Muscarinic regulation of phospholipase A2 and iodide fluxes in FRTL-5 thyroid cells.

FRTL-5 thyroid cells express a muscarinic receptor which inhibits the phospholipase C activity in a pirenzepine-insensitive manner. We here report that the cholinergic agonist carbachol decreases in these cells the steady-state iodide content, an effect correlated with the iodination of thyroglobulin and with thyroid hormone formation. Several signal pathways may be involved in this phenomenon since carbachol in addition to inhibiting phospholipase C, increased the arachidonic acid release and modified the adenylyl cyclase activity. In FRTL-5 cells, arachidonic acid is released via the direct stimulation of phospholipase A2 by a pirenzepine-sensitive muscarinic receptor coupled to a GTP binding protein sensitive to pertussis toxin. Regarding adenylyl cyclase, carbachol potentiated the thyrotropin-induced stimulation of the enzyme, whereas it did not affect the basal levels of cAMP. In vitro binding studies revealed the presence of two muscarinic binding sites. To summarize, the analysis of signal pathways and of in vitro binding sites indicates a complex muscarinic regulation of thyroid function, which includes the modulation of iodide fluxes.     

Synergistic effect of thyroid hormone and thyrotropin on iodothyronine 5'-deiodinase in FRTL-5 rat thyroid cells

The effects of T3 and T4 on the iodothyronine 5'-deiodinase (5'-D) activity in FRTL-5 rat thyroid cells were investigated. T3 and T4 stimulated the 5'-D activity in a dose-dependent manner. Kinetic studies showed that the stimulation of the 5'-D by T3 was associated with an increase in maximum velocity (Vmax) in [11.9 +/- 0.2 (mean +/- SE) and 25.4 +/- 0.9 pmol I-released/mg protein.min, respectively, in control and cultured with 10(-9) M T3 for four days] but without a change in apparent Michaelis-Menten constant (Km) (94.8 +/- 5.3 nM and 105.4 +/- 12.1 nM, respectively). Furthermore, cycloheximide (5 microM) completely abolished the stimulatory effect of T3 on the 5'-D activity. T3 and T4 also enhanced the 5'-D activity stimulated by TSH in a dose-dependent manner. Kinetic studies showed that the stimulatory effect of T3 on the 5'-D stimulated by TSH was again associated with an increase in Vmax (86.0 +/- 4.0 and 166.5 +/- 1.9 pmol I- released/mg protein.min, respectively, cultured with 0.3 U/liter TSH and cultured with TSH plus 10(-9) M T3 for four days) without a change in apparent Km (114.0 +/- 7.4 nM and 111.6 +/- 12.5 nM, respectively). Cycloheximide (5 microM) completely abolished the stimulatory effect of TSH plus T3 on the 5'-D activity. There were no significant differences observed between cells cultured with TSH and with TSH plus T3 in either the intra- or extracellular cAMP contents. Furthermore, T3 enhanced the 5'-D activity stimulated by (Bu)2 cAMP. These results strongly suggest that T3 or T4 was synergistic with TSH in stimulating the 5'-D activity in FRTL-5 cells, and that cAMP production would be an important component of the synergism.     

Ghrelin enhances the proliferating effect of thyroid stimulating hormone in FRTL-5 thyroid cells

Ghrelin regulates cell proliferation through the growth hormone secretagogue receptor (GHS-R). We confirmed the expression of GHS-R in FRTL-5 thyroid cells and investigated the effects of ghrelin in thyrocytes using FRTL-5 cells. Ghrelin increased intracellular calcium levels but not intracellular cyclic AMP levels. Ghrelin activated Erk within 2min, then activated Akt and STAT3. Erk phosphorylation was inhibited by the calcium inhibitor cyclopiazonic acid (CPA). Ghrelin alone did not stimulate FRTL-5 cell proliferation but enhanced the effects of thyroid stimulating hormone (TSH). Pretreatment with TSH potentiates the growth effects of ghrelin in thyroid cells, and p66Shc, a growth factor receptor adaptor protein, might mediate these synergistic effects. Ghrelin phosphorylated TSH-induced p66Shc, which was inhibited by CPA. Ghrelin did not affect the proliferation of ARO cells, which showed no increased expression of p66Shc after TSH treatment. Thus, ghrelin-induced intracellular calcium signaling enhanced the TSH-induced proliferation of thyrocytes, possibly mediated by the p66Shc pathway.     

The effects of lysophosphatidate on thyrotropin-mediated differentiated thyroid function in FRTL-5 thyroid cells.

Lysophosphatidate (LPA; 1-acyl-sn-glycero-3-phosphate) is a novel lipid mediator with diverse biological activity. The intracellular mechanisms that mediate the actions of LPA include activation of phospholipase C and protein kinase C (PKC), increases in intracellular Ca2+, inhibition of adenylyl cyclase, and activation of phospholipase D (PLD). We have shown that thyrotropin (TSH) mediated PLD activation involves both the cyclic adenosine monophosphate (cAMP) and PKC pathways. We determined the effects of LPA (10 or 50 microM; 30 minutes) on TSH- and forskolin-mediated cAMP production in FRTL-5 thyroid cells. Basal cAMP was unaffected by LPA. However, both 10 microM and 50 microM LPA inhibited TSH-mediated cAMP production by 66% and 64%, respectively (p < 0.01, ANOVA). A similar inhibition of forskolin-mediated cAMP production was observed following LPA (p < 0.01, ANOVA). After 30-minutes exposure to 50 microM LPA, TSH-mediated iodide uptake (IU) was unaffected. However, 50 microM LPA enhanced TSH-IU after 24-hour exposure by 23%+/-8% (p < 0.03, ANOVA) and inhibited TSH-IU following 72-hour exposure by 43%+/-10% (p < 0.02, ANOVA). There was no effect of LPA on basal IU. To determine whether PLD activation mediated the effects of LPA, PLD activity was examined in FRTL-5 thyroid cells 30 minutes after LPA exposure. While PLD was increased 3.5-fold compared to control values following 50 microM LPA (p < 0.05, ANOVA), no increase in PLD activation was seen following treatment with 10 microM LPA. Preliminary evidence revealed no effect of a protein kinase C inhibitor on LPA inhibition of cAMP generation. To examine the products of PLD activation, we measured the production of phosphatidate (PA) and diacylglycerol (DAG) in FRTL-5 thyroid cells following treatment with 50 microM LPA or 100 microU/mL TSH. Within 1 minute following LPA, a rapid spike of DAG production was observed (1.5- +/- 0.2-fold above basal, p < 0.05, ANOVA). No similar increases in PA or bisPA were demonstrated. However, TSH caused a steady increase in PA and DAG that reached a maximum after 30 minutes. In summary, the effects of LPA on differentiated thyroid function in FRTL-5 thyroid cells are complex. LPA inhibits TSH- and forskolin-mediated cAMP generation most likely via a direct inhibition of adenylyl cyclase, whereas its effects on TSH-IU involve other mechanisms, possibly including PLD activation.     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for Ca2+ entry after stimulation with ATP

The relationship between ATP-induced uptake of 45Ca2+ and the ATP-induced changes in [Ca2+]i was investigated in rat FRTL-5 thyroid cells. Addition of 1 microCi 45Ca2+/ml together with ATP induced a time- and dose-dependent increase in uptake of 45Ca2+, the uptake being still significantly above control after 30 min. Resting intracellular free Ca2+ levels ([Ca2+]i), measured using Fura-2, was determined to be 60 +/- 14.3 nM (mean +/- SE). ATP induced a rapid, transient increase in [Ca2+]i (785 +/- 56.2 nM) followed by a plateau phase (127 +/- 34.3 nM). In a Ca(2+)-free buffer, the ATP-induced transient was significantly decreased (357 +/- 57.4 nM, p less than 0.05), and the plateau phase was abolished. The results suggested that stimulating FRTL-5 cells with ATP induced an influx of Ca2+, possibly by a mechanism dependent on a transient increase in [Ca2+]i. To further test this possibility, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was tested. In cells loaded with BAPTA, the ATP-induced uptake of 45Ca2+ was greatly enhanced, while the ATP-induced transient increase in [Ca2+]i was almost totally abolished. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after termination of the ATP response induced a rapid increase in [Ca2+]i. Furthermore, addition of Mn2+ to cells stimulated with ATP induced a more rapid quenching of Fura-2, compared to that seen in control cells. The results indicate that stimulating FRTL-5 cells with ATP induces a rapid release of Ca2+ from intracellular stores, followed immediately by an increase in plasma membrane conductance and influx of extracellular Ca2+. The ATP-induced change in [Ca2+]i may function as a signal enhancing influx of extracellular Ca2+, although some other unknown mechanism(s) is also needed.     

The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells

The effect of iodide on growth of rat thyroid cells (FRTL-5) was studied. TSH-stimulated cell growth was inhibited by iodide in a concentration-dependent manner, and an effect of iodide was detected at 10(-6) mol/l. KClO4 or 1-methylimidazole-2-thiol blocked the effect of iodide, suggesting that iodide uptake and its organification are required to produce the inhibitory effect of iodide on cell growth. Iodide not only decreased TSH-stimulated cAMP production in FRTL-5 cells but also cell growth induced by cAMP. These observations suggest that iodide inhibits TSH-stimulated growth of the cells by attenuating cAMP production and also by acting on the step(s) distal to cAMP generation. The inhibitory effect of iodide was also seen in growth stimulated by insulin, insulin-like growth factor-I or 12-O-tetradecanoyl phorbol 13-acetate, suggesting multiple sites of action of iodide in the process of growth of FRTL-5 cells.     

Iodine suppression of iodide uptake in FRTL-5 thyroid cells

Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action.     

The characterization of phospholipase D in FRTL-5 thyroid cells

We previously demonstrated that TSH activates phospholipase D (PLD) in Fischer rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosphatidylcholine-specific PLD cDNAs, designated as PLD-1 and PLD-2, have been cloned. The present study determined the PLD isoform composition in FRTL-5 thyroid cells and which isoform is regulated by TSH. PLD-1 is activated by small molecular weight G-proteins, such as ADP-ribosylation factor (ARF) and RhoA family members, while PLD-2 is relatively independent of such stimuli. We established the presence of PLD-1 and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in the presence and absence of GTPgammaS. The membrane fraction showed very little activity in the absence of GTPgammaS, but this activity increased approximately 5-fold (P<0.05, ANOVA) in the presence of GTPgammaS. Maximal PLD activity was seen with the combination of membrane plus cytosolic fractions (which contained ARF and RhoA) where the addition of GTPgammaS increased PLD activity approximately 8-fold (P<0.05, ANOVA). To determine the relative activities of PLD-1 and PLD-2 in FRTL-5 thyroid cells, cell-free PLD assays were performed in the presence of GTPgammaS or GDPbetaS with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP(2)). PLD-2 contributed only approximately 19% of the total amount of PLD activity in the membranes and PLD-1 was the predominant PLD isoform. TSH stimulated PLD-1 activity by up to 2. 3-fold over control values (P<0.01, ANOVA). To establish the dependence of PLD-1 on small molecular weight G-proteins, the translocations of ARF and RhoA to the membrane fractions was determined after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 microU/ml TSH by approximately 1.7- and 2.3-fold over control values, respectively (P<0.02 and P<0.03, ANOVA). It is concluded that TSH stimulates PLD-1 activity in FRTL-5 thyroid cells and this is accompanied by the translocation of ARF and RhoA to the membrane fraction.     

Methimazole increases thyroid-specific mRNA concentration in human thyroid cells and FRTL-5 cells.

Methimazole (1-methyl-2-mercaptoimidazole; MMI) increases thyroglobulin mRNA and thyroid peroxidase mRNA concentration in human thyroid cells and in FRTL-5 cells. MMI (1-10,000 microM) gives a dose-dependent increase of thyroglobulin concentration in the medium of human thyroid cells and FRTL-5 cells. The stimulation by MMI has no effect on the TSH-induced cAMP production and occurs in the presence or absence of thyrotropin (TSH). TSH increases the thyroglobulin and thyroid peroxidase mRNA synthesis in human thyroid cells and FRTL-5 cells. The accumulation of thyroglobulin in the medium has an optimum at 100 microU TSH/ml in FRTL-5 cells. This optimum can also be found in most human thyroid cell cultures.     

Characterization of a 1,25-dihydroxy-vitamin D3 receptor in FRTL-5 cells. Evidence for an inhibitory effect of 1,25-dihydroxy-vitamin D3 on thyrotropin-induced iodide uptake.

When FRTL-5 cell cytosol was incubated with increasing amounts of [3H]1,25-dihydroxy-vitamin D3 [( 3H]1,25-(OH)2D3), saturation of specific hormone binding occurred. Scatchard analysis of specific binding of [3H]1,25-(OH)2D3 to the macromolecule yielded an apparent Kd value of 0.41 +/- 0.08 X 10(-10) M and a single maximum binding capacity of 42.8 +/- 8.8 fmol/mg protein. Sucrose gradient analysis revealed substantial [3H]1,25-(OH)2D3 association with a macromolecule sedimentating slightly faster than ovalbumin (3.7 S). [3H]1,25-(OH)2D3 was completely displaced by excess 1,25-(OH)2D3. The 1,25-(OH)2D3-receptor complex bound to DNA cellulose columns in low salt buffer, and eluted as a single peak at 0.15-0.20 M KCl. Thus, we have shown for the first time the existence of a functional 1,25-(OH)2D3 receptor in thyroid follicular cells. Furthermore, 1,25-(OH)2D3 inhibited the thyrotropin (TSH)-stimulated iodide uptake in a dose-dependent manner, indicating that 1,25-(OH)2D3 has an effect on the physiological function of rat thyroid follicular cells in culture.     

Thyrotropin upregulates alpha 1-adrenergic receptors in rat FRTL-5 thyroid cells.

FRTL-5 rat thyroid cells grown and maintained in a medium containing 0.05 nM thyrotropin have a 10-fold higher number of alpha 1-adrenergic receptors on their cell surface than FRTL-5 cells maintained in the absence of thyrotropin in their medium. The increased number of alpha 1-adrenergic receptors, measured as increased specific [3H]prazosin binding per microgram of DNA, is not associated with any changes in Kd values for prazosin. Thyrotropin increases the number of alpha 1-adrenergic receptors by inducing their biosynthesis, as evidence by the inhibitory effects of cycloheximide or actinomycin D; the effect on biosynthesis is cAMP-mediated, since 8-bromoadenosine 3',5'-cyclic monophosphate, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine can mimic the thyrotropin effect in both extent and time course. The alpha 1-adrenergic receptors on FRTL-5 thyroid cells have been functionally linked to iodide efflux into the follicular lumen and to the iodination of thyroglobulin--i.e., to the formation of thyroid hormones; the alpha 1-adrenergic receptor signal is mediated by Ca2+ rather than by cAMP and involves arachidonic acid intermediates. The present data thus describe a unique upregulation phenomenon wherein the sequential expression of two receptors (thyrotropin and alpha 1-adrenergic) and two distinct signal systems (cAMP and Ca2+) are apparently a necessary prelude to thyroid hormone homoeostasis.     

Protein kinase-C activation during thyrotropin-stimulated proliferation of rat FRTL-5 thyroid cells.

We have reported previously that TSH and insulin-like growth factor-I (IGF-I) synergistically stimulate DNA synthesis and elevate the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 thyroid cells and have suggested that protein kinase-C (PKC) may mediate the growth-promoting effects of these hormones. We now present evidence that the effects of TSH on 1,2-DG content are associated with commensurate changes in PKC activity. We measured 1,2-DG content and PKC activity in TSH-deprived growth-arrested cells when TSH was readded. Cells were maintained in medium containing a high dose of insulin (which interacts with IGF-I receptors) and no TSH. When cells incubated in the absence of TSH were reexposed to TSH for 24 h, the 1,2-DG content increased to 234 +/- 22% of the control value, and the ratio of PKC activity in membrane and cytosol fractions of cell homogenates, an index of the state of activation of PKC in situ, increased to 323 +/- 42% of the control value. In cells growing under the influence of TSH in medium containing a high dose of insulin, we found that PKC activity varied during growth. Total cellular PKC activity (3.2 +/- 0.1 nmol/min.micrograms DNA) and the ratio of membrane/cytosol PKC activity (0.24 +/- 0.002) were high during exponential proliferation and fell progressively to 1.1 +/- 0.08 nmol/min.micrograms DNA and 0.12 and fell progressively to 1.1 +/- 0.08 nmol/min.micrograms DNA and 0.12 +/- 0.01, respectively, as cells attained confluence. The specific activity of membrane-associated PKC was 3.0 +/- 0.37 nmol/mg.min in early exponential growth and declined to 0.72 +/- 0.14 nmol/mg.min as cell proliferation ceased. The 1,2-DG content also varied during growth, with a peak occurring during exponential growth, followed by a decline as cells attained a confluent state. These data are consistent with the hypothesis that the growth-promoting effects of TSH in FRTL-5 cells are mediated, at least in part, by 1,2-DG activation of PKC. Since we have demonstrated previously that the effect of TSH to elevate 1,2-DG is, in turn, mediated by cAMP, this represents a special example of the interaction of these two signal transduction systems in regulation of cell proliferation.     

Autoinduction of tumor necrosis factor-alpha in FRTL-5 rat thyroid cells

Tumor necrosis factor-alpha (TNFalpha) may play a role in the development of autoimmune thyroiditis such as Hashimoto's thyroiditis. In the present study, we examined whether TNFalpha induced its own expression in FRTL-5 rat thyroid cells. Lipopolysaccharide (LPS) markedly increased TNFalpha mRNA levels in FRTL-5 cells as assessed by semiquantitative RT-PCR. In addition, LPS-stimulated cells released TNFalpha protein into the culture medium. Similarly, TNFalpha induced its own gene and protein expression in FRTL-5 cells as assessed by RT-PCR and metabolic labeling and immunoprecipitation of TNFalpha. The autoinduction of TNFalpha gene was also observed in TNFalpha-stimulated human thyroid epithelial cells. TNFalpha induction was specific to LPS and TNFalpha since interferon-alpha or amiodarone failed to increase TNFalpha mRNA levels in FRTL-5 cells. Human TNFalpha induced rat TNFalpha gene expression, indicating that type 1 TNF receptor (TNF-R) is involved in the autoinduction. TNFalpha did not increase either type 1 or type 2 TNF-R mRNA levels, suggesting that upregulation of TNF receptors is not involved in the autoinduction of TNFalpha. Although the biological significance of autoinduction of TNFalpha remains unclear, our results suggest that thyroid epithelial cells may participate in the development of autoimmune thyroiditis through production of TNFalpha. Furthermore, inhibition of TNFalpha production in the thyroid may represent a novel approach to mitigating inflammation in autoimmune thyroiditis.     

Thyrotropic activity of crude hCG in FRTL-5 rat thyroid cells

The presence of thyroid stimulating activity in partially purified hCG was investigated using, as bioassay system, iodide uptake in rat thyroid FRTL-5 cells. The biological responses evoked by hCG were tested after neutralisation with monoclonal and polyclonal antisera to hTSH and hCG, and after fractionation on Sephadex G-100. The molar amounts of TSH and hCG in respective preparations were calculated assuming an activity of 30 IU/mg and 19 IU/mg, respectively, for bTSH and hTSH, and of 14,000 IU/mg for hCG. A dose-dependent response, paralleling that evoked by bTSH, was observed in a concentration range of 0.1-4 mumol/l hCG; 1 mumol of hCG was equivalent to 50 pmol of bTSH and 132 pmol of hTSH. The thyrotropic activity coeluted with hCG immunoactivity on Sephadex G-100. Incubation with monoclonal anti-hTSH antibodies did not affect the stimulatory ability of hCG preparation, indicating that it was not due to hTSH contamination. Similarly, a pretreatment with monoclonal and polyclonal anti-hCG antibodies did not significantly alter the iodide uptake response induced by hCG. These results indicate that the thyrotropic activity in partially purified hCG is not due to the presence of aspecific contaminants, but to a substance structurally related to hCG in terms of molecular weight. However, it appeared to differ from hCG immunologically, suggesting the hypothesis that minor modifications in the molecular structure may confer thyrotropic activity on hCG, altering its immunoreactive potency.     

Short-term effects of 1,25-dihydroxycholecalciferol on disordered calcium metabolism of renal failure

The short-term effects of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) on disordered calcium metabolism were studied in 15 trials in patients with advanced renal failure and, for comparison, in 20 trials in normal persons. The steroid was given orally in doses of 0.027, 0.14, 0.68 and 2.7 μg/day for 7 to 15 days. Calcium absorption and urinary calcium increased after the administration of 0.14 to 2.7 μg/day in normal subjects, but 0.68 to 2.7 μg/day was needed to augment calcium absorption in the patients with uremia. Fecal calcium decreased significantly during metabolic balance studies. Serum calcium increased markedly only in the patients with uremia, and hypercalcemia occurred at a dosage level of 2.7 μg/day. With the increase in serum calcium, the blood levels of immunoreactive parathyroid hormone (iPTH) decreased. Urinary calcium increased substantially only in normal subjects. The steroid corrected the “vitamin D resistance” of uremia, suggesting that the renal production of 1,25-(OH)₂D₃ in uremia is defective.     

Effect of sunitinib on growth and function of FRTL-5 thyroid cells

BACKGROUND: Sunitinib, a multitargeted vascular endothelial growth factor and receptor tyrosine kinase inhibitor, causes hypothyroidism in patients who take it for treatment of cancer. Although the pathophysiologic mechanism of the hypothyroidism is unclear, it has been claimed that it is due to inhibition of iodide uptake. METHODS: To evaluate the pathologic mechanism of induction of the hypothyroidism, we studied the effect of sunitinib on FRTL-5 rat thyroid cells. We measured the effect of sunitinib on cell growth, (125)I-iodide uptake and efflux, TSH receptor (TSH-R), and sodium-iodide symporter (NIS) message. RESULTS: At 48 hours, sunitinib caused a dose-related inhibition of growth with LC(50) of 14.6 muM, but there was no apparent inhibition of growth at 24 hours at concentrations of 0.1-25 microM. Preincubation with sunitinib did not impair the response to TSH, indicating that it did not affect the TSH-R. Incubation with sunitinib for 24 hours caused a dose-related increase of (125)I-iodide uptake and did not reduce iodide efflux or NIS mRNA expression. CONCLUSION: The data indicate that sunitinib is unlikely to cause hypothyroidism by inhibition of iodide uptake.     

Effects of 1,25-dihydroxycholecalciferol and calcium on calcitonin mRNA levels in suckling rats

The chronic administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) to 9-day-old suckling rats induced no change on day 13 in the calcitonin (CT) mRNA steady-state level of thyroid glands measured by Northern hybridization. Thyroidal CT contents were decreased in relation to increased plasma calcium levels in animals treated with 0.1 or 1 microgram 1,25-(OH)2D3/kg. Using a lower dose (0.01 microgram/kg), neither plasma calcium, nor thyroidal CT contents were changed. No correlation was found between CT mRNA levels and thyroidal CT contents as well as for plasma CT levels and thyroidal CT contents since hormone in blood remained unchanged after treatment by the active vitamin D3 metabolite. Intraperitoneal calcium administration in fasted 13-day-old rats was associated with a 5-fold increase in plasma CT 30 min after injection, but CT mRNA levels were unchanged within 240 min. By contrast, stomach gavage with calcium in fasted 13-day-old rats induced a sustained increase in plasma CT (X2), and a 4-fold increase in the steady-state level of CT mRNA. Calcium per se is a potent stimulator of CT release in suckling rats, but did not change the amount of CT mRNA. However, gastrointestinal factors may be implied directly or indirectly in the increased CT mRNA level after calcium gavage. In conclusion, 1,25-(OH)2D3 which is known to affect CT gene expression in adult rats is ineffective in 13-day-old suckling rats. This observation may be related to developmental changes in the amount of 1,25-(OH)2D3 receptors of C cells.     

Regulation of sodium iodide symporter gene expression in FRTL-5 rat thyroid cells.

The sodium iodide symporter (NIS), first identified in FRTL-5 cells, plays a critical role in iodide transport in the thyroid gland and in the production of the iodine-containing thyroid hormones. The aim of our study was to examine the regulation of NIS RNA steady-state levels and protein expression as well as functional activity in FRTL-5 cells. FRTL-5 cells cycling in media containing thyrotropin (TSH) were incubated for 48 hours with dexamethasone (10(-8)-10(-5) M), triiodothyronine (T3; 10(-9)-10(-6) M), methimazole (100 microM), propylthiouracil (PTU; 100 microM), perchlorate (10 microM) and potassium iodide (40 microM). In other experiments, cells were treated for 48 hours with various cytokines including interleukin-6 (IL-6) (100 U/mL), interferon-gamma (IFN-gamma) (100 U/mL), tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml), IL-1alpha (100 U/mL), and IL-1beta (100 U/mL). Northern blot analysis using a 32P-labeled rat NIS-specific cDNA probe (nucleotides 1397-1937) revealed NIS mRNA as a single species of approximately 3 kb. When normalized for beta-actin mRNA signal intensities, NIS RNA steady-state levels in viable FRTL-5 cells were suppressed by approximately 80% after incubation with dexamethasone and T3 in a concentration-dependent manner. Iodide accumulation was decreased by up to 40% after incubation with dexamethasone and T3, respectively, in a concentration-dependent manner. Using a rabbit polyclonal rNIS-specific antibody, Western blot analysis of FRTL-5 cell membranes revealed a 60% and 70% suppression of NIS protein expression after treatment with T3 (0.1 microM) and dexamethasone (1 microM), respectively. In additon, NIS RNA steady-state levels were decreased by approximately 50% after treatment of monolayers with methimazole, PTU, and potassium iodide, respectively. Incubation with methimazole and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. Treatment of FRTL-5 cells with IL-6 and IL-1beta resulted in a 30% decrease of NIS RNA steady-state levels. IL-6 did not alter NIS functional activity, but IL-1beta suppressed iodide accumulation by approximately 25%. IFN-gamma and perchlorate failed to alter NIS RNA steady-state levels. In contrast to IFN-gamma that had no effect on iodide accumulation, perchlorate almost completely suppressed iodide accumulation. TNF-alpha and IL-1alpha failed to alter NIS RNA steady-state levels in higher passage numbers of FRTL-5 cells, whereas treatment with TNF-alpha and IL-1alpha of early passages of FRTL-5 cells (<20 cell passages) resulted in a 70% and 40% decrease of NIS RNA steady-state levels, respectively, and in a 20% suppression of NIS functional activity. In conclusion, our data suggest that various agents known to affect iodide transport are capable of differentially altering NIS gene expression and function in cultured thyroid cells. Suppression of NIS gene expression and function by certain cytokines may be responsible, at least in part, for the impaired radioiodine uptake by thyroid tissue in certain forms of thyroiditis.