Role of BK potassium channels shaping action potentials and the associated [Ca(2+)](i) oscillations in GH(3) rat anterior pituitary cells

Measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch clamped GH(3) cells to determine the contribution of large-conductance calcium-activated K(+) (BK) channels to action potential repolarization and size of the associated Ca(2+) oscillations. By examining the dependence of action potential (AP) duration on extracellular Ca(2+) levels in the presence and the absence of the specific BK channel blocker paxilline, it is observed that plateau-like action potentials are associated to low densities of paxilline-sensitive currents. Extracellular Ca(2+) increases or paxilline additions are not able to largely modify action potential duration in cells showing a reduced expression of BK currents. Furthermore, specific blockade of these currents with paxilline systematically elongates AP duration, but only under conditions in which short APs and/or prominent BK currents recorded under voltage-clamp mode are present in the same cells. Our data indicate that in GH(3) cells, BK channels act primarily ending the action potential and suggest that by contributing to fine-tuning cellular electrical properties and hence intracellular Ca(2+) variations, BK channels may play an important role on time- and cell-dependent modulation of physiological outputs in adenohypophyseal cells.     

The mechanism of 17 beta-estradiol uptake into prolactin-producing rat pituitary cells (GH3 cells) in culture

Estrogens stimulate PRL synthesis in cultured GH3 cells, which are clonal strains derived from the rat pituitary gland. This model system was used to study the mechanism by which 17 beta-estradiol (E2) enters target cells. The cellular uptake of [3H]E2 was rapid at 37 C and reached half-maximal values within 10-15 sec. Maximal uptake was observed in less than 2 min at 37 C. The initial rates of E2 uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]E2 in intact cells and the binding to cytosol studied at different temperatures resulted in linear Arrhenius plots, and the energies of activation were 39.0 and 33.5 kJ mol-1 degree-1, respectively. Purified GH3 cells membrane fractions, which showed specific binding sites for [3H]TRH, displayed the same maximal binding of [3H]E2 in the absence or presence of cold hormone. The amount of membrane-associated [3H]E2 increased linearly with temperature and extra-cellular hormone concentration. The effect of temperature on binding of E2 to membrane fractions occurred gradually without phase transitions and was not saturable. We suggest that the mechanism by which E2 is taken up by target cells at physiological temperature involves instantaneous dissolution in the cell membrane from where it diffuses passively into the cell. E2 binds thereafter to specific receptors in an energy-dependent step.     

Activities of bone morphogenetic proteins in prolactin regulation by somatostatin analogs in rat pituitary GH3 cells

Involvement of the pituitary BMP system in the modulation of prolactin (PRL) secretion regulated by somatostatin analogs, including octreotide (OCT) and pasireotide (SOM230), and a dopamine agonist, bromocriptine (BRC), was examined in GH3 cells. GH3 cells are rat pituitary somato-lactotrope tumor cells that express somatostatin receptors (SSTRs) and BMP system molecules including BMP-4 and -6. Treatment with BMP-4 and -6 increased PRL and cAMP secretion by GH3 cells. The BMP-4 effects were neutralized by adding a BMP-binding protein Noggin. These findings suggest the activity of endogenous BMPs in augmenting PRL secretion by GH3 cells. BRC and SOM230 reduced PRL secretion, but OCT failed to reduce the PRL level. In GH3 cells activated by forskolin, BRC suppressed forskolin-induced PRL secretion with reduction in cAMP levels. OCT did not affect forskolin-induced PRL level, while SOM230 reduced PRL secretion and PRL mRNA expression induced by forskolin. BMP-4 treatment enhanced the reducing effect of SOM230 on forskolin-induced PRL level while BMP-4 did not affect the effects of OCT or BRC. Noggin treatment had no significant effect on the BRC actions reducing PRL levels by GH3 cells. However, in the presence of Noggin, OCT elicited an inhibitory effect on forskolin-induced PRL secretion and PRL mRNA expression, whereas the SOM230 effect on PRL reduction was in turn impaired. It was further found that BMP-4 and -6 suppressed SSTR-2 but increased SSTR-5 mRNA expression of GH3 cells. These findings indicate that Noggin rescues SSTR-2 but downregulates SSTR-5 by neutralizing endogenous BMP actions, leading to an increase in OCT sensitivity and a decrease in SOM230 sensitivity of GH3 cells. In addition, BMP signaling was facilitated in GH3 cells treated with forskolin. Collectively, these findings suggest that BMPs elicit differential actions in the regulation of PRL release dependent on cellular cAMP-PKA activity. BMPs may play a key role in the modulation of SSTR sensitivity of somato-lactotrope cells in an autocrine/paracrine manner.     

Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells.

The involvement of nitric oxide (NO) in human GH-releasing hormone (hGHRH)-induced GH secretion was studied with freshly dissociated male rat pituitary cells. The cells were packed in a column of Bio-Gel-P2 and continuously perifused at 37 C. Hemoglobin (Hb; 10 microM), which is known to strongly bind NO, potentiated 0.01, 0.1, and 1 nM hGHRH-induced GH secretion by 73%, 52%, and 39%, respectively, without affecting the basal secretion of GH. As reported previously, 1-nM or higher concentrations of hGHRH elicit an increase in GH secretion during the application of hGHRH (on-response) and also a transient increase after the cessation of hGHRH application (off-response). It was found that Hb potentiated only the off-response in 1 nM hGHRH-induced GH secretion, and the same concentration of Hb had no effect on 10 nM hGHRH-induced GH secretion. N-Methyl-L-arginine (MeArg; 500 microM), a competitive inhibitor of NO synthase, also potentiated both the on- and off-responses of 1 nM hGHRH-induced GH secretion by 39% without affecting basal GH secretion. Since cAMP is thought to be an intracellular messenger of hGHRH action, the effects of Hb and MeArg on 1 mM (Bu)2AMP-induced GH secretion were examined. Their actions were found to be greater than those in hGHRH-induced GH secretion. Excess K+ (15 and 50 mM)-induced GH secretion, which does not involve cAMP, however, was not affected by either Hb or MeArg. In contrast, 3 mM sodium nitroprusside, which releases NO, suppressed the 1 nM hGHRH-induced off-response by 18%. The same concentration of sodium nitroprusside had no effect on excess K(+)-induced GH secretion. The effect of 8-bromo-cGMP on hGHRH-induced GH secretion was also examined, since NO is thought to exert its action through cGMP by activating guanylate cyclase in neural tissue. The application of 8-bromo-cGMP, however, did not affect 1 nM hGHRH-induced GH secretion. These observations suggest that hGHRH stimulates the synthesis of NO at least partly through cAMP, thereby partially inhibiting hGHRH-induced GH secretion.     

Modulation of diaphragm action potentials by K(+) channel blockers

K(+) channels regulate diaphragm contractility. The present study examined the electrophysiological mechanisms accounting for diversity among K(+) channel blockers in their inotropic actions on the diaphragm. Rat diaphragmatic muscle fibers were recorded intracellularly in vitro at 37 degrees C. Apamin and charybdotoxin (Ca2+)-activated K(+) channel blockers) did not alter resting membrane potential or action potentials. Glibenclamide (ATP-sensitive K(+) channel blocker) slowed action potential repolarization by 12% (P<0.05) and increased action potential area by 25% (P<0.005). Tetraethylammonium (which blocks several types of K(+) channels) increased action potential overshoot by 20% (P<0.01) and prolonged action potential rise time by 17% (P<0.02). 4-Aminopyridine and 3,4-diaminopyridine (which also block several types of K(+) channels) slowed action potential repolarization by 163% (P<0.0001) and 253% (P<0.0001), and increased action potential area by 183% (P<0.0001) and 298% (P<0.0001), respectively. Slowing of repolarization for the aminopyridines was especially marked at voltages approaching resting membrane potential, thereby changing action potential repolarization from a first to a second order decay. Previously reported variability in inotropic effects among K(+) channel blockers correlated significantly with the extent to which they slowed action potential repolarization and increased action potential area, but not with changes in other action potential properties.     

Arginine increases growth hormone gene expression in rat pituitary and GH3 cells

The effect of arginine (Arg) and Ornitargin (OT) [a compound containing the aminoacids Arg, citrulline (Cit) and ornithine (Orn)] administration upon growth hormone (GH) gene expression was studied both in vivo and in vitro (hemipituitaries and GH3 cells) by Northern blot analysis. For in vivo studies, adult male Wistar rats were anesthetized, subjected to i.v. infusion of 200 microl of 150 mM NaCl (control group), Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) at a rate of 20 microl/min, and killed 50 min thereafter. For the in vitro studies, hemipituitaries or GH3 cells were incubated in 1 ml of appropriate medium containing Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) for 60 min. The pituitaries of the in vivo and in vitro studies and GH3 cells were subsequently processed for RNA extraction. Total RNA was subjected to electrophoresis in agarose (1%)/formaldehyde gel, transferred to a nylon membrane and subjected to hybridization with a rat GH (32)P-cDNA, and (32)P-18S rRNA probe to correct for the variability in RNA loading. After autoradiography of the membrane, the abundance of GH mRNA and 18S rRNA bands was quantified by densitometry. The in vivo study demonstrated that Arg and OT infusion induced a 2.3-fold increase in GH mRNA expression, which could result from the Arg-mediated inhibition of somatostatin release. In addition, in vitro Arg, but not OT, induced GH gene expression in hemipituitaries and GH3 cells, indicating that the aminoacid can act per se at the pituitary somatotrope level. In conclusion, our data show for the first time that arginine stimulates GH gene expression in parallel to its recognized GH-releasing activity.     

Thyroid hormone action: induction of morphological changes and stimulation of cell growth in rat pituitary tumor GH3 cells

The effects of thyroid hormones on morphology and growth were studied in rat pituitary tumor GH3 cells using medium containing serum depleted of thyroid hormones. T3 and T4 induce the cells to change from a flattened fibroblastic morphology to a rounded or spindle-shaped morphology. The induction in morphological changes is T3 and T4 specific and dose dependent. Thyronine and rT3 are ineffective in inducing morphological changes; the half-maximal effective concentrations for T3 and T4 are 0.3 and 2 nM, respectively. Concomitantly, T3 stimulates cell growth, as indicated by a 2-fold reduction in doubling time and a 2-fold increase in mitotic rate. The growth-stimulating effect has the same analog specificity and dose dependency as the morphological changes. The morphological changes could be potentially useful for evaluating the biological effects of T3 and its analogs and in studying the mechanism of thyroid hormone action.     

Vitamin D-induction of secretory responses in rat pituitary tumour (GH4C1) cells

1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) selectively enhances prolactin gene expression in GH4C1 clonal rat pituitary tumour cells. Because this effect requires extracellular Ca2+, we studied the effect of 1,25-(OH)2D3 on another Ca2+-dependent process, agonist-induced hormone secretion. Pretreatment with 1,25-(OH)2D3 (1 nmol/l) caused at least 25-fold sensitization of GH4C1 cells to the voltage-sensitive Ca2+ channel agonist BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine -5-carboxylate) as a prolactin secretagogue. This inductive effect of 1,25-(OH)2D3 followed a similar time-course to the enhancement of prolactin production. 1,25-(OH)2D3 had no effect on basal or BAY K 8644-induced 45Ca2+ uptake. The Ca2+-selective divalent cation ionophore 11,19,21-trihydroxy-4,6,8,12,14,18,20- heptamethyl-9-oxo-22-(tetrahydro-5 methyl-5-tetra hydro-5-(1-hydroxyethyl)-5-methyl-2-furanyl)-10,16-docosadienoic acid (ionomycin; 12 nmol/l-1.2 mumol/l) caused no significant increase in prolactin secretion in the absence of 1,25-(OH)2D3, but in cells treated with 1,25-(OH)2D3-(1 nmol/l), it increased prolactin secretion by 73% at 12 nmol/l and by a maximum of 98% at 0.12 mumol/l. These data demonstrate that vitamin D markedly enhances the responsiveness of GH4C1 functional pituitary tumour cells to two secretagogues which acts primarily through Ca2+-dependent mechanisms. They support the proposal that 1,25-(OH)2D3 acts in this cultured cell model either by effecting a redistribution of intracellular Ca2+ or by increasing the response of a Ca2+-sensitive effector system, but not by enhancing agonist-induced Ca2+ uptake.     

Secretogranin I (chromogranin B) mRNA accumulation is hormonally regulated in GH3B6 rat pituitary tumor cells

Secretogranin I (SgI; chromogranin B) belongs to a class of acidic tyrosine-sulfated secretory proteins believed to play a role in the secretory process of endocrine cells. Our aim here was to compare the levels of SgI mRNA to that of prolactin (PRL) and growth hormone (GH), using rat pituitary cell lines. As far as the constitutive expression is concerned, we found a positive correlation between SgI mRNA and PRL mRNA levels. However, the neuropeptide TRH (50 nM) inhibited the accumulation of SgI mRNA in GH3B6 cells whereas, as expected, it induced a rapid and sustained increase in PRL mRNA accumulation. By contrast, 17β-estradiol (1 nM) stimulated the accumulation of both SgI and PRL mRNAs, with the same EC₅₀ (18–59 pM). Reciprocally, treatment with dexamethasone (100 nM) reduced the level of SgI and PRL mRNAs to 23% and 29% of control, respectively, but led to a 2.1-fold increase in the GH mRNA level. Altogether, the present work shows that SgI gene expression is subject to multiple hormonal regulations and occasionally parallels the regulation of the PRL gene but never that of the GH gene, under the conditions tested.     

Melatonin suppresses cyclosporine A‐induced autophagy in rat pituitary GH3 cells

Abstract: Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0–100 μm ) decreased cell survival of rat pituitary GH3 cells in a dose‐dependent manner. At concentrations ranging from 1.0 to 10 μm , CsA induced a dose‐dependent increase in the expression of microtubule‐associated protein 1 light chain 3 (LC3)‐I and LC3‐II. Cells treated with 2.5 μm CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0–10 μm CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol‐requiring enzyme 1 alpha (IRE1α), increased as compared those levels in untreated cells. These results suggested that CsA‐induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA‐induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 μm ) suppressed autophagy induced by 2.5 and 5 μm CsA. Furthermore, co‐treatment with 100 μm melatonin inhibited LC3‐II expression, and increased catalase and phosphorylated p‐ERK levels in the presence of 2.5 and 5 μm CsA. BiP and IRE1α expression in melatonin‐co‐treated cells was superior to that in cells treated with 2.5 and 5 μm CsA alone. Thus, melatonin suppresses CsA‐mediated autophagy in rat pituitary GH3 cells.     

Role of voltage-dependent and Ca(2+)-activated K(+) channels on the regulation of isometric force in porcine coronary artery

We investigated the role of K(+) channels in the regulation of vascular tone in de-endothelialized porcine coronary artery. Isometric force and intracellular Ca(2+) ([Ca(2+)](i)) under resting conditions were increased by treatment with 4-aminopyridine (4-AP, 1 mM), an inhibitor of voltage-dependent K(+) (K(v)) channels, but not by tetraethylammonium chloride (TEA, 1 mM) or charybdotoxin (100 nM), both inhibitors of Ca(2+)-activated K(+) (K(Ca)) channels, or glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels. Under stimulated conditions with 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619), 4-AP as well as TEA or charybdotoxin increased isometric force and [Ca(2+)](i), but not glibenclamide. 4-AP was the most potent in terms of depolarization of membrane potential compared with TEA or glibenclamide in the presence or absence of EGTA. In the presence of U46619, a high concentration of 4-AP (10 mM) caused a further contraction with oscillations. The force oscillations induced by 4-AP were inhibited by diltiazem (10 microM), an inhibitor of voltage-dependent Ca(2+) channels, or TEA (1 mM), but not by glibenclamide (10 microM). These force oscillations may be associated with the periodic activation of K(Ca) channels. These findings suggested that 4-AP-sensitive K(v) channels play an important role in the control of vascular tone in both resting and stimulated conditions. Moreover, under stimulated conditions, K(Ca) channels also have an important role in the regulation of vascular tone. Dysfunction of these channels induces abnormal vasoconstriction and may be implicated in vascular diseases such as hypertension and vasospasm.     

Thyroid hormone and glucocorticoid regulation of receptor and target gene mRNAs in pituitary GH3 cells

We have examined the effects of triiodothyronine (T₃), in dose-response and time-course studies, on T₃ receptor (T₃R) and β and glucocorticoid receptor (GR) mRNAs in rate pituitary GH₃ cells, in parallel with T₃ actions on expression of the growth hormone (GH) target gene. Modulatory influences of dexamethasone (dex) on T₃ action were studied by treatment with dex before and during T₃ treatment. T₃ treatment (1–100 nM) for 24 h reduced T₃R mRNA, while the presence of dex (1 μM) enhanced the T₃ effect on T₃R mRNA and induced T₃ inhibition of T₃R β mRNA. Stimulatory effects of T₃ treatment on GH mRNA and release were seen in the face of inhibition of T₃R mRNAs; these effects on GH were also enhanced by the presence of dex. T₃ treatment for 24 h increased GR mRNA; this effect was inhibited by the presence of dex. We next examined the influence of dex on GR and T₃R and β mRNAs, in parallel with effects of dex on the prolactin (PRL) target gene. Modulatory influences of T₃ on dex action were studied by treatment of cells with T₃ before and during dex treatment. Treatment with dex (0.1–10 μM) for 24 h reduced GR mRNA, an action enhanced by the presence of T₃ (100 nM). Dex treatment resulted in inhibition of PRL mRNA and release despite parallel inhibition of GR mRNA by dex; these effects were enhanced by the presence of T₃. In contrast to actions on GR, dex has no effect on T₃R mRNAs. These effects of T₃ and dex on receptor mRNAs suggest that glucocorticoid modulation of T₃ action is not related to direct actions on T₃R synthesis. In contrast, the mechanism of T₃ modulation of glucocorticoid action may be due in part to alteration of GR mRNA expression. Effects of T₃ and dex on target gene expression were observed in the presence of parallel reduction of their respective receptor mRNAs. This provides new evidence that interactions between these hormones are likely to be mediated by mechanisms other than regulation of receptor gene expression.     

Reciprocal regulation by estradiol 17-beta of ezrin and cadherin-catenin complexes in pituitary GH3 cells

The antiestrogen, ICI 182780, and estradiol-17β (E₂) regulate cadherin-mediated cell adhesion in pituitary GH₃ cells. Using a cDNA expression array to screen for E₂-regulated genes that are associated with the cytoskeleton, we observed that E₂ stimulated ezrin gene expression and confirmed that ezrin gene expression is regulated pretranslationally by ICI 182780 versus E₂. E₂ increased ezrin protein levels in whole-cell lysates and in the cytoskeletal-associated, detergent-insoluble fraction. Confocal microscopy revealed that ezrin was associated with free apical membranes of E₂-treated cells. E₂ decreased N-cadherin and β-catenin levels and induced a redistribution of p120^ctn to the cytoplasm. In GH₃ transfectants overexpressing E-cadherin, E₂ had no effect on adhesiveness or on E-cadherin and p120^ctn distribution, but increased levels of active ezrin. Ezrin was concentrated at free and apical membranes. These studies provide the first demonstration of the regulation of ezrin by E₂ and show that the ER signaling pathway coordinately regulates two cytoskeletal-associated protein complexes, with mutually exclusive cellular distributions, in a reciprocal manner. These findings indicate that E₂ enriches the cell membrane with ezrin-membrane protein complexes by both increasing ezrin expression and by enlarging the relative area of nonadhesive membrane to which ezrin is targeted.     

Ceramide enhances growth hormone (GH)-releasing hormone-stimulated cyclic adenosine 3',5'-monophosphate accumulation but inhibits GH release in rat anterior pituitary cells

In this study, the effect of ceramide on GH-releasing hormone (GHRH)-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. C2-, C6-, and C8-ceramide were found to enhance GHRH-stimulated cAMP accumulation. In contrast, their effects on GHRH-stimulated GH release were inhibitory. Treatment with a glucosylceramide synthase inhibitor produced a similar enhancing effect on cAMP accumulation and an inhibitory effect on GH release. To identify the pathway through which ceramide mediated its effect, it was found that ceramide inhibited GH release stimulated by KCl, BayK 8644, and a GH-releasing peptide, but not that stimulated by ionomycin or an activator of protein kinase C. Direct measurement of intracellular Ca2+ revealed that C2-ceramide inhibited GHRH- and KCl-mediated increases in intracellular Ca2+, suggesting that ceramide probably inhibits GH release through inhibition of the L-type Ca2+ channels. As for its mechanism on cAMP accumulation, the enhancing effect of ceramide on GHRH-stimulated cAMP accumulation was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine, suggesting that ceramide enhances the cAMP response through inhibition of its metabolism. Taken together, our results suggest that ceramide plays an important role in the regulation of GHRH-stimulated responses in somatotrophs. By reducing GH secretion while enhancing cAMP accumulation, ceramide may promote the synthesis and storage of GH in rat anterior pituitary cells.     

Benzodiazepines modulate voltage-sensitive calcium channels in GH3 pituitary cells at sites distinct from thyrotropin-releasing hormone receptors

Benzodiazepines (BZs) have been shown to modulate voltage-sensitive Ca2+ channels in a number of neuronal and nonneuronal cell types and to competitively antagonize TRH binding to receptors on cells of the nervous system and anterior pituitary gland. Because interaction of TRH with its receptor is known to cause enhanced influx of Ca2+ through voltage-sensitive channels in rat pituitary GH3 cells, it was determined whether BZs and TRH were interacting with the same binding site on these cells. The potencies of three BZs, Ro5-4864, diazepam (DZP), and chlordiazepoxide (CDE), were compared as modulators of Ca2+ channels and as inhibitors of TRH binding in GH3 cells. Modulation of Ca2+ channel activity was measured as the inhibition by BZs of K+ depolarization-induced Ca2+ influx using intracellularly trapped quin 2 or 45Ca2+ uptake. The three BZs caused dose-dependent inhibition of Ca2+ influx with an order of potency of Ro 5-4864 greater than DZP greater than CDE. In contrast, the order of potency of the three BZs to inhibit [3H]TRH binding was CDE greater than DZP much greater than Ro 5-4864. The concentrations of BZs needed to inhibit Ca2+ influx and TRH binding were in the micromolar range. These data show that BZs can modulate Ca2+ channel activity in endocrine cells and that these sites are distinct from those that modulate TRH binding on pituitary cells.     

Effect of calcium on membrane potential behavior in a rat pituitary cell line (GH3)

The effects of intracellular calcium buffering and increasing bath Ca2+ on spontaneous membrane depolarizations expressed by the clonal rat pituitary cell line GH3 were examined by use of the whole-cell patch-clamp technique. Increasing intracellular calcium buffering capacity caused the duration of spontaneous depolarizations to increase without altering other parameters of membrane potential activity. Increasing bath Ca2+ caused a decrease in duration. These results suggest that the duration of spontaneous membrane depolarization in GH3 cells is regulated by the accumulation of free intracellular Ca2+. The behavior of spontaneous depolarizations measured with the perforated-patch variation of whole-cell patch-clamp techniques closely resembled that obtained in standard whole-cell patch-clamp measurements with an intracellular calcium buffer of 200 microM EGTA with free Ca2+ adjusted to 100 nM.