Kid Törnquist

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL‐5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of 3H‐thymidine in a dose‐ and time‐dependent manner in the cells. At least a 24‐h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of 3H‐thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH‐mediated incorporation of 3H‐thymidine. The effect of ATP was apparently mediated via a G‐protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL‐5 cells. In addition, ATP stimulated the expression of a 62 kDa c‐fos dependent protein in a dose‐ and time‐dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c‐fos.

Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells

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.

Importance of arachidonic acid metabolites in regulating ATP-induced calcium fluxes in thyroid FRTL-5 cells.

Stimulating rat thyroid FRTL-5 cells with the purinergic agonist ATP activates both the inositol phosphate signal-transduction pathway and the phospholipase A2 pathway. In the present study we wanted to investigate the possible inter-relationships between these two systems during ATP-induced changes in intracellular free calcium ([Ca2+]i). Pretreatment of Fura-2 loaded cells with 4-bromophenylacyl, an inhibitor of phospholipase A2, had no effect on the ATP-induced entry of Ca2+ but inhibited the release of sequestered Ca2+. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, and 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of cytochrome P-450 enzymes, attenuated the ATP-evoked transient increase in [Ca2+]i. Furthermore, the capacitative entry of Ca2+ was also attenuated in NDGA- and ETYA-treated cells stimulated with ATP. Similar results were obtained using econazole, an inhibitor of cytochrome P-450 enzymes. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the ATP-evoked response in [Ca2+]i. We also showed that stimulation of intact or permeabilized FRTL-5 cells with arachidonic acid released sequestered calcium. This calcium originated, at least in part, from an IP3 sensitive calcium pool. In addition, arachidonic acid rapidly acidified the cytosol. The results suggest that metabolism of arachidonic acid by a non-cyclooxygenase pathway is of importance in supporting agonist-induced calcium fluxes evoked via stimulation of the inositol phosphate pathway in FRTL-5 cells. Furthermore, arachidonic acid per se may modify agonist-induced calcium fluxes in these cells.

Inhibitory action of fatty acids on calcium fluxes in thyroid FRTL-5 cells

In the present study, we wanted to investigate the action of fatty acids on agonist-evoked changes in intracellular free calcium ([Ca2+]i) in thyroid FRTL-5 cells. Stimulating Fura 2 loaded cells with long chain unsaturated fatty acids increased [Ca2+]i in a dose-dependent manner. This increase was in part dependent on extracellular calcium. Long chain saturated fatty acids and short chain fatty acids had no effects on [Ca2+]i per se. Pretreatment of the cells with long chain unsaturated fatty acids almost totally inhibited both the ATP- and thapsigargin-evoked release of sequestered calcium and the entry of extracellular calcium. Long chain saturated fatty acids also attenuated the ATP-evoked increase in [Ca2+]i, while short chain fatty acids had no effects on the ATP-evoked change in [Ca2+]i. The inhibitory effect of long chain unsaturated fatty acids on agonist-evoked changes in [Ca2+]i was not dependent on activation of protein kinase C, and was not due to an enhanced efflux of calcium. These fatty acids rapidly acidified the cytosol in the cells, which could, in part, explain the inhibitory effect of the long chain unsaturated fatty acids on agonist-evoked changes in [Ca2+]i. Addition of bovine serum albumin to the cells rapidly reversed the inhibitory effect of the fatty acids on [Ca2+]i, and restored pHi. Thus, fatty acids could be potential modulators of calcium signaling in FRTL-5 cells, possibly by modulating calcium entry at the level of the plasma membrane.

PROTEIN TYROSINE PHOSPHORYLATION AND CALCIUM SIGNALING IN THYROID FRTL-5 CELLS

We examined the importance of tyrosine kinase(s) on the ATP‐evoked Ca2+ entry and DNA synthesis of thyroid FRTL‐5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72‐kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca2+ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca2+, but the capacitative Ca2+ or Ba2+ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca2+ ([Ca2+]i), whereas the Ba2+ entry was not increased. Phorbol 12‐myristate 13‐acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP‐evoked phosphorylation of MAP kinase and attenuated both the ATP‐ and the PMA‐evoked DNA synthesis. However, genistein did not inhibit the ATP‐evoked expression of c‐fos. Furthermore, genistein enhanced the ATP‐evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca2+‐dependent mechanism. A genistein‐sensitive mechanism participates, in part, in the ATP‐evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca2+ entry in FRTL‐5 cells. J. Cell. Physiol. 175:211–219, 1998.

Purinergic agonists stimulate the secretion of endothelin-1 in rat thyroid FRTL-5 cells

The aim of the present study was to investigate the mechanisms regulating endothelin‐1 (ET‐1) secretion in rat thyroid FRTL‐5 cells. ET‐1 was found to be secreted after stimulation with adenosine and ATP. The release of ET‐1 was sensitive to pertussis toxin, indicating a role of G‐proteins in the stimulus‐secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P1‐receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine‐ and ATP‐mediated ET‐1 secretion was abolished. These evidences suggest a role of a P1‐adenosine receptor in the secretion of ET‐1. Increasing cyclic AMP with forskolin decreased the adenosine‐mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni2+ prevented the response. Protein kinase C (PKC) is also partly involved in ET‐1 secretion in FRTL‐5 cells. Activation of PKC with the phorbol ester phorbol 12‐myristate 13‐acetate (PMA) stimulated the secretion of ET‐1 in a time‐ and dose‐dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET‐1 stimulated by adenosine. In conclusion, ET‐1 secretion in FRTL‐5 cells is stimulated via a pertussis toxin‐sensitive P1‐receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca2+, and PKC.

Regulatory effect of 1,25-dihydroxycholecalciferol on calcium fluxes in thyroid FRTL-5 cells

The aim of the present study was to investigate the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) on the regulation of calcium fluxes in rat thyroid FRTL-5 cells. The ATP-induced uptake of 45Ca2+ was decreased in cells pretreated with 1,25(OH)2D3 for 48 h. No effect was seen on basal uptake of 45Ca2+. At least a 24 h incubation period was required for the effect of 1,25(OH)2D3 to be expressed. Pretreatment with 1,25(OH)2D3 for 48 h did not change resting intracellular Ca2+ ([Ca2+]i) in fura-2-loaded FRTL-5 cells. However, the ATP-induced increase in [Ca2+]i was significantly enhanced in cells preincubated with 1,25(OH)2D3. The effect of 1,25(OH)2D3 was abolished in Ca(2+)-free buffer. No difference in the ionomycin-induced increase in [Ca2+]i was observed between control cells and cells pretreated with 1,25(OH)2D3. However, in Ca(2+)-free buffer the ionomycin response was decreased in cells incubated with 1,25(OH)2D3. The ATP-induced change in [Ca2+]i was decreased when ATP was added after ionomycin to cells treated with 1,25(OH)2D3. The results suggest that 1,25(OH)2D3 has a regulatory effect on Ca2+ fluxes in FRTL-5 cells, possibly by acting on Ca2+ sequestration.

TRH-evoked entry of extracellular calcium in GH4C1 cells: possible importance of arachidonic acid metabolites.

Previous studies have shown that stimulating pituitary GH4C1 cells with thyrotropin-releasing hormone (TRH) evoked a biphasic change in cytosolic free Ca2+ concentration ([Ca2+]i): a rapid release of sequestered Ca2+ due to the production of inositol-1,4,5-trisphosphate, and Ca2+ entry via both voltage-operated Ca2+ channels and a presently unknown voltage-independent influx pathway. The aim of the present study was to further evaluate to which extent the TRH-evoked changes in [Ca2+]i were dependent on entry of extracellular Ca2+, and which mechanisms participated in regulating this Ca2+ entry. Pretreatment of the cells with 4-bromophenylacylbromide (an inhibitor of phospholipase A2), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and econazole (an inhibitor of both lipoxygenase and cytochrome P-450 enzymes), attenuated the TRH-evoked increase in [Ca2+]i, suggesting that noncyclooxygenase metabolites of arachidonic acid or cytochrome P-450 metabolites may participate in regulating the TRH-evoked entry of extracellular Ca2+. Both nordihydroguaiaretic acid and econazole showed a similar inhibition of the Ca2+ entry, as did SKF 96365, a compound previously shown to inhibit receptor-activated Ca2+ entry. We also showed that arachidonic acid per se increased [Ca2+]i, and acidified the cytosol in GH4C1 cells in a dose-dependent manner. The effects of arachidonic acid was reversed by addition of BSA to the cell suspension. The calcium entry and the activation of the metabolism of arachidonic acid may thus be important components of the TRH-evoked signal-transduction pathway in GH4C1 cells.

Influence of Hepes- and CO2HCO3−-buffer on Ca2+ transients induced by TRH and elevated K+ in rat pituitary GH4C1 cells

The influence of two buffer systems (Hepes and CO2/HCO3-) on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH and by elevated K+ were studied in single, and small clusters of, clonal rat pituitary GH4C1 cells using Fura 2. The steady-state level of [Ca2+]i was virtually identical in Hepes and CO2/HCO3-. In both buffers, addition of TRH induced a transient increase in [Ca2+]i which attained a significantly higher peak in Hepes (357 +/- 43 nM) when compared with values measured in the presence of CO2/HCO3- (184 +/- 21 nM). In Hepes, the basal IP3-level was higher than in CO2/HCO3-. The TRH-evoked increase in IP3 was higher in magnitude in Hepes than in CO2/HCO3-, although the stimulated/basal ratio was not different between the two buffers. The buffer composition had no effect on the specific binding of 3H-TRH to the cells. Furthermore, the amplitude of the increase in [Ca2+]i evoked by 50 mM K+ was identical in both buffers. TRH and K+ had no effect on pHi in either buffer. The present results indicate that HCO3- has an influence on TRH-induced Ca2+ transient, at least in part by modifying the TRH-evoked production of IP3.

1,25-Dihydroxyvitamin D3 reduces the number of α1-andrenergic receptors in FRTL-5 rat thyroid cells

Abstract Noradrenaline and ATP evokes a transient increase in the intracellular calcium concentration ([Ca2+]i) in FRTL-5 cells. In a previous study, we showed that 1,25-dihydroxyvitamin-D3 (1,25(OH)2-D3) increases the ATP evoked changes in [Ca2+]i. In the present paper, we found that pre-incubating the cells with 10 nM 1,25(OH)2-D3 for 48 h did not affect the noradrenaline-evoked increase in [Ca2+]i. We subsequently examined if this could be due to an effect of 1,25(OH)2-D3 on α1-adrenergic receptor number, or receptor affinity. Pretreatment with 10 nM 1,25(OH)2-D3 for 48 h decreased the binding of the α1-adrenergic specific antagonist [3H]prazosin by 55% (Bmax for 1,25(OH)2-D3 treated = 27.6 ± 5.0 fmol/mg protein, untreated = 61.7 ± 5.4 fmol/mg protein). No effect of 1,25(OH)2-D3 on the affinity for [3H]prazosin was observed. The effect of 1,25(OH)2-D3 on the [3H]prazosin binding was both time- and dose-dependent and could first be seen after 8–12 h of 1,25(OH)2-D3 treatment, indicating a genomic effect. The effect of 1,25(OH)2-D3 could be abolished with the protein synthesis inhibitor cycloheximide. No effect on the [3H]prazosin binding could be seen after a 48 h preincubation with 100 nM of either 24,25-dihydroxyvitamin D3 and 25-dihydroxyvitamin D3, indicating that the effect of 1,25(OH)2-D3 was specific. The cellular cAMP concentration was decreased after 48 h treatment with 10 nM 1,25(OH)2-D3. When TSH was replaced with dibutyryl cAMP or forskolin the [3H]prazosin binding increased. 1,25(OH)2-D3 also reduced the dibutyryl cAMP and forskolin stimulated [3H]prazosin binding. In addition, 1,25(OH)2-D3 reduced the [3H]prazosin binding in TSH deficient media. Our results suggest that 1,25(OH)2-D3 reduces the number of α1-adrenergic receptors via a dual mechanism: by reducing cAMP production and by affecting some mechanism(s) downstream from the production of cAMP.