Pharmacological Blockade of ERG K Channels and Ca Influx through Store-Operated Channels Exerts Opposite Effects on Intracellular Ca Oscillations in Pituitary GH3 Cells

In the present study, the effects on intracellular calcium concentration ([Ca]i) oscillations of the blockade of ether-a-gogo-related gene (ERG) K channels and of Ca influx through store-operated channels (SOC) activated by [Ca]i store depletion have been studied in GH3 cells by means of a combination of single-cell fura-2 microfluorimetry and whole-cell mode of the patch-clamp technique. Nanomolar concentrations (1–30 nM) of the piperidinic second-generation antihistamines terfenadine and astemizole and of the class III antiarrhythmic methanesulfonanilide dofetilide, by blocking ERG K channels, increased the frequency and the amplitude of [Ca]i oscillations in resting oscillating GH3 cells. These compounds also induced the appearance of an oscillatory pattern of [Ca]i in a subpopulation of nonoscillating GH3 cells. The effects of ERG K channel blockade on [Ca]i oscillations appeared to be due to the activation of L-type Ca channels, because they were prevented by 300 nM nimodipine. By contrast, the piperazinic second-generation antihistamine cetirizine (0.01–30 mM), which served as a negative control, failed to affect ERG K channels and did not interfere with [Ca]i oscillations in GH3 cells. Interestingly, micromolar concentrations of terfenadine and astemizole (0.3–30 mM), but not of dofetilide (10–100 mM), produced an inhibition of the spontaneous oscillatory pattern of [Ca]i changes. This effect was possibly related to an inhibition of SOC, because these compounds inhibited the increase of [Ca]i achieved by extracellular calcium reintroduction after intracellular calcium store depletion with the sarcoplasmic or endoplasmic reticulum calcium ATPase pump inhibitor thapsigargin (10 mM) in an extracellular calcium-free medium. The same inhibitory effect on [Ca]i oscillations and SOC was observed with the first-generation antihistamine hydroxyzine (1–30 mM), the more hydrophobic metabolic precursor of cetirizine. Collectively, the results of the present study obtained with compounds that interfere in a different concentration range with ERG K channels or SOC suggest that 1) ERG K channels play a relevant role in controlling the oscillatory pattern of [Ca]i in resting GH3 cells and 2) the inhibition of SOC might induce an opposite effect, i.e., an inhibition of [Ca]i oscillations. It is widely recognized that pituitary cells display spontaneous changes in intracellular Ca (Cai) concentrations ([Ca]i), which vary considerably from cell to cell, with a periodic range from 3 to 30 s and a peak amplitude ranging from 40 to 400 nM. These spontaneous fluctuations are defined as [Ca]i oscillations (Charles et al., 1999). It has been proposed that frequency and amplitude of these [Ca]i oscillations play a role in the regulation of anterior pituitary hormone secretion and gene expression (Berridge, 1997; Dolmetsch et al., 1998). [Ca]i oscillations may be related to changes in plasma membrane potential and action potential frequency. In fact, the sum of sodium, potassium, and chloride currents flowing through the respective plasma membrane channels expressed in pituitary cells at each time determines the cell membrane potential, which forms the basis of the plasma membrane oscillator (Stojilkovic and Catt, 1992; Stojilkovic, 1996). Changes in the plasma membrane oscillator regulate the opening of L-type voltage-dependent Ca channels, which are also modulated by tyrosine (Cataldi et al., 1996) and serine-threonine kinases (Cataldi et al., 1999). Furthermore, the participation of a cytoplasmic Ca oscillator, composed of intracellular calcium-storing organelles, has also been suggested. This cytoThe study was supported by the following grants: Telethon 1058, National Research Council 97.04512. CT04, 97.01230. PF49, and 98.03149. CT04 (M.T.); Istituto Superiore di Sanità, Roma, Italy (Progetto sulle proprietà chimicofisiche dei medicamenti e loro sicurezza d’ uso), National Research Council 96.02074, 97.04559, 98.01048. CT04, and 98.00062. PF31 (PS Biotecnologie 5%), Murst Cofinanziamento 1998, and Regione Campania (P.O.P. and Legge 41) (L.A.). ABBREVIATIONS: ERG, ether-a-go-go-related gene; [Ca]i, intracellular calcium concentration; Ca 21 e, extracellular calcium; Ca 21 i, intracellular calcium; SOC, store-operated channels; GH, growth hormone; DMSO, dimethyl sulfoxide. 0026-895X/00/051115-14$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 5 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 91/861093 Mol Pharmacol 58:1115–1128, 2000 Printed in U.S.A. 1115 at A PE T Jornals on D ecem er 8, 2017 m oharm .aspeurnals.org D ow nladed from plasmic oscillator releases its Ca content upon activation of IP3-generating mechanisms and is refilled from the extracellular space by specific plasma membrane channels named store-operated channels (SOC) (Stojilkovic, 1996). K channels play an important role in the regulation of membrane potential in pituitary cells. In the rat growth hormone (GH)and prolactin-secreting pituitary clonal cell line GH3, in particular, Ca -dependent, ATP-dependent, outwardly, and classical inwardly rectifying K channels have been described (Barros et al. 1994; Nelson et al., 1996; Jakab et al., 1997). All of these channels contribute to the shaping of the electrophysiological properties of these cells. More recently, a novel type of voltage-dependent K current has been suggested to contribute to the resting membrane potential control in pituitary GH cells (Bauer, 1998; Bauer et al., 1999), as well as in primary lactotrophs (Corette et al., 1996). The molecular basis for this novel K current has recently been identified as the gene product of the ether-agogo-related gene (ERG; Warmke and Ganetzky, 1994), which encodes for K channels expressed in several tissues such as the heart, the central nervous system, and tumor cell lines of different histogenesis, including GH3 cells (Bianchi et al. 1998). Considering the relevant role played by the membrane potential in the modulation of [Ca]i oscillations, the present study investigated the possible involvement of ERG K channels and of SOC in [Ca]i oscillatory pattern in GH3 cells. [Ca]i oscillations and the activity of ERG K 1 channels were studied using single-cell fura-2 microfluorimetry and the whole-cell mode of the patch-clamp technique, respectively. Because it has been shown that the piperidinic secondgeneration antihistamines terfenadine and astemizole (Roy et al. 1996; Suessbrich et al.1996; Taglialatela et al. 1998), as well as the class III antiarrhythmic dofetilide (Kiehn et al., 1996; Snyders and Chaudhary, 1996), block with elevated affinity constitutively and heterologously expressed ERG K channels, we studied the effect of terfenadine and astemizole on IERG in GH3 cells; subsequently, the effect of nanomolar concentrations of these second-generation antihistamines and of dofetilide were studied on [Ca]i oscillations in these cells. Furthermore, because it has been also shown that astemizole may inhibit store-operated Ca fluxes when used at micromolar concentrations in rat basophilic leukemia cells (RBL-2H3) (Fischer et al. 1997, 1998a), higher (micromolar) concentrations of this agent were studied on [Ca]i increase induced by [Ca]i store depletion and subsequent refilling. In addition, to rule out the possibility that these secondgeneration antihistamines can interfere with [Ca]i oscillations by inhibiting Ca channels, the effect of astemizole on high-voltage-activated Ca channel currents was also investigated. Finally, with the help of selective inhibitors, the role played by other K channel subtypes different from ERG in [Ca]i oscillations in GH3 cells was also studied. The results obtained suggest that the inhibition of ERG K channels achieved by nanomolar concentrations of terfenadine, astemizole, and dofetilide is able to increase the frequency and the amplitude of [Ca]i oscillations in GH3 cells. However, when micromolar concentrations of astemizole, terfenadine, and hydroxyzine, but not of dofetilide, were used, an inhibition of the spontaneous oscillatory pattern of [Ca]i changes was observed. This inhibitory effect seems to be related to an inhibition of the SOC channels activated upon depletion of [Ca]i stores. Finally, the piperidinic secondgeneration antihistamine cetirizine, which is devoid of any inhibitory action on ERG K channels and SOC, did not interfere with [Ca]i oscillations in GH3 cells. Materials and Methods Cell Culture. GH3 cells were obtained from Flow Laboratories (Irvine, Scotland) and grown on plastic dishes in Ham’s F10 medium (Gibco-BRL, San Giuliano Milanese, Italy) composed of 15% horse serum (Flow), 2.5% fetal calf serum (Hyclone, Logan, UT), 100 I.U. penicillin/ml, and 100 mg streptomycin/ml. The cells were cultured in a humidified 5% CO2 atmosphere, and the culture medium was changed every 2 days. For microfluorimetric studies, the cells were seeded on glass coverslips (Fisher, Springfield, NJ) coated with polyL-lysine (30 mg/ml) (Sigma, St. Louis, MO) and used at least 12 h after seeding. [Ca]i Measurements and Quantification of [Ca ]i Oscillations. [Ca]i was measured using a microfluorimetric technique, as previously reported (Cataldi et al., 1996). Briefly, the cells grown on glass coverslips were loaded with 5 mM 1-[2-(5-carboxyoxazol-2-yl)-6aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N, N,N,N-tetraacetic acid pentaacetoxymethyl ester (fura-2 AM) in Krebs-Ringer saline solution (5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4) for 1 h at room temperature. At the end of the fura-2 AM-loading period, the coverslip was introduced into a microscope chamber (Medical System Co., Greenvale, NY) on an inverted Diaphot fluorescence microscope (Nikon, Tokyo, Japan). The cells were kept in Krebs-Ringer saline solution throughout the experiment. All of the drugs tested were introduced into the microscope chamber by fast injection. A 100-W Xenon lamp (Osram, Frankfurt, Germany) with a computer-operated filter wheel bearing two different interference filters (340 and 380 nm) illuminated the microscopic field with UV light, alternating the wavelengths at an interval of 500 ms. The interval between each pair of illuminations ranged from 1 to 3 s, and the interval between filter movements was 1 s. Emitted light was passed through a 400-nm dichroic mirror, filtered at 510 nm, and collected by a charge-coupled device camera (Photonic Science, Robertsbridge, East Sussex, UK) connected to a light amplifier (Applied Imaging Ltd., Dukesway Gateshead, UK). Images were digitized and analyzed with a Magiscan image processor (Applied Imaging Ltd.). Using a calibration curve, the Tardis software (Applied Imaging Ltd.) calculated the [Ca]i corresponding to each pair of images from the ratio between the intensity of the light emitted when the cells were illuminated at both 340 and 380 nm. [Ca]i oscillations were defined as an increase of [Ca ]i above the mean of the basal value 62 S.D., occurring with a frequency higher than one peak/3 min. According to Villalobos et al. (1998), two different parameters were used for the quantification of [Ca]i oscillations: the oscillation index and the mean [Ca]i value. The oscillation index was calculated by adding all of the absolute differences in [Ca]i between each [Ca ]i measurement and the previous value; this parameter represents the rate of [Ca]i changes during the measurements and the frequency and/or amplitude of [Ca]i oscillations and is independent of the actual [Ca]i value. Instead, the mean [Ca]i value was obtained by adding all of the [Ca ]i values measured during the experimental period divided by the number of all of the experimental points measured. This parameter provides a mean value of [Ca]i over time. Each experiment was divided into three periods of equal duration (i.e., 100 s when the acquisition time was 1 s and 300 s when the acquisition time was 3 s), and both the oscillation index and the mean [Ca]i value were calculated for each period. In control conditions (i.e., no pharmacological treatment), no significant change in the oscillation index or the mean [Ca]i value occurred (data not shown) during these three successive periods. This allowed us to use 1116 Secondo et al. at A PE T Jornals on D ecem er 8, 2017 m oharm .aspeurnals.org D ow nladed from the first experimental period of acquisition, in which no experimental maneuver was performed, as a reference control for the following two periods in which the pharmacological treatment was applied. The quantification of the effects of the drugs on [Ca]i oscillations was performed by comparing the last period of drug application with the first control period. Patch-Clamp Recordings. Currents from GH3 cells were recorded at room temperature using a commercially available amplifier (Axopatch 200A, Axon Instruments, Foster City, CA). The wholecell configuration of the patch-clamp technique (Hamill et al., 1981) was used with glass micropipettes of 3 to 7 MV resistance. No compensation was made for pipette resistance and cell capacitance. For the experiments on ERG K channels, the relatively small density of the current required the use of a high (100 mM) external K concentration as a charge carrier. Therefore, GH3 cells were perfused with an extracellular solution containing (in mM): 100 KCl, 10 EGTA, and 10 HEPES, pH 7.3, with KOH, and the pipettes were filled with (in mM): 110 CsCl, 10 tetraethylammonium-Cl, 2 MgCl2, 10 EGTA, 8 glucose, 2 Mg-ATP, 0.25 cAMP, and 10 HEPES, pH 7.3. For Ca current recordings, the cells were perfused with an extracellular solution containing (in mM): 10 BaCl2, 125 NaCl, 1 MgCl2, 10 HEPES, and 300 nM tetrodotoxin, pH 7.3. The pipettes were filled with (in mM): 110 CsCl, 10 tetraethylammonium-Cl, 2 MgCl2, 10 EGTA, 8 glucose, 2 Mg-ATP, 0.25 cAMP, and 10 HEPES, pH 7.3. The Ba current through Ca channels was obtained by subtracting the current elicited in the presence of 50 mM CdSO4. Drugs and Chemicals. Chemicals were of analytical grade and were purchased from Sigma Italia (Milan, Italy). fura 2-AM was obtained from Calbiochem (La Jolla, CA). Astemizole and dofetilide were kindly provided by Janssen-Cilag (Rome, Italy) and Pfizer, Inc. (Sandwich, UK), respectively. Cetirizine was generously donated by UCB Pharma (Bruxelles, Belgium). All of the drugs were dissolved in 10 mM DMSO), and stock solutions were kept at 220°C. Appropriate dilutions were prepared daily. The maximal DMSO concentration (0.3%) did not affect [Ca]i oscillations in GH3 cells. Statistical Analysis of the Data. All of the data are expressed as the means 6 S.E.M. The statistical analysis was performed using the Student’s t test for paired or unpaired data, where required. The threshold for statistical significance was set at P , .05. The data reported in the present study are the means 6 S.E.M. of single-cell determinations obtained by the analysis of all the cells recorded in each of the different experimental sessions. For each pharmacological treatment, at least five cells in at least three experimental sessions were evaluated.