Antithyroid effects in vivo and in vitro of babassu and mandioca: a staple food in goiter areas of Brazil.

Babassu (Orbignya phalerata), a palm-tree coconut fruit, mixed with mandioca (Manihot utilissima) is the staple food of people living in the endemic goiter area of Maranhao in Brazil, where goiter prevalence among schoolchildren was still 38% in 1986 despite an adequate iodine intake in most of the population. Therefore, the question arose as to whether or not the ingestion of babassu alone or mixed with mandioca contributed to the persistence of endemic goiter in this area of Brazil. In this investigation we examined the potential antithyroid effects of babassu and mandioca by means of in vivo studies in Sprague-Dawley rats, in vitro studies in porcine thyroid slices and using a purified porcine thyroid peroxidase (TPO) system. Samples of various edible parts of babassu and mandioca flour were homogenized and extracted in goitrogen-free water (GFW) for in vivo experiments, and in methanol (100 g/l), GFW or 0.06 mol/l phosphate buffer (pH 7.0) for in vitro experiments. The edible parts of babassu produced significant in vivo antithyroid effects (p < 0.05- < 0.001) in rats on a high iodine intake (14 micrograms I- day-1.rat-1), as well as distinct and reproducible antithyroid and anti-TPO activities in both in vitro systems, their action being similar to that of the thionamide-like antithyroid drugs propylthiouracil and methimazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Blocking type immunoglobulins in patients with nongoitrous primary hypothyroidism in area of iodine deficiency.

We have evaluated the role of circulating serum immunoglobulins (IgG) which inhibit the growth of thyroid in the etiology of thyroid atrophy in endemic cretinism. Twenty nongoitrous cretins (13 women and 7 men, age range: 9-33) were classified on the basis of clinical criteria for cretinism in China. They were born and living in an iodine deficient area, Xinjiang, northwest China. Antimicrosomal antibody titers were negative in all serum. Nine patients (seven women and two men; age range: 11-23) were biologically primary hypothyroid. Seven subjects were of a myxedematous form and two subjects were of a mixed form. We have studied thyroid-growth inhibiting immunoglobulin (TGII) activity that was measured as an inhibitory effect of 4 mg/ml IgG on TSH-induced [3H]-thymidine incorporation into the DNA of a rat thyroid follicular cell line, FRTL5 cells. Six (five women and one man) out of the nine patients with primary hypothyroidism (66.7 percent) had TGII. We also measured other growth-blocking IgG that inhibited [3H]-thymidine incorporation into DNA stimulated by insulin-like growth factor-I (IGF-I), a growth factor working through a cAMP-independent pathway. Five (three women and two men) out of nine patients (55.6 percent) with nongoitrous primary hypothyroidism had IGF-I-blocking IgG. These results indicate that TGII plays an important role in atrophy of the thyroid in spite of increased serum TSH concentrations, and IgG which inhibits thyroid growth stimulated by IGF-I also might play a role in thyroid atrophy in some endemic cretins.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of surgical stress on the in vitro metabolism of thyroxine by rat liver, kidney, and brain.

In man, acute stress, like extensive surgery, leads to a rapid and prolonged decrease in serum T3 concentrations. The present study was carried out to investigate the mechanisms that underly the abrupt decrease in T3-neogenesis that occurs in response to acute surgical stress. Male Sprague-Dawley rats, surgically thyroidectomized, and treated with 1.2 micrograms T4/100 g BW/day, underwent wide vertical and horizontal incisions extending into the abdominal cavity while receiving light ether anesthesia. Different dietary manipulations were performed to investigate the superimposed influence of reduced carbohydrate and caloric intake on T3-neogenesis. The metabolism of 125I T4 labeled in its outer (phenolic) ring was investigated in liver, kidney, and brain homogenates of animals killed 48 h after surgery. In liver, values for the proportion of T4 degraded and the percent generation of T3 and iodide were unaffected by laparotomy. The percent T3 generation in experiments with 25 nM T4 concentration was 3.7 +/- 1.24% (mean +/- SD) in fed control animals given free access to 5% glucose, 3.4 +/- 0.67% in unoperated controls given a restricted amount of chow and 5% glucose, and 3.8 +/- 0.67% in operated animals given free access to chow and 5% glucose. As expected, T3 neogenesis in livers from unoperated animals was significantly reduced in rats fasted for 48 h and this reduction was similar in laparotomized rats fasted for 48 h after surgery. As in the liver, no effect of laparotomy on T4 metabolism in kidney and brain homogenates was observed. Finally, serum total T4 and T3 concentrations were not affected by surgery. It is concluded that acute surgical stress in thyroidectomized T4 replaced rats does not influence T4 metabolism in liver, kidney, and brain homogenates or affect the serum T4 and T3 concentrations. Since thyroid secretion of T4 (and T3) was eliminated and careful attention was paid to caloric intake in this rat model, previously reported abnormalities in serum thyroid hormone concentrations and T3-neogenesis in various states of nonthyroidal illness in man and rat, including surgery, are probably contributed to by thyroid secretion of T4 (and T3) and caloric deprivation, especially carbohydrate.

Thyroid atrophy in myxedematous endemic cretinism: possible role for growth-blocking immunoglobulins.

We have examined the ability of IgGs obtained from 8 endemic cretins to inhibit TSH-stimulated thyroid cell growth in culture. Clinical and laboratory evidence for hypothyroidism was present in six subjects; the two remaining patients had borderline low serum T4, normal T3 and exaggerated TSH response to TRH. In six patients 2 mg IgG exhibited an inhibitory effect in the cellular growth expressed by a diminished incorporation of 3H-thymidine into the DNA of TSH-stimulated FRTL-5 cells (range: 26-87% inhibition). Seven patients presented clinically with thyroid atrophy of relatively small thyroid enlargements for the degree of chronic iodine deficiency that was present in the area. The remaining subject had a large multinodular goiter and IgG purified from this patient had no inhibitory effect in the FRTL-5 cellular growth. A direct relationship was noted between the degree of thyroid growth inhibition (%) and the basal serum TSH concentration. We conclude that the presence of thyroid growth inhibiting immunoglobulin may be related to the absence of thyroid growth or even thyroid atrophy in endemic cretins.

Immunoglobulins of patients recovering from Yersinia enterocolitica infections exhibit Graves' disease-like activity in human thyroid membranes.

Substantial evidence suggests a link between infections with Yersinia enterocolitica (YE) and Graves' disease. We have now examined the sera of 72 patients recovering from YE infection for immunoglobulins that interacted with the TSH receptor in human thyroid membranes. Compared with controls, in concentrations between 1 and 4 mg/mL, patient IgG produced a significant, concentration-dependent inhibition of TSH binding (p less than 0.001) and stimulation of adenylate cyclase activity (p less than 0.005-0.05). Whereas IgG from normal individuals caused no stimulation of adenylate cyclase, IgG from controls caused some concentration-dependent displacement of TSH, as previously reported. However, IgG from convalescents of YE infections was significantly more potent than normal IgG in reducing the binding of TSH to the membrane. Thus, at each examined concentration, YE patients' IgG displaced more TSH than IgG from normal controls. For each milligram per milliliter increment of IgG in the assay, patients' IgG caused a 10.2% inhibition of TSH binding (r -0.90, p less than 0.001), significantly greater than that seen with normal IgG (p less than 0.02). The present studies provide the first demonstration that IgG of patients recovering from YE infections react with the human TSH receptor. The antibodies presumably are produced against the TSH-binding protein present in YE. However, in view of lack of evidence for thyroid dysfunction in the sera of patients recovering from yersiniosis and the presence of TSH-binding proteins in other bacteria, we postulate that infection with YE is neither necessary nor sufficient to cause thyroid autoimmune disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodide-induced hypothyroidism in patients after thyroid resection.

The purpose of this investigation was to determine whether an intrinsic defect in thyroid hormone production is required for the development of iodide-induced hypothyroidism or does it also develop in TSH-stimulated normal thyroid tissue. To answer this question, we studied the response to iodine administration (180 mg iodide daily for 3-4 months) in eight euthyroid patients who had had partial thyroidectomies 2 months to 10 years previously for benign thyroid nodules, and in three euthyroid control subjects. In all 11 euthyroid patients, basal serum TSH concentrations increased during iodide administration. In six of the eight patients who had previous thyroid operations and in two of the three control patients, basal serum TSH concentrations increased into the abnormal range (greater than 6 U ml-1). Increased serum TSH concentrations were noted as early as 1 week after potassium iodide had been started and the increased levels persisted during the period of iodide administration. Although basal values for serum TSH concentration were initially within the normal range, those patients with highest basal serum TSH values developed the greatest increase in TSH in response to potassium iodine. Among the eight patients treated by partial thyroidectomy, serum T4 concentrations decreased in five, serum T3 concentration decreased in three and all five developed mild symptoms of hypothyroidism while receiving iodide. Serum T4 concentrations also decreased slightly in two of the three control patients. Serum total iodine levels increased from 7.0 +/- 0.5 to 315.7 +/- 108.6 g dl-1 (mean-+/- standard error) during potassium iodide administration, but there was no correlation between the level of serum iodide concentration achieved and inhibition of thyroid function.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors determining the differential tissue response of Na/K-ATPase to thyroid hormone in the neonatal rat.

The concentration of ouabain binding sites (OBS), a measure of the abundance of sodium/potassium-dependent ATPase (Na/K-ATPase), was measured in cerebral cortex (CC), cerebellum (CBL), brown adipose tissue (BAT), and heart of neonatal rats with congenital hypothyroidism. In euthyroid rats, the concentration of OBS was 50- to 100-fold greater in the nervous tissue than in the nonnervous tissue. Congenital hypothyroidism resulted in a significant reduction in the number of OBS in all four tissues. Although in absolute terms (pmol/mg protein) the reduction was greater in the nervous tissues, in relative terms it was much greater in the nonnervous tissues. The restoration of OBS concentration was much more sensitive to T4 in CC, CBL, and BAT than in heart. In contrast, the response of OBS concentration to T3 in hypothyroid rats was greater in the heart, followed by the BAT and CBL, being minimal in CC. The sensitivity to T4 replacement correlated with the degree of the stimulation of the type II 5'deiodinase (5'D-II) by hypothyroidism, whereas the response to exogenous T3 correlated with the fraction of the tissue T3 that derives from plasma T3 and inversely with the plasma T3 concentration required to saturate 50% of the nuclear receptors as reported in previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodothyronine deiodinase activities in FRTL5 cells: predominance of type I 5'-deiodinase.

FRTL5 cells, a thyroid follicular cell line derived from normal rat thyroid, has been extensively used as a model system to study various aspects of the physiology of the thyroid epithelium. The capacity of these cells to metabolize iodothyronines and to generate T3 from T4 has not been previously examined. Here we studied the deiodination of T4, T3, and rT3 in homogenates of FRTL5 cells. By far, these homogenates were more potent catalyzing the 5'-deiodination (outer ring) of T4 and rT3 than the inner ring deiodination of T4 or T3. Both the production of rT3 and the degradation of newly formed T3 from T4 were very limited. Thus, when T4 was used as a substrate, T3 and iodide accumulated in a linear fashion with time, and initially the amounts of iodide and T3 were approximately equal. rT3 and 3,3'-diiodothyronine were rapidly deiodinated by these homogenates, with the 3'-deiodination of 3,3'-diiodothyronine occurring at a slower rate than the 5'-deiodination of rT3. The iodothyronine 5'-deiodinase activity corresponded to type I, as indicated by the following: the Km for T4 and T3 was in the micromolar range; rT3 was a better substrate than T4 (maximum velocity = 101 vs. 19 pmol/min.mg protein; Km = 0.83 vs. 3.1 microM, respectively); and the kinetics of inhibition by 6n-propyl-2-thiouracil were uncompetitive and substrate dependent, suggesting ping-pong kinetics. The type I 5'-deiodinase activity of FRTL5 cells was distinctly stimulated by TSH. This stimulation seems to be mediated by cAMP and requires serum as a permissive factor. In conclusion, 1) FRTL5 cells exhibit both inner and outer ring iodothyronine-deiodinating activities; 2) iodothyronine 5'-deiodination is by far more active; 3) the 5'-deiodination has been characterized as type I deiodinase based on substrate preference, enzyme kinetics, and inhibitors; 4) in all respect iodothyronine deiodination by FRTL5 cell homogenates proceeded with marked similarity to that in homogenates or microsomes of thyroid glands from several species; and 5) the FRTL5 type I deiodinase is more active than that reported in thyroid tissue and as active as that reported in liver and kidney, the prototype of type I deiodinase-containing tissues. The present studies indicate that FRTL5 cells are an excellent model system to study cellular and biochemical aspects of the regulation of this enzyme as well as its regulation by TSH and putative serum factors.

Influences of thyroid status and sympathoadrenal system on extrarenal potassium disposal.

Effects of hyper- and hypothyroidism on the ability of rats to transfer acute intravenous loads of potassium from the extracellular to the intracellular milieu (extrarenal potassium disposal, ERPD) were studied. We also examined the effects of the sympathoadrenal system on ERPD, as well as the manner in which it interacts with thyroid status. Experiments were performed in thyroidectomized (hypothyroid), sham-operated (euthyroid), or 3,5,3'-triiodo-L-thyronine-treated (thyrotoxic) rats. In anesthetized, acutely nephrectomized animals given a constant infusion of KCl over a 90-min period, ERPD was assessed as an inverse function of the increase in plasma potassium concentration. Some animals were subjected to chemical sympathectomy, adrenalectomy, the administration of adrenergic antagonists, or the infusion of adrenergic agonists. The effects of these treatments in various combinations on ERPD in animals of differing thyroid status were determined and the following conclusions could be drawn: 1) beta 2-adrenergic influences increase ERPD; 2) alpha 1- and alpha 2-adrenergic influences decrease ERPD; 3) these influences of the sympathoadrenal system on ERPD are qualitatively independent of thyroid status, and in all three thyroid states, beta-adrenergic enhancement predominates over alpha-adrenergic inhibition; 4) thyrotoxicosis increases and hypothyroidism decreases ERPD, and these effects are qualitatively independent of the presence of sympathoadrenal activity; 5) the intrinsic effect of thyroid hormone insufficiency and increased alpha-adrenergic tone and/or responsiveness together account for the decreased ERPD observed in hypothyroid animals; and 6) the intrinsic effect of thyroid hormone excess and increased beta-adrenergic tone and/or responsiveness, as well as decreased alpha-adrenergic tone and/or responsiveness, together account for the increased ERPD found in thyrotoxic animals.

3,5,3'-tri-iodothyronine enhances sugar transport in rat thymocytes by increasing the intrinsic activity of the plasma membrane sugar transporter.

We have shown that 3,5,3'-tri-iodothyronine (T3) produces a prompt increase in sugar transport in rat thymocytes by increasing the maximal velocity without changing the Michaelis-Menten constant of the plasma membrane sugar transport system. To elucidate further the mechanism of this effect, we have now assessed the influence of T3 on the number and affinity of sugar transporters in thymocytes, measured as the sugar (2-deoxyglucose; dGlc)-displaceable binding of cytochalasin B. Cytochalasin B inhibited in a dose-related manner the uptake of dGlc by rat thymocytes with inhibition constant values of 0.19 and 0.22 mumol/l in the presence and absence of T3 respectively. Binding of cytochalasin B by the sugar-displaceable sites was rapid and saturable, demonstrating a single class of sites having an apparent dissociation constant of 0.33 +/- 0.02 (S.D.) mumol/l and maximal binding capacity of 3.73 +/- 0.48 pmol/20 x 10(6) cells (11.2 +/- 1.4 x 10(4) sites/thymocyte). In the rat thymocyte, sugar transporters were found to be located in two major subcellular pools, the plasma membrane and microsomes, the latter being about twice the size of the former. In these subcellular compartments, as well as in the intact cell, binding of [3H]cytochalasin B by the sugar-displaceable sites constituted about 40% of total cytochalasin B binding. 3,5,3'-Tri-iodothyronine in concentrations that stimulated uptake of dGlc by thymocytes had no effect on [3H]cytochalasin B binding (total and sugar-displaceable) in the intact cell and in the plasma membrane and microsomal compartments, nor did it influence the affinity and number of sugar transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat.

Thyroid hormones influence growth in part by altering the secretion and effects of GH. GH, in turn, mediates its effects by regulating the synthesis and secretion of insulin-like growth factor-I (IGF-I). IGF-I is a pleiotropic growth factor that is synthesized by many tissues and acts on many tissues to regulate both cellular replication and differentiated function. We have studied the direct effects of thyroid hormones and the combined effects of thyroid hormones and GH on the regulation of IGF-I synthesis and secretion in hypophysectomized (hypox) rats in vivo. All rats, except normal littermates and a hypox control group, received 100 micrograms hydrocortisone/100 g BW for 10 days. Circulating IGF-I was measured by specific RIA (normal rats, 1 U/ml), and hepatic IGF-I mRNA was measured by Northern blot hybridization with an antisense cRNA probe. 1) Hypox rats treated with hGH (75 micrograms, ip, twice daily) for 10 days gained 17 g BW vs. 70 g for normal littermates. GH markedly increased hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 U/ml 12 h after the last GH injection vs. 0.03 +/- 0.02 for hypox controls). 2) T4 (1 micrograms/100 g BW, ip) for 10 days increased neither weight, hepatic IGF-I mRNA, nor circulating IGF-I. 3) Rats treated with T4 for 10 days followed by a single injection of 1 mg GH, ip, increased hepatic IGF-I mRNA and circulating IGF-I levels comparably as in rats receiving acute GH alone (IGF-I, 12 h, 0.31 +/- 0.09 vs. 0.36 +/- 0.06 U/ml). 4) Hypox rats treated with a single injection of T3 (1.5 micrograms/100 g BW, ip) had slightly increased hepatic IGF-I mRNA, but showed no significant change in circulating IGF-I levels. 5) A single injection of T3 plus GH to hypox rats increased IGF-I mRNA levels above those in rats injected with GH alone and increased serum IGF-I levels to 0.48 +/- 0.12 U/ml compared to 0.36 +/- 0.06 U/ml for GH alone. 6) After 10 days of GH treatment, a single injection of T3 lowered both hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 to 0.16 +/- 0.06 U/ml, 6 h after T3). These studies demonstrate that thyroid hormones have relatively little direct effect on IGF-I synthesis but can have major effects on GH-stimulated IGF-I synthesis and secretion. The pattern of these effects depends on the integrity of the pituitary gland, prior exposure of the liver to GH and/or thyroid hormones, and the temporal relationship between GH and thyroid hormone administration.

Stimulation of calcium-ATPase activity by 3,5,3'-tri-iodothyronine in rat thymocyte plasma membranes. A possible role in the modulation of cellular calcium concentration.

We have previously demonstrated that 3,5,3'-tri-iodo-L-thyronine (T3) produces a very rapid and transient increase in calcium uptake and cytoplasmic free calcium concentration in the rat thymocyte, and have postulated that Ca2+-ATPase may contribute to the overall effect of T3 on cellular calcium metabolism. In the present study, we show that in the rat thymocyte, T3 increased plasma membrane Ca2+-ATPase activity. This effect of T3 was very rapid, seen at 30 s after the addition of the hormone, and was concentration-related, evident at a physiological concentration as low as 1 pM. Evaluation of the effect of several thyronine analogues on Ca2+-ATPase activity revealed the following order of potency: D-T3 greater than or equal to 3'-isopropyl-L-T2 = L-T3 = L-T4 = D-T4 greater than L-rT3 greater than 3,5-L-T2 greater than DL-thyronine. Studies with the calmodulin antagonist trifluoperazine demonstrated that thymocyte Ca2+-ATPase activity and its stimulation by T3 are influenced by calmodulin. Other studies showed that several adrenergic agents, agonists and antagonists, had no effect on thymocyte Ca2+-ATPase activity and its stimulation by T3. From these and previous observations, we would suggest that in the rat thymocyte, the T3-induced increase in Ca2+-ATPase activity, which enhances the expulsion of calcium from the cell, plays a role in the diminution and transiency of the stimulatory effect of T3 on thymocyte calcium metabolism.

Transferrin in FRTL5 cells: regulation of its receptor by mitogenic agents and its role in growth.

Transferrin, a serum iron-binding protein, delivers iron to the cell after binding to specific receptors on the cell surface and is an important component of culture medium for virtually all cell lines, including the FRTL5 line of rat thyroid follicular cells. Therefore, we undertook studies in FRTL5 cells to examine the regulation of the transferrin receptor, the effects of transferrin on growth and differentiated functions, and the interactions of transferrin with several mitogenic pathways. FRTL5 cells possess one class of saturable transferrin receptors (Ka, 0.7 x 10(9) M-1). Binding of 125I-labeled transferrin was highest in actively growing cells and declined progressively, reaching minimal values when confluence was achieved. Removal of transferrin from culture medium caused a rapid increase in transferrin binding. TSH, acting within 5 min, induced a modest increase in transferrin binding, due to a cycloheximide-resistant increase in binding sites. Binding of transferrin after a 24-h incubation was also increased by other mitogenic agents, (Bu)2cAMP, forskolin (FK), insulin, insulin-like growth factor-I (IGF-I), and the phorbol ester TPA. Transferrin alone stimulated growth only minimally, but enhanced the mitogenic effect of TSH, (Bu)2cAMP, and FK, all of which act through the cAMP pathway. In contrast, transferrin did not alter the cAMP-independent mitogenic effects of insulin and IGF-I. Transferrin did not affect TSH-induced cAMP generation. Desferoxamine, an iron chelator, inhibited the mitogenic effects of all of the agents tested. Desferoxamine had no significant effect on TSH-induced cAMP accumulation. We conclude that FRTL5 cells contain saturable receptors for transferrin whose abundance varies with the rate of cell replication. Transferrin down-regulates its own receptors, while stimulation of growth by various mitogens is accompanied by increased binding of transferrin. Transferrin enhances the mitogenic effect of the cAMP-dependent mitogens, TSH, (Bu)2cAMP, and FK, without modifying basal or stimulated cAMP generation. In contrast, transferrin fails to affect the mitogenic responses to IGF-I and insulin, which are cAMP independent. Iron is required for the mitogenic response to various mitogens, especially those that are cAMP dependent.

Iodine inhibits the proliferation of rat thyroid cells in culture.

We have explored the mechanisms whereby iodine inhibits thyroid growth using as models both the FRTL5 line of rat thyroid follicular cells that require TSH for growth and the M12 line of mutant cells that grow in the absence of TSH. Between 0.01-1.0 mM, NaI produced a dose-dependent inhibition of TSH stimulation of [3H]thymidine incorporation and replication in FRTL5 cells as well as spontaneous growth in M12 cells. Iodide also inhibited the cAMP-dependent growth of FRTL5 cells induced by forskolin and (Bu)2cAMP, as well as the cAMP-independent mitogenesis induced by insulin-like growth factor-I. The effect of iodide to inhibit both TSH- and insulin-like growth factor-I-stimulated growth in FRTL5 cells was abolished by concomitant culture with methimazole, and no iodide inhibition of growth was observed in L6 myoblasts and BRL 30E hepatocytes. Exposure of cells to iodide under conditions that resulted in inhibition of TSH-stimulated growth did not significantly alter the ability of TSH to increase the intracellular cAMP concentration, nor did iodide alter two responses to TSH in FRTL5 cells that depend upon an increase in cAMP concentration: down-regulation of TSH receptor and cytoskeletal reorganization. We conclude that iodide exerts its inhibitory action on the growth of thyroid cells at multiple loci related to both the cAMP-dependent and cAMP-independent pathways of mitogenic regulation.

3,5,3'-Triiodothyronine increases cellular adenosine 3',5'-monophosphate concentration and sugar uptake in rat thymocytes by stimulating adenylate cyclase activity: studies with the adenylate cyclase inhibitor MDL 12330A.

We have previously demonstrated that T3 increases adenylate cyclase activity in preparations of plasma membranes from rat thymocytes. On the basis of this and other evidence, we have postulated that the increased cAMP concentration and consequent increase in 2-deoxyglucose (dGlc) uptake that T3 induces in the intact thymocyte is the consequence of a similar stimulation of adenylate cyclase activity. To obtain further evidence to this point, we have now conducted experiments with MDL 12330A [N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride], a compound that inhibits adenylate cyclase activity in several other tissues. In thymocyte plasma membrane preparations, MDL 12330A induced a concentration-dependent inhibition of both basal enzyme activity (activity in the absence of hormone) and the increase in activity induced by T3 and epinephrine. In the intact thymocyte, MDL 12330A greatly limited the marked increase in cellular cAMP concentration induced by maximally effective concentrations of the phosphodiesterase inhibitor 3'-isobutyl-1'-methylxanthine. This indicates that MDL 12330A inhibits adenylate cyclase activity in the intact thymocyte as it does in thymocyte plasma membrane preparations. Further, in intact thymocytes incubated with MDL 12330A, there occurred small but significant decreases in basal cAMP concentration and dGlc uptake, and the T3-induced enhancement of these functions was reduced or abolished. These data provide additional evidence that the increase in dGlc uptake in rat thymocytes that T3 induces is linked to an antecedent increase in cellular cAMP concentration, and that the latter results from a T3-induced enhancement of adenylate cyclase activity.

Evidence that an increase in cytoplasmic calcium is the initiating event in certain plasma membrane-mediated responses to 3,5,3'-triiodothyronine in rat thymocytes.

The present studies were undertaken to explore further the mechanism by which T3 increases adenylate cyclase activity and the uptake of the sugar analog 2-deoxyglucose (2-DG) in freshly isolated rat thymocytes. In studies of cells preloaded with the fluorescent probe quin-2, whose fluorescence intensity increases linearly with increases in cytoplasmic free calcium concentration [( Ca2+]i), we have now demonstrated that T3 increases [Ca2+]i in thymocytes suspended in buffer containing 1 mM Ca2+. This effect was extremely prompt, becoming evident much less than 1 min after the addition of T3 and reaching maximal values in about 5-8 min. The subsequent time course of the T3 effect was obscured by an increase in fluorescence intensity in control thymocytes not exposed to T3, beginning after about 8 min of incubation. However, the T3 effect, after reaching its peak, appeared to remain stable for about 5 min and then to decline, abating completely in 18-30 min. No effect of T3 on [Ca2+]i was observed when thymocytes were suspended in Ca2+-free medium. The effect of T3 on [Ca2+]i was concentration dependent, and as with its actions on thymocyte adenylate cyclase activity, cAMP concentration and 2-DG uptake, the lowest effective concentration of T3 was 1 nM. Among several thyronine analogs studied, L-T3 was the most potent, followed in decreasing order of potency by L-T4, D-T3, 3,5-diiodo-3'-isopropyl-L-thyronine, and D-T4. rT3, 3,5-diiodo-L-thyronine, and D,L-thyronine were without effect. l-Alprenolol alone (10 microM) produced a modest increase in thymocyte [Ca2+]i, but, as it does with the effect of T3 on cellular cAMP concentration and 2-DG uptake, it markedly inhibited or abolished the stimulatory effect of T3 on [Ca2+]i. From these observations we conclude that T3 initiates the increase in thymocyte [Ca2+]i by enhancing the influx of extracellular calcium, though the possibility that it also releases calcium from an intracellular calcium pool cannot be excluded. Since the effects of T3 on thymocyte adenylate cyclase activity, cAMP concentration, and 2-DG uptake occur subsequent to these effects on calcium metabolism and require the presence of Ca2+ in the extracellular fluid, we suggest that an increase in [Ca2+]i, due at least partly to an influx of extracellular calcium, is the initiating event in these plasma membrane-mediated responses of the rat thymocyte to T3.

Antithyroid and goitrogenic effects of millet: role of C-glycosylflavones.

Pearl millet [Pennisetum millet (L.) leeke] is the main source of food energy for the rural poor in many areas of the semiarid tropics. Epidemiological evidence suggests that millet may play a role in the genesis of endemic goiter in these areas, and sparse experimental data in rats support this suspicion. This study was undertaken to determine in vivo in rats and in vitro using porcine thyroid slices and a thyroid peroxidase (TPO) assay the goitrogenic and antithyroid effects of millet diets, extracts of millet, and certain pure compounds contained therein. For use in these studies, whole grain millet was progressively dehulled to yield successively four bran and four flour fractions in which direct analyses revealed progressively lower concentrations of C-glycosylflavones. In vivo feeding of bran fraction 1, that richest in C-glycosylflavones, led to a significant increase in thyroid weight and antithyroid effects. Feeding of bran fraction 2, the next richest in C-glycosylflavones, produced similar, but less marked, changes. In vitro studies of 125I metabolism using porcine thyroid slices indicated that extracts of bran fractions 1 and 2 were most potent, producing changes similar to those produced by methimazole (MMI). At a concentration of 60 mumol/L, glucosylvitexin, the major C-glycosylflavone present in millet, had effects comparable to those of 1 mumol/L MMI. Similarly, in studies of porcine TPO, extracts of bran fraction 1 caused pronounced (85%) inhibition of enzyme activity, and progressively less inhibition was induced by extracts of bran fractions 2, 3, and 4. Overall, the TPO-inhibiting activities of the various millet fractions closely correlated with their C-glycosylflavone concentrations. Three C-glycosylflavones present concentrations. Three C-glycosylflavones present in millet, glucosylvitexin, glycosylorientin, and vitexin, also inhibited TPO activity. Thus, in vivo and in vitro studies revealed that millet diets rich in C-glycosylflavones produce goitrogenic and antithyroid effects similar to those of certain other antithyroid agents and small doses of MMI. We conclude that in areas of iodine deficiency in which millet is a major component of the diet, its ingestion may contribute to the genesis of endemic goiter.

Design of a long-lived thyrotropin antagonist from derivatives of human chorionic gonadotropin.

Previous studies have revealed that desialylated forms of hCG have a high potency to inhibit both the binding of bovine TSH (bTSH) to human thyroid membranes and the bTSH-stimulated adenylate cyclase activity therein. The biological activities of these desialylated forms, however, are greatly reduced in vivo due to the high affinity of asialo-glycoproteins for hepatic receptors and their consequent rapid clearance from the circulation. Intact purified hCG (hCGp), on the other hand, has little affinity for hepatic receptors and has a relatively long half-life in vivo, but displays only negligible inhibitory potency in the human thyroid. In the present studies, we have sought to obtain one or more compounds derived from hCG that are able to inhibit binding to TSH to the high affinity receptor in human thyroid membranes, and, consequently, could inhibit the stimulatory effect of TSH and Graves' immunoglobulin G and which also display a relatively low affinity for the hepatic asialoglycoprotein receptor and, as a consequence, have a reasonable survival in the circulation. Two hCG forms isolated by sequential chromatography of crude hCG on DEAE-52 and Sephadex G-100 were of interest, since they displayed some degree of selectivity in binding to thyroid and liver receptors. The first form (hCGv), whose isolation we described previously, was comprised of partially desialylated variant forms of intact hCG. The second material, as judged from its immunoreactivity, elution behavior on Bio-Gel P-100, and migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contained a fragment of the beta-subunit (BF), similar to the beta-core fragment described by others. However, BF differed from the beta-core fragment in having a higher sialic acid content. Interestingly, we found that BF as well as the enzymatically desialylated form of BF displayed a much lower affinity for mouse liver receptors than did asialo-hCGp or the free asialo-beta- and asialo-alpha-subunits. Further, the activity of BF to inhibit the binding of [125I]bTSH to human thyroid membranes exceeded that of the desialylated subunits of hCGp as well as that of intact hCGp, but was only exerted at the low affinity binding site and was not accompanied by inhibition of TSH-stimulated adenylate cyclase. In an attempt to shift the locus of BF action from the low to the high affinity TSH receptor, we recombined BF with either an intact alpha-subunit of hCG or an asialo-alpha-subunit. This led to the creation of two novel forms of hCG with properties of the type that we were seeking.(ABSTRACT TRUNCATED AT 400 WORDS)