P. Cochaux

A mechanism in the control of intracellular CAMP level: the activation of a calmodulin-sensitive phosphodiesterase by a rise of intracellular free calcium

The intracellular concentration of cAMP results from the balance between its synthesis by adenylate cyclase and its catabolism by the system of cyclic nucleotide phosphodiesterases. Positive regulation of the cAMP system by activation of adenylate cyclase has been the focus of most of the research on the regulation of the level of this nucleotide. With regard to negative controls, a direct inhibition of adenylate cyclase by a-adrenergic, cholinergic agonists and several peptides is now well characterized and accounts for multiple negative controls of cAMP generation (Jakobs et al., 1980). It is thus often assumed now that intracellular concentration of cAMP is only controlled at the level of its synthesis. Such a concept is an oversimplification as it neglects the potential of controls at the level of cAMP catabolism. Phosphodiesterases are rather complex enzymes consisting of multiple soluble and particulate forms (reviewed by Beavo et al., 1982). It has been demonstrated that they are distinct non-intercon-

Stimulation by forskolin of the thyroid adenylate cyclase, cyclic AMP accumulation and iodine metabolism

Forskolin, a diterpene hypotensive drug, activates adenylate cyclase in brain and in some other tissues (Seamon et al., 1981). Forskolin activated adenylate cyclase in particulate preparations and enhanced cyclic AMP accumulation in slices of dog thyroid. These effects were maximal within minutes and remained constant afterwards. The action of forskolin on intact cells disappeared rapidly after washing. It reproduced two known cyclic AMP-mediated TSH effects: the activation of secretion and of protein iodination. Forskolin thus provides a very convenient tool for the study of the action of defined elevations of cyclic AMP level in thyroid cells. The activation by forskolin of adenylate cyclase was not reduced by Mn2+ which uncouples TSH and PGE1 action. This suggests that in the thyroid also, forskolin acts beyond the receptor level. The effect of forskolin on cyclic AMP accumulation was inhibited by the known negative regulators of this system in the thyroid, acetylcholine, iodide, norepinephrine, PGF1 alpha and adenosine. On the other hand, forskolin potentiated the effects of TSH, PGE1 and cholera toxin. These data show that, though it does not require the receptors for its action, forskolin does not uncouple them from the catalytic unit of adenylate cyclase.

Further characterization of the iodide inhibitory effect on the cyclic AMP system in dog thyroid slices.

Iodide inhibits cyclic AMP accumulation in the thyroid by a process which is prevented by inhibition of iodide uptake and of thyroid peroxidase. By a similar process, it also exerts other independent effects such as the enhancement of iodinated protein release. Iodide inhibited the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin and forskolin. The action of iodide was not relieved by phosphodiesterase inhibitors and was not additive with the effect of norepinephrine or adenosine. Iodide did not decrease the cellular level of ATP. The data are compatible with an inhibition of adenylate cyclase beyond the level of the receptor, presumably at the level of the catalytic unit or its interaction with the positive transducing unit NS. The effect of iodide required TSH for its expression but not for its installation. It was decreased under all conditions in which iodide organification was decreased: decreased iodide or increased methimazole concentration, absence of calcium in the medium, etc. However, the relation between iodide binding to proteins and effect was not linear. The effect was not relieved by washing in the absence of iodide and in the presence of perchlorate, but it was partly reversible in the presence of methimazole propylthiouracyl or thiourea. It was not relieved by cooling to 20 degrees C and cytochalasin b, which block stimulated thyroglobulin hydrolysis and iodothyronine release, nor by actinomycin D, cycloheximide, puromycin, mepacrine or indomethacin. The data suggest that iodide binds to a saturable cell component by a reaction which is reversible only in the presence of thiol-containing drugs.

Negative control of norepinephrine on the thyroid cyclic AMP system

Negative control on the thyroid cyclic AMP system has been studied. The increase of cyclic AMP levels induced by TSH in dog thyroid slices and its consequent secretion were inhibited by norepinephrine. This effect was different from the previously described activation of cyclic AMP disposal by acetylcholine: it was not calcium-dependent, was observed in the presence of isobutyl methylxanthine and was not inhibited by atropine. The inhibitory action of norepinephrine was abolished by phentolamine but not by L-propranolol. Clonidine and epinephrine also markedly inhibited the elevation of cyclic AMP levels, but phenylephrine did not. The inhibitory effect of norepinephrine and clonidine was abolished by yohimbine but not by prazosin. These results suggest that the effect of adrenergic agents on dog thyroid follicular cells is mediated by alpha 2-receptors. Similar results were obtained on dog thyroid adenylate cyclase activity: norepinephrine diminished the activation of adenylate cyclase induced by TSH, in a sodium-dependent process. This inhibition was abolished by phentolamine and yohimbine, but not by L-propranolol and and prazosin. This shows that the negative alpha 2-adrenergic effect bears directly on adenylate cyclase.

Islet-activating protein discriminates between different inhibitors of thyroidal cyclic AMP system

TSH‐induced cyclic AMP accumulation in dog thyroid slices is inhibited by norepinephrine through an α2‐adrenergic receptor, by carbamylcholine through a muscarinic cholinergic receptor, and by iodide. The inhibitory effect of iodide bears on the adenylate cyclase, but the exact mechanism of its action is still unknown. It is known that norepinephrine acts through activation of the N1 subunit of the cyclase, and that carbamylcholine, activating a phosphodiesterase, acts independently of Ni IAP (islet‐activating protein) has been shown to inactivate the Ni subunit. We studied the effect of IAP on the inhibitory action of iodide, norepinephrine, and carbamylcholine on cyclic AMP accumulation in TSH‐stimulated thyroid slices. Incubations of 15 or 22 h, and relatively high concentrations of IAP (250 ) were necessary to demonstrate an effect of IAP on thyroid slices. We report here that, under those conditions, inhibition of cyclic AMP accumulation by norepinephrine, but not by carbamylcholine or iodide, was suppressed by IAP treatment. These results indicate that the cyclase inhibition by iodide, is either not mediated by Ni or if mediated by Ni involves a mode of regulation of this coupling protein that is different from that by which the other Ni‐mediated inhibitory hormones act on the enzyme.

Effect of thyrotropin-releasing hormone on dog thyroid in vitro

The in vitro action of thyrotropin-releasing hormone (TRH) on the cyclic AMP level and iodine metabolism in dog thyroid, has been studied. TRH inhibited cyclic AMP accumulation and subsequent secretion in slices stimulated by thyrotropic hormone (TSH), prostaglandin E1, cholera toxin and to a lesser extent forskolin. The effect of TRH was suppressed in a medium deprived of calcium or in the presence of isobutylmethylxanthine. TRH also stimulated iodide binding to proteins, but not cyclic GMP accumulation. Although all these characteristics of TRH action on dog thyroid fit those of prostaglandin F1 alpha in this tissue, TRH effects were not relieved by indomethacine. The possibility of a TRH action through other known inhibitors of the cyclic AMP system in dog thyroid such as: acetylcholine, alpha-adrenergic agents, adenosine, iodide were checked and ruled out. The possible involvement of other neurotransmitters, such as ATP or vasoactive intestinal peptide were studied but could not be substantiated. Our data suggest the existence of a direct negative action of TRH on the thyroid itself besides its stimulatory role at the pituitary level. The great variability of the TRH effect was overcome by pretreatment of the dog by pyridostigmine, an acetylcholinesterase inhibitor.

A pitfall in the computer-aided quantitation of autoradiograms

Computer-aided quantitation of autoradiograms is now available as a result of recent developments in optical scanners and microcomputers. Data expressed as optical density values, however, are based on the unverified assumption that optical density and radioactivity density are linearly correlated. This article demonstrates the need to construct a calibration curve which should be used to calculate radioactivity density values more precisely.

Diadenosine 5′,5′′′-P1,P4-tetraphosphate (AP4A) levels under various proliferative and cytotoxic conditions in several mammalian cell types

Diadenosine 5,5-P1,P4-tetraphosphate (Ap4A) has been proposed as an intracellular signal for growth. In order to test this hypothesis Ap4A levels were followed in several cell types under various conditions. Quiescent dog thyroid cells in a primary culture were induced to proliferate by addition of a mixture of epidermal growth factor, thyrotropin and foetal calf serum; V79 cells were synchronized by serum depletion then stimulated to proliferate by addition of foetal calf serum. Protein and DNA synthesis increased in both cases, although no significant changes in Ap4A levels per cell could be demonstrated. HeLa D98/AH2 and L929 cells were treated with human recombinant tumour necrosis factor alpha which caused marked cell death. This was measured by a decrease in DNA content and a release into extracellular medium of incorporated radioactive precursor. No concomitant variations in Ap4A concentrations could be observed under these conditions. The data from these various systems do not support the hypothesis that changes in Ap4A levels regulate cellular proliferation.

IODIDE ORGANIFICATION DEFECT IN A COLD THYROID NODULE: ABSENCE OF IODIDE EFFECT ON CYCLIC AMP ACCUMULATION

A follicular adenoma of the thyroid was ‘hot’ one hour after 99mTc pertechnetate administration, but ‘cold’24 h after 131I iodide administration. Incubation of the tissue in vitro demonstrated a defect in iodide binding to proteins that was abolished by addition of an H2O2 generating system. In this tissue iodide failed to inhibit TSH‐induced cyclic AMP accumulation. The results show that iodide oxidation is required for its inhibitory action on cyclic AMP accumulation in human thyroid.

Inhibition of the cyclic AMP-adenylate cyclase system and of secretion by high concentrations of adenosine in the dog thyroid

Adenosine inhibits cyclic AMP accumulation in stimulated slices and adenylate cyclase in acellular preparations of dog thyroid. The onset of this inhibition is rapid, requires relatively high adenosine concentrations (greater than or equal to 10 microM) and occurs with all activators tested (TSH, PGE1, forskolin and cholera toxin). The manganous ion, which uncouples receptor and cyclase, enhances the inhibition by adenosine. The effect of 2,5-dideoxyadenosine, the high concentration of adenosine needed, the Mn2- effect and the lack of reversal by methylxanthines all suggest that this effect bears on the P-site, i.e. on the cyclase itself. Adenosine also inhibits thyroid secretion, which shows that its effect bears on the follicular cells. However the fact that cyclic AMP and DB cyclic AMP induced secretion are also reduced by adenosine suggests that adenosine also inhibits cyclic AMP action.