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⁺]_iwas 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 P_2p-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. © 1995 Wiley-Liss, Inc.     

Depolarization of the membrane potential decreases the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of membrane depolarization on ATP-induced changes in intracellular Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL-5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonal, significantly reduced the ATP-induced uptake of 45Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP-induced efflux of 45Ca2+ was obtained between control cells and depolarized cells. The ATP-induced transient increase in [Ca2+]i in Fura-2 loaded cells was not altered by depolarization, whereas the ATP-induced plateau in [Ca2+]i was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]i in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by first stimulating the cells with noradrenaline (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+, the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL-5 cells.     

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. J. Cell. Physiol. 175:211–219, 1998. © 1998 Wiley-Liss, Inc.     

Phosphatase inhibition reveals a calcium entry pathway dependent on protein kinase A in thyroid FRTL-5 cells: comparison with store-operated calcium entry

Calcium entry through store-operated calcium channels is an important entry mechanism. In the present report we have described a novel calcium entry pathway that is independent of depletion of intracellular calcium stores. Treatment of the cells with the phosphatase inhibitor calyculin A (caly A), which blocked thapsigargin-evoked store-operated calcium entry (SOCE), induced a potent concentration-dependent calcium entry. In a calcium-free buffer, acute addition of caly A evoked a very modest increase in cytosolic free calcium ([Ca(2+)](i)). This increase was not from the agonist-mobilizable calcium stores, as the thapsigargin-evoked increase in [Ca(2+)](i) was unaltered in caly A-treated cells. The caly A-evoked calcium entry was not blocked by Gd(3+) or 2-APB, whereas SOCE was. Caly A enhanced the entry of barium, indicating that the increase in intracellular calcium was not the result of a decreased extrusion of calcium from the cytosol. Jasplakinolide and cytochalasin D had only marginal effects on calcium entry. The protein kinase A (PKA) inhibitor H-89 and an inhibitory peptide for PKA abolished the caly A-evoked entry of both calcium and barium. The SOCE was, however, enhanced in cells treated with H-89. In cells grown in the absence of thyrotropin (TSH), the caly A-evoked entry of calcium was smaller compared with cells grown in TSH-containing buffer. Stimulation of cells grown without TSH with forskolin or TSH restored the calyculin A-evoked calcium entry to that seen in cells grown in TSH-containing buffer. SOCE was decreased in these cells. Our results thus suggest that TSH, through the production of cAMP and activation of PKA, regulates a calcium entry pathway in thyroid cells. The pathway is distinctly different from the SOCE. As TSH is the main regulator of thyroid cells, we suggest that the novel calcium entry pathway participates in the regulation of basal calcium levels in thyroid cells.     

Release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.     

Thyrotropin regulation of malic enzyme in FRTL-5 rat thyroid cells

TSH-induced increases in malic enzyme mRNA levels in FRTL-5 rat thyroid cells are paralleled by increases in malic enzyme activity and are mimicked by 8-bromo-cAMP. Apparent approximately 4 h after TSH challenge and maximal after 16 h, they decline by 24 h and are at basal levels by 48 h. The increase occurs in the absence of a measurable effect of TSH on DNA synthesis related to cell growth, since [3H] thymidine incorporation into DNA is still at basal levels 24 h after TSH challenge and is maximal only at 48 h. A protein(s) whose formation is inhibited by cycloheximide appears to be critical to the ability of TSH to increase malic enzyme mRNA levels. Thus, cycloheximide given 30 min before TSH prevents the hormone-induced increase in malic enzyme mRNA; also, when given 24 h after TSH, cycloheximide accelerates the loss of the TSH-induced increase in malic enzyme mRNA. In neither case does cycloheximide affect beta-actin mRNA levels. A second factor(s) whose formation is prevented by actinomycin-D appears to be important for the decrease in malic enzyme mRNA levels seen 24 and 48 h after TSH challenge. Thus, in experiments in which it is given 24 h after TSH, actinomycin-D preserves the hormone-induced increase in malic enzyme mRNA levels rather than accelerating the decrease, as does cycloheximide. In the same experiment, beta-actin mRNA levels decrease to less than 10-20% of control values over the same period; this factor also, therefore, appears to exhibit some degree of specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of immunoreactive ANF from FRTL-5 rat thyroid cells: regulation by calcium ionophore A23187.

Atrial natriuretic factor-like immunoreactivity (ir-ANF) was characterized in a continuous line of rat thyroid follicular cells (FRTL-5) and the influence of the calcium ionophore A23187 on ir-ANF secretion was examined. Ir-ANF was identified by immunohistochemical staining as primarily reticular and juxtanuclear in short-term cultures, and more peripheral and granular in longer-term cultures, suggesting a process of ir-ANF packaging into secretory granules. The accumulation of ir-ANF granules was dependent upon the presence of thyrotropin (TSH) in the medium. Secreted ir-ANF was characterized using reversed-phase, high-performance liquid chromatography (RP-HPLC) and radioimmunoassay as a single peak eluting one fraction earlier than 125I-labeled rat ANF (99-126) (i.e., circulating atrial ANF) included as an internal standard. A23187 treatment of cells exhibiting primarily reticular ir-ANF caused a change to a pattern of more distinct, peripherally localized granules. This change occurred within 1 h after A23187 treatment and was dependent on the presence of Ca2+ in the medium. In cultures containing primarily ir-ANF granules, A23187 (0.5 micrograms/ml) induced a peripheral translocation of the granules at 30 min and a complete degranulation by 7 h. Enzyme-linked immunoadsorbent assay (EIA) confirmed a dose-dependent effect of A23187 on ir-ANF release into the medium. These results suggest that some of the effects of Ca2+ in the thyroid could be ascribed to its mobilization and release of ir-ANF, which in turn may have autocrine effects on thyroid follicular cells.     

Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.     

Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.     

Uptake of Ca2+ and refilling of intracellular Ca2+ stores in Ehrlich-ascites-tumour cells and in rat thymocytes.

We have studied the uptake of Ca2+ and its redistribution between the cytoplasm and the intracellular stores in Ehrlich-ascites-tumour cells and rat thymocytes previously depleted of Ca2+ by incubation in Ca2(+)-free medium. Measurements included changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), uptake of 45Ca2+ and uptake of Mn2+, a Ca2+ surrogate for Ca2+ channels. Refilling of the Ca2+ stores in thymocytes was very fast (half-filling time: 4 s at 37 degrees C) and very sensitive to temperature (10 times slower at 20 degrees C). It was always preceded by increase of [Ca2+]i. In the Ehrlich cell, both refilling and increase of [Ca2+]i were about one order of magnitude slower. The increase of [Ca2+]i and the refilling of the intracellular stores were both almost completely blocked by Ni2+ in thymocytes, but only partially in the Ehrlich cell. The rates of 45Ca2+ and Mn2+ uptake varied consistently with temperature and the kind of cell. These results suggest that the intracellular stores are refilled by Ca2+ taken up from the cytoplasm. We also find that filling of the Ca2+ stores decreases by about 90% the rate of Mn2+ uptake in thymocytes. This is direct evidence of modulation of the plasma-membrane Ca2+ entry by the degree of filling of the intracellular stores. This modulation occurs in the absence of agonists, suggesting some kind of signalling between the intracellular stores and the Ca2+ entry pathways of the plasma membrane.     

Thyrotropin stimulation of lysosomal tyrosine transport in rat FRTL-5 thyroid cells

Tyrosine countertransport was used to demonstrate the hormonal stimulation of neutral amino acid transport across the lysosomal membrane of FRTL-5 cells. Cells grown with thyrotropin (1 X 10(-10) M) had 7-fold (+/- S.E.) higher tyrosine countertransport activity in their lysosomes than cells grown without thyrotropin. Thyrotropin also stimulated the uptake into tyrosine-loaded lysosomes of other neutral amino acids recognized by the tyrosine carrier, namely, phenylalanine (3-fold) and leucine (6-fold). In contrast lysosomal cystine countertransport was not affected by thyrotropin. Addition of thyrotropin to cells grown without thyrotropin showed that the stimulation of tyrosine counter-transport (a) required at least 48 h to reach the level of the thyrotropin-supplemented cells, (b) depended upon protein synthesis, since cycloheximide (20 microM) was inhibitory, and (c) depended upon RNA synthesis, since actinomycin D (1 nM) was inhibitory. Cells grown without thyrotropin but with dibutyryl cyclic AMP (1 mM) or cholera toxin (1 nM) exhibited enhanced lysosomal countertransport of tyrosine, suggesting that cyclic AMP may act as a messenger. This represents the first demonstration of hormonal responsiveness in a lysosomal transport system and may reflect the importance of salvage and reutilization of lysosomal degradation products for the thyroid epithelial cell.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

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. © 1994 Wiley-Liss, Inc.     

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.     

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. © 1996 Wiley-Liss, Inc.     

Na(+)-ionophore, monensin-induced rise in cytoplasmic free calcium depends on the presence of extracellular calcium in FRTL-5 rat thyroid cells

Calcium is an important regulator of cell function, and may be influenced by the intracellular sodium content. In the present study, the Na(+)-ionophore, monensin, was used to investigate the interrelationship between changes in intracellular Na+ concentration ([Na+]i) and elevation of cytosolic Ca2+ concentration ([Ca2+]i) in FRTL-5 thyroid cells. Cytoplasmic Ca2+ levels were measured using the fluorescent dye, indo-1. Monensin induced a dose-dependent increase in [Ca2+]i in FRTL-5 cells. Inhibitors of intracellular Ca2+ release, TMB-8 and ryanodine, were unable to prevent the monensin effect on [Ca2+]i. The alpha 1-receptor antagonist, prazosin, did not block the monensin-stimulated increase in [Ca2+]i. In the absence of extracellular calcium there was a marked diminution in the monensin effect on [Ca2+]i, yet calcium channel antagonists (nifedipine, diltiazem and verapamil) did not inhibit the response. Replacement of Na+ by choline chloride in the medium depressed the monensin-evoked rise in [Ca2+]i by up to 84%. Furthermore, addition of the Na(+)-channel agonist, veratridine, elicited an increase in [Ca2+]i, even though less dramatic than that caused by monensin. Ouabain increased the resting cytosolic Ca2+ concentration as well as the magnitude of the monensin effect on [Ca2+]i. The absence of any effect on the Na(+)-ionophore evoked increase in [Ca2+]i upon addition of tetrodotoxin (TTX) excluded a possible involvement of TTX-sensitive Na+ channels. These data show that the rise in [Ca2+]i induced by increasing [Na+]i is largely dependent on both external Na+ and Ca2+. Calcium entry appears not to involve voltage-dependent or alpha 1-receptor sensitive Ca2+ channels, but may result from activation of an Na(+)-Ca2+ exchange system.     

Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells

The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.