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.     

Binding of thyrotropin releasing hormone and prolactin release by synchronized GH3 rat pituitary cell line.

GH3 cells were synchronized by growing them in a low serum concentration (1%). They were thereafter put back in normal medium (17.5% serum) (time 0 of synchronization). Four parameters were then examined every two hours for up to 40 hours : rate of [³H] thymidine incorporation, cell number, binding of [³H] Thyrotropin Releasing Hormone (TRH) after a 30 min exposure, and prolactin (PRL) content of culture medium and cell extract.The rate of thymidine incorporation presented a 10–20 fold increase in S phase, beginning on 12–16 hours and lasting at 26 hours. The cell population was doubled at 28 hours. [³H] TRH binding to attached cells was observed throughout the cell cycle, but presented a significant increase (40–80%) during the S phase. In contrast, the % increase of PRL release in response to TRH was optimum (300% of control) in G₁ phase. Variations of the PRL cell content as well as of the PRL spontaneous release ability of the cell do not account for the variations of TRH responsiveness. The discrepancy between the two parameters of the TRH-GH3 cells interaction strongly suggest a morphological or functional heterogeneity of the TRH-binding sites.     

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line

Our aim was to determinewhether the expression of K⁺ currents is related to thecell cycle in the excitable GH3 pituitary cell line. K⁺currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cellcycle. We show that the peak density of the transient outward K⁺ current (I_to) was 33% lower incells in S phase (BrdU+) than in cells in other phases of the cellcycle (BrdU). The voltage-dependence of I_towas not modified. However, of the two kinetic components ofI_to inactivation, the characteristics of thefast component differed significantly between BrdU+ and BrdU cells.Recovery from inactivation of I_to showedbiexponential and monoexponential function in BrdU and BrdU+ cells,respectively. This suggests that the molecular basis of this currentvaries during the cell cycle. We further demonstrated that4-aminopyridine, which blocks I_to, inhibited GH3cell proliferation without altering the membrane potential. These datasuggest that I_to may play a role in GH3 cellproliferation processes.

     

Expression and stability of insulin-like growth factor-I (IGF-I) mRNA splicing variants in the GH3 rat pituitary cell line

We have employed Northern blot analyses and solution hybridization/RNase protection assays to evaluate the presence and stability of IGF-I mRNA splicing variants in the GH3 rat pituitary cell line. All of the IGF-I mRNA size classes and IGF-I mRNAs with alternately-spliced 5'-untranslated regions and E-peptide coding regions seen in adult rat liver also were present in GH3 cells, although the proportions of the 5' splicing variants were significantly altered. In actinomycin D-treated cells, all IGF-I mRNA splicing variants were equally stable; thus, changes in the levels of some splicing variants were not due to differential mRNA stability. Additionally, all IGF-I mRNA size classes seen on Northern blots were equally stable; this data suggests that the large IGF-I mRNA species is not a precursor of the smaller species.     

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.

     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH 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.     

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)

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.     

Characterization of bombesin receptors in a rat pituitary cell line

Bombesin is a tetradecapeptide which stimulates prolactin secretion in rats and man and in cultures of GH4C1 cells, a clonal strain of rat pituitary tumor cells. We have utilized [125I-Tyr4]bombesin to identify and characterize specific high affinity receptors in GH4C1 cells. Scatchard analysis of equilibrium binding data at 4 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (RT = 3600 +/- 500 sites/cell). The value for the equilibrium dissociation constant (Kd = 1.2 +/- 0.4 nM) agreed closely with the ED50 (0.5 nM) for bombesin stimulation of prolactin release. [125I-Tyr4]Bombesin binding at steady state at 37 degrees C was inhibited by increasing concentrations of unlabeled bombesin in a dose-dependent manner, with an ID50 = 1.4 +/- 0.2 nM. However, binding of [125I-Tyr4] bombesin was not inhibited by 100 nM thyrotropin-releasing hormone, vasoactive intestinal peptide, epidermal growth factor, or somatostatin. Therefore, [125I-Tyr4]bombesin binds to a receptor distinct from the receptors for other peptides which regulate hormone secretion by GH4C1 cells. The analog specificity for high affinity binding showed that the receptors for bombesin recognize the COOH-terminal octapeptide sequence in the molecule. Among five pituitary cell strains tested, two which contained saturable binding sites for [125I-Tyr4]bombesin (GH4C1 and GH3) had previously been shown to respond to bombesin with increased hormone secretion, whereas three which lacked receptors (GC, F4C1, and AtT20/D16v) were unresponsive. Therefore, the [125I-Tyr4]bombesin binding sites appear to be necessary for the biological actions of bombesin. Examination of the processing and metabolism of receptor-bound peptide demonstrated that at 4 degrees C [125I-Tyr4]bombesin binds to receptors on the surface of GH4C1 cells. At 37 degrees C, receptor-bound peptide is rapidly internalized and subsequently degraded in lysosomes. In summary, we have characterized for the first time specific, high affinity pituitary bombesin receptors which are necessary for the biological action of bombesin.     

Immunolocalization of vascular endothelial growth factor in the GH3 cell line

The question of whether vascular endothelial growth factor (VEGF) is expressed in the GH3 cell line was investigated using immunocytochemistry, immunoelectron microscopy, and Western blotting. Using immunocytochemistry, VEGF was demonstrated in approximately 90% of the cells. Immunopositivity was localized mainly in the paranuclear Golgi region. In a small minority of cells, diffuse cytoplasmic immunostaining was also noted. By immunoelectron microscopy VEGF was evident in the secretory granules, cytoplasmic vesicles, rough endoplasmic reticulum, and the Golgi apparatus. Western blotting confirmed the results of the morphologic studies. It can be concluded that VEGF, which is know to induce angiogenesis and to increase vascular permeability, is produced in the prolactin- and growth hormone (GH)-secreting GH3 cell line. The functional role of VEGF in the GH3 cells is unknown. It is possible that this growth factor affects endocrine activity of GH3 cells by a paracrine mechanism.     

Solubilization and characterization of a membrane 3, 3', 5-triiodo-L-thyronine binding protein from rat pituitary tumor GH3 cells

To understand the mechanism by which T3 enters cells and carries out its biological functions membrane binding sites for 3, 3', 5-triiodo-L-thyronine were solubilized from rat pituitary tumor GH3 cells by detergents. Among three detergents tested, CHAPS is the best in preserving hormonal binding affinity and specificity. Least square analysis of the binding data show one class of binding site with a Kd of (6.35 +/- 1.27) nM and Bmax of (0.84 +/- 0.056) pmoles/50 micrograms protein. Hormone binding activity is lost by heating, pronase digestion and in the absence of NaCl. The pH optimum for binding is 7.0 and the binding activity is enhanced by dithiothreitol. The solubilization of membrane-associated thyroid hormone binding proteins will facilitate further characterization and exploration of their biological functions.     

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.     

Phosphorylation of the endogenous thyrotropin-releasing hormone receptor in pituitary GH3 cells and pituitary tissue revealed by phosphosite-specific antibodies

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.     

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.