Dog Thyroid Cells In Culture Research Articles

Biosynthesis and Metabolism of 2-Iodohexadecanal in Cultured Dog Thyroid Cells

2-Iodohexadecanal (2-IHDA) is a major thyroid iodolipid. It mimics the main regulatory effects of iodide on thyroid metabolism: inhibition of H2O2 production and of adenylyl cyclase. The biosynthesis of 2-IHDA and its metabolism have been investigated in cultured dog thyroid cells maintained in a differentiated state by forskolin. Incubation of these cells with [9,10-3H]hexadecan-1-ol or [9,10-3H]palmitic acid labeled several phospholipids, but [9, 10-3H]hexadecan-1-ol was selectively incorporated into plasmenylethanolamine. In the presence of an exogenous H2O2 generating system (glucose oxidase), iodide induced the production of [9,10-3H]2-IHDA from [9,10-3H]hexadecan-1-ol-labeled cells but not from [9,10-3H]palmitic acid-labeled cells. 2-IHDA was also generated during the lactoperoxidase-catalyzed iodination of brain and heart plasmalogens, and of ethyl hexadec-1-enyl ether, a synthetic vinyl ether-containing compound. Taken together, these results show that thyroid 2-IHDA is derived from plasmenylethanolamine via an attack of reactive iodine on the vinyl ether group. 2-Iodohexadecan-1-ol (2-IHDO) was also detected in these studies; it was formed later than 2-IHDA, and thyroid cells converted exogenous 2-IHDA into 2-IHDO in a time-dependent way. The ratio of 2-IHDO/2-IHDA increased with H2O2 production and decreased as a function of iodide concentration. An aldehyde-reducing activity was detected in subcellular fractions of the horse thyroid. No formation of 2-iodohexadecanoic acid could be detected. Reduction into the biologically inactive 2-IHDO is thus a major metabolic pathway of 2-IHDA in dog thyrocytes.

Inhibition of human thyroid adenylyl cyclase by 2-iodoaldehydes

2-Iodohexadecanal (IHDA), which can be formed upon addition of iodine to the vinyl ether group of plasmalogens, has been identified as a major thyroid iodolipid (Pereira et al. (1990) J. Biol. Chem. 265,17018–17025). In this study, we have investigated the possibility that it would be a mediator of the inhibitory effect of iodide on thyroid adenylyl cyclase. In human thyroid membranes, IHDA inhibited the adenylyl cyclase activity stimulated by thyrotropin (TSH), GTP-γ-S or forskolin (FSK), whereas it did not decrease the specific binding of TSH to its receptors. The inhibitory effect on the cyclase reached a maximum after a 1-h-pre-incubation of the membranes with IHDA at 30°C and was poorly reversible. It was also observed following a 4-h incubation with IHDA at 4°C, a condition in which adenylyl cyclase is protected against heat inactivation. IHDA decreased the V max of adenylyl cyclase, but had no effect on the K m for ATPMg 2−. IHDA also inhibited the FSK-stimulated adenylyl cyclase activity in liver and kidney cortex membranes, but had no effect on the Mg 2+-ATPase activity of thyroid membranes. The inhibitory effect of IHDA has also been demonstrated in intact cells. As in membranes, IHDA decreased the rise in cAMP induced by TSH in cultured dog thyroid cells and this inhibition was maintained following pretreatment of the cells with pertussis toxin. In order to evaluate the specificity of the IHDA action, various analogs have been synthesized. This study has permitted the identification of two major structural features required for the inhibition of human thyroid adenylyl cyclase; the terminal aldehyde function and an iodine atom at C2, other halogens being ineffective. In conclusion, we have shown that IHDA exerts a direct inhibitory effect at or near adenylyl cyclase; all the properties of this effect characterized so far are identical to those of the adenylyl cyclase inhibition obtained following the exposure of thyroid tissue to iodide.

Inhibition of H 2O 2 production by iodoaldehydes in cultured dog thyroid cells

2-Iodohexadecanal (IHDA) has been identified as a major thyroid iodolipid which can be formed upon addition of iodine to the vinyl ether group of plasmalogens (Pereira et al., 1990). In order to test whether IHDA plays a role in the thyroid autoregulation by iodide, we have investigated its effects on the production of H 2O 2 by cultured dog thyroid cells. IHDA inhibited the formation of H 2O 2 in dog thyroid cells stimulated by carbamylcholine (CCHOL). In the presence of BSA, which potentiated its action, the effect of IHDA was maximal after 2 h and had an IC 50 around 5 μM. The effect of IHDA was not decreased by methimazole, which abolished the inhibition by iodide. IHDA also inhibited the stimulatory effect of bradykinin, but had only a marginal effect on the production of H 2O 2 induced by ionomycin or phorbol 12-myristate 13-acetate (PMA). The accumulation of inositol phosphates in CCHOL-stimulated thyroid cells was decreased by IHDA. As evaluated by measurements of 51Cr release and [ 3H]thymidine incorporation into DNA, IHDA had no adverse effect on thyroid cell viability. Several analogs of IHDA, of which the synthesis is described, have been tested for their inhibitory activity. This allowed the identification of two major structural features required for the biological activity: the carbonyl group at C1 and an halogen atom at C2, with iodine confering a greater activity than bromine, while chlorine and fluorine were inactive. In conclusion, IHDA inhibits the production of H 2O 2 in CCHOL-stimulated dog thyroid cells by decreasing the phospholipase C cascade activity. This effect involves both the aldehyde function and the iodine atom. These results suggest that IHDA might be the mediator of some of the regulatory actions of iodide on the thyroid gland.

Protein Kinase C Activation Mimics but Does Not Mediate Thyrotropin-Induced Desensitization of Adenylyl Cyclase in Cultured Dog Thyroid Cells*

The mechanism and site(s) of the defect responsible for desensitization to hormone stimulation of adenylyl cyclase (AC) vary with cell type. Plasma membrane preparations were assayed after treatment of primary cultured dog thyroid cells to determine the role of the TSH receptor, stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi), and catalytic unit in AC desensitization. Exposure of cells to TSH or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused time dependent decreases in TSH-stimulated AC and [125I]TSH binding with approximately 50% decreases seen after 18 h; Bt2cAMP was unable to reproduce the TSH effect. Whereas TSH treatment caused concomitant decreases (approximately 25%) in both cyclase activity and [125I]TSH binding after 2 h, TPA treatment decreased AC activity after 6 h and binding only after 18 h. The protein kinase C inhibitor, H-7, prevented TPA-induced but not TSH-induced effects on AC and hormone binding. Membrane AC activation by cholera toxin or forskolin was not altered by 18 h pretreatment of cells with TSH or TPA, indicating that these agents had no apparent effect on intrinsic functionality of either Gs or the catalytic unit. TSH or TPA pretreatment of cells reduced subsequent toxin-mediated AD[32P]-ribosylation of Gs and Gi in isolated membranes. However, the TSH- and TPA-induced decreases in AD[32P]-ribosylation and desensitization do not appear to be due to endogenous ribosylation of G proteins, since treatment of cells with pertussis toxin, for example, to endogenously ribosylate Gi, both increased TSH-stimulated AC activity and failed to affect the ability of TSH or TPA to desensitize. Thus, in this system, although specific hormone-induced AC desensitization and receptor down-regulation conform to several aspects of classic homologous processes, similar effects are also induced by a nonreceptor (phorbol ester) pathway; desensitization, however, can precede down-regulation, possibly due to receptor-Gs uncoupling.

Amiodarone Effects on Thyrotropin Receptors and Responses Stimulated by Thyrotropin and Carbachol in Cultured Dog Thyroid Cells*

Amiodarone is an antiarrhythmic drug that often induces thyroid disorders. Its effects on several aspects of thyroid function were studied using cultured dog thyroid cells. Within 5-60 min of incubation of cell membranes with amiodarone, there were profound changes in adenylate cyclase activity and TSH receptor binding. Amiodarone specifically decreased TSH-stimulated adenylate cyclase activity, but not the basal or forskolin-stimulated activities, while it increased the binding of 125I-labeled TSH to its receptors. Significant effects were seen with 5-10 microM amiodarone, with maximal effects at 50-100 microM, when TSH-stimulated adenylate cyclase activity was completely blocked and the labeled TSH binding increased 4- to 5-fold over control. These effects of amiodarone were reversible, since membranes exposed to 50 microM amiodarone for 1 h exhibited normal binding and cyclase activities, when amiodarone was removed by washing before the assay. The above effects of amiodarone were also observed when cells, instead of membranes, were treated with the drug, although the magnitude of changes was less than in membranes. Lower concentrations of amiodarone (10-25 microM) caused significant inhibition of iodide organification, without affecting iodide uptake, while higher concentrations (50-100 microM) inhibited organification by nearly 75% and uptake by about 20%. Amiodarone (10-100 microM) also inhibited [3H]2-deoxy-glucose uptake and the increase in intracellular calcium concentration in response to TSH and carbachol. In contrast to membranes, treatment of cells with amiodarone caused persistent inhibition of TSH-stimulated cAMP formation and iodide organification even 24-48 h after removal of the drug. However, amiodarone had no effect on cell viability, as judged by trypan blue exclusion and ability to remain attached to the culture dishes. These results suggest that amiodarone has specific inhibitory effects on agonist-stimulated functions in thyroid cells, possibly by interfering with TSH-receptor interactions and also at the level of cholinergic receptors.

Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells.

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Dephosphorylation of 19K and 21K polypeptides in response to thyroid-stimulating hormone in cultured thyroid cells.

Cultured dog thyroid cells incubated with [32P] phosphate contain at least two phosphoproteins of 19 and 21 kDalton (K), as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Myosin light chain appears to be a component of the 19K and 21K phosphoproteins by the following criteria: 1) coextraction with myosin heavy chain from Triton-insoluble cytoskeletons with KCl-ATP, 2) coisolation with myosin heavy chain by immunoprecipitation, and 3) purification of undenatured myosin with pyrophosphate-agarose gel electrophoresis. The phosphorylation state of these proteins is decreased by incubation of cells with TSH. In the basal state, the 19K and 21K proteins from Triton-insoluble cytoskeleton fractions contain 0.86 +/- 0.07 (+/- SE) mol phosphate/mol protein, which is reduced to 0.34 +/- 0.03 in TSH-treated cells. TSH-induced dephosphorylation occurs in 1 min with 2.5 mU/ml TSH and reaches a maximum at 15 min. This TSH effect appears to be mediated by cAMP, since it is mimicked by (Bu)2cAMP, forskolin, cholera toxin, and prostaglandin E1 and is potentiated by isobutylmethylxanthine. Carbamylcholine, ionophore A23187, and norepinephrine, which inhibit TSH stimulation of cAMP, have no effect on basal phosphorylation of the 19K and 21K proteins, but do inhibit the effect of TSH.

Modulation of differentiated function in cultured thyroid cells: thyrotropin control of thyroid peroxidase activity.

The activity of thyroid peroxidase (TPO) in primary dog thyroid cell cultures was measured by both guaiacol oxidation and iodide oxidation assays. Whether cultures were initiated in the absence or presence of 50 mU/ml TSH, TPO activity fell in the first 24 h of culture to approximately 10% of the activity in freshly isolated follicles. After 5 days in culture, TPO activity almost completely disappeared in the absence of TSH, whereas in the presence of TSH, TPO activity rebounded to approximately 30% of that in freshly isolated follicles. TSH similarly induced TPO activity in cells that had lost this activity during a 1- to 6-day preincubation period in the absence of hormone. The half-time for the induction of TPO activity was approximately 3 days. Whether TSH was present from the start of culture or added after 5 days of culture without TSH, the half-maximal dose for reinduction of TPO activity was 0.3-0.4 mU TSH/ml. (Bu)2cAMP, 8-bromo-cAMP, forskolin, and cholera toxin all mimicked, either completely or in part, the ability of TSH to induce TPO activity in cells preincubated without hormone. We conclude that, in cultured dog thyroid cells, TPO activity is modulated by chronic TSH stimulation, and that this effect is mediated by cAMP. However, even though TSH stimulates TPO activity in cultured thyroid cells, the fact that there is no comparable restoration of organic iodine formation (as found in previous studies) makes it likely that other aspects of the iodide organification mechanism are altered.

Lack of prostaglandin involvement in the mitogenic effect of TSH on canine thyroid cells in primary culture

The production of prostaglandin E 2 (PGE 2) by cultured dog thyroid cells was high in a serum-containing medium and low in a serum-free, completely defined medium. Thyrotropin (TSH) and epidermal growth factor (EGF), two mitogenic factors for these cells, did not stimulate PGE 2 release. Indomethacin, at a concentration which completely inhibited PGE 2 production, had no effect on thyroid cell multiplication and DNA synthesis stimulated by TSH and EGF. It is concluded that cyclooxygenase products are not involved in the proliferation of canine thyroid cells and its control by TSH.

Effects of Thyrotropin on Iodine Metabolism of Dog Thyroid Cells in Tissue Culture*

When grown in the presence of thyrotropin, dog thyroid cells in culture from follicle-like structures, take up labeled iodide, and iodinate macromolecular components in the cell. When grown in the absence of thyrotropin, dog thyroid cells in culture form a monolayer, take up only 6% of the iodide of follicular cells, and do not iodinate macromolelcular components in the cell. The iodide uptake in monolayer cells does, however, reflect an incorporation process unique to thyroid cells because hepatocytes and fibroblasts do not have the capacity of the monolayer cells to take up iodide. Thyrotropin stimulation of monolayer cells for a prolonged period (3-8 days) causes the cAMP levels of these cells to return to levels identical to those in follicular cells. The increased cAMP levels are not due to the induction of an adenylate cyclase enzyme, because homogenates of monolayer cells have a thyrotropin-stimulable adenylate cyclase activity. The low level of cAMP, thus, seems to be a problem of receptor coupling to the adenylate cyclase enzyme. The return of cAMP to normal levels is accompanied by an increase in iodide uptake and by macromolecular organification; the return of cAMP levels to normal values is not accompanied by follicular development. The majority (75%) of the iodinated macromolecular product accumulated by follicular thyroid cells, by monolayer thyroid cells stimulated with thyrotropin for a prolonged period, or by thyroid cells treated with dibutyryl cAMP from the onset of culture has the characteristics of 19 S thyroglobulin. The remainder appears to be low mol wt material which may be thyroglobulin-related i.e., be either precursor or biodegraded material.

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.

Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes

The thyrotropin receptor from bovine thyroid plasma membranes has been solubilized using lithium diiodosalicylate, and an assay to measure thyrotropin binding to the solubilized receptor has been developed. Both the solubilized thyrotropin receptor and the thyrotropin receptor on thyroid plasma membranes have effectively identical nonlinear Scatchard plots and negatively sloped Hill plots, i.e. both preparations have receptors which appear to exhibit a similar negatively cooperative relationship. Although the pH optimum of thyrotropin binding to the solubilized receptor is the same as that of the thyroid plasma membrane receptor, pH 6.0, the pH dependency curve of the solubilized receptor is slightly different in its outline. Thyrotropin binding to the solubilized receptor is less sensitive to salt inhibition than is binding to the thyroid plasma membrane receptor; however, optimal binding remains at 0 degrees. The relative affinities of thyrotropin and two glycoprotein hormones which can be considered structural analogs, luteinizing hormone and human chorionic gonadotropin, are 100:10:5, respectively, toward plasma membrane receptors, but 100:25:40 toward the solubilized receptors. The solubilized receptor preparation is heterogeneous in size in that it has binding components with molecular weights of 286,000, 160,000, 75,000, and 15,000 to 30,000. Tryptic digestion converts all three higher molecular weight components to the 15,000 to 30,000 molecular weight species, and the 15,000 to 30,000 molecular weight receptor component has all of the binding properties of the solubilized receptor preparation before tryptic digestion including an identical nonlinear Scatchard plot. It has the same size as and coelutes from Sephadex G-100 with a 15,000 to 30,000 molecular weight receptor released by tryptic digestion of bovine thyroid plasma membranes or tryptic digestion of bovine or dog thyroid cells in culture. The tryptic fragment of the solubilized receptor or preparations has been purified almost 250-fold by affinity chromatography on thyrotropin-Sepharose columns. The binding activity is lost when the solubilized thyrotropin receptor preparation is exposed to beads of neuraminidase-Sepharose or conconavalin A-Sepharose.