Intracellular Colloid Droplet Formation Research Articles

Ionophore A23187 inhibits the release of thyrotropin-stimulated 3, 5, 3'-triiodothyronine from perifused rat thyroid glands.

We have studied the effect of ionophore A23187 on the release of T3 and cAMP from perifused rat thyroid glands and on the morphological changes of the follicle epithelial cells. Ionophore A23187 at a concentration of 5 microM significantly inhibited TSH-stimulated T3 release. Depletion of Ca2+ in the infusion buffer completely abolished the inhibitory effect of ionophore A23187 on TSH-stimulated T3 release. In the presence of TSH and 3-isobutyl-1-methylxanthine, ionophore A23187 at 5 microM did not significantly affect either the release of cAMP during a 3-h perifusion or the cAMP content in the thyroid tissues after 1-h perifusion. By electron microscopy, the follicle epithelial cells of the rat thyroid tissues showed marked responses to TSH after 3-h stimulation. By the addition of 5 microM ionophore A23187, the ultrastructural changes such as phagocytotic uptake of luminal colloid and the formation of intracellular colloid droplets were rarely observed in spite of the presence of TSH. The present study indicates that ionophore A23187 at a concentration of 5 microM inhibits both TSH-induced morphological changes of the follicle epithelial cells and TSH-stimulated T3 release without reducing cAMP level stimulated by TSH in rat thyroid glands.

Progressive Recruitment of Follicular Cells with Graded Secretory Responsiveness during Stimulation of the Thyroid Gland by Thyrotropin*

One of the earliest responses of the thyroid cells to TSH is macropinocytosis with formation of intracellular colloid droplets. We demonstrate here that increasing stimulation with TSH not only elicits a highly individual macropinocytotic response among different follicular cells but that the fraction of TSH-responsive cells is also a function of the TSH dose. After pretreatment with T4, mice and rats were injected ip with bovine TSH and killed 2 h later. The macropinocytotic response to TSH was evaluated on periodic acid-Schiff-stained 3-microns sections of the thyroids in terms of droplet number per 25 follicles and, in addition, by assessing recruitment, i.e. percentage of droplet-containing cells. Both variables increased with increasing TSH stimulation until they reached a plateau at about 9 mU TSH in mice and at about 300 mU TSH in rats: the percentage of droplet-containing cells gradually increased in mice from 2% (no TSH) to 67% (9 mU TSH) and in rats from 11% (no TSH) to 54% (300 mU TSH). Overall pinocytotic response as well as thyrocyte recruitment could be modified by extra- and intrathyroidal factors: for example, pretreatment of the mice with an iodine-deficient diet increased the maximal percentage of droplet containing cells to nearly 90%. Obviously, two separate components of the macropinocytotic response of the thyroid gland to TSH can be distinguished: the first is the gradually increasing fraction of droplet-containing cells, the second is the well known increase of the number of colloid droplets in each TSH-responsive cell with progressive TSH stimulation. Recruitment of thyrocytes with a gradually increasing natural threshold to a hormonal stimulus appears to be a fundamental mechanism in the thyroid gland and possibly in other organs.

LATS-Protector Activity in Thyrotoxicosis Measured by Thyroidal Intracellular Colloid Droplet Formation*

A modified assay method of LATS and LATS protector (LATSP) was devised employing murine thyroidal intracellular colloid droplet formation. This method is 4- to 8-fold more sensitive to stimulators than is the regular McKenzie bioassay. For the assay of LATSP, IgG to be tested was incubated with human thyroid homogenate, then LATS-IgG was added and it was further incubated. The potency of LATS in the mixture was assayed. The adequacy of the present method for the assay of LATSP was supported by the facts that the LATSP activity was roughly related to the dose of IgG tested and that the inactivation of LATSP was observed when the IgG was pretreated with human thyroid particulate fraction and then assayed. With this method, 7 out of 32 patients with thyrotoxicosis showed LATS and 18 showed LATSP. Changes in LATSP were followed up in 7 cases out of the 18. LATSP activity decreased and then disappeared in 6 out of the 7 cases, when they had been euthyroid for a certain period of time. At 12-20 months after the treatment either by thionamide medication or by subtotal thyroidectomy. LATSP and 131I-thyroid uptake were measured in these 6 patients. In all of the 6, LATSP was negative regardless of thyroid suppressibility.

Characterization of T3 immunoreactivity release from thyroid gland in vitro a reflection of colloid droplet formation.

T3 immunoreactivity release from the thyroid gland in vitro was shown to be increased by TSH. In the present study, we sought to determine whether the T3 immunoreactivity release is an indicator of thyroid hormone secretion or due to hormone synthesis. When thyroid glands from mice were incubated with TSH, T3 immunoreactivity release was increased in parallel with intracellular colloid droplet formation in a dose related manner. When colchicine, a known inhibitor of colloid droplet formation, was added, both T3 immunoreactivity release and colloid droplet formation were inhibited, whereas MMI, an inhibitor of hormone synthesis, failed to influence both aspects. Thus T3 immunoreactivity release as a reflection of colloid droplet formation was demonstrated. The analysis of the released immunoreactivity by Sephadex column and subsequent radioimmunoassay suggested that the T3 immunoreactivity was, to a considerable extent, due to macromolecule instead of T3 itself. The effect of I- or Li+ to inhibit thyroid hormone secretion was shown to be on the step prior to, but not subsequent to, colloid droplet formation.

Effect of Triiodothyronine or Iodide on the Thyroidal Secretion In Vitro: Inhibition of TSH-and Dibutyryl-Cyclic-AMP Induced Endocytosis

T3 or iodide, at the concentration of 10- minus 3M, inhibits TSH- or dibutyryl-cyclic AMP-induced thyroidal intracellular colloid droplet formation, while leaving the TSH-induced increase in cyclic AMP levels intact. The effect of T3 appeared not to be mediated through the iodide derived from deiodination of T3. Those results clearly demonstrate that T3 or iodide acts at the point beyond cyclic AMP generation and prior tothe formation of intracellular colloid droplets. This is also the site at which microtubule active reagents exert their influence. Whether T3 or iodide fits this category or acts in as yet undetermined manner remains to be established.

Alteration of Thyroidal Responsiveness to TSH Under the Influence of Circulating Thyroid Hormone: Short Feed-Back Regulatory Effect

In order to examine the hypothesis that the thyroidal responsiveness to TSH is under the influence of thyroid hormone, following the T3 injection to the mice, serum T3 concentrations and the response of thyroid tissue to a fixed dose of TSH in terms of intracellular colloid droplet formation was studied. The colloid droplets induced by TSH was significantly reduced when serum T3 was decreasing, while it was significantly increased when serum T3 was increasing. This results demonstrate for the first time the existence of short feed-back loop regulating intra-thyroidal function by circulating T3. To delineate the possible mechanism of action of T3, the thyroid gland of mouse whose serum T3 concentration was elevated by injecting 50 mug T3, was incubated with TSH in vitro. TSH-induced cyclic AMP generation was not inhibited at all but colloid droplet formation was significantly inhibited in the thyroid tissue of the animal whose serum T3 concentration was enormously high. Thus, it was demonstrated that the site at which T3 affects is beyond cylcic AMP generation but prior to endocytosis, being consistent with our previous results.

Effect of dose of thyroid hormone on the sensitivity of the McKenzie bioassay.

Florsheirn reported that large doses of thyroid hormone, when employed for suppression of intrinsic TSH in McKenzie assay mice, reduced the response of the animal to TSH or LATS. Accelerated radioiodine clearance was proposed for the mechanism of this reduction in the response. We sought to determine whether thyroidal responsiveness per se is influenced by the dose of thyroid hormone, adopting radioiodine release and intracellular colloid droplet formation as parameters of thyroid response. A daily dose of T3, 0.5, 10, 25 μg, or T4, 0.5 or 25 μg was injected SC into mice for three days after 125I injection. On the day after the last thyroid hormone injection, they received 0.075 mU TSH or LATS-IgG. Blood radioactivity and response index were obtained on the specimens taken before, 2½ or 8 hr after the injection. Colloid droplet counts were obtained on the thyroid gland removed at ½ hr after TSH or 2 hr after LATS-IgG. The dose of TSH or LATS, which caused sizable and significant extent of radioiodine rele...

Effect of repeated administration of excess iodide on thyroid hormone secretion in rats, with special reference to the escape from the iodide effect.

Two hundred μg to 2 mg KI were injected every 12 or 24 hr ip for up to 7 days in rats, and thyroidal radioiodine release and the increase of intracellular colloid droplet formation induced by TSH were measured to assess the effect of repeated injections of excess iodide. A single injection of excess iodide blocked thyroidal radioiodine release, but this effect continued for about 34 hr in spite of repeated injections of excess iodide. An increase of colloid droplet formation induced by TSH was blocked by excess iodide, but 2 to 3 injections of excess iodide were required to produce maximal inhibition. This iodide effect on colloid droplet formation disappeared almost completely by the Sth day of iodide administration. However, a huge dose of KI again depressed the increase of TSH-induced colloid droplet formation in the thyroid which had escaped from the inhibitory effect of excess iodide. It is suggested that excess iodide blocks the release of thyroid hormone in rats, but only for a short time. (Endocri...

New in vitro tests to detect the thyroid stimulator in sera from hyperthyroid patients by measuring colloid droplet formation and cyclic AMP in human thyroid slices.

In an attempt to detect a thyroidstimulating substance in the sera of hyperthyroid patients, human and guinea pig thyroid slices were incubated directly in the sera of untreated hyperthyroid patients. The intracellular colloid droplets were enumerated in 25 follicles of the incubated slices. Eighty-two % of LATS-negative sera obtained from untreated hyperthyroid patients did stimulate intracellular colloid droplet formation in normal human thyroid slices. More than 0.1 mU/ml of bovine TSH was required to produce the increase of colloid droplets similar to that produced by LATS-negative sera. A similar increase in colloid droplet formation was also produced by LATS-IgG in human thyroid slices of normal tissue and Graves' disease as well. The sera from normal subjects were without effect in this respect. Both the sera from untreated hyperthyroid patients and LATS-IgG also stimulated the accumulation of cyclic AMP in human thyroid slices in vitro. Although LATS-IgG stimulated colloid droplet formation in gui...

Direct evidence for human thyroidal stimulation by LATS-protector.

To examine the hypothesis that LATS-protector may cause thyroidal hyperfunction in Graves' disease, the effect of LATS-protector on the intracellular colloid droplet formation in human thyroid tissue was studied in vitro. The IgG's from 9 untreated patients with Graves' disease, 4 thyrotoxic patients who were being treated hut were still thyrotoxic, and 4 normal subjects not showing LATS were chosen to study the presence of LATS-protector and its human thyroid stimulating activity. LATS-protector was assayed as described by Adams with slight modifications. LATS-protector was demonstrated in the IgG's of 6 out of 9 untreated patients with Graves' disease. Their IgG significantly inhibited the inactivation of LATS by the thyroid extract, recovering approximately 30% of the LATS-potency. The IgG from the treated, but thyrotoxic patients or from the normal individual did not show such an inhibition. When sliced normal human thyroid tissue was incubated with the IgG from thyrotoxic patients, an obvious elevati...

Evidence for stimulation of thyroidal secretion by iodoaminoacids or iodide.

Iodoaminoacids or iodide, in a dose containing 50μg iodine stimulated intracellular colloid droplet formation and radioactive iodothyronine release in mouse prepared for McKenzie bioassay. Theophyllin enhanced those effects on both aspects. Those results may explain thyroglobulin-induced enhancement of thyroidal secretion currently drawing attention of many investigators.

Effect of inhibitors of microtubules and microfilaments on dog thyroid slices in vitro

In order to test the role of microtubules and microfilaments in thyroid secretion, agents interfering with microtubules and with microfilaments were added with TSH to the incubation medium of dog thyroid slices. Electron microscopy of the slices demonstrated microtubules and microfilaments in follicular cells; it is suggested that microfilaments can be divided into four classes. The effects of microtubule inhibitors and the microfilament inhibitor cytochalasin B are similar to those observed in other systems; they were compatible with the postulated mechanism of action of these agents. The TSH-stimulated thyroid secretion from the slices was measured by the BE 131I release 1 1 Abbreviations: BE 131I, butanol extractable 131I iodine (iodide and thyroid hormones); cAMP, cyclic 3′,5′-adenosine monophosphate; DBcAMP, N 6-2-0′-dibutyryl cyclic adenosine monophosphate; NBE 131I, butanol non extractable 131I iodine ( 131I radioactivity remaining in the protein precipitate after butanolic extraction). in the medium; it was inhibited both by microtubule- and microfilament-inhibitors. Electron microscopy of the slices demonstrated that the inhibitors completely abolished pseudopod formation and intracellular colloid droplet formation in stimulated cells. This suggests that the inhibition of thyroid secretion by these agents bears on the first step of secretion: the endocytosis of colloid. As these agents blocked endocytosis and secretion in both TSH and in dibutyryl cyclic 3′,5′-AMP stimulated slices, it is concluded that they affect cyclic 3′,5′-AMP action rather than cyclic 3′,5′-AMP formation. The effects of the microtubule and microfilament inhibitors on the activity of the pentose phosphate pathway were divergent or non-existent, which is incompatible with the hypothesis of a common action on the NADPH supply of the follicle cell. The inhibition of colloid endocytosis and secretion by D 2O and cytochalasin was reversible, which contradicts the possibility of a non-specific toxic effect. It is suggested that both microfilaments and microtubules are required for the TSH-stimulated thyroid secretion process: the first (microfilaments) as active contractile agents would constitute newly formed pseudopods; the more passive microtubules would play a role of scaffolding in the cells and in the pseudopods, thus supporting the pseudopods in the engulfing process.

Significance of thyroid mast cells in thyroid hormone secretion.

The significance of thyroid mast cells in thyroid hormone secretion was investigated in 131I-injected, thyroxine-blocked rats and mice, by a combination of histochemical, light microscopical and bio-assay procedures. In the rat thyroid, numerous 5-hydroxytryptamine-(S-HT) and histamine-containing mast cells were present. A single injection of compound 48/ 80 released S-HT, histamine and metachromatic material from the mast cells. This was accompanied by a formation of intracellular colloid droplets and an increase in the blood 131I levels. In the mouse, virtually no mast cells were found in the thyroid, and treatment with 48/80 did not induce any colloid droplet formation or blood 131I increase. In both species, 5-HT evoked a blood 131I increase, whereas histamine yielded no such response. In rats, in which mast cells had been depleted by multiple, gradually increasing doses of 48/80, the blood 131I response to TSH was smaller than in intact controls, whereas similar pretreatment of mice did not affect th...

Further studies on the acute stimulatory effect of cold on thyroid activity and its mechanism.

In an attempt to study the nature, magnitude, duration, and temporal sequence of hypothalamo-pituitary-thyroid responses to cold, intact guinea pigs and rats, guinea pigs with midbrain transection or intact rats injected with 3-adrenergic blocking agent were exposed to cold (4 C) for 30 min. For comparison, TSH, thyrotropin releasing factor (TRF), and three hormones were administered to intact mice and guinea pigs or midbrain-transected guinea pigs. An increase of intracellular colloid droplets in the thyroid was apparent within 10 min, maximal at 30 min, and gradually subsided by 120 min when the animals were acutely exposed to cold. Propranolol increased intracellular colloid droplet formation and this effect was additive to that of cold. Midbrain transection completely abolished an increase of colloid droplets in response to cold, without affecting pituitary responsiveness to TRF. Since this increase of colloid droplet was produced by TSH and TRF but not by hydrocortisone, norepinephrine, and epinephri...

Sympathetic activation of thyroid hormone secretion in mice.

The influence of the sympathetic nervous system on the secretion of thyroid hormone was studied in mice, in which TSH secretion had been blocked by exogenous thyroxine. Stimulation of the right cervical sympathetic trunk for 5–60 sec induced a formation of intracellular colloid droplets and an increase in blood 131I. The droplet formation was restricted to the thyroid regions supplied by this nerve, as judged from the distribution of catecholamine-containing nerve terminals in thyroids of animals with a left-side sympathectomy. Iv L-norepinephrine (NE) induced a symmetrical formation of colloid droplets and an increase in blood 131I. Phentolamine inhibited all these effects. Neither uni- nor bilateral occlusion of the carotid blood flow for 60 sec was followed by any colloid droplet formation or blood 131I increase. The results suggest that sympathetic stimulation induces endocytosis of thyroglobulin and release of thyroid hormone, through a direct action on alpha adrenergic receptors in thyroid follicle ...

Dissociation of thyrotropin-stimulated hormone secretion and glucose oxidation in thyroid glands by lithium and colchicine

Li + and colchicine, which inhibit hormone secretion by the thyroid gland, were studied for their effects on glucose oxidation in this tissue. Both 2–25 mM Li + and colchicine (3·10 −6 M or more) enhanced the thyrotropin (TSH)-stimulated oxidation of [I- 14C]glucose in beef thyroid slices. Lithium also potentiated the TSM-stimulated oxidation of [6- 14C]glucose and [I- 14C]pyruvate as well as the incroporation of 32P into phospholipid. Colchine, however, was without effect on [6- 14C]glucose and pyruvate oxidation. Methods were adapted to permit measurement of [I- 14C]glucose oxidation by mouse thyroid lobes. Neither Li + nor colchicine had a significant effect on basal or TSH-stimulated glucose oxidation in this species at concentrations which almost totally abolish secretion. In dog thyroid slices also, TSH-stimulated 14CO 2 production was unaffected by Li + and colchicine at levels which inhibited intracellular colloid droplet formation. The results provide evidence for Li +- and colchicine-induced dissociation of the response to TSH and suggest a lack of direct coupling between hormone secretion and glucose oxidation.

Stimulation in vitro by thyrotropin, cyclic 3′,5′-AMP, dibutyryl cyclic 3′,5′-AMP and prostaglandin E 1, of secretion by dog thyroid slices

The release of 131I radioactivity extracted by butanol (thyroid hormones and iodide) in vitro from slices of prelabeled thyroids removed from pretreated dogs is measured. Results are compared with measurements of intracellular colloid droplet formation. Several arguments support the hypothesis that the in vitro release corresponds to secretion. Secretion in vitro was apparently delayed. Secretion in vitro as well as intracellular colloid droplet formation was enhanced by thyroid stimulating hormone (TSH), cyclic AMP (cAMP), N 6-2- O-dibutyryl cyclic AMP (b 2cAMP), and prostaglandin E 1, but not by caffeine, AMP or ATP. The kinetics of these effects were similar for the 4 stimulating agents. Concentration effect relationships for the 4 compounds are presented. Orders of magnitude of equieffective concentrations were TSH 0.25 mU/ml, cAMP 3.5–10 mM, b 2cAMP 0.1 mM. The concentration effect curve of prostaglandin E 1 was biphasic. The results support the hypothesis that the induction by TSH of thyroid secretion is mediated by cAMP. For low concentrations of TSH intracellular colloid droplet formation varied greatly from cell to cell, thus suggesting a marked intercellular functional heterogeneity. Several facts suggest that in the in vitro system thyroglobulinolysis may become the limiting step of secretion at high levels of thyroid stimulation.

Endocytosis of Thyroglobulin and Release of Thyroid Hormone in Mice by Catecholamines and 5-Hydroxytryptamine

The effects of L-epinephrine (E), L-isoproterenol (IPNE), 5-hydroxytryptamine (5-HT) and some other aromatic monoamines on the in vivo endocytosis of thyroglobulin and the release of thyroid hormone in mice were studied by light and electron microscopy, and by measurements of blood 131I levels. In hypophysectomized and in T4-blocked mice single iv doses of E, IPNE and 5-HT all induced a formation of intracellular colloid droplets. These appeared within 10 min, were most numerous at 30 min, and were disappearing within 60 min after treatment. This and other morphological signs of thyroid activation were followed by increased blood 131I levels, which reached peak values about 2 hr after treatment. All these effects closely resembled those induced by a small dose of TSH (0.02–0.04 mU). A less pronounced colloid droplet formation occurred with L-norepinephrine, dopamine and tryptamine. In animals whose thyroids were not sensitized to exogenous stimulation (i.e., having an intact TSH secretion) no colloid drop...

Stimulation by prostaglandin E-1 of endocytosis and glucose oxidation in canine thyroid slices.

Prostaglandin E1 (PGE1) stimulated canine thyroid tissue in vitro, as exemplified by a 2- to 4-fold increase in glucose oxidation and a 40- to 100-fold increase in the number of intracellular colloid droplets. The effect on colloid droplets was detectable with a concentration of PGE1 as low as 10−7m and that on glucose oxidation at 10−6m. Both actions of PGE1 were inhibited by the lysosome stabilizer, chlorpromazine, and were additive with submaximal doses of TSH. The similarity of PGE1 action to that of TSH was also manifest in vivo; PGE1 stimulated both intracellular colloid droplet formation and 131I release. (Endocrinology86: 423, 1970)

Effects of chlorpromazine and propranolol on in vitro thyroid activation by thyrotropin, long-acting thyroid stimulator and dibutyryl cyclic-AMP.

Thyrotropin (TSH), long-acting thyroid stimulator (LATS), and dibutyryl cyclic- AMP (DBcAMP) markedly stimulated intracellular colloid droplet formation (endocytosis) in canine thyroid slices. The addition of chlorpromazine (Cpz) or propranolol (Ppl) to the incubation medium inhibited or completely eliminated the response to each of the 2 stimulators. Isoproterenol did not induce endocytosis, nor did it potentiate the endocytotic effect of TSH. Cpz (2×10-4M) or Ppl (5×10-4M) also blocked TSH-induced or LATS-induced stimulation of the oxidation of glucose-l-14C to 14CO2. This concentration of Cpz also inhibited TSH-induced 32P incorporation into thyroid slice phospholipid, although causing mild stimulation of this process by itself. Ppl alone (5×10-4m) produced marked stimulation of phospholipogenesis; in lower concentration (10-4M) it caused mild stimulation and, when combined with TSH, it was neither fully additive nor inhibitory. These studies are interpreted as evidence that Ppl, chosen for study as an...