J. Mockel

Dual activation by thyrotropin of the phospholipase C and cyclic AMP cascades in human thyroid

In human thyroid slices prelabeled with myo-[2-3H]inositol, thyrotropin (TSH, 3-30 mU/ml) stimulated IP3, IP2 and IP1 generation over a prolonged time course. The cAMP response was much more sensitive to TSH, peaking between 1 and 5 mU/ml. Forskolin (10(-5) M) and isoproterenol had no effect on basal IP levels, while carbamylcholine (10(-5) M, 10(-4) M) also increased IP accumulation. These data suggest that in the human thyroid, TSH activates a phospholipase C generating IP3 and diacylglycerol independently of the well-known adenylate cyclase stimulation. They validate in the human model a dual mode of action of the hormone previously proposed on the basis of indirect observations.

Carbachol and sodium fluoride, but not TSH, stimulate the generation of inositol phosphates in the dog thyroid

In dog thyroid slices prelabeled with myo‐[2‐3H]inositol, carbachol (10−710−4 M) and NaF (10‐20 mM) stimulated IP1, IP2 and IP3 generation. These effects did not require the presence of extracellular calcium. Atropine and PDBu inhibited the action of the cholinergic agonist. No effect of TSH (1–100 ) could be detected on PIP2 hydrolysis and IP production. These results suggest that (i) IP3 could play a role in the metabolic actions of carbachol in the thyroid; (ii) a G‐protein coupling the hormone‐receptor binding to phospholipase C activation exists in the thyroid membrane; (iii) the well known TSH‐induced increased PI turnover does not result in IP3 accumulation.

Importance of sulfhydryl groups for the uncoupling activity of non-steroidal acidic anti-inflammatory drugs and valinomycin in oxidative phosphorylation

Abstract In media at pH 7·4, the uncoupling activity on oxidative phosphorylation with sodium succinate (5 mM) as substrate was studied in liver mitochondria for many antiinflammatory drugs (e.g., ibuprofen, 0·25 mM; flufenamic acid, 0·20 mM; pyrazinobutazone, 0·25 mM; indomethacin, 0·20 mM; and methiazinic acid, 1 mM) Mersalyl (0·035 mM) doesnt inhibit the respiration induced by 2,4-dinitrophenol, but inhibits respiration induced by all these drugs. This inhibition was not reversed by 2,4-dinitrophenol (20 μM), Janus Green B (50 μM) or any other uncoupling agents in media with or without phosphate. This is the proof that this action is independent of the ability of Mersalyl to prevent phosphate penetration into mitochondria. Meanwhile, cysteine (0·15 mM) was able to reverse the inhibition of Mersalyl in media with as well as without phosphate and restored the uncoupling activity of the anti-inflammatory drugs. This competition between cysteine and Mersalyl is “dose-dependent”. A similar action of Mersalyl and cysteine was also observed on the uncoupling activity of valinomycin (0·2 μg). These results suggest that acidic anti-inflammatory drugs and valinomycin exert their uncoupling activity by the way of fixation on membrane sites rich in sulfhydryl groups. These sites might be similar for the two kinds of drugs. There is a slight inhibition of the uncoupling activity of valinomycin and acidic anti-inflammatory drugs observed with nupercaine (0·6 mM).

Regulation of calcium fluxes in the thyroid

Calcium (Ca2+) exchanges were studied in dog thyroid slices incubated in vitro. With 45Ca2+-prelabeled slices, carbamylcholine 10(-7)-10(-5) M (Cchol) induced an important transitory spike efflux, inhibited by procaine and atropine while the stimulated efflux obtained with high concentrations of TSH (10 mU/ml) was progressive and sustained over time. The effects observed with both agents did not require extracellular Ca2+ and were insensitive to verapamil 10(-6)-10(-4) M. Neither dibutyryl (Bu2)-cAMP, nor any agent raising intracellular cAMP (prostaglandin E2, choleratoxin, inhibitors of phosphodiesterases with low concentrations of TSH) were able to reproduce the action of TSH 10 mU/ml, forskolin 10(-5) M being the only exception. Replacement of sodium by choline (+ atropine) in the incubation medium decreased the basal efflux and inhibited the TSH effect. Ouabain 10(-3) M also abolished the TSH-induced Ca2+ efflux, while having no influence on carbamylcholine action. TSH 10 mU/ml and 1 mU/ml, Bu2-cAMP 10(-3) M, choleratoxin and prostaglandin E2 with inhibitors of phosphodiesterase decreased the total 45Ca2+ uptake of the slices, while no effect of Cchol could be detected on this parameter. The results obtained suggest that (1) Cchol and TSH stimulate 45Ca2+ efflux from dog thyroid slices with different kinetics, by mobilization of intracellular Ca2+ stores; (2) this effect of TSH is not mediated by cAMP; (3) independently TSH at low concentrations (1 mU/ml), through cAMP, decreased 45Ca2+ uptake; this suggests that increased 45Ca2+ efflux and decreased uptake result from different mechanisms, as has been described for iodide exchange in FRTL-5 cells.

The Control of Human Thyroid Cell Function, Proliferation and Differentiation

The study of thyroid regulation at the cellular level is the main interest of our laboratory since many years. The complex picture emerging from these studies leads to conclusions of general relevance. The regulation of the thyroid cell was once a classical example of the concept one hormone — one cell type — one intracellular secondary messenger with its pleiotypic effects. It should now rather be considered as a network of crosslinked regulatory steps where the extracellular and intracellular signal-molecules act on their receptors as bits of information in an electronic circuit, i.e., express on/off regulations with no definite general physiological meaning per se. Such networks differ from one cell type to another and for a given cell type from one species to another. In the case of the thyroid, many apparent discrepancies in the literature are explained if this is taken into account. In this presentation, we wish to draw mainly on the results of our group to illustrate this point with regard to the regulation of function, proliferation and differentiation of the thyroid cell.

Regulatory Networks in the Acute Control of Thyroid Function

Two main regulatory pathways are involved in the acute control of dog thyroid cell function: TSH activates thyroid adenylate cyclase and, through cyclic AMP, stimulates both thyroid hormone synthesis and secretion. TSI, E prostaglandins, and norepinephrine, through β receptors, all act similarly. Norepinephrine, through α receptors and the inhibitory pathway of cyclase (NI), directly inhibits adenylate cyclase and the cyclic AMP-mediated effects (Fig. 1). Acetylcholine, through muscarinic receptors, enhances calcium influx and phosphatidylinositol (PI) turnover. The latter process releases in the cell two other intracellular signal molecules, diacylglycerol (DAG) and myoinositol 1,4,5-phosphate (IP3). IP3 induces the intracellular release of Ca++, while DAG activates protein kinase C (Fig. 2). Increased cytosolic calcium enhances H2O2 generation and iodination, cyclic GMP accumulation, and arachidonate release; it activates calmodulin-dependent cyclic nucleotide phosphodiesterase and, thus, lowers cyclic AMP levels and thyroid hormone secretion (Fig. 1). These effects are mimicked by high extracellular calcium and ionophore A23187.

CYCLIC NUCLEOTIDES IN THYROID HORMONE SECRETION

Publisher Summary This chapter describes the cyclic nucleotides in thyroid hormone secretion. The main function of the thyroid gland is to secrete thyroid hormones. The particularities of the thyroid secretory process are linked to the scarcity of iodide in nature, and the ensuing constraint to trap and store it. The existence of an active pumping at the basal membrane of thyroid cells, the inclusion of thyroid hormones in a high-molecular-weight precursor, thyroglobulin, the storage of thyroglobulin inside the lumen of cell-lined follicles can all be rationalized as consequences of this basic requirement. Two main factors that regulate the activity of the normal thyroid gland by a direct action at the level of thyroid cells are thyrotropin and iodide. The thyroid gland receives a cholinergic and an adrenergic innervation, the action of which may involve either α- or β-receptors.