A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures

1984 ◽  
Vol 4 (8) ◽  
pp. 695-702 ◽  
Author(s):  
C. A. Ollis ◽  
R. Davies ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Intracellular Calcium ◽  
Cell Monolayer ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Human Thyroid ◽  
Free Calcium ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Calmodulin Antagonist ◽  
Low Concentrations

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T3. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intraceliular calcium mobilization, TMB-8, was found to inhibit the release of T3 in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T3 secretion by TSH and itself stimulated the release of T3 to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23197 produced a biphasic effect on T3 secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T3 secretion but above a concentration of 1 μM, T3 secretion was increased. The calmodulin antagonist W7 was found to inhibit T3 release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T3 secretion.

Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

1987 ◽  
Vol 58 (02) ◽  
pp. 737-743 ◽  
Author(s):  
Frarnçois Lanza ◽  
Alain Beretz ◽  
Martial Kubina ◽  
Jean-Pierre Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fluorescent Indicators ◽  
Fura 2 ◽  
Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

Forskolin perturbs cGMP as well as cAMP levels in human thyroid cells

1984 ◽  
Vol 107 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Maria Luisa Brandi ◽  
Carlo M. Rotella ◽  
Andrea Lopponi ◽  
Leonard D. Kohn ◽  
Salvatore M. Aloj ◽  
...  
Keyword(s):  
Guanylate Cyclase ◽  
Cyclase Activity ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Camp Production ◽  
Thyroid Cells ◽  
Guanylate Cyclase Activity ◽  
Low Concentrations ◽  
Camp Levels ◽  
Stimulation Of

Abstract. Forskolin, at 10−11 m, stimulates guanylate cyclase activity in primary human thyroid cell cultures, but does not modify cAMP accumulation. At a 10-fold higher concentration it still stimulates guanylate cyclase activity and becomes an inhibitor of cAMP production. Above 10−9 m, forskolin stimulation of cGMP decreases, while it also becomes a stimulator of cAMP production. There is an additive effect of TSH and forskolin on cAMP production at concentrations of the diterpene which are stimulatory. Concentrations of forskolin which are inhibitory for cAMP, but stimulatory for cGMP, are inhibitory for TSH stimulation of cAMP. The addition of 8-bromo-cGMP duplicates the forskolin effect at low concentrations.

INCREASED AGGREGATION AND SECRETION RESPONSES OF HUMAN PLATELETS WHEN LOADED WITH THE CALCIUM FLUORESCENT PROBES QUIN2 AND FURA-2

1987 ◽  
Author(s):  
F Lanza ◽  
A Beretz ◽  
M Kubina ◽  
J-P Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fura 2 ◽  
Low Concentrations ◽  
Platelet Shape

Incubation of human platelets with the fluorescent dye esters quin2-AM (10 μM) or fura-2-AM (1 μM) makes possible the direct measurement of intracellular free calcium ([Ca2+1).Underthese conditions, basal levels of [Ca2+]i of 120 ± 16 nM (n=23) using quin2 and 137 ± 15 nM (n=5) using fura-2 can be measured. Both probes record comparable increases of [Ca2 ]i after stimulation with ADP, thrombin, PAF, or U-46619. Incorporation into human platelets of quin2 or fura-2 at the concentrations used to monitor [Ca2+]i leads to the activation of platelets. This was shown by increased aggregation and secretion responses of quin2or fura-2 loaded platelets after stimulationwith ADP (5 μM), PAF (1 μM) and with low concentrations of thrombin (0.015U/ml), collagen (0.5 μg/ml), the endoperoxide analog U-46619 (0.5 μM) or the calcium ionophore A 23187 (1 μM). Quin2 and fura-2 mediated platelet activation could be due to altered arachidonic acid metabolism, since it was partly inhibited by prior treatment with the cyclooxygenase inhibitor acetylsalicylate (1 μM). In contrast, platelets loaded with higher concentrations of calcium chelators (20 to 100 μM quin2-AM)exhibited diminished aggregation responses to all aggregating agents. Thislatter effectwas accompanied by increased fluidity of theplatelet plasma membrane bilayer and by the exposure of a new pool of membranes at the outer surface of platelets, as monitored withtrimethylammonium-diphenylhexatriene (TMA-DPH) in platelets loaded with thenon-fluorescent calcium probe analog MAPT. Platelet shape change, as measured in the aggregometer, was dose-dependently inhibited after loading of quin2 (10-50 μM quin2-AM), even at concentrations which potentiated aggregation. We conclude that incorporation of intracellular calcium chelators alters platelet responses, including at concentrations used to monitor intracellular calcium changes.

An investigation of the ability of TSH and Graves' immunoglobulin G to increase intracellular calcium in human thyroid cells, rat FRTL-5 thyroid cells and eukaryotic cells transfected with the human TSH receptor

1994 ◽  
Vol 143 (3) ◽  
pp. 527-540 ◽  
Author(s):  
S MacNeil ◽  
D S Munro ◽  
R Metcalfe ◽  
S Cotterell ◽  
L Ruban ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Immunoglobulin G ◽  
Chinese Hamster ◽  
Dermal Fibroblasts ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Cell Monolayers ◽  
Low Concentrations ◽  
Normal Human

Abstract The purpose of this study was to determine if immunoglobulin G preparations (IgGs) from patients with Graves' disease can increase intracellular calcium in thyroid cells, as has been reported for TSH. Both TSH and Graves' IgGs (prepared by protein G affinity chromatography) increased calcium in a range of thyroid cells; however, the response seen, using Fura-2-loaded coverslips of cell monolayers, varied considerably. Chinese hamster ovary (CHO/JPO9) cells transfected with a high number of human TSH receptors showed the greatest response: TSH (10 mU/ml) increased calcium in 46% of experiments and 18 out of 25 (72%) Graves' IgGs increased calcium at 0·1 mg/ml (significantly greater, P<0·001, than for control IgGs where cells responded to 2 out of 13 preparations). Rat FRTL-5 cells only responded to TSH in 22% of experiments and to 2 out of 8 (25%) of Graves' IgGs. Similarly, human thyroid cells responded to TSH in 22% of experiments and to 2 out of 9 (22%) of Graves' IgGs. (When studying cyclic AMP responses in JPO9 cells, much higher concentrations of Graves' IgGs were required (1–3 mg/ml).) However, higher concentrations (03 mg/ml) of both Graves' IgGs, and to a lesser extent of control IgGs, were capable of increasing calcium in cells both with and without TSH receptors (control CHO cells and normal human dermal fibroblasts). We conclude that relatively low concentrations of patient IgGs can be distinguished from control IgGs in JPO9 cells on the basis of their ability to increase calcium, but that additionally all IgG preparations possibly contain another factor which can increase calcium in a range of cells independent of the presence of the TSH receptor. Journal of Endocrinology (1994) 143, 527–540

scholarly journals Modulation of ACE-2 mRNA by inflammatory cytokines in human thyroid cells: a pilot study

Endocrine ◽  
2021 ◽  
Author(s):  
Francesca Coperchini ◽  
Gianluca Ricci ◽  
Laura Croce ◽  
Marco Denegri ◽  
Rubina Ruggiero ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Combination Treatment ◽  
Primary Cultures ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Mrna Levels ◽  
Thyroid Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Abstract Introduction Angiotensin-converting-enzyme-2 (ACE-2) was demonstrated to be the receptor for cellular entry of SARS-CoV-2. ACE-2 mRNA was identified in several human tissues and recently also in thyroid cells in vitro. Purpose Aim of the present study was to investigate the effect of pro-inflammatory cytokines on the ACE-2 mRNA levels in human thyroid cells in primary cultures. Methods Primary thyroid cell cultures were treated with IFN-γ and TNF-α alone or in combination for 24 h. ACE-2 mRNA levels were measured by RT-PCR. As a control, the levels of IFN-γ inducible chemokine (CXCL10) were measured in the respective cell culture supernatants. Results The mean levels of ACE-2 mRNA increased after treatment with IFN-γ and TNF-α in all the thyroid cell preparations, while the combination treatment did not consistently synergically increase ACE-2-mRNA. At difference, CXCL10 was consistently increased by IFN-γ and synergically further increased by the combination treatment with IFN-γ + TNF-α, with respect to IFN-γ alone. Conclusions The results of the present study show that IFN-γ and, to a lesser extent TNF-α consistently increase ACE-2 mRNA levels in NHT primary cultures. More interestingly, the combined stimulation (proven to be effective according to the synergic effect registered for CXCL10) produces different responses in terms of ACE-2 mRNA modulation. These results would suggest that elevated levels of pro-inflammatory cytokines could facilitate the entering of the virus in cells by further increasing ACE-2 expression and/or account for the different degree of severity of SARS-COV-2 infection. This hypothesis deserves to be confirmed by further specific studies.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

AN INVESTIGATION OF THE PATHOGENESIS OF HASHIMOTO'S DISEASE BY THYROID TISSUE CULTURE

1960 ◽  
Vol 20 (2) ◽  
pp. 83-NP ◽  
Author(s):  
W. J. IRVINE
Keyword(s):  
Tissue Culture ◽  
Alternative Hypothesis ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
Rabbit Serum ◽  
Thyroid Cell ◽  
Thyroid Extract ◽  
Thyroid Cells ◽  
Hashimoto’S Disease ◽  
Hashimoto's Disease

SUMMARY Human thyroid cells were grown in tissue culture in media containing normal human serum, Hashimoto serum, and rabbit sera containing antibodies to purified human thyroglobulin and to crude thyroid extract, respectively. The thyroid cells grew equally well in all media, with the exception of the rabbit serum containing antibodies to crude thyroid extract. Intact thyroid cells obtained from tissue culture failed to fix Hashimoto antibodies in the presence of complement, whereas the constituents of disrupted thyroid cells gave a strongly positive complement-fixation test with Hashimoto serum. It is therefore suggested that the intact thyroid cell is impermeable to complement-fixing Hashimoto antibody. The evidence afforded by the present work adds further weight to the belief that Hashimoto's disease may not be due to a simple auto-immunizing process consequent upon the interaction of thyroid antigen and the known circulating auto-antibodies. Evidence in support of an alternative hypothesis involving 'cell-bound' antibodies with disruption of the follicular basement membrane is discussed.

Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium

1992 ◽  
Vol 103 (2) ◽  
pp. 389-396 ◽  
Author(s):  
C. Vincent ◽  
T.R. Cheek ◽  
M.H. Johnson
Keyword(s):  
Cell Cycle Progression ◽  
Polar Body ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Mouse Oocyte ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.

scholarly journals Detection of SARS-COV-2 receptor ACE-2 mRNA in thyroid cells: a clue for COVID-19-related subacute thyroiditis

2020 ◽  
Author(s):  
M. Rotondi ◽  
F. Coperchini ◽  
G. Ricci ◽  
M. Denegri ◽  
L. Croce ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Thyroid Tissue ◽  
Subacute Thyroiditis ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Type I ◽  
Rt Pcr ◽  
Follicular Cells ◽  
Thyroid Cells ◽  
Tissue Samples

Abstract Purpose SARS-COV-2 is a pathogenic agent belonging to the coronavirus family, responsible for the current global world pandemic. Angiotensin-converting enzyme 2 (ACE-2) is the receptor for cellular entry of SARS-CoV-2. ACE-2 is a type I transmembrane metallo-carboxypeptidase involved in the Renin-Angiotensin pathway. By analyzing two independent databases, ACE-2 was identified in several human tissues including the thyroid. Although some cases of COVID-19-related subacute thyroiditis were recently described, direct proof for the expression of the ACE-2 mRNA in thyroid cells is still lacking. Aim of the present study was to investigate by RT-PCR whether the mRNA encoding for ACE-2 is present in human thyroid cells. Methods RT-PCR was performed on in vitro ex vivo study on thyroid tissue samples (15 patients undergoing thyroidectomy for benign thyroid nodules) and primary thyroid cell cultures. Results The ACE-2 mRNA was detected in all surgical thyroid tissue samples (n = 15). Compared with two reporter genes (GAPDH: 0.052 ± 0.0026 Cycles−1; β-actin: 0.044 ± 0.0025 Cycles−1; ACE-2: 0.035 ± 0.0024 Cycles−1), the mean level of transcript expression for ACE-2 mRNA was abundant. The expression of ACE-2 mRNA in follicular cells was confirmed by analyzing primary cultures of thyroid cells, which expressed the ACE-2 mRNA at levels similar to tissues. Conclusions The results of the present study demonstrate that the mRNA encoding for the ACE-2 receptor is expressed in thyroid follicular cells, making them a potential target for SARS-COV-2 entry. Future clinical studies in patients with COVID-19 will be required for increase our understanding of the thyroid repercussions of SARS-CoV-2 infection.

scholarly journals Rapid ultrastructural changes in the dense tubular system following platelet activation

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 718-723 ◽  
Author(s):  
L Ebbeling ◽  
C Robertson ◽  
A McNicol ◽  
JM Gerrard
Keyword(s):  
Intracellular Calcium ◽  
Platelet Activation ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ultrastructural Changes ◽  
Ionophore A23187 ◽  
Tubular System ◽  
Protein Kinase C Activators ◽  
Morphologic Changes

Abstract The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5′-dimethyl-bis-(0- aminophenoxy)-ethane-N,N,N′,N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

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