REDOX MODULATION OF CALCIUM-ENTRY AND RELEASE OF INTRACELLULAR CALCIUM BY THIMEROSAL IN GH(4)C(1) PITUITARY-CELLS

In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate i ntracellular free Ca2+ concentration ([Ca2+](i)) in GH(4)C(1) pituitar y cells. In intact Fura-2 loaded cells, low concentrations of thimeros al potentiated the spike phase of the TRH-induced (thyrotropin-releasi ng 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 relea se (IICR) was studied in permeabilized cells using the Ca2+ indicator Fluo-3. A low concentration of thimerosal (10 mu M) stimulated IICR: t he Ca2+ release induced by 300 nM inositol-1,4,5-trisphosphate (IP3) w as enhanced in cells treated with thimerosal for 1 or 6 min (67+/-11 n M and 34+/-5 nM, respectively) as compared to control cells (17+/-2 nM ). On the other hand, a high concentration of thimerosal (100 mu M) in hibited IICR: when IP3 (10 mu M) was added after a 5 min preincubation with thimerosal, the IP3-induced rise in [Ca2+], (46+/-14 nM) was 57% smaller as compared with that seen in control cells (106+/-10 nM). Th e effect of thimerosal on the voltage-operated Ca2+ channels (VOCCs) w as studied by depolarizing intact Fura-2 loaded cells by addition of 2 0 mM K+ to the cuvette, The depolarization-evoked increase in [Ca2+](i ) was inhibited in a dose-dependent manner by thimerosal. Direct evide nce for an inhibitory effect of thimerosal on VOCCs was obtained by us ing the whole-cell configuration of the patch-clamp technique: thimero sal (100 mu M) potently inhibited the Ba2+ currents through VOCCs. In addition, our results indicated that thimerosal inhibited the caffeine -induced increase in [Ca(2+)](i), and activated a capacitative Ca2+ en try pathway. The actions of thimerosal were apparently due to its oxid izing activity because the effects were mostly reversed by the thiol-r educing agent dithiothreitol (DTT). We conclude that, in GH(4)C(1) pit uitary cells, the mobilization of intracellular calcium and the differ ent Ca2+ entry pathways are sensitive to redox modulation.