Nanoparticles carrying fingolimod and methotrexate enables targeted induction of apoptosis and immobilization of invasive thyroid cancer.

Metastatic tumors are the main cause of cancer-related death, as the invading cancer cells disrupt normal functions of distant organs and are nearly impossible to eradicate by traditional cancer therapeutics. This is of special concern when the cancer has created multiple metastases and extensive surgery would be too dangerous to execute. Therefore, combination chemotherapy is often the selected treatment form. However, drug cocktails often have severe adverse effects on healthy cells, whereby the development of targeted drug delivery could minimize side-effects of drugs and increase the efficacy of the combination therapy. In this study, we utilized the folate antagonist methotrexate (MTX) as targeting ligand conjugated onto mesoporous silica nanoparticles (MSNs) for selective eradication of folate receptor-expressing invasive thyroid cancer cells. The MSNs was subsequently loaded with the drug fingolimod (FTY720), which has previously been shown to efficiently inhibit proliferation and invasion of aggressive thyroid cancer cells. To assess the efficiency of our carrier system, comprehensive in vitro methods were employed; including flow cytometry, confocal microscopy, viability assays, invasion assay, and label-free imaging techniques. The in vitro results show that MTX-conjugated and FTY720-loaded MSNs potently attenuated both the proliferation and invasion of the cancerous thyroid cells while keeping the off-target effects in normal thyroid cells reasonably low. For a more physiologically relevant in vivo approach we utilized the chick chorioallantoic membrane (CAM) assay, showing decreased invasive behavior of the thyroid derived xenografts and an increased necrotic phenotype compared to tumors that received the free drug cocktail. Thus, the developed multidrug-loaded MSNs effectively induced apoptosis and immobilization of invasive thyroid cancer cells, and could potentially be used as a carrier system for targeted drug delivery for the treatment of diverse forms of aggressive cancers that expresses folate receptors.

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Towards an ADL taxonomy for occupational therapists. Previously published in Scandinavian Journal of Occupational Therapy 1994; 1:69-76.

One of the main concepts in occupational therapy is human occupation. In occupational therapy there is a need for a common conceptual framework to assess and describe the ability of patients to perform occupational activities of daily living. The aim of this report was to develop a taxonomy concerning the activities of daily living (ADL). In the taxonomy, occupation has been defined and related to common concepts of disability. Ordinary ADL terms have been categorized into three levels: occupational forms, activities and actions. Different actions are components of and subordinated to superior activities. Experience shows that the ADL taxonomy contributes to a valid (content and construct) assessment of ADL, a common language for OTs and to a clearer picture of the patient's performance in daily life activities.

Sphingosine 1-phosphate, sphingosine kinase and autocrine calcium signalling in thyroid cells.

In many cell types metabolites of sphingomyelin have a profound role in cellular signalling. One particular field where these derivatives have obtained a crucial role is calcium signalling. This is an interesting aspect on how lipids may wield their physiological role, as calcium is probably one of the most versatile signalling molecules in the cell, and modulation of calcium signalling may have profound effects on cellular physiology. In this review we discuss a novel aspect of sphingolipid signalling, i.e. the autocrine role of sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid cells. Although many investigations have highlighted the importance of S1P as a regulator of both calcium release from the endoplasmic reticulum and calcium entry through plasma membrane channels, the autocrine mechanism presented here introduces a new aspect of S1P signalling in thyroid cells. This mechanism may be physiologically relevant in many other cell types, including cancer cells.

Sphingosine kinase as an oncogene: autocrine sphingosine 1-phosphate modulates ML-1 thyroid carcinoma cell migration by a mechanism dependent on protein kinase C-alpha and ERK1/2.

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

The coumarin osthol attenuates the binding of thyrotropin-releasing hormone in rat pituitary GH4C1 cells.

The influence of two plant coumarins, osthol and xanthotoxin, on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH were studied in clonal rat pituitary GH4C1 cells. Osthol, but not xanthotoxin, decreased the TRH-induced transient increase in [Ca2+]i in Fluo-3 loaded cells incubated in Ca(2+)-free buffer. Binding experiments with [3H]TRH showed that osthol decreased the binding of TRH to its receptor, whereas the affinity of the receptor for TRH increased. This resulted in a decreased TRH-evoked production of IP3 in cells treated with osthol, and a decreased mobilization of sequestered calcium. Osthol did not inhibit the release of calcium evoked by exogenous IP3 in permeabilized cells. Furthermore, osthol decreased the uptake of 45Ca2+ in response to high K+. Xanthotoxin had no effects in these experiments. The results show that osthol modulates TRH-evoked responses by interacting with the TRH receptor.

Mechanisms of P2 receptor-evoked DNA synthesis in thyroid FRTL-5 cells.

The expression of the P2 receptors and their functional responses were studied in rat thyroid FRTL-5 cells. RT-PCR analysis revealed transcripts for the G protein-coupled P2Y(2), P2Y(4) and P2Y(6) receptors, and for the transmitter-gated ion channel P2X(3), P2X(4) and P2X(5) subunits. In Fura-2-loaded cells, UTP, ATP, ATPgammaS or UDP increased [Ca(2+)](i), and behaved as potent full agonists, while 2-Methylthio-ATP (2-MeSATP), alpha,beta-methylene-ATP (alpha,beta-meATP) and pure ADP were weak agonists. The agonist-mediated [Ca(2+) ](i) increases were diminished in Ca(2+) -free buffer, and by pertussis toxin (PTX) or suramin treatments. ATP, UTP, UDP and ATPgammaS increased (3)H-thymidine incorporation into DNA and expression of the protooncogenes c-Fos and c-Jun, while 2-MeSATP was ineffective, and alpha,beta-meATP gave a response only at 100-microM dose. The ATP-stimulated expression of c-Fos and c-Jun was dependent on Ca(2+), and protein kinase C, but not on calmodulin or Ca(2+)/calmodulin-dependent protein kinase II. Extracellular signal-regulated kinases (ERK1 and ERK2) are also involved as the MEK inhibitor, PD98059, reduced both ATP-evoked (3)H-thymidine incorporation and c-Fos and c-Jun expression. These results indicate that multiple P2Y receptor subtypes and at least the P2X(5) subtype are functionally expressed in FRTL-5 cells, and that nucleotides acting via P2 receptors are involved in the regulation of DNA-synthesis.

Effects of simple aromatic compounds and flavonoids on Ca2+ fluxes in rat pituitary GH(4)C(1) cells.

The biological activity of phenolic compounds from plants is well documented in vitro, but little is known about the possible effect of simple aromatic compounds and flavonoids on voltage-operated Ca2+ channels (VOCCs). In pituitary cells, several intracellular pathways may regulate the activity of VOCCs. In this study, we investigated the effect of nine phenylpropanes and metanes, and 20 flavonoids on high K(+)-induced 45Ca2+ entry in clonal rat pituitary GH(4)C(1) cells. At the highest dose tested (20 microg/ml), flavone (a flavone) inhibited 45Ca2+ entry by 63.5%, naringenin (a flavanone) by 56.3% and genistein (an isoflavone) by 54.6%. The phenylmetane derivative octyl gallate was the most potent compound tested, with an IC(50) value of 15.0 microg/ml. The IC(50) value for the reference compound verapamil hydrochloride was 3.0 microg/ml. In sharp contrast to the above, the flavonols quercetin and morin potentiated 45Ca2+ entry. At 20 microg/ml, quercetin increased 45Ca2+ entry by 54.1% and morin by 48.0%. Quercetin increased the cellular cAMP content in a concentration-dependent manner. H 89, an inhibitor of protein kinase A, inhibited the effect of quercetin on 45Ca2+ entry. The results thus suggest that the effect of quercetin is the result of a protein kinase A-mediated activation of VOCCs. Quercetin induced a rapid and marked increase in both the transient (143.1+/-4.2%) and delayed (198.8+/-10.0%) Ca2+ currents, measured by the whole cell patch clamp technique. The onset of the inhibitory effect of octyl gallate was slow, but resulted in an almost complete inhibition of both Ca2+ currents.

Do occupational therapy and physiotherapy students care about research? A survey of perceptions and attitudes to research.

Do occupational therapy and physiotherapy students care about research? A survey of perceptions and attitudes to research. In this cross-sectional study, we have used a questionnaire to study perceptions and attitudes to research-related activities of Swedish occupational therapy and physiotherapy first (T1) and final term (T6) students. Two hundred and eleven students from programmes employing either problem-based learning (PBL) or traditional education methods participated. The results showed that students had a positive attitude towards research, particularly for the activity 'read research literature to update knowledge' and 'apply research findings to improve practice'. When T1 were compared with T6 students, the ability to perform research-related activities was rated significantly higher by T6 students regardless of educational methods. A comparison of PBL and the traditional educational method resulted in moderate but significant differences. The PBL students had a more positive attitude towards research and to a greater extent intended to engage in research activities in the future. The results give hope for a positive future development of the occupational therapy and physiotherapy professions and a PBL education method may be more conducive to the shaping of a research consumer than a traditional method, although more research is needed to substantiate these claims.

Hydrogen peroxide attenuates store-operated calcium entry and enhances calcium extrusion in thyroid FRTL-5 cells.

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca(2+)](i)) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca(2+)](i) regulate several important functions, including the production of H(2)O(2) (hydrogen peroxide). As H(2)O(2) is of crucial importance for the production of thyroid hormones, we investigated the effects of H(2)O(2) on [Ca(2+)](i) in thyroid FRTL-5 cells. H(2)O(2) itself did not modulate basal [Ca(2+)](i). However, H(2)O(2) attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H(2)O(2) was abrogated by the reducing agent beta-mercaptoethanol. H(2)O(2) also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H(2)O(2) was, at least in part, mediated by activation of protein kinase C (PKC), as H(2)O(2) enhanced the binding of [(3)H]phorbol 12,13-dibutyrate. H(2)O(2) also stimulated the translocation of the isoenzyme PKCepsilon from the cytosolic fraction to the particulate fraction. Furthermore, H(2)O(2) did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H(2)O(2) depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H(2)O(2)-evoked inhibition of calcium entry. In addition, H(2)O(2) enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent beta-mercaptoethanol. In conclusion H(2)O(2) attenuates an increase in [Ca(2+)](i). As H(2)O(2) is produced in thyroid cells in a calcium-dependent manner, our results suggest that H(2)O(2) may participate in the regulation of [Ca(2+)](i) in these cells via a negative-feedback mechanism involving activation of PKC.

Thyrotropin regulates adenosine A(1) receptor expression in rat thyroid FRTL-5 cells.

The effect of thyrotropin (TSH), on adenosine A(1) receptor expression in thyroid FRTL-5 cells was examined by [(3)H]-1, 3-dipropyl-,8-cyclopentyl xanthine ([(3)H]-DPCPX) binding on cells in suspension and on membrane preparation, and by in situ mRNA labelling. The estimated K(D) for intact cells was 0.19 nM and about 47,000 binding sites per cell were found in cells constantly grown in the presence of TSH. Three days deprivation of TSH decreased the number of [(3)H]-DPCPX binding sites without any significant effect of K(D). Reintroduction of TSH to the cells returned the higher level of A(1) receptors both in suspension binding studies on whole cells and on membrane preparations. In situ hybridization revealed that TSH evoked an increase in the number of cells densely labelled with a probe against A(1) receptor mRNA. The potency of the A(1) receptor agonist N(6)-cyclohexyladenosine (CHA) as an inhibitor of cyclic AMP formation induced by forskolin was increased in TSH-treated cells, with a shift in the IC(50) from 2.05 nM in TSH-deprived cells to 0.14 nM in TSH-treated cells. Since the activation of A(1) receptors inhibits TSH-mediated cyclic AMP signalling, our results suggest a regulatory feedback mechanism between signalling via adenosine A(1) receptors and TSH receptors.

Cotinine and nicotine inhibit each other's calcium responses in bovine chromaffin cells.

Cotinine is the major metabolite of nicotine. It has some biological activity, but its pathophysiological effects are largely unclear. We studied whether cotinine initiates calcium transients or affects those induced by nicotine. In bovine adrenal chromaffin cells labeled with the fluorescent calcium indicator Fura 2, cotinine (0. 32-3.2 mM) concentration-dependently increased the intracellular Ca(2+) concentration ([Ca(2+)](i)). The effect was abolished by omitting extracellular Ca(2+) during the stimulations. Also nicotinic receptor channel blockers hexamethonium (10 microM-1 mM) and chlorisondamine (100 microM), as well as a competitive nicotinic receptor antagonist dihydro-beta-erythroidine (10-100 microM), inhibited the response. Cotinine (0.32-3.2 mM) preincubation for 2 min inhibited both the nicotine-induced and the cotinine-induced increases in [Ca(2+)](i). Also nicotine (3.2-10 microM) inhibited the cotinine-induced increase in [Ca(2+)](i). Tetrodotoxin (1 microM) and thapsigargin (1 microM) pretreatments did not affect the responses to cotinine, while 300 nM nimodipine partially inhibited the cotinine-induced increase in [Ca(2+)](i). The results indicate that cotinine has nicotine-like effects on chromaffin cells. It may also desensitize the nicotinic cholinergic receptors, possibly by acting as a low-affinity agonist at these receptors.

The role of adenosine A(1) receptors in the ATP-evoked Ca(2+) response in rat thyroid FRTL-5 cells.

The effect of adenosine A(1) receptor activation on the ATP-induced increase in intracellular free Ca(2+) was studied in control and protein kinase C down-regulated Fisher rat thyroid (FRTL-5) cells. Long-term phorbol ester treatment, which leads to protein kinase C down-regulation, enhanced the ATP-evoked extracellular Ca(2+) influx. The increased Ca(2+) influx was antagonized by the adenosine A(1) receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). [3H]DPCPX binding studies revealed that phorbol ester-treatment increased the number of adenosine A(1) receptors. The adenosine A(1) receptor-mediated inhibition of the cyclic AMP formation was not affected by the increased receptor number. We conclude that the enhanced ATP-evoked Ca(2+) influx in protein kinase C down-regulated cells is mediated by adenosine formed by hydrolysis of ATP, and that this adenosine interacts with the increased number of A(1) receptors. The mechanism by which adenosine enhances Ca(2+) entry is not known. Thus, the larger number of adenosine A(1) receptors broadens the spectrum of adenosine A(1) receptor affected signaling systems in FRTL-5 cells.

Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase.

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process.

Monensin and hypo-osmolar medium cause calcium-independent beta-endorphin secretion from melanotropes.

Monensin has been shown to cause nonexocytotic release of catecholamines from adrenal medullary and PC12 cells. We examined the effect of monensin on peptide secretion with cultured melanotropes from the rat pituitary as a model. 1 microM monensin caused an immediate, transient increase in beta-endorphin secretion. The effect was still seen in a calcium-free medium, but was totally abolished in a sodium-free medium. Intracellular calcium concentration was measured with Fura 2: no increase was observed during monensin stimulation. Hypo-osmolar medium mimicked the effect of monensin, causing a 12-fold transient increase in beta-endorphin secretion. This effect was not abolished in either calcium-free or sodium-free medium. No increase in the number of exocytotic figures captured by tannic acid incubation was observed during 5 min of incubation with 1 microM monensin or hypo-somolar medium. We thus show that monensin causes beta-endorphin secretion from the melanotrope and that this effect is due to sodium influx and resultant cell swelling. The calcium independency and lack of increase of exocytotic figures suggest that swelling-induced secretion is nonexocytotic, possibly via transient exocytotic pore opening.

Sphingosylphosphorylcholine activates Gq, Gi-2, and Gi-3 in thyroid FRTL-5 cells: implications for the activation of calcium fluxes and Na+-H+ exchange.

In the present investigation of rat thyroid FRTL-5 cells, we show using reverse-transcriptase PCR that these cells express both Edg-1 and Edg-5. We show using a [35S]GTPgammaS-binding assay that sphingosylphosphorylcholine (SPC), which binds to both Edg-1 and EDG-5, activates Gq, Gi-2, and Gi-3 proteins. SPC potently increases intracellular free calcium concentrations ([Ca2+]i). This effect is mediated through both Gq and Gi proteins, as the mobilization of sequestered calcium was insensitive to pertussis toxin (i.e., mediated by Gq), while the SPC-evoked calcium entry was inhibited by pretreatment with pertussis toxin (i.e., mediated by Gi). Furthermore, SPC in a concentration-dependent manner increases intracellular pH in acidified cells via a Na+-H+ exchange mechanism. The enhanced activation of Na+-H+ exchange is independent of both an increase in [Ca2+]i and an activation of protein kinase C. The effect of SPC on Na+-H+ exchange is insensitive to pertussis toxin, suggesting an effect mediated via Gq.

Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium.

Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent beta-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12, 13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase.

Tumor necrosis factor-alpha, sphingomyelinase, and ceramide inhibit store-operated calcium entry in thyroid FRTL-5 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inhibitor of proliferation in several cell types, including thyroid FRTL-5 cells. As intracellular free calcium ([Ca2+]i) is a major signal in activating proliferation, we investigated the effect of TNF-alpha on calcium fluxes in FRTL-5 cells. TNF-alpha per se did not modulate resting [Ca2+]i. However, preincubation (10 min) of the cells with 1-100 ng/ml TNF-alpha decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-alpha did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF-alpha on calcium entry was mediated via the sphingomyelinase pathway, the cells were pretreated with sphingomyelinase (SMase) prior to stimulation with Tg. SMase inhibited the Tg-evoked calcium entry in a concentration-dependent manner. Furthermore, an inhibition of calcium entry was obtained after preincubation of the cells with the membrane-permeable C2-ceramide and C6-ceramide analogues. The inactive ceramides dihydro-C2 and dihydro-C6 showed only marginal effects. Neither SMase, C2-ceramide, nor C6-ceramide affected the release of sequestered calcium. C2- and C6-ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF-alpha and SMase was inhibited by the kinase inhibitor staurosporin and by the protein kinase C (PKC) inhibitor calphostin C but not by down-regulation of PKC. However, we were unable to measure a significant activation of PKC using TNF-alpha or C6-ceramide. The effect of TNF-alpha was not mediated via activation of either c-Jun N-terminal kinase or p38 kinase. We were unable to detect an increase in the ceramide (or sphingosine) content of the cells after stimulation with TNF-alpha for up to 30 min. Thus, one mechanism of action of TNF-alpha, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.

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.