Biomedical subjects

K Törnquist

Publications and source records attributed to K Törnquist.

At least 37 records · Page 2Linked to original sources

PubMed1997

Sphingosine 1-phosphate stimulates Na+/H+ exchange in thyroid FRTL-5 cells.

Sphingosine derivatives are potent mitogens in several cell types. Many mitogens activate the Na+/H+ exchange, although the interrelationships between Na+/H+ exchange and mitogenesis are unclear. The present investigation in thyroid FRTL-5 cells shows that sphingosine 1-phosphate (SPP) activates Na+/H+ exchange in a dose-dependent manner in acid-loaded cells. The effect of SPP was abolished in a Na+-free buffer and by pretreatment of the cells with ethylisopropylamiloride. SPP did not affect basal intracellular pH (pHi). SPP stimulated the release of sequestered Ca2+ and a substantial entry of Ca2+. The effect of SPP on pH(i) was abolished in cells incubated in a Ca2+-free buffer, and in cells loaded with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Furthermore, the effect of SPP was abolished in pertussis toxin (PTX)-treated cells. PTX decreased Ca2+ entry only, without affecting the release from intracellular stores. Phosphatidic acid (PA) did not activate Na+/H+ exchange, suggesting that the effect of SPP was not mediated via activation of phospholipase D and the production of PA. Thus one mechanism of action of SPP in FRTL-5 cells appears to be to activate Na+/H+ exchange. This action is mediated via a G protein-dependent mechanism and requires an increase in intracellular free Ca2+.

Animals↗

PubMed1997

Sphingosine 1-phosphate mobilizes sequestered calcium, activates calcium entry, and stimulates deoxyribonucleic acid synthesis in thyroid FRTL-5 cells.

Sphingosine 1-phosphate (SPP) potently mobilizes sequestered calcium and is a mitogen in several cell types. In the present investigation, we have evaluated the effect of SPP on intracellular free calcium concentration ([Ca2+]i) and synthesis of DNA in thyroid FRTL-5 cells. SPP rapidly and transiently mobilized sequestered calcium and stimulated entry of extracellular calcium. The entry of calcium, but not the mobilization, was in part inhibited by pretreatment with pertussis toxin (Ptx), and by activation of protein kinase C. SPP did not stimulate the production of inositol 1,4,5-trisphosphate. SPP stimulated the incorporation of 3H-thymidine in a time- and dose-dependent manner. The effect was not inhibited by Ptx. Furthermore, SPP stimulated the activation of the proto-oncogene c-fos. SPP rapidly tyrosine-phosphorylated an approximately 66 kDa protein. This phosphorylation persisted for at least 1 h. Pretreatment of the cells with genistein abolished the SPP-evoked tyrosine phosphorylation, and attenuated the SPP-evoked increase in [Ca2+]i. Furthermore, the SPP-evoked activation of Na+-H+ exchange was inhibited by genistein. The phosphorylation was not attenuated by pretreatment of the cells with Ptx. SPP per se did not affect cellular cAMP levels but attenuated the TSH-evoked increase in cAMP. As the effect of SPP might be due to activation of phospholipase D, we tested whether phosphatidic acid (PA) mobilized calcium or stimulated the incorporation of 3H-thymidine. PA mobilized sequestered calcium but did not stimulate calcium entry. PA very modestly enhanced the incorporation of 3H-thymidine. Our results suggest, that SPP stimulates DNA synthesis and activates entry of calcium in FRTL-5 cells. The effect on calcium entry appears to be dependent, at least in part, on one or several tyrosine kinases.

Animals↗

PubMed1996

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.

Adenosine Triphosphate↗

PubMed1996

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.

Adenosine↗

PubMed1996

On the possible origin of giant or slow-rising miniature end-plate potentials at the neuromuscular junction.

Giant or slow-rising miniature end-plate potentials (GMEPPs) caused by vesicular release of acetylcholine (ACh) occur at any time in about 50% of mouse diaphragm neuro muscular junctions, but generally at frequencies less than 0.03 s-1. Their frequency is, unlike that of miniature end-plate potentials (MEPPs), not affected by nerve terminal depolarization. Unlike MEPPs and stimulus-evoked end-plate potentials, GMEPPs have a prolonged time-to-peak and show an increase in time-to-peak with amplitude. By using these differences in amplitude and time course, GMEPPs can be separated from MEPPs. In contrast to MEPPs, GMEPPs are not blocked by botulinum neurotoxin type A. GMEPPs have a greater temperature sensitivity than MEPPs, disappearing at temperatures below 15 degrees C. Long-term paralysis by botulinum toxin and certain drugs which inhibit protein kinase C or affect actin filament polymerization (cytochalasins) enhance the frequency of GMEPPs. End-plate current recordings show that similar postsynaptic ACh receptors are activated by MEPPs and GMEPPs. It is suggested that GMEPPs are not caused by mechanisms involved in regulated neurotransmitter release but are generated by constitutive secretion.

Acridine Orange↗

PubMed1996

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.

Animals↗

PubMed1996

Inhibition of agonist-mediated calcium entry by calmodulin antagonists and by the Ca2+/calmodulin kinase II inhibitor KN-62. Studies with thyroid FRTL-5 cells.

Calmodulin and calmodulin-dependent mechanisms are probably important in regulating thyroid cell function. However, calmodulin antagonists may directly modify calcium fluxes in cells. In the present investigation the effects of several calmodulin inhibitors and of KN-62, a specific calcium/calmodulin kinase II inhibitor, on the ATP- and thapsigargin-evoked changes in intracellular free calcium ([Ca2+]i) were investigated in Fura-2 loaded thyroid FRTL-5 cells. All of the inhibitors tested attenuated agonist-evoked calcium entry. The inhibitor calmidazolium per se potently released sequestered calcium followed by enhanced calcium entry. Pretreatment of the cells with calmidazolium inhibited both the thapsigargin-and the ATP-evoked calcium entry. Our results show that calmodulin antagonists are potent inhibitors of calcium entry in thyroid cells, possibly by directly inhibiting the calcium entry pathway. This inhibition may explain, in part, the results obtained with calmodulin inhibitors in previous studies.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Caffeine inhibits the binding of thyrotropin-releasing hormone in GH4C1 pituitary cells.

Caffeine can modulate intracellular Ca2+ concentration ([Ca2+]i) by triggering the mobilization of Ca2+ from intracellular Ca2+ stores. In the present study we show that in Fura 2 loaded GH4C1 cells, caffeine inhibited, in a dose-dependent manner, the Ca2+ response induced by a submaximally effective dose (3 nM) of thyrotropin-releasing hormone (TRH). We also show that caffeine decreased the specific binding of [3H]TRH. Equilibrium binding studies with [3H]TRH and Scatchard analysis of the binding data showed that caffeine increased the dissociation constant (Kd) from 8 +/- 1 nM to 26 +/- 3 nM, while the maximum amount of [3H]TRH bound to the cells was increased by 32%. Thus, caffeine inhibited the TRH-evoked increase in [Ca2+]i by inhibiting the binding of TRH to its receptor.

1-Methyl-3-isobutylxanthine↗

PubMed1995

1,25-Dihydroxyvitamin D3 reduces the number of alpha 1-adrenergic receptors in FRTL-5 rat thyroid cells.

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 alpha 1-adrenergic receptor number, or receptor affinity. Pretreatment with 10 nM 1,25(OH)2-D3 for 48 h decreased the binding of the alpha 1-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.(ABSTRACT TRUNCATED AT 250 WORDS)

24,25-Dihydroxyvitamin D 3↗

PubMed1995

Evidence for a pertussis toxin sensitive calcium entry pathway in thyroid FRTL-5 cells.

Receptor-mediated calcium entry was investigated in Fura 2 loaded FRTL-5 cells. The purinergic agonist ATP activated the release of sequestered calcium and the entry of extracellular calcium. Downregulation of protein kinase C (PKC) substantially enhanced the ATP-evoked calcium entry. Pretreatment of the cells with pertussis toxin (Ptx) decreased the ATP-evoked calcium entry by 56% and the release of sequestered calcium by 34%. In PKC-downregulated cells, the effect of Ptx treatment on the ATP-evoked increase in [Ca2+]i was 73% and 44%, respectively. Phorbol myristic acetate (PMA) decreased the ATP-evoked calcium entry to the same extent as Ptx. In Ptx-treated cells, the ATP-evoked influx of 45Ca2+ was attenuated. Stimulation of the cells with P2p-purinergic agonist GTP evoked no entry of calcium, although GTP released the same amount of sequestered calcium as did ATP. PKC downregulation or pretreatment with Ptx had no effects on the GTP-evoked responses, whereas PMA decreased the GTP-evoked release of calcium. We conclude that the ATP-activated rapid calcium entry pathway is a second messenger-operated calcium channel.

Adenosine Triphosphate↗

PubMed1995

Influence of Hepes- and CO2/HCO(3-)-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.

Animals↗

PubMed1995

Sphingosine derivatives inhibit depolarization-evoked calcium entry in rat GH4C1 cells.

Several investigations have suggested that sphingosine (SP) derivatives are potent inhibitors of protein kinase C. In GH4C1 cells, protein kinase C is a potent modulator of voltage-operated calcium channels (VOCCs). The aim of the present study was to investigate whether SP derivatives could modify calcium entry via VOCCs. Using fura-2-loaded cells and 45Ca2+ flux studies, we show that several SPs potently and rapidly inhibit depolarization-evoked calcium entry in a dose-dependent manner. The effect was not due to an enhanced efflux of calcium from the cells, as the depolarization-evoked entry of Ba2+ was inhibited by the SPs. A similar inhibition was observed with 1,2-dioctanoylglycerol, an activator of sphingomyelinase in GH3 cells. Phorbol myristate acetate and 1-oleyl-2-acetylglycerol had only a modest inhibitory effect. Furthermore, whole cell patch-clamp experiments showed that sphingosinephosphorylcholine (SPC) potently attenuated calcium entry via VOCCs. In experiments using cells grown on coverslips, we found that the inhibitory effect of SPC on calcium entry was reversible. The addition of sphingomyelinase or hexanoyl ceramide, a cell-permeable ceramide, only modestly inhibited the depolarization-evoked entry of calcium, whereas arachidonic acid and phosphatidic acid had no effect. The SP metabolite sphingosine-1-phosphate had no effect on the entry of calcium. The results suggest that the effects of the SPs were probably not the result of a conversion to ceramide or of the production of other lipid second messengers. In cells with down-regulated protein kinase C, SPC, SP, and 1,2-dioctanoylglycerol inhibited depolarization-evoked calcium entry, suggesting that the inhibition was independent of an action mediated via protein kinase C. The SPs per se did not induce any changes in intracellular free calcium, and they did not inhibit the TRH-evoked release of sequestered calcium in the cells. However, TRH-evoked calcium entry was inhibited. The results suggest that SPs are potential regulators of calcium entry mediated by VOCCs in GH4C1 cells.

Animals↗

PubMed1994

Effects of 5,8,11,14-eicosatetraynoic acid on thapsigargin-induced calcium entry, and intracellular pH in thyroid FRTL-5 cells.

The effect of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of lipoxygenase and cytochrome P-450 epoxygenase enzymes, on calcium fluxes was investigated in Fura 2 loaded rat thyroid FRTL-5 cells. ETYA per se released sequestered calcium. ETYA also inhibited calcium influx in thapsigargin-stimulated cells in dose-dependent manner. Addition of calcium to cells treated with ETYA and stimulated with thapsigargin in a calcium-free buffer resulted in a blunted increase in intracellular free calcium compared with the response in control cells. In addition, ETYA per se acidified the cytosol in a dose-dependent manner. Acidification of the cytosol with the K+/H+ ionophore nigericin also decreased thapsigargin-induced calcium entry, but not to the same extent as that seen in cells treated with ETYA. The results suggest that ETYA is a potent modulator of calcium entry, and that part of the inhibitory effect of ETYA may be due to the ETYA-induced acidification of the cytosol.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1994

Effect of sphingosine derivatives on calcium fluxes in thyroid FRTL-5 cells.

The effects of sphingosine derivatives on Ca2+ fluxes were investigated in thyroid FRTL-5 cells labelled with Fura 2. Addition of sphingosylphosphocholine (SPC) or sphingosine (SP) increased intracellular free Ca2+ ([Ca2+]i) in a dose-dependent manner. At the highest dose tested (30 microM), the response was biphasic: a rapid transient increase in [Ca2+]i, followed by a new, elevated, level of [Ca2+]i. Both phases of the SPC-evoked increase in [Ca2+]i were dependent on extracellular Ca2+, whereas only the SP-evoked elevated level of [Ca2+]i was dependent on the influx of Ca2+. Both compounds released sequestered Ca2+ from thapsigargin- and inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools. In addition, the increase in [Ca2+]i in response to SPC, but not to SP, was attenuated in cells treated with phorbol myristate acetate or with the putative Ca(2+)-channel blocker SKF 96365, and in cells pretreated with pertussis toxin for 24 h. SPC did not activate the production of IP3. Furthermore, both SPC and SP released sequestered Ca2+ from permeabilized cells. We observed that SPC, but not SP, stimulated release of [3H]arachidonate from cells prelabelled with [3H]arachidonate for 24 h. Both SPC and SP stimulated the incorporation of [3H]thymidine into DNA in cells grown in the absence of thyroid-stimulating hormone (TSH). The results suggest that sphingosine derivatives are putative regulators of Ca2+ fluxes in FRTL-5 cells, and that SP and SPC may act on [Ca2+]i via different mechanisms. Furthermore, both SP and SPC may be of importance in modulating thyroid-cell proliferation.

Animals↗

PubMed1994

Thapsigargin-induced calcium entry in FRTL-5 cells: possible dependence on phospholipase A2 activation.

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A2 (PLA2) is activated. Since PLA2 is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA2 activity and the entry of calcium. Stimulating 3H-arachidonic acid (3H-AA)-labelled cells with thapsigargin resulted in a substantial release of 3H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA2 activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA2 in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA2 activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca2+ in FRTL-5 cells.

Acetophenones↗

PubMed1994

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.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1994

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.

Adenosine Triphosphate↗

PubMed1994

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.

Animals↗