Iodotyrosine deiodination in the normal and acutely TSH-stimulated thyroid.

The deiodination of L-MIT-125I was measured in rat thyroid homogenates and slices before and after acute TSH stimulation. Slices and homogenates were incubated with identical concentrations of tissue and substrate in the presence and absence of NADPH. 1 USP unit TSH added in vitro to thyroid slices failed to stimulate deiodination; a single in vivo ip injection of 3 USP units TSH was also unable to raise deiodinating activity. In contrast to TSH, NADPH added to homogenates and slices enhanced deiodination significantly. However, several arguments, including a review of the literature, strongly militate against the hypothesis of an increased intracellular concentration of the coenzyme NADPH being the prerequisite to enhanced deiodination. The results suggest that deiodinase activity in acutely stimulated thyroids is not limited by the intracellular concentration of the enzyme itself nor by the availability of co-enzyme. Therefore, the increased iodide release induced by acute TSH stimulation is a mere consequence of the enhanced thyroglobulin proteolysis and does not require higher enzyme concentration. It will be shown subsequently that a different conclusion must be drawn in experiments with chronic TSH stimulation.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Mitotic Response to Wounding In Goitrous and Normal Rat Thyroid: Implications For Thyroid Growth Control

Abstract. Thyroid growth in the rat in response to a sustained elevation of serum thyrotropin (TSH) is limited by a progressive desensitization of the follicular cells to the mitogenic action of TSH which is not reversed by withdrawal of stimulation or by reduction of cell number. This study shows that, in thyroids which have reached a 'plateau' of growth, wounding induces a marked local follicular cell mitotic response which is equal in magnitude to that seen in control glands. This demonstrates that these cells, which are refractory to TSH, have not lost the capacity to divide. It is concluded that limitation of TSH-induced thyroid growth is not due to a non-specific loss of mitotic capacity resulting from severe hypothyroidism or goitrogen toxicity, or to an inherent limitation of the number of divisions which a follicular cell can undergo. the implications of these findings are discussed.     

Mitotic response to wounding in goitrous and normal rat thyroid: implications for thyroid growth control

Thyroid growth in the rat in response to a sustained elevation of serum thyrotropin (TSH) is limited by a progressive desensitization of the follicular cells to the mitogenic action of TSH which is not reversed by withdrawal of stimulation or by reduction of cell number. This study shows that, in thyroids which have reached a 'plateau' of growth, wounding induces a marked local follicular cell mitotic response which is equal in magnitude to that seen in control glands. This demonstrates that these cells, which are refractory to TSH, have not lost the capacity to divide. It is concluded that limitation of TSH-induced thyroid growth is not due to a non-specific loss of mitotic capacity resulting from severe hypothyroidism or goitrogen toxicity, or to an inherent limitation of the number of divisions which a follicular cell can undergo. The implications of these findings are discussed.     

Iodide modulation of Ca2+ efflux from mouse thyroid

In a preceding report, we showed evidence that thyrotropin (TSH) stimulates Ca2+ efflux from mouse thyroid gland and that TSH stimulation of Ca2+ efflux is inhibited by acute administration of excess iodide to mice fed a low iodine diet (Hashizume et al., 1984). The observations suggested that iodide inhibits Ca2+ efflux through an inhibition of TSH-sensitive adenylate cyclase activity. We found further, that iodide inhibits dibutyryl cyclic AMP (DBC)-stimulated Ca2+ efflux. The results suggested that iodide influences the step subsequent to the generation of cyclic AMP. In this report, we studied whether iodide can inhibit Ca2+ efflux by a mechanism which is distinct from adenylate cyclase inhibition. The acute administration of excess iodide to mice fed a regular diet did not decrease the basal Ca2+ efflux rate in the thyroid. TSH-induced stimulation of Ca2+ efflux in thyroids obtained from regular diet-treated mice was not modified by iodide administration. Iodide injection to mice fed a low iodide diet, however, decreased the basal Ca2+ efflux rate though the content of cyclic AMP in the thyroids was not altered by this treatment. The decreased-Ca2+ efflux rate induced by iodide in the low iodine diet-treated thyroids was not modified by TSH in vitro. The results indicate that an acute administration of excess iodide in thyroid inhibits Ca2+ efflux not only by an inhibition of adenylate cyclase but also by an inhibitory action which is distinct from the adenylate cyclase inhibiting action of iodide.     

Prior exposure of macrophages to a low temperature enhances their cyclic AMP responses due to microtubule disruption

Prior exposure of intact macrophages to a low temperature (4 degrees C) resulted in tremendous increases in their cAMP-generating responses to prostaglandin (PG) E1, epinephrine, adenosine, forskolin, and cholera toxin. The extent of the enhancement was dependent on the site of stimulation by these agents. The effect of cold exposure was (a) completely reversed by reexposure of the cold-treated cells to 37 degrees C; (b) mimicked by antimicrotubule agents, colchicine and vinblastine; (c) not further increased by colchicine or vinblastine treatment; and (d) efficiently antagonized by D2O and taxol, microtubule stabilizers. These results demonstrated that enhancement of cAMP generation by cold exposure was mediated through microtubule disruption. The effects of cold exposure and microtubule-disrupting agents on hormone-induced refractoriness was also studied. Macrophages stimulated at 37 degrees C by PGE1 became refractory to the subsequent stimulation by PGE1, regardless of whether the cells had been, or were subsequently, exposed to 4 or 37 degrees C. In contrast, cells stimulated by PGE1 in the presence of colchicine, or cells pretreated with PGE1 at 4 degrees C, responded to PGE1 rechallenge normally. The results suggested that disassembly of microtubules provides a condition unfavorable for development of receptor refractoriness, but never favors recovery therefrom.     

Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells.

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.     

Dynamics of hormone release from the perfused canine thyroid during cyclic AMP infusion.

In order to try to characterize the sequence of processes leading to hormone secretion from the stimulated thyroid, the effect of cyclic 3'5' adenosine monophosphate (cAMP) and related compounds were examined in 15 two-sided perfusions of canine thyroids isolated in situ. T4 and T3 concentrations in the effluent were measured radioimmunologically. cAMP 5 mM and TSH 100 muU per ml induced the same pattern of hormone release from the thyroid. After a latency period of 15--25 minutes a steep increase occurred in both T4 and T3 release. During the initial part of the stimulation the rise in T4 relase was somewhat slower than that of T3 release. The prolonged latency period before response earlier recorded in the same preparation during infusions of low concentrations of TSH was not observed during infusions of decreasing concentrations of cAMP (1, 0.8, 0.5 and 0.2 mM) or theophylline (5 and 1 mM). Either there was no response or the latency period was of the same length as that observed after a strong stimulus. These findings suggest that the latency period can be divided in two parts: () a variable, dose dependent satency period confined to the early part of the process sequence leading to secretion--i.e. before cAMP exerts its effect, and 2) an obligatory latency period related to the processes taking place after the formation of pseudopods. The duration of these late processes seems to be independent of the degree of stimulation.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.     

Evaluation of thyroid gland activity by hormone assays and flow cytometry in rats

Spontaneous variations in the activity of thyroids and changes induced by methimazole, phenobarbital and 2,4--diaminoanisole sulfate were assessed in rats by repeated measurements of serum T3, T4 and TSH concentrations and cell cycle analysis in isolated nuclei of the thyroids. Methimazole caused a decrease in T3 and T4, and elevation in TSH, reduction of cells in G1 and increase in the percentage of cells in S and G2 phases. With phenobarbital T4 concentrations were decreased, but a mitogenic effect was only seen after 8 weeks. With 2,4-diaminoanisole sulfate the most prominent change observed was a decrease in T3 concentrations.     

Effects of endogenous and exogenous prostaglandin in neurotransmission in canine trachea

The effect of PGE2 on neurotransmission in the canine tracheal strip dissected free of epithelium was studied in the single sucrose gap and organ bath. PGE2 was a potent inhibitor of the initiation of excitatory junction potentials (ejps) by just submaximal nerve stimulation. In a concentration of 10(-9) or 10(-8) M PGE2 nearly or completely abolished them. Contractile responses to field stimulation in the sucrose gap at 27 degrees C or in muscle baths at 37 degrees C were also reduced or abolished by PGE2 in the same dose range; reductions were greater at low frequency. Responses to acetylcholine were also depressed but significantly less than to field stimulation. These are consistent with major presynaptic as well as some postsynaptic inhibitory actions of PGE2. No evidence was obtained that endogenous PGE2 affected excitatory junction potentials and contractions; i.e. they were stable for hours and unaffected by indomethacin 10(-6) and 10(-5) M under our conditions. Post-stimulus potentiation of ejps amplitude, maximum at 10 s, was observed and became more marked after the first ejp had been markedly reduced or abolished by PGE2. This potentiation was unaffected by indomethacin. It was suggested that a presynaptic process inhibited by PGE2 might participate in this potentiation. The canine trachea is a useful preparation when studied under the experimental condition used here for study of effects of products of arachidonate on neurotransmission.     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

TSH stimulation of iodine organification in early foetal rat thyroid in vitro

Thyroid glands from 15 day-old rat foetuses were incubated in Eagle's medium containing Na 125I and supplemented, or not, with TSH for 4 or 24 hours. Electron microscopic radioautographic study shows silver grains mainly in follicular cavities only in the thyroids submitted to TSH during 24 hr. A functional differentiation must therefore take place in thyroid cells under TSH stimulation.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production.

The ability of prostaglandin I2 (PGI2) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE2. However, a concentration of 15 microgram/ml PGI2 was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 microgram/ml PGE2, which produced the maximum response. It therefore appears that PGI2 is not more effective than PGE2 in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI2 appeared to be able to modify the stimulation of cyclic AMP production by follicle-stimulating hormone (FSH), but whether or not PGI2 plays any role in follicular function remains to be established.     

Cyclic AMP level of human thyroid cells in monolayer culture. TSH induced refractoriness to TSH action.

Thyroid cells from euthyroid patients with Graves' disease were cultured in a chemically defined medium. The cells preserved the ability to respond to TSH with 8-fold increase in cyclic AMP concentration. This cyclic AMP response to TSH was diminished by prior exposure of cells to TSH. The decrease in cyclic AMP response to TSH induced to TSH was reversible, was not associated with a similar decrease to cyclic AMP response to PGE1, and could not be attributed to increased phosphodiesterase activity or to decreased adenyl cyclase activity. The partial resistence to TSH stimulation of thyroid cells previously exposed to TSH may be due to changes in the TSH receptor, possibly caused by TSH itself.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells.

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e. protein iodination, [1-14C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20 degrees C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect. This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex) and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20 degrees C and 16 degrees C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH acetivate adenylate cyclase by a mechanism identical to cholera toxin.     

Further studies on prostaglandins. III. Effect of prostaglandins on thyroid activity as assessed by 125I.

The effect of crude prostaglandins extraced from prostate glands of camels as well as that PGE1, on the thyroid activity of male immature Boskat rabbits, was investigated. The iodinated amino acid fractions (T1, T2, T3 and T4) in the serum of the injected groups were determined in control and treated animals. Cytological work on sections of the thyroid glands of the treated and control rabbits were performed to study the effect on subcellular level. The results indicate that both crude and pure prostaglandins have a stimulating effect on thyroid activity. This was indicated by the increased 125I uptake of the thyroids of rabbits and evidenced histologically. PGE1, was found to be more effective as compared with the crude prostaglandins. The indinated amino acid and hormone fractions in the serum of the treated groups showed that T2 was increased as a result of injection of the crude prostaglandins, and T4 was increased after PGE1 injection. Tables and figures explained diversed effect.     

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells

Summary Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing the intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.