Effects of lithium on stimulated metabolic parameters in dog thyroid slices

Thyroid abnormalities may develop during chronic lithium therapy for affective disorders. Lithium, like iodide, inhibits TSH stimulation of adenylate cyclase and thyroid hormone release. The present study examined the effect of lithium on stimulation of intrathyroidal intermediary metabolism by several agonists. LiCl (5 mmol/l) did not inhibit basal cAMP, glucose oxidation or 32P incorporation into phospholipids in dog thyroid slices. Although LiCl inhibited TSH stimulation of cAMP, it did not abolish the hormone's effect on cAMP-dependent protein kinase. The stimulation of iodide organification, glucose oxidation or 32P incorporation into phospholipids by TSH, carbachol and phorbol esters was not inhibited by lithium. This is in contrast to the effects of iodide, which inhibited stimulation of glucose oxidation and 32P incorporation into phospholipids by various agonists. Thus, although both lithium and iodide inhibited TSH-stimulated cAMP formation, they act differently on intrathyroidal intermediary metabolism.     

Effects of phorbol esters on metabolic variables in the thyroid

Since 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reproduced some of the effects of TSH on phosphorylation of polypeptides in the thyroid, its effects on several thyroid metabolic variables were investigated. Like TSH, TPA stimulated glucose oxidation, iodide organification, and 32P incorporation into phospholipids in thyroid slices. However, in contrast to TSH, it did not augment cAMP accumulation. An inactive phorbol ester, 4 alpha-phorbol, did not reproduce any of the effects of TPA. An initial incubation of thyroid slices with TPA decreased the stimulation of cAMP, glucose oxidation, and colloid droplet formation induced by TSH. However, an initial incubation with TPA did not modify the subsequent stimulation of glucose oxidation induced by (Bu)2 cAMP. TPA potentiated the ability of TSH to desensitize the adenylate cyclase system. Although both TPA and TSH increased 32P incorporation into phospholipids, the patterns were different when individual phospholipids were examined. These results indicate another regulatory mechanism for thyroid cell functions independent of cAMP.     

Effect of iodide on glucose oxidation and 32P incorporation into phospholipids stimulated by different agents in dog thyroid slices

Since iodide (I-) inhibits TSH stimulation of cAMP formation, which mediates most of the effects of the hormone, it has been assumed that this accounts for the inhibitory action of iodide on the thyroid. However, TSH stimulation of 32P incorporation into phospholipids and stimulation of thyroid metabolism by other agonists, such as carbachol, phorbol esters, and ionophore A23187, is not cAMP mediated. The present studies examined the effect of iodide on stimulation of glucose oxidation and 32P incorporation into phospholipids by TSH and other agonists to determine if the inhibition of cAMP formation was responsible for the action of iodide. Preincubation of dog thyroid slices for 1 h with iodide (10(-4) M) inhibited TSH-, (Bu)2cAMP-, carbachol-, methylene blue-, 12-O-tetradecanoyl phorbol-13-acetate-, ionophore A23187-, prostaglandin E1-, and cholera toxin-stimulated glucose oxidation. I- also inhibited the stimulation by TSH, 12-O-tetradecanoyl phorbol-13-acetate, carbachol, and ionophore A23187 of 32P incorporation into phospholipids. The inhibition was similar whether iodide was added 2 h before or simultaneously with the agonist. I- itself sometimes stimulated basal glucose oxidation, but had no effect on basal 32P incorporation into phospholipids. The effects of iodide on basal and agonist-stimulated thyroid metabolism were blocked by methimazole (10(-3) M). When dog thyroid slices were preloaded with 32PO4 or [1-14C]glucose, the iodide inhibition of agonist stimulation disappeared, suggesting that the effect of iodide involves the transport process. In conclusion, I- inhibited stimulation of glucose oxidation and 32P incorporation into phospholipids by all agonists, indicating that the effect is independent of the cAMP system and that iodide autoregulation does not only involve this system. Oxidation and organification of iodide are necessary for the inhibition. The ability of iodide to decrease glucose and 32PO4 transport may play an important role in thyroid autoregulation.     

On the mechanism of inhibition by iodine of the thyroid adenylate cyclase response to thyrotropic hormone.

Rats maintained on a low-iodine diet were hypophysectomized, and their diet was than enriched with iodide. Cyclic AMP (cAMP) concentrations achieved in their thyroids following in vitro TSH stimulation were significantly lower than those in the thyroids of control animals that did not receive dietary iodide enrichment. The addition of 0.1% methimazole (MMI) or 1% KC1O4 to the diet abolished this inhibitory effect of iodide. The administration of triiodothyronine in the died did not reproduce the inhibitory effect of iodide. The effect of iodide in vitro on the thyroid cAMP response to TSH was then investigated using paired thyroid lobes obtained from intact rats fed a low-iodine diet. During a 15-min incubation period, concentrations of iodide up to 10(-3)M, together with TSH (125 mU/ml), did not affect the thyroid cAMP response to TSH. In contrast, the preincubation of the lobes in 5 X 10(-5)M Nal for 2 h preceding a final 15-min incubation in medium containing TSH alone resulted in final cAMP concentrations significantly lower than those in paired lobes not exposed to iodide. Basal cAMP concentrations in thyroids not subjected to TSH stimulation were unaffected by preincubation in iodide. The inclusion of TSH during the preincubation period augmented the inhibitory effect of iodide on the final thyroid cAMP concentration achieved. The inclusion of MMI together with iodide during the preincubation period abolished the inhibitory effect of iodide on the final cAMP concentration achieved by TSH stimulation. Direct measurement of newly formed organic iodine in vitro demonstrated it to be inversely proportional to the final cAMP concentration achieved by TSH stimulation. The preincubation of thyroid lobes in iodide was without effect on the subsequent stimulation of cAMP by PGE1, or on the stimulation by F- of adenylate cyclase activity in the thyroid homogenate. The data support the concept of an as yet unknown organic form of iodine that limits thyroid adenylate cyclase responsiveness to TSH stimulation. This may, in part, explain the diverse, and generally inhibitory, actions of iodide on thyroid function.     

TSH-responsive adenylate cyclase activity in thyroid tissue from patients with Graves' disease

Basal adenylate cyclase activity of thyroid plasma membranes obtained from six patients with Graves' disease was slightly but not significantly lower than normal (83.3 +/- 13.9 pmol cAMP/10 min/mg of protein versus 120.9 +/- 19.5 pmol cAMP/10 min/mg of protein). In five of these patients the adenylate cyclase activity was stimulated by bovine TSH with an apparent Km value similar to that of normal thyroid (3.1 +/- 0.5 X 10-9 M versus 3.4 +/- 0.6 X 10-9 M). The response to prostaglandin E2 was also normal. In the sixth patient adenylate cyclase activity was stimulated by prostaglandin E2 but not by bovine TSH. The distribution of basal adenylate cyclase activity in various gradient layers was studied in two TSH-responsive patients. A relative increase of this activity was found in the denser layer when compared to normal thyroid tissue. This could be the expression of an altered ratio between the protein and lipid components of the plasma membranes in patients with Graves' disease.     

Alteration in the transmission of TSH-message to thyroid target in a transplantable rat thyroid tumor

Plasma membranes derived from a transplantable rat thyroid tumor (line 1-5G in Wollman's classification), which is unresponsive to thyrotropin (TSH) but is responsive to dibutyryl 3', 5' cAMP, have been evaluated to localize the defect. TSH binding in tumor plasma membrane is slightly lower than in normal rat thyroid membranes. No change in affinity, but simply a lower capacity was observed. The glycoprotein component of the TSH receptor exhibits similar binding and solubilization properties to the glycoprotein component derived from normal rat thyroid. Analogously to normal rat thyroid membranes, gangliosides more complex than N-acetylneuraminylgalactosylglucosyl-ceramide (GM3) are also present in tumor line 1-5G membranes. Phospholipid content of tumor line 1-5G is 50% lower than that of normal rat thyroid. At variance also with normal rat thyroid, 32P incorporation in tumor line 1-5G phospholipids such as phosphatidylserine and phosphatidylethanolamine is not modified after in vitro incubation with TSH. An even more pronounced effect by TSH on 32P incorporation into phosphatidylinositol is evident in tumor line 1-5G by comparison to normal. The 1-5G thyroid tumor membranes has a 12-fold higher basal adenylate cyclase activity than that of rat thyroid membranes. The high basal adenylate cyclase activity is associated with high ADP ribosylation activity. Both enzymes of tumor are only slightly responsive to TSH. These results suggest that the block in the transmission of TSH message to the cell machinery is localized to the regulatory domains between TSH receptor and adenylate cyclase catalytic subunit.     

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.     

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.     

Thyroid hormone inhibition of intermediary metabolism in dog thyroid slices stimulated by different agonists.

The role of thyroid hormones in a short loop feedback in the thyroid is controversial. This process was studied in dog thyroid slices stimulated by TSH, carbachol and phorbol esters. Incubation of thyroid slices with T3 and T4 for 1 hour inhibited the subsequent stimulation of glucose oxidation induced by carbachol and phorbol esters but not by TSH. T3 also inhibited the stimulation of 32P incorporation into phospholipids stimulated by these two agonists. Glucose oxidation stimulated by TSH, carbachol and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by rT3 and the inhibition was not reversed by methimazole, which did abolish the inhibition induced by iodide, MIT and DIT. TSH stimulation of cAMP was not blocked by T3 or T4 but was by rT3 and MIT- and DIT. The mechanism of such inhibition appears to be complex, possibly involving formation of iodide from rT3, MIT and DIT but also dependent on the intact iodothyronine. Moreover, our data suggest that T3 and T4 exert their inhibition on the thyroid through the phospholipids cascade and this mechanism is probably independent on the release of iodide from these iodocompounds.     

Cultured thyroid cell adenosine 3',5'-cyclic monophosphate response to thyrotropin: loss and restoration of sensitivity to iodide inhibition.

Unlike in all other thyroid preparations, exposure of dog thyroid cells in long-term monolayer culture to iodide (10(-7) to 10(-3) M for up to 19 h did not blunt the subsequent adenosine 3', 5'-cyclic monophosphate (cAMP) response to thyrotropin (TSH) stimulation. This lack of effect of iodide was observed even when confluent thyroid cells were "follicularized" by the action of TSH in the culture medium. Preincubation of these cells in thyroxine (T4) and triiodothyronine (T3) was similarly without effect on the subsequent cAMP response to TSH. Study of thyroid cells during the early phase of primary culture demonstrated that inhibition by iodide (10(-4) M) of the cAMP response to TSH occurred after 7 h but was lost after 48 h of cell culture. This inhibitory effect of iodide was prevented by the inclusion of methimazole in the preincubation medium. As with iodide-insensitive cells, T4 and T3 were without effect on the cAMP response to TSH in iodide-sensitive thyroid cells. Exposure of iodide-insensitive thyroid cells to iodide-containing medium obtained after 2 h of incubation with dog thyroid slices, as well as to medium enriched with the 100,000 g supernatant fraction of homogenates prepared from these thyroid slices, did not restore the inhibitory action of iodide. However, iodide-sensitivity of the cAMP response to TSH was restored by preincubation of iodide-insensitive cells in 10(-4) M iodide plus an H2O2-generating system (glucose-glucose oxidase). These data suggest that T4 and T3 are not organic iodine inhibitors of the thyroid cAMP response to TSH. In addition, they provide evidence against the existence of a soluble, freely diffusible, organic iodine inhibitor of thyroid adenylate cyclase. The loss of sensitivity to iodide inhibition of adenylate cyclase that occurs in thyroid cells shortly after initiation of primary culture appears to be related to a defect in the cellular organification mechanism, possibly the H2O2-generating system.     

Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.     

Effects of thyrotropin and cholera toxin on the thyroidal adenylate cyclase-adenosine 3',5'-monophosphate system

The present experiments examined the relationship between cholera toxin and TSH stimulation of the adenylate cyclase system in bovine thyroid tissue. Preincubation of thyroid slices for 20 min at 4 C with a maximal concentration of cholera toxin (100 microgram/ml) did not impair the subsequent stimulation of cAMP by submaximal amounts of TSH (1 mU/ml) during a 5-min incubation at 37 C. Incubation of cholera toxin or TSH with mixed gangliosides, followed by the addition of thyroid slices resulted in inhibition of the cholera toxin but not the TSH stimulation of cAMP formation. Previous exposure of thyroid slices to TSH induced refractoriness to subsequent stimulation of cAMP formation by TSH, but the response to cholera toxin was unchanged. NAD is necessary for cholera toxin, but not TSH, stimulation of adenylate cyclase. In the absence of NAD, cholera toxin inhibited the effect of maximal concentrations of TSH and prostaglandin E1 on adenylate cyclase activity but had no effect on NaF stimulation. In the presence of NAD, the stimulation of adenylate cyclase activity of bovine thyroid plasma membranes by a maximal amount of TSH was not influeced by maximal amounts of cholera toxin. Cholera toxin had a biphasic action on the binding of [125I]iodo-TSH, with low concentrations enhancing and high concentrations inhibiting binding. TSH augmented the binding of [125I]iodo-cholera toxin over the range of 1-100 mU/tube. Cholera toxin at 10 microgram/ml maximally inhibited binding. In addition to the requirement for ribosylation of adenylate cyclase, the present results indicate that the mechanisms of action of TSH and cholera toxin on cAMP formation are different.     

Effects of diamide on basal and thyrotropin-stimulated thyroid metabolism.

The effect of diamide on basal and TSH-stimulated thyroid metabolism was studied using bovine and dog thyroid slices. Diamide (10 mM) inhibited basal and TSH-stimulated net cAMP production, basal and cAMP-stimulated protein kinase activity, and stimulation by TSH of colloid droplet formation and organification of iodide and abolished basal and TSH-stimulated uptake of 32P into phospholipids. In thyroid slices incubated with 0.1 mM diamide, none of these activities, whether basal or TSH-stimulated, was affected. However, 0.1 mM diamide increased the net basal production of cAMP and potentiated the effect of TSH on this process. These results demonstrate that the previously reported inhibition of protein kinase by 2-20 mM diamide is not specific and that this compound cannot be used to determine which of the metabolic effects of TSH are dependent upon cAMP activation of protein kinase. While 0.1 mM diamide increased cAMP in thyroid slices, it did not reproduce any of the other effects of TSH.     

Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells

hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.     

Functional properties of porcine-thyroid follicles in suspension

Porcine thyroid follicle cells were isolated (about 10(7) cells per gram of tissue) and cultured in small aggregates in agarose-coated culture dishes. The aggregates became arranged into follicle-like structures capable of iodide uptake and organification. In the presence of TSH (0.2 mU/ml), the aggregation of follicles was enhanced, and iodide uptake as well as TSH-stimulated organification of iodide was increased compared with that in the control. In culture, the active iodide metabolism was gradually lost over a 7-day period. This was not due to a disappearance of the TSH-adenylate cyclase system, since cAMP production was retained and stimulated by TSH (half-maximal effect at about 1 mU/ml). Acutely TSH stimulated iodide efflux and iodide organification (half-maximal effect at about 20 microU/ml). The stimulatory effect on organification was transient: within an hour further organification proceeded as in the absence of hormone. The effects on efflux and organification were already maximal at low TSH concentrations, whereas cAMP production was stimulated with up to 50-fold higher TSH levels, i.e. the findings were typical of spare receptors. In the continued presence of epidermal growth factor, a potent mitogen for thyroid cells, the follicles increased in size and contained one single large lumen. Their capability to take up and organify iodide was reduced.     

Thyrotrophin-responsive adenylate cyclase activity in thyroid toxic adenoma.

The adenylate cyclase system was studied in hyperfunctioning autonomous nodules in comparison with normal thyroid tissue. The basal, TSH- and NaF-stimulated adenylate cyclase activities were tested in purified plasma membrane preparations. Basal enzyme activity in membranes from hyperfunctioning nodules was variable and the response to TSH was either normal, low or absent. The present study demonstrates that an intact adenylate cyclase activity, hyporesponsive to TSH, may exist in the cell membrane of the adenoma.     

Inhibition of cyclic AMP formation by iodide in suspension cultures of porcine thyroid follicle cells

In the present study porcine thyroid cells in suspension cultures were employed to investigate the suppressive effect of iodide on adenylate cyclase under basal conditions and following incubation with TSH, PGE1, cholera toxin and forskolin. Within 30 min of incubation with iodide (half-maximal effect 10(-5) M), inhibition was established and remained unchanged up to 40 h of culture. The inhibitory action was abolished by methimazole. TSH, PGE1, cholera toxin and forskolin stimulated cAMP accumulation 10-, 3-, 24- and 22-fold, respectively. Iodide pretreatment reduced basal cAMP levels and also made the cells less sensitive to stimulation by the various agents. High concentrations of TSH or PGE1 could not overcome the suppressive influence of iodide, whereas with high concentrations of cholera toxin and forskolin the reduction in cAMP levels in iodide-treated cultures was less pronounced. Membranes isolated from iodide-treated cultures produced significantly lower amounts of cAMP compared to control membranes. Furthermore, iodide did not inhibit basal or forskolin-stimulated cAMP production in human fibroblasts. The results demonstrate that iodide via an iodination-dependent mechanism influences cAMP generation in thyroid cells. It is suggested that the inhibitory activity, which has a long half-life, involves stable modification of the membrane-localized catalytic unit of adenylate cyclase such that its activation by the regulatory unit is rendered less efficient.     

Thyroid-stimulating immunoglobulin bioassay using cultured human thyroid cells

Modifications are described in the cultured thyroid cell cAMP assay for TSH which make it suitable for the measurement of thyroid-stimulating immunoglobulins. Comparison was made between this assay and two others measuring cAMP responsiveness in human thyroid tissue, namely the thyroid slice and thyroid plasma membrane adenylate cyclase assays, all performed with the same tissue sample. Of immunoglobulin G (IgG) samples from 7 unselected patients with untreated hyperthyroidism associated with Graves' disease, 5 produced significant stimulation of cAMP content in cultured thyroid cells when compared to pooled normal IgG. None of these 7 produced a statistically significant increase in thyroid slice cAMP content when assayed in triplicate, the same replicate number used in the cultured thyroid cell assay. Similarly, none of the same Graves' IgG samples produced significant stimulation (vs. control IgG) in the membrane adenylate cyclase assay, in which sensitivity to TSH stimulation was very poor. With a scaled-down modification of the assay using microtiter wells and acetylation to enhance detection of cAMP in the RIA, significant TSI activity was observed in 15 of 18 (83%) IgG samples from patients with untreated Graves' disease. The data indicate the excellent sensitivity and precision of the thyroid cell cAMP assay, as well as its convenience.     

Recovery from thyroid-stimulating hormone-induced refractoriness in thyroid slices: effect of removal of hormone and new protein synthesis

An initial incubation of bovine thyroid slices with TSH causes decreased responsiveness to the subsequent addition of the hormone when the adenylate cyclase -cAMP system and other metabolic parameters are measured. After the initial incubation with TSH, refractoriness persists despite incubation of thyroid slices for 24 h in the absence of added TSH. Removal of persistently bound TSH by trypsin or antibody to TSH did not reverse the refractoriness during a subsequent 2 h incubation without added TSH. However, normal TSH responsivity was restored by the removal of TSH bound during the first incubation by the addition of either trypsin or antibody to TSH at the beginning of a 24-h second incubation. Restitution of TSH responsiveness after treatment with trypsin or antibody to TSH requires new protein synthesis. While TSH-induced refractoriness does not modify stimulation of cAMP by cholera toxin, its effect on glucose oxidation is significantly diminished. Menadiol stimulation of glucose oxidation is not inhibited in thyroid slices refractory to TSH. Thus, the effect of menadiol is subsequent to the block induced by TSH, whereas that of cholera toxin is proximal to it.     

Norepinephrine and thyrotropin effects on the thyroid in vitro: simultaneous stimulation of iodide organification and antagonism of thyroxine release

Norepinephrine (NE), which has previously been shown to inhibit TSH-induced T4 release by mouse thyroids in vitro, was found to stimulate iodide organification. The concentration of NE (6 X 10(-7) M) necessary to stimulate organification of iodide was 10 times less than the concentration (6 X 10(-6) M) required for inhibition of TSH-induced T4 release. Both actions of NE were exerted through an alpha-adrenergic receptor, since they were inhibited by phentolamine but not by l-propranolol. One milliunit of TSH maximally stimulated T4 release only, but larger amounts (100 mU) also stimulated organification. TSH stimulation of T4 release and organification was not affected by adrenergic antagonists and therefore was not mediated by adrenergic receptors. N6, O2-Dibutyryl cAMP and isobutylmethylxanthine, like TSH, stimulated T4 release. Their actions were inhibited by NE. However, both compounds, unlike TSH, failed to enhance organification in mouse thyroids. The effects of TSH and NE on the cAMP content of incubated mouse thyroids were also studied. TSH induced a prolonged increase in thyroidal cAMP during the 90-min incubation; this increase was unaffected by alpha- or beta-adrenergic antagonists. In contrast, NE (6 X 10(-5) M) produced a transient but significant increase in cAMP only within the first 5 min. Unlike the action of NE on organification, this short term stimulatory effect on cAMP production was mediated by a beta-adrenergic receptor, since it was blocked by l-propranolol but not by phentolamine. The following conclusions were reached: 1) stimulation of iodide organification and thyroid hormone release involves different sensitivity thresholds for TSH and NE; 2) TSH stimulation of iodide organification, hormone release, and cAMP formation is not exerted through adrenergic receptors; 3) NE stimulates organification and inhibits TSH-stimulated T4 release through alpha-adrenergic receptors, but stimulates cAMP production through beta-receptors; and 4) cAMP may not be the mediator of all TSH actions on the thyroid.