Chemically modified tetracycline (CMT)-3 inhibits histamine release and cytokine production in mast cells: possible involvement of protein kinase C.

OBJECTIVE: To find novel inhibitors of mast cell function we have studied the effect of a potent, non-antimicrobial, chemically modified tetracycline, CMT-3 or COL-3, on key functions of mast cells. METHODS AND RESULTS: In the presence of 25 microM CMT-3, the 48/80-induced histamine release from rat serosal mast cells was inhibited significantly, to 43.0 +/- 7.3% of control. Similarly, the activation-induced secretion of TNF-alpha and IL-8 by HMC-1 cells were decreased in the presence of 25 microM CMT-3 to 13.5 +/- 4.1% and 9.7 +/- 1.1% of control, respectively. CMT-3 did not cause intracellular accumulation of TNF-alpha but instead it reduced the expression of TNF-alpha mRNA in HMC-1 cells. Moreover, CMT-3 was found to significantly inhibit the protein kinase C (PKC) activity with IC(50) value of 31 microM. CMT-3 inhibited effectively both human recombinant PKCalpha and PKCdelta isoforms. In comparison to doxycycline, CMT-3 was more effective as an inhibitor of both cytokine production and PKC activity. CONCLUSIONS: Considering the central role of PKC in mast cell activation, PKC inhibition could, at least partially, explain the observed inhibitory effects of CMT-3. The inhibition of the key proinflammatory functions of mast cells by CMT-3 suggests its potential clinical usefulness in the treatment of allergic and inflammatory disorders.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Intracellular free sodium concentrations in GH4C1 cells.

In the present investigation, intracellular sodium ([Na+]i) levels were determined in GH4C1 cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na+]i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na+. Resting [Na+]i in GH4C1 cells was 26 +/- 6.2 mM (mean +/- SD). In cells incubated in Na(+)-free buffer [Na+]i decreased to 3 +/- 3.6 mM. If Na+/K+ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na+]i increased to 47 +/- 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetate, or thapsigargin had no effect on [Na+]i. Incubating the cells in Ca(2+)-free buffer rapidly increased [Na+]i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca2+, nimodipine, or Ni2+ to these cells immediately decreased [Na+]i, whereas Bay K 8644 enhanced the influx of Na+. In cells where [Na+]i was increased the TRH-induced increase in intracellular free calcium ([Ca2+]i) was decreased compared with control cells. Our results suggest that Na+ enters the cells via Ca2+ channels, and [Na+]i may attenuate TRH-induced changes in [Ca2+]i in GH4C1 cells.