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.     

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.     

Unusual antagonistic actions of mixtures of vasoactive intestinal peptide, thyroid-stimulating antibodies, and cholera toxin on adenosine 3',5'-monophosphate accumulation in normal human thyroid cell cultures

When individually tested, vasoactive intestinal peptide (VIP), cholera toxin, and monoclonal thyroid-stimulating antibodies (TSAbs), but not TSH binding-inhibiting antibodies (TBIAbs), elevate cAMP levels in cultured human thyroid cells. In this study, we tested the effect on cAMP levels in human thyroid cells of the simultaneous presence of VIP and the other ligands noted. Monoclonal TBIAbs (11E8 and 122G3), which interact with a membrane glycoprotein containing the high affinity binding site of the TSH receptor, did not alter VIP-induced cAMP accumulation in the thyroid cells. These data indicate that VIP and TSH bind to distinct sites on the cell membrane. In contrast, monoclonal TSAbs 208F7 and 307H6, which interact with a portion of the TSH receptor other than its high affinity binding site, not only did not have their usual agonist activity, but, instead, caused a marked inhibition of human thyroid cAMP accumulation when VIP was also present. Mutual antagonism by two agonists, i.e. inhibition evident when TSAbs and VIP were mixed, was also found when cholera toxin was coincubated with VIP. The inhibitory effect of mixing cholera toxin and VIP was nearly immediate and was duplicated with mixtures of the beta-subunit of cholera toxin and VIP. The inhibition evident in mixing VIP and the TSAbs or cholera toxin was not duplicated in mixtures of isoproterenol and VIP or in mixtures of forskolin and VIP. These results suggest that the mutual inhibition of VIP and either TSAbs or cholera toxin is at a step that couples the TSH and VIP high affinity receptor-binding sites to the adenylate cyclase complex.     

Loss of adrenergic regulation of cAMP production in the FRTL-5 cell line

Rat thyroid cells in primary culture augment cAMP production when challenged with beta-adrenergic agonists; at 10(-5) M the potency is isoproterenol greater than norepinephrine greater than epinephrine. In analogy with human thyroid cells, rat thyroid primary cultures display alpha-adrenergic-stimulated cGMP production which inhibits TSH and norepinephrine stimulation of cAMP. Adrenergic regulation of cyclic nucelotide production is lost in the cloned thyroid cell line of rat origin known as FRTL-5. Also the potentiating effect of phentolamine on TSH stimulation of cAMP production in thyroid primary culture becomes an inhibitory one in the FRTL-5 cells. Neither 'soluble factors' nor contamination of other cell populations could account for the different behaviour of the primary culture and the cell line toward adrenergic regulation. The reported activation by norepinephrine of iodide efflux in FRTL-5 cells rules out the loss of specific adrenergic receptors in the FRTL-5 cells. It is proposed that the cloning of FRTL-5 cells from primary cultures causes an 'alteration' in the coupling of adrenergic receptors to the adenylate cyclase system. This alteration does no affect those mechanism of message transduction that do not involve cAMP as the signal.     

Stimulation of thyroid adenylate cyclase activity by sera from patients with non-thyroidal illness

We have previously shown that sera from many hypothyroid patients stimulated adenylate cyclase activity as measured by serum bioactive TSH concentrations produced by FRTL-5 cell line. This TSH-stimulating activity did not correlate with serum immunoreactive TSH. IgG fractions of these sera did not stimulate FRTL-5 cells. The present study was, therefore, undertaken to investigate the thyroid stimulating activity of sera from patients with non-thyroidal illness. Studies were performed in 36 patients with various non-thyroidal illness. In these patients, serum concentrations of T4, free thyroxine, T3, and TSH were determined. In addition, sera were incubated with FRTL-5 cells or porcine thyroid cells in primary culture in the presence of 0.4 mM MIBX, and medium cAMP concentrations were determined by radioimmunoassay. Sera obtained from some patients with various non-thyroidal illness increased cAMP concentrations in culture media of FRTL-5 cells as well as that of porcine thyroid cells. The thyroid stimulating effects of sera were not disease specific and significantly correlated inversely with serum T3 and T4 concentrations. Serum TSH concentrations in these patients were within the normal range even by the newly developed ultrasensitive assay. Although the nature of substance(s) present in sera of patients with low T3 syndrome which stimulates thyroid adenylate cyclase is not entirely known, it is conceivable that there exist mechanisms independent of TSH to compensate the decreased serum T3 levels in low T3 syndrome.     

Transient lack of response to TSH of human cultured thyroid cells obtained from hyperfunctioning tissue

TSH-induced cAMP accumulation in cells obtained from normal and pathological thyroid tissue was studied during the first 12 days of primary culture. In normal thyroid tissue cultures (N = 7), the response of cAMP to TSH was present from the second day of culture and reached its maximum after 8 days. A similar behaviour was observed in cultures obtained from euthyroid sporadic goitres (N = 8), even if the rate of response was slightly lower than that of normal tissue. Similarly, cultured cells from euthyroid 'autonomous' nodules (N = 8) appeared to be responsive to TSH during the period of study, but the rate of response was also lower than in the controls. On the contrary, in cultures obtained from toxic adenomas (N = 5) and from diffuse toxic goitres (N = 5) the response to TSH was absent during the first 4 days of culture. The cells became sensitive to TSH from 6 and 6 day onwards, with the rate of response increasing progressively and reaching its maximum on day 12. Finally, in cultured cells obtained from different areas of multinodular toxic goitres (N = 4), the response to TSH was similar to that of euthyroid goitres in cells prepared from 'cold' areas, and to that of toxic adenomas in cells obtained from 'hot' areas. The present data demonstrate the existence of an inhibitory action of unknown factors, possibly iodothyronines or thyroglobulin, on the TSH effect in short-term cultures obtained from thyrotoxic tissues. A normal TSH responsiveness can be restored when the culture is prolonged.     

Intralysosomal hydrolysis of thyroglobulin. II. Different fates of poorly and fully iodinated Tg and specific activation by TSH of the degradation of fully iodinated Tg

Even though adrenergic nerve terminals between and around thyroid follicles and catecholamine stimulation of thyroid adenylate cyclase have been reported, there is no uniform concept on catecholamine interaction with thyrotrophin (TSH) receptors. Therefore, the effect of catecholamines on TSH-stimulated cyclic AMP (cAMP) accumulation in human follicular thyroid cells has been investigated, to thus eliminating the extrathyroidal actions of catecholamines. Epinephrine, norepinephrine and isoproterenol appeared to be rapid and potent stimulators of intracellular cAMP accumulation, the half maximum increase doses being 4 X 10(-7)M, 1 X 10(-5)M and 5 X 10(-7)M, respectively. While propranolol (1 X 10(-5)M) prevented the stimulatory effect of catecholamines and failed to inhibit the effect of bovine TSH, phentolamine (1 X 10(-5)M) enhanced the potency of norepinephrine and bovine TSH, leaving that of epinephrine unchanged. The effects of epinephrine (2 X 10(-8)M) and isoproterenol (2 X 10(-8)M) were additive to that of bovine TSH (0.5 mU/ml), but the effect of simultaneous stimulation with norepinephrine (5 X 10(-7)M) and bovine TSH (0.5 mU/ml) was lower than expected. Prenalterol, a selective beta 1-agonist, did not stimulate cAMP accumulation, while terbutaline, a selective beta 2-agonist, exerted a potent stimulation. Metoprolol, a selective beta 1-adrenergic blocker, did not affect the response of thyroid follicular cells to isoproterenol. These results demonstrate the existence of beta-adrenergic receptors in human thyroid follicular cells, mainly of the type beta 2, apparently not correlated with TSH receptor. The existence of alpha-adrenergic receptors which counter-regulate TSH functional responses in human thyroid follicular cells is suggested.     

Desensitization of the thyroid cyclic AMP response to thyroid stimulating immunoglobulin: comparison with TSH

Studies were conducted to examine the characteristics of thyroid cell cAMP stimulation by thyroid stimulating immunoglobulins (TSI) and to compare the cAMP response to TSI and TSH in desensitized human thyroid cells. In terms of cAMP production, preexposure (eight hours) of the cells to TSI induced a desensitization very similar to TSH-induced desensitization: both TSH- and TSI-desensitized cells showed a normal response to cholera toxin and forskolin stimulation; TSH and TSI desensitization was interchangeable in that desensitization by either stimulator affected the action of the other; the time of recovery from either TSH and TSH desensitization was identical; the cycloheximide (10(-4) mol/L) prevented both TSI- and TSH-induced desensitization; preexposure of the cells to iodine, which affects mainly the adenylate cyclase catalytic unit, or to epinephrine, which activate the inhibitory regulatory protein Ni by the alpha 2-adrenergic stimulation, induced a similar inhibition of the subsequent stimulation by both TSH or TSI. The remarkable similarities between TSH and TSI in stimulating and desensitizing thyroid cells strongly support the concept that TSI activates thyroid adenylate cyclase by interacting with the TSH receptor and not through an allosteric mechanism.     

CHARACTERISTICS OF PATIENTS WITH NORMAL T3 AND T4 AND A LOW TSH RESPONSE TO TRH

The nature of the thyroid disorder presented by patients with normal T4 and T3 but blunted TSH response to TRH has not been clarified. In this study, we compared thyroid function tests in 16 such patients with those of 14 controls and 10 hyperthyroid patients. Basal total T4, free T4, total T3, iodine uptake and cholesterol of the study group were similar to controls but significantly (P less than 0.001) lower than in hyperthyroid patients, except for cholesterol which was higher. In contrast, the basal TSH, increase in TSH after TRH stimulation, and decrease of T4 during T3 suppression tests were similar to data obtained in hyperthyroid patients but significantly (P less than 0.001) lower than in controls. Pulse rate was mid-way between the control and the hyperthyroid group. Thyroid stimulating antibody (TSAb) was measured with human thyroid cells in culture; the assay was positive in four subjects in the 16-patient group and in all hyperthyroid patients tested. TSH stimulation test showed a hyporesponse in iodine uptake in the four patients with positive TSAb (26 +/- 29%), as well as in hyperthyroid patients (6 + 5%). However, there was a hyper-response to TSH (213 +/- 52%) in the remaining 12 patients in the group, none of whom had TSAb. Thus TSAb is not seen as responsible for the thyroid disorder in the majority of patients with normal T3 and T4 and absent or blunted TSH response to TRH; surprisingly, most of these patients have thyroid hypersensitivity to TSH. These two characteristics, absence of TSAb and hypersensitivity to TSH, delineate a thyroid disorder clearly different from Graves' disease.     

TGFbeta1 effects on functional activity of porcine thyroid cells cultured in suspension

Thyrotropin (TSH) and transforming growth factor beta 1 (TGFbeta1) have major roles in the regulation of folliculogenesis and differentiation in thyroid cells. Isolated porcine thyroid cells cultured in the presence of TSH on a plastic surface recover a follicular architecture and exhibit normal functional properties. The addition of TGFbeta1 to the culture medium induces important morphological changes and extracellular matrix remodelling. Similarly, thyroid cells lose their ability to organify iodine and their responsiveness to adenylate cyclase. The aim of this study was to analyse the influence of TGFbeta1 on the functional activity of thyrocytes in suspension culture, independent of follicle disruption. In this system, we demonstrate that TGFbeta1 inhibits expression of thyroperoxidase, NADPH oxidase activity, iodine uptake and, consequently, iodine organification. Moreover, TGFbeta1 decreases basal and TSH-stimulated cAMP production and TSH receptor expression. Taken together, these data converge to demonstrate an essential role of TGFbeta1 in the regulation of the thyroid cell function.     

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.     

Influences of cholera toxin on thyroid stimulation by thyrotropin and thyroid-stimulating antibody

Thyroid-stimulating antibody (TSAb) is an immunoglobulin G (IgG) occurring in the blood in hyperthyroid Graves' disease patients; it stimulates the thyroid in a manner analogous to the action of TSH, i.e. by activation of adenylate cyclase. Since cholera toxin is also known to stimulate thyroid adenylate cyclase, we studied possible interaction of the enterotoxin on effects of TSAb and TSH using slices of canine thyroid in vitro and an increase in the concentration of cAMP as endpoint. Normal human IgG, known to inhibit the binding of [125I]-iodo-TSH to thyroid membranes, decreased stimulation of thyroid slices by TSH; this inhibitory effect occurred also with preparations of TSAb (inevitably comprised mainly of normal IgG) that were themselves stimulatory. The cholera toxin effect was not prevented by normal IgG and, by factorial analysis of variance, was shown to potentiate the action of subsequently added TSAb or TSH. There was also positive interaction of the effect of TSAb with the combination of TSH and cholera toxin. The data indicate that responses of thyroid tissue to TSAb and TSH are readily influenced by effects of other membrane-active agents (in the present context, normal IgG and cholera toxin).     

Thyroid-stimulating antibody bioassay using porcine thyroid cells cultured in follicles

Isolated porcine thyroid cells were cultured in agarose-coated dishes and allowed to reform follicles with normal polarity. The thyroid cells reaggregated into follicles were compared with cells cultured in monolayer for cAMP responsiveness to TSH and thyroid-stimulating antibody (TSAb). The cells in follicles were sensitive to TSH stimulation and responded to the hormone at concentrations as low as 3.3-10 microU/ml with an increase in cAMP production. In contrast, 10-50 microU/ml TSH were required to elicit a cAMP response in cells cultured in monolayer using identical conditions. cAMP responsiveness to TSAb also was greater in the cells organized into follicles. TSAb was detected in serum from 89.4% of 66 untreated patients with hyperthyroid Graves' disease using thyroid follicles, but TSAb was detected in serum from only 60% of the patients when assayed using cells in monolayer. The assay using thyroid follicles was used to measure TSAb in 27 euthyroid patients who were euthyroid while receiving thionamide therapy and compared with 20-min thyroid 131I uptake after T3 suppression. TSAb was present in 11 of 12 nonsuppressible patients and in 5 of 15 suppressible patients. TSAb was positively correlated with 20-min 131I thyroid uptake. We conclude that thyroid cells cultured in follicles are suitable for measuring TSAb.     

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 thyroid-stimulating hormone on human thyroid carcinoma and adjacent normal tissue.

Effects of TSH on the adenylate cyclase-cAMP system and some parameters of intermediary metabolism were investigated in human thyroid carcinoma and adjacent normal thyroid tissue. Basal adenylate cyclase activity and cAMP concentrations were significantly higher in carcinomatous tissue. Basal [1-14C]glucose oxidation, 32Pi incorporation into phospholipids, and organification of iodide were similar in both tissues. Stimulation of cAMP by TSH was significantly greater in normal compared to carcinomatous tissue. In neither tissue was there a good correlation between TSH stimulation of adenylate cyclase activity and cAMP concentrations. The TSH stimulation of 32Pi incorporation into phospholipids by TSH was significantly greater in normal tissue. The mean effect of TSH on iodide organification and glucose oxidation was similar in normal and carcinomatous tissue. Although specific binding of TSH was demonstrated in both normal and carcinomatous tissue, it did not correlate very well with stimulation of adenylate cyclase activity. Hormones other than TSH also augmented adenylate cyclase activity in two of the carcinomas. In individual patients, the relative responsivity of carcinomatous tissue compared to normal was not always consistent when all of the metabolic parameters were considered.     

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.     

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.     

Thyrotropin-independent mutant clones from FRTL5 rat thyroid cells: hormonal control mechanisms in differentiated cells

Mutant cells varying in the pathways of their responses to hormonal stimulation are useful in defining the subcellular steps in the mechanisms of hormone action. FRTL5, a strain of normal and differentiated cells originally derived from adult rat thyroids, which depends on TSH for growth in vitro, was used to produce five TSH-independent mutants, after chemical mutagenesis and selection in medium lacking TSH. Their characterization and comparison with wild type cells demonstrated full retention of differentiated thyroid function markers such as thyroglobulin production and active iodide transport, and a slower growth rate. Characterization of cAMP metabolism in mutants revealed levels of basal cAMP and adenylate cyclase and phosphodiesterase activities similar to those of wild type cells kept in a nonproliferative state in medium lacking TSH. Adenylate cyclase responsiveness to very low doses of TSH (10(-12) M) was fully retained in all mutant clones, but the TSH-dependent cAMP elevation, although comparable to that reported in wild type cells, was not followed by significant growth stimulation in mutants. These findings demonstrate that the persistence of functional TSH receptors in these cells and that of growth regulation in them is independent of cAMP elevation.     

Adenylate cyclase system responsive to thyroid stimulating hormone (TSH) of porcine thyroid cells in primary monolayer cultures. Potential effect of forskolin on TSH-mediated adenylate cyclase stimulation

The TSH-responsive adenylate cyclase system was studied using porcine thyroid cells in a primary monolayer culture. Isolated porcine thyroid cells treated with collagenase were inoculated into 96 wells at the density of 5 X 10(4) viable cells/0.25 ml Ham F-12 containing 10% fetal bovine serum and cultured for 4 days in a humidified atmosphere with 5% CO2. Adenylate cyclase activities in the cells treated or non-treated with protein synthesis inhibitor were assayed in Hanks/20 mM Hepes buffer (pH 7.4) containing 1% BSA, 1 mM IBMX and various stimulants at 37 degrees C for 30 or 60 min. The reaction was stopped by adding ice-cold TCA, and cAMP content in the extract was measured by radioimmunoassay after treatment with water-saturated ether. The cultured thyroid cells had an adenylate cyclase system responsive to TSH, cholera toxin and forskolin. TSH (50 mU/ml) stimulated the activity about eight fold over the basal activity. Cholera toxin (1 microgram/ml) and forskolin (100 microM), however, were much stronger activators of the adenylate cyclase system. In the cells pretreated with cyclo-heximide (5 micrograms/ml) up to 24 hours, cAMP formation by TSH was potentiated 200 approximately 170% compared to that in non-treated cells, suggesting a suppression of an inhibitory mechanism dependent upon new protein synthesis. In contrast, forskolin (100 microM)-stimulation was greatly reduced to 30% of the control after 24-hour treatment. Cholera toxin (1 microgram/ml)-stimulation was significantly lessened or slightly reduced by the treatment. Although the ability of forskolin to act synergistically with TSH or cholera toxin was observed in non-treated cells, it was clearly unaffected and demonstrated in the cells treated with protein synthesis inhibitor. The mechanism(s) and site(s) of forskolin action still remain unclear. However, these observations are compatible with a two-site model of forskolin action. The direct activating site of forskolin appears to reside in a protein which is closely associated with the catalytic unit of adenylate cyclase system and has a relatively shorter half-life than other components of the system. The potential action of forskolin may reside in a more stable complex of an activated stimulatory guanine nucleotide binding component and catalytic unit of the adenylate cyclase system. Based on these results, it is likely that the primary monolayer culture of porcine thyroid cells is a good model to investigate the adenylate cyclase system in the thyroid, and that forskolin may potentiate the TSH-mediated stimulation of adenylate cyclase.     

A mechanism additional to cyclic AMP accumulation for vasoactive intestinal peptide-induced prolactin release

Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor which has been proposed to exert its secreting property by activating the adenylate cyclase enzyme. The present study shows that the omission of external Ca2+ did not affect the ability of VIP to induce PRL release while it completely abolished the VIP stimulatory effect on adenylate cyclase. We found that VIP (500 nM) stimulated PRL secretion in a time-dependent manner reaching a plateau at 3 min. This pattern was not changed when Ca2+ was omitted from the incubation medium. When tested at different concentrations, VIP stimulated PRL release with EC50 values of 1.3 nM in the presence of Ca2+ and 30 nM in the absence of Ca2+. On the other hand, Ca2+ removal completely suppressed the VIP-induced cAMP formation. VIP (200 nM) was also found to activate Ca2+ influx into pituitary cells. The increase in Ca2+ permeability showed a peak at 5 s and remained significantly higher than control values until 1 min. In conclusion, in an experimental condition where Ca2+ was omitted from the medium, VIP was found to induce PRL release without stimulating cAMP production. This cAMP-independent PRL release was blocked by preincubation of the cells with 1 microgram/ml pertussis toxin. An additional mechanism other than adenylate cyclase activation or Ca2+ entry is proposed to sustain VIP-induced PRL release.