao, T. C. (1980) J. Biol. Chem. 255, 2499-2508). In this study, we compared the n-butyrate and propionate modulation of receptor levels to regulation of the growth hormone and prolactin response by 3,5,3'-triiodo-L-thyronine (L-T3). n-Butyrate (0.1-10 mM) did not stimulate growth hormone production. L-T3 stimulated the growth hormone response 4- to 5-fold and n-butyrate (0.5-1 mM) increased L-T3 stimulation of growth hormone production 1.5- to 2-fold compared to L-T3 alone. L-T3 stimulation of growth hormone production at higher n-butyrate concentrations decreased in parallel with the n-butyrate-mediated reduction of receptor levels. In contrast with the growth hormone response, n-butyrate (0.5 mM) increased basal prolactin production about 5-fold. Prolactin production, which is inhibited 25 to 50% by L-T3, was stimulated between 20- and 70-fold by L-T3 + n-butyrate (0.5-1 mM) and this decreased at higher n-butyrate levels. Prolactin mRNA and growth hormone mRNA levels paralleled the changes in prolactin and growth hormone production rates. These effects of L-T3, n-butyrate, or L-T3 + n-butyrate appeared unrelated to changes in cAMP levels or global changes in DNA methylation of the growth hormone or prolactin genes. Propionate elicited the same effects as n-butyrate but at a 5- to 10-fold higher concentration consistent with their relative effect on stimulating acetylation of chromatin proteins. These results suggest that prolactin gene expression is under partial regulatory repression which is reversed by a carboxylic acid-mediated postsynthetic modification event which allows for stimulation of the prolactin gene by thyroid hormone.     

Phytohemagglutinin alters cell morphology and decreases prolactin production in GH cells

Phytohemagglutinin (PHA) produced morphological and functional alterations in a clonal strain of rat pituitary tumor cells (GH₄C₁). Addition of PHA (2–5 μg/ml) results in a decrease in the proportion of elongated cells from 20% in control cell cultures to less than 10% in the presence of PHA. This effect can be observed after exposure of cells to PHA for 2–3 h and requires 4 days to be reversed after removing PHA from the culture medium. A specialized cell function, the production of the peptide hormone prolactin (PRL), is also affected by PHA treatment. Exposure of cells to 2 μg/ml PHA results in greater than 50% inhibition of PRL production. The above effects of PHA occur without any apparent alteration in total protein per culture dish, the rate of protein synthesis or the overall growth characteristics of the cells.     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Autocrine-paracrine inhibition of growth hormone and prolactin production by GH3 cell-conditioned medium

Summary In previous work we have shown that perifused GH₃ cells exhibit spontaneously accelerating growth hormone (GH) and prolactin (PRL) secretory rates. This behavior contrasts with GH and PRL secretion rates that are decreasing or stable over the same 3-d period in static cell culture. We now report that GH₃ cells maintained in serum-supplemented medium produce an autocrine-paracrine factor(s) which inhibits GH secretion in plate culture; PRL release is frequently reduced as well. The inhibitory effect of conditioned medium on GH secretion was concentration dependent, whereas PRL release was stimulated at low and inhibited at high concentrations over the same range. Extensive dialysis of conditioned medium using membranes with a molecular weight cut-off of 12 000–14 000 did not remove GH inhibition but produced a retentate that stimulated PRL secretion. Heat-inactivation of conditioned medium did not abolish inhibition of GH release but did remove the PRL-stimulatory effect. IGF-I added to fresh culture medium did not reproduce the GH-inhibitory effects of conditioned medium. We conclude that GH₃ cell secretory behavior in perifusion and plate culture systems may be partially explained by the production of an autocrine-paracrine factor: its accumulation in plate culture inhibits GH and PRL secretion whereas its removal, by perifusing medium, allows GH and PRL secretion to accelerate. Supported by grant DK33388 to M. E. S. from the National Institute of Health, Bethesda, MD, and in part by the Medical Research Service of the Veterans Administration, Washington, DC.     

Intracellular free sodium concentrations in GH4C1 cells

In the present investigation, intracellular sodium ([Na⁺]_i) levels were determined in GH₄C₁ cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na⁺]_i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na⁺. Resting [Na⁺]_i in GH₄C₁ cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na⁺-buffer [Na⁺]_i decreased to 3 ± 3.6 mM. If Na⁺/K⁺ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na⁺]_i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na⁺]_i. Incubating the cells in Ca²⁺-buffer rapidly increased [Na⁺]_i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca²⁺, nimodipine, or Ni²⁺ to these cells immediately decreased [Na⁺]_i, whereas Bay K 8644 enhanced the influx of Na⁺. In cells where [Na⁺]_i was increased the TRH-induced increase in intracellular free calcium ([Ca²⁺]_i) was decreased compared with control cells. Our results suggest that Na⁺ enters the cells via Ca²⁺ channels, and [Na⁺]_i may attenuate TRH-induced changes in [Ca²⁺]_i in GH₄C₁ cells. © 1993 Wiley-Liss, Inc.     

Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.

TRH regulates PRL secretion and synthesis in GH4C1 rat pituitary cells. TRH responses are associated with activation of protein kinase C (PKC) isozymes and elevation of cytosolic calcium. To determine which PKC isozymes are involved in TRH-directed responses, we evaluated the effect of TRH on GH cell alpha-, beta-, delta-, and epsilon-PKC isozymes. Immunoblot analysis demonstrated that TRH caused rapid redistribution of all isozymes to a Triton X-100-insoluble (i.e. cytoskeletal) fraction. Corollary immunocytofluorescence studies demonstrated that redistributed PKCs accumulate in cell peripheries. Exocytosis involves reorganization of the cytoskeleton, therefore, each of the GH cell PKCs is appropriately located to phosphorylate proteins important for cytoskeleton organization. To determine the relative contributions of calcium and PKC signal transduction pathways in mediating TRH responses, the effects of potassium depolarization (which increases cytosolic calcium) and phorbol dibutyrate (which activates all PKC isozymes without increasing calcium) were compared. The data indicate that TRH-mediated reorganization of vinculin proceeds via a calcium-mediated pathway, whereas fragmentation of actin filaments proceeds via a PKC-dependent pathway. Selective down-modulation of epsilon-PKC with prolonged TRH-treatment was used to demonstrate that epsilon-PKC is not necessary for certain TRH-stimulated biological responses.     

Bombesin stimulates prolactin secretion from cultured rat pituitary tumour cells (GH4C1) via activation of phospholipase C

Bombesin (BBS) stimulated prolactin (PRL) secretion from monolayer cultures of rat pituitary tumour cells (GH4C1) in a dose-dependent manner with half maximal and maximal effect at 2 nM and 100 nM, respectively. No additional stimulatory effect on PRL secretion was seen when BBS was combined with thyroliberin (TRH) used in concentrations known to give maximal effects, while the effects of BBS and vasoactive intestinal peptide (VIP) were additive. Using a parafusion system, BBS (1 microM) was found to increase PRL secretion within 4 s and the secretion profiles elicited by BBS and TRH (1 microM) were similar. Both BBS and TRH increased inositoltrisphosphate (IP3) as well as inositolbisphosphate (IP2) formation within 2 s. BBS also induced the same biphasic changes in the electrical membrane properties of GH4C1 cells as TRH, and both peptides caused a rapid and sustained increase in intracellular [Ca2+]. These results suggest that BBS stimulates PRL secretion from the GH4C1 cells via a mechanism involving the immediate formation of IP3 thus resembling the action of TRH.     

G protein preferences for dopamine D2 inhibition of prolactin secretion and DNA synthesis in GH4 pituitary cells

Dopamine is the primary inhibitory regulator of lactotroph proliferation and prolactin (PRL) secretion in vivo, acting via dopamine D2 receptors (short D2S and long D2L forms). In GH4C1 pituitary cells transfected with D2S or D2L receptor cDNA, dopamine inhibits PRL secretion and DNA synthesis. These actions were blocked by pertussis toxin, implicating G(i)/G(o) proteins. To address roles of specific G(i)/G(o)4 proteins in these actions a series of GH4C1 cell lines specifically depleted of individual Galpha subunits was examined. D2S-mediated inhibition of BayK8644-stimulated PRL secretion was primarily dependent on G(o) over G(i), as observed for BayK8644-induced calcium influx. By contrast, inhibitory coupling of the D2S receptor to TRH-induced PRL secretion was partially impaired by depletion of any single G protein, but especially G(i)3. Inhibitory coupling of D2L receptors to PRL secretion required G(o), but not G(i)2, muscarinic receptor coupling was resistant to depletion of any G(i)/G(o) protein, whereas the 5-HT1A and somatostatin receptors required G(i)2 or G(i)3 for coupling. The various receptors also demonstrated distinct G protein requirements for inhibition of DNA synthesis: depletion of any G(i)/G(o) subunit completely uncoupled the D2S receptor, the D2L receptor was uncoupled by depletion of G(i)2, and muscarinic and somatostatin receptors were resistant to depletion of G(i)2 only. These results demonstrate distinct receptor-G protein preferences for inhibition of TRH-induced PRL secretion and DNA synthesis.     

Bombesin stimulates inositol polyphosphate production in GH4C1 pituitary tumor cells: comparison with TRH

The hormones bombesin and thyrotropin-releasing hormone (TRH) stimulated formation of inositol- monophosphate, bisphosphate, trisphosphate and tetrakisphosphate with parallel time courses in GH4C1 cells, while a more polar inositol polyphosphate peak, consisting of inositol-pentakisphosphate and perhaps also inositol-hexakisphosphate, was unaffected by either hormone. Although bombesin and TRH had similar potencies in stimulating inositol trisphosphate production (Km = 30 nM and 40 nM, respectively), TRH was significantly more efficacious than bombesin. Maximal stimulation of inositol-1,4,5-trisphosphate formation by TRH was not further increased by addition of a maximally effective dose of bombesin, suggesting that the two hormones act through stimulation of a common pool of phospholipase C, and this enzyme pool can be fully stimulated by TRH, alone.     

Muscarinic inhibition of prolactin production in cultures of rat pituitary cells

Acetylcholine inhibits prolactin production from cell cultures of rat pituitary glands with a half-maximal effect at about 0.2 μM, and from GH-cells, clonal strains of rat pituitary cells, with a half-maximal effect at about 1 μM. The inhibition ranges between 80 and 40 % of control values. Inhibition is detectable at 2 hours, and continues for days in the presence of the anticholinesterase, eserine. Muscarinic agonists mimic the cholinergic inhibition and nicotinic agonists do not. The inhibition is blocked by atropine, a muscarinic antagonist, and not by hexamethonium, a nicotinic antagonist.     

Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells.

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, Bmax = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp-dependent shifts in [3H]N-0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1-10 microM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Ionomycin inhibits thyrotropin-releasing hormone-induced translocation of protein kinase C in GH4C1 pituitary cells

Thyrotropin-releasing hormone (TRH) induces rapid and transient conversion of protein kinase C (Ca2+/phospholipid-dependent enzyme) from a soluble to a particulate-bound form in GH4C1 rat pituitary cells. Ionomycin (200 nM), a calcium ionophore, had no effect by itself on the subcellular distribution of protein kinase C. However, pretreatment of the cells with 200 nM ionomycin inhibited by greater than 50% the ability of TRH to cause translocation of protein kinase C from the cytosol to the particulate cell fraction. Inhibition by ionomycin required that the cells be incubated with the ionophore for at least 10 s before TRH addition. Ionomycin pretreatment did not alter the kinetics of TRH-induced protein kinase C redistribution. Incubation of the cells with 43 mM potassium prior to TRH addition almost completely reversed the inhibition induced by ionomycin. We propose that the mechanism by which ionomycin attenuates TRH action on protein kinase C may involve the capacity of the ionophore to empty the intracellular calcium reservoir which normally releases calcium into the cytosol in response to TRH. Our result provides evidence that the rise in intracellular calcium, which accompanies diacylglycerol formation following TRH action on polyphosphatidylinositide hydrolysis, may be required to achieve maximal conversion of protein kinase C to its presumed active, membrane-bound form in these cells.     

Thyrotropin-releasing hormone induces redistribution of protein kinase C in GH4C1 rat pituitary cells

Thyrotropin-releasing hormone (TRH) affects hormone secretion and synthesis in GH4C1 cells, a clonal strain of rat pituitary cells. Recent evidence suggests that the intracellular mediators, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which are generated as a result of TRH-induced hydrolysis of the polyphosphatidylinositols, may be responsible for some of the physiological events regulated by TRH. Because diacylglycerol is an activator of protein kinase C, we have examined a role for this enzyme in TRH action. The subcellular distribution of protein kinase C in control and TRH-treated cells was determined by measuring both enzyme activity and 12,13-[3H]phorbol dibutyrate binding in the cytosol and by measuring enzyme activity in the particulate fraction. Acute exposure of GH4C1 cells to TRH resulted in a decrease of cytosolic protein kinase C, and an increase in the level of the enzyme associated with the particulate fraction. The redistribution of protein kinase C induced by TRH was dose- and time-dependent, with maximal effects occurring within the first minute of TRH treatment. Analogs of TRH which do not bind to the TRH receptor did not induce redistribution of protein kinase C, while the active analog, methyl-TRH, did promote redistribution. Treatment of GH4C1 cells with phorbol myristate acetate also resulted in a shift in protein kinase C distribution, although the response was slower than that produced by TRH. TRH-induced redistribution of protein kinase C implies translocation of the enzyme from a soluble to a membrane-associated form. Because protein kinase C requires a lipid environment for activity, association with the membrane fraction of the cell suggests activation of the enzyme; thus, protein kinase C may play a role in some of the actions of TRH on GH4C1 cells.