title: Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells.
abstract: In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca2+ concentration ([Ca2+]i) in GH4C1 pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca2+]i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory. The effect of thimerosal on the IP3-induced calcium release (IICR) was studied in permeabilized cells using the Ca2+ indicator Fluo-3. A low concentration of thimerosal (10 microM) stimulated IICR: the Ca2+ release induced by 300 nM inositol-1,4,5-trisphosphate (IP3) was enhanced in cells treated with thimerosal for 1 or 6 min (67 +/- 11 nM and 34 +/- 5 nM, respectively) as compared to control cells (17 +/- 2 nM). On the other hand, a high concentration of thimerosal (100 microM) inhibited IICR: when IP3 (10 microM) was added after a 5 min preincubation with thimerosal, the IP3-induced rise in [Ca2+]i (46 +/- 14 nM) was 57% smaller as compared with that seen in control cells (106 +/- 10 nM). The effect of thimerosal on the voltage-operated Ca2+ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K+ to the cuvette. The depolarization-evoked increase in [Ca2+]i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 microM) potently inhibited the Ba2+ currents through VOCCs. In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca2+]i, and activated a capacitative Ca2+ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT). We conclude that, in GH4C1 pituitary cells, the mobilization of intracellular calcium and the different Ca2+ entry pathways are sensitive to redox modulation.
authors.affiliation: Department of Biosciences, University of Helsinki, Finland.
authors.forename: L
authors.initials: L
authors.lastname: Karhapää
authors.forename: A
authors.initials: A
authors.lastname: Titievsky
authors.forename: K
authors.initials: K
authors.lastname: Kaila
authors.forename: K
authors.initials: K
authors.lastname: Törnquist
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001464:Barium / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D010084:Oxidation-Reduction; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D013849:Thimerosal / Q000494:pharmacology*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D013439:Sulfhydryl Reagents; D013849:Thimerosal; D001464:Barium; D002110:Caffeine; D013973:Thyrotropin-Releasing Hormone; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D011188:Potassium; D002118:Calcium; D004229:Dithiothreitol