Effect of energy deprivation on intracellular transport and secretion of thyroglobulin

Summary The effect of energy deprivation on the intracellular transport and secretion of thyroglobulin was studied in open follicles isolated from porcine thyroids. Follicles were pulse-labeled with ³H-leucine or ³H-galactose. Labeled thyroglobulin was secreted into the incubation medium where it was isolated by means of immunoprecipitation. Secretion was followed in chase incubations under various experimental conditions using CCCP (carbonyl-cyanide-mchlorophenylhydrazone) or DNP (dinitrophenol), both uncouplers of oxidative phosphorylation, or CN^–, which inhibits respiration. CCCP (1 M) was shown to inhibit exocytosis by about 80%, DNP (0.1–5 mM) by 45–85%, and CN^– (0.5–1.1 mM) by 5–55%. By combining CN^– with the ionophore monensin, which blocks transport through the Golgi complex but does not essentially interfere with exocytosis, evidence was obtained that CN^– also inhibits transport of thyroglobulin from the Golgi cisternae to the exocytic vesicles by 40%. Electron-miroscopic autoradiography of isolated thyroid lobes from the rat also indicated that transport of ³H-leucine label into the follicle lumen is inhibited in the presence of CCCP or CN^–. Intracellular ATP content was found to be about 40% of the control level in follicles incubated with CCCP (1 uM) or CN^– (0.9 mM). The results show that the transport of thyroglobulin from the Golgi complex to the exocytic vesicles as well as from the exocytic vesicles into the follicle lumen is dependent upon metabolic energy. The transport blocks are probably associated with inhibited membrane fusions and fissions.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DNP dinitrophenol     

Effect of cooling on intracellular transport and secretion of thyroglobulin

Summary The effect of cooling to 20° C on the intracellular transport and secretion of thyroglobulin was studied by incubating open thyroid follicles isolated from porcine thyroid tissue. Follicles were labeled with ³H-leucine or ³H-galactose and the secretion of labeled thyroglobulin into the incubation medium was followed by chase incubations under various experimental conditions. The observations indicate that the transport of thyroglobulin is inhibited at three sites of the intracellular pathway by cooling to 20° C, i.e., between the RER cisternae and the Golgi cisternae, between the latter and the exocytic vesicles, and between these vesicles and the extracellular space (corresponding to the follicle lumen). The secretion of ³H-leucine-labeled thyroglobulin decreased linearly between 37° and 20° C; within this temperature range the activation energy for secretion, calculated from Arrhenius plots, was found to be 37 kcal/mol. Below 20° C the secretion was scarcely measurable. It is suggested that the three transport blocks at 20° C result mainly from inhibition of membrane fission and fusion due to phase transition in membrane lipids.     

Localization of the incorporation of 3H-galactose and 3H-sialic acid into thyroglobulin in relation to the block of intracellular transport induced by monensin

Summary The Na⁺/K⁺ ionophore monensin is known to arrest the intracellular transport of newly synthesized proteins in the Golgi complex. In the present investigation the effect of monensin on the secretion of ³H-galactose-labeled and ³H-sialic acid-labeled thyroglobulin was studied in open thyroid follicles isolated from porcine thyroid tissue.Follicles were incubated with ³H-galactose at 20° C for 1 h; at this temperature the labeled thyroglobulin remains in the labeling compartment (Ring et al. 1987a). The follicles were then chased at 37° C for 1 h in the absence or presence of 1 M monensin. Without monensin substantial amounts of labeled thyroglobulin were secreted into the medium, whereas in the presence of the ionophore secretion was inhibited by 80%. Since we have previously shown (Ring et al. 1987 b) that monensin does not inhibit secretion of thyroglobulin present on the distal side of the monensin block we conclude that galactose is incorporated into thyroglobulin on the proximal side of this block.Secretion was also measured in follicles continuously incubated with ³H-galactose for 1 h at 37° C in the absence or presence of monensin. In these experiments secretion of labeled thyroglobulin was inhibited by about 85% in the presence of monensin. Identically designed experiments with ³H-N-acetylmannosamine, a precursor of sialic acid, gave similar results, i.e., almost complete inhibition of secretion of labeled thyroglobulin in the presence of monensin. The agreement between the results of the galactose and sialic acid experiments indicates that sialic acid, like galactose, is incorporated into thyroglobulin on the proximal side of the monensin block.Considering observations made in other cell systems the present results suggest that galactosylation and sialylation of thyroglobulin are completed within the Golgi complex.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

Effects of the monovalent ionophore monensin on the intracellular transport and processing of pro-opiomelanocortin in cultured intermediate lobe cells of the rat pituitary

Detailed studies on the effects of the ionophore monensin upon synthesis, maturation, and intracellular transport of pro-opiomelanocortin in cultures of rat pituitary intermediate lobe cells have been carried out. When added at concentrations larger than 5 X 10(-8) M monensin significantly inhibited protein synthesis by cultured intermediate lobe cells. Pro-opiomelanocortin synthesis was also reduced proportionally to the overall rate of protein synthesis. During pulse-chase experiments, monensin when added at a concentration of 10(-5) M at the beginning of the chase incubation completely inhibited the proteolytic processing of pro-opiomelanocortin. Using a subcellular fractionation procedure of intermediate lobe cell extracts on Percoll gradients, we were able to show that after the addition of monensin (10(-5) M), labeled pro-opiomelanocortin molecules synthesized during a 15-min pulse-incubation were recovered intact after a 2-h chase, in the fractions of the density gradient corresponding to the rough endoplasmic reticulum and Golgi elements. No maturation products or precursor molecules entered the granule fractions as observed in nontreated cells. Taken together these results strongly suggest that monensin blocks the intracellular transport of newly synthesized pro-opiomelanocortin molecules at the Golgi level and that inhibition of proteolytic processing is due to the failure of the prohormone to enter the cell compartment (probably the secretion granules) where maturation proteases are located.     

Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.     

Thyrotropin effects on vesicle transfer and thyroid follicle morphogenesis: a stereological study in the rat

Incubation in a culture medium with and without TSH of 16 day-old foetal thyroid glands induces hypertrophy of the Golgi apparatus which may be correlated with a considerable increase in the number of secretory vesicles. A stereological study performed during the first 6 hr of incubation showed that: vesicle secretion was biphasic; vesicle secretion was heterogeneous with two different populations of vesicles; When TSH (20 mU and 80 mU) was added to the medium, the volume density of the follicular lumina increased; at least during the first 6 hr TSH seemed to be necessary to the formation of follicular lumina.     

A BIOCHEMICAL AND RADIOAUTOGRAPHIC ANALYSIS OF PROTEIN SECRETION BY THYROID LOBES INCUBATED IN VITRO

In this study we analyzed several aspects of protein secretion by thyroid follicular cells. The study was carried out on intact thyroid lobes obtained from newborn rats and incubated in vitro. The fate of leucine-³H incorporated into protein within follicular cells of untreated and thyrotropic hormone (TSH)-treated lobes was traced by quantitative electron microscope radioautography. Our findings indicate that protein synthesized by the rough-surfaced endoplasmic reticulum during a pulse exposure to leucine-³H is released relatively slowly by this organelle. Approximately 1 hr after onset of the pulse, a peak of radioactive protein appears in the Golgi region. The significance of this peak is not clear. Newly synthesized secretory protein passes through the apex of follicular cells without being concentrated or temporarily stored there in the form of large secretory droplets. Passage probably takes place via small vesicles which are intermingled among diverse small vesicles at the apex of the cells as well as in the Golgi region. Exposure of the lobes to TSH in the incubation medium for 45 or 90 min does not stimulate incorporation of leucine-³H into protein. Acute stimulation with TSH does, however, modify the movement of secretory protein within the exocrine secretory apparatus of the follicular cell. It accelerates the arrival of the protein at the apex of follicular cells, and it accelerates the release of the protein into the follicular lumen.     

The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes.

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.     

Process of iodination of thyroglobulin and its maturation. I. Properties and distribution of thyroglobulin labeled with radioiodine in pig thyroid slices.

Pig thyroid slices were incubated with Na131I and the 17--19S 131I-labeled thyroglobulin isolated was subjected to dissociation with 0.3 mM sodium dodecyl sulphate SDS) on sucrose density gradient centrifugation and to iodoamino acid analysis. During the incubation, initially dissociable thyroglobulin was gradually altered to 0.3 mM SDS-resistant species with increasing incorporation of iodine. Microsome-bound, poorly iodinated thyroglobulin and preformed thyroglobulin were chemically iodinated and then subjected to analysis of dissociability and iodoamino acid contents with newly incorporated iodine. The results indicated that the behavior of the former thyroglobulin resembled that of 131I-thyroglobulin obtained from the slices. Then, thyroid slices were incubated for 3 min with Na131I and 3H-leucine with or without 10-min chase incubation. The sucrose density gradient centrifugation patterns of 131I and 3H-radioactivity of cytoplasmic extracts indicated that 131I-thyroglobulin is contained in particulates, especially in vesicles with low density(d=1.12) and that some of them are released into the soluble fraction within 10 min. The vesicles contained peroxidase and NADH-cytochrome c reductase, and are probably exocytotic vesicles in the apical area of cytoplasm of follicular cells. No positive evidence was obtained that plasma membranes participate in the iodination of thyroglobulin under the present experimental conditions. These results suggest that, in the incubation of thyroid slices, iodine atoms are preferentially incorporated into newly synthesized, less iodinated thyroglobulin, rather than preformed thyroglobulin, and that the iodination occurs, at least to a certain degree, in apical vesicles before the thyroglobulin is secreted into the colloid lumen.     

Dilatation of Golgi vesicles by monensin leads to enhanced accumulation of sugar nucleotides

Incubation of mouse thymocytes with 10M monensin for 1 hour induces morphological alterations characterized by the extensive dilatation and vacuolization of the Golgi complex. This effect is used to study the transport and utilization of labelled sugar nucleotides into intracellular vesicles by using thymocytes whose plasma membrane has been permeabilized by ammonium chloride treatment. It is demonstrated that monensin stimulates the incorporation of labelled sialyl, fucosyl, galactosyl, and N-acetylglucosaminyl residues. This enhanced incorporation is not due to a direct effect of monensin on glycosyltransferase activities themselves but is a consequence of a higher entry and accumulation of labelled sugar nucleotides in the dilated vesicles.Laboratoire de Chimie Biologique and Laboratoire Associé au CNRS no. 217.     

Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes

We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at 1 microgram/ml and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha 1-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at 10 micrograms/ml which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of 125I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.     

Rate of protein transport in thyroid follicle cells of normal, thyroxine-treated and TSH-injected rats

The transport of labeled protein in thyroid follicles was studied with quantitative electron microscopic autoradiography in normal and T4-treated rats (2d) injected with 3H-leucine 1 to 6 h before perfusion fixation. During this time interval the total amount of labeled protein in either group was unchanged, although T4-treatment caused a reduction by about 30% of the amount of 3H-leucine incorporated into protein. The autoradiographic data were corrected for the effect of scatter of radioactivity. The relative amounts of labeled, exportable protein in the compartments Er-Golgi and exocytotic vesicles were then estimated. The half-lives of labeled, exportable protein in these compartments were calculated with non-linear regression analysis. In normal rats the half-life of labeled, exportable protein in ER-Golgi was 28 min and in the exocytotic vesicles 18 min. Inhibition of TSH-secretion by injection of thyroxine decreased the rate of protein transport through the follicle cell and increased the half-lives to 63 min (ER-Golgi) and 62 min (exocytotic vesicles). TSH given to thyroxine-treated rats 20 min or 1.5 h before fixation reduced the half-lives of labeled, exportable protein in ER-Golgi to 25 to 33 min and in exocytotic vesicles to 9 min. The findings indicate that TSH regulates the rate of intracellular protein transport in rat thyroid follicle cells at the exocytotic step as well as at an earlier step in the pathway of intracellular protein transport. The mechanism and exact location of the latter TSH regulated step is at present unknown.     

Process of iodination of thyroglobulin and its maturation. II. Properties and distribution of thyroglobulin labeled in vitro or in vivo with radioiodine, 3H-tyrosine, or 3H-galactose in rat thyroid glands.

With the aim of obtaining information on the process of iodination of thyroglobulin, the properties and subcellular distribution of thyroglobulin labeled with radioiodine, 3H-tyrosine, or 3H-galactose were studied. The following results were obtained for 17-19S thyroglobulin isolated from rat thyroid lobes labeled in vitro. (a) The effect of sodium dodecyl sulfate (SDS) concentration (0.1-2.0 mM) on the dissociability of the proteins into 12S subunits showed that 3H-labeled, 131I-labeled, and preformed thyroglobulin behaved very differently; their dissociability decreased in that order. In addition, 0.3 mM SDS is most suitable for discriminating among these species. (b) The amount of 0.3 mM SDS-resistant 131I-thyroglobulin increased with the time of incubation of the lobes or with the amount of iodine atoms incorporated by chemical iodination. (c) Digestion of 3H-tyrosine-labeled thyroglobulin showed that 3H-monoiodotyrosine and 3H-diiodotyrosine were present after incubation of the lobes for 180 min. (d) The dissociability of 3H-galactose-labeled 17-19S thyroglobulin was higher than that of 131I-labeled protein, but its elution pattern on DEAE-cellulose chromatography resembled that of the latter. (e) 131I-Thyroglobulin was scarcely found in the incubation medium, although a considerable amount of 19S thyroglobulin was released into the medium during the incubation. As for the lobes, a significant amount of 131I-radioactivity as well as 3H-radioactivity was found in cytoplasmic particulates, especially in fractions containing apical vesicles and rough microsomes. On the other hand, the following results were obtained for 17-19S thyroglobulin isolated from rats injected with 125I. (a) Dissociability of the protein by 0.3 mM SDS and analysis of 125I-iodoamino acids of pronase digest showed that the iodination process was essentially similar to the case of in vitro incorporation, but was faster. (b) The effect of cyclohiximide treatment showed that the relative reduction of 0.3 mM SDS dissociable species was probably due to a shortage of newly synthesized proteins. All the results obtained in the present experiments are compatible with the view that iodine atoms are incorporated selectively into newly synthesized, less iodinated thyroglobulin, and that the iodination occurs intracellularly, at least to a certain degree, after carbohydrate attachment, probably in the apical vesicles. The possibility that iodination also occurs to some extent in the endoplasmic reticulum and in the colloid lumen of thyroglobulin-stimulated thyroids is discussed.     

The effects of monensin on Golgi complex of adrenal cortex and steroidogenesis.

The ultrastructural and biochemical changes produced by monensin on zona fasciculata cells of the rat adrenal cortex are described. In this study we used adrenal cells in culture, adrenal slices and the intact animal. Monensin (1 microM) was added to the culture medium containing the cells, and to the incubation medium containing the adrenal slices, and was injected intravenously to the intact animal (0.65 mg/kg body weight). The ultrastructural alterations were similar in the three experimental conditions, and consisted of Golgi complex disorganization with dilated cisternae or large smooth vesicles. Quantitative analysis showed a significant increase of the relative volume of the Golgi area. The biochemical study demonstrated a significant decrease of corticosterone concentrations in culture medium after monensin addition, and in adrenal glands from treated rats. These results showed that monensin alters the fine structure of adrenal cortex Golgi complex and inhibits corticosteroidogenesis, which supports the probable role of the Golgi complex in the regulation of steroidogenesis.     

Inhibition of very-low-density lipoprotein secretion by chloroquine, verapamil and monensin takes place in the Golgi complex

The effects of chloroquine, verapamil and monensin on secretion of very-low-density lipoproteins (VLDLs) were studied in cultured rat hepatocytes. Maximum inhibition of VLDL-triacylglycerol secretion by 50–90% of control was reached at 200 μM chloroquine, 200 μM verapamil and 5 μM monensin, whereas no effect on cellular triacylglycerol synthesis was observed. The inhibition could be seen within 15 min and was reversible after washout of the drugs. Chloroquine and verapamil inhibited both cellular protein synthesis and protein secretion, whereas monesin reduced protein secretion without any effect on protein synthesis. Control experiments with cycloheximide revealed that intact protein synthesis was not necessary for secretion of VLDL-triacylglycerol during 2 h. Electron micrographs of cells treated with chloroquine, verapamil or monensin showed swollen Golgi cisternae containing VLDL-like particles. By morphometry, a more than 2-fold increase in volume fractions and size indices of Golgi complexes and secondary lysosomes was observed, except that monensin had no significant effect on these parameters of secondary lysosomes. These results suggest that the inhibition of VLDL secretion by chloroquine, verapamil and monensin which takes place in the Golgi complex might be due to disruption of trans-membrane proton gradients. An increase in pH of acidic Golgi vesicles may cause swelling and disturb sorting and membrane flow through this organelle.     

Role of microtubules in the intracellular transport of growth hormone

Summary Pulse-chase experiments utilising(³H)leucine have been used to study the effects of colchicine and vinblastine on intracellular transport and secretion of newly synthesised growth hormone from rat anterior pituitary fragments. Growth hormone was isolated from medium and fragments by polyacrylamide gel electrophoresis. When colchicine or vinblastine, which disrupt microtubules, were added immediately after pulse labelling, inhibition of the subsequent secretion of newly synthesised growth hormone was detected throughout the succeeding 5 h. Similar inhibition was seen if the drugs were added after a 1 h delay. However, if colchicine or vinblastine were added only after a 2 h chase incubation, then no significant effect on subsequent release of labelled growth hormone was seen. The results suggest that these agents may inhibit the transport of newly formed growth hormone storage granules from the Golgi complex to the cytoplasmic pool. Microtubules do not appear to be involved in the mechanism of the final secretion of newly synthesised hormone by exocytosis.These studies were supported by grants from the Medical Research Council and British Diabetic Association     

Cyclic AMP release from perfused dog thyroid lobes as a sensitive indicator of TSH activation of the secretion process. Evidence for two types of thyroid secretion latency

It has been demonstrated in various types of thyroid tissue preparations that cyclic AMP (cAMP) released into the medium reflects the amount of cAMP in the cells. In the present study employing perfused dog thyroid lobes the dynamics of cAMP release were compared to those of thyroxine (T4) and triiodothyronine (T3) release. The experiments gave evidence that even the lowest concentrations of TSH which stimulate hormone release (in this study 1 microU/ml) also activate the cAMP system; the very high levels of cAMP obtained by stimulation with high concentrations of TSH (in this study 10,000 microU/ml) are not accompanied by corresponding high increases in hormone release. On the contrary the T4 and T3 release is lower than during stimulation with more moderate concentrations of TSH (100 microU/ml). Hence studies employing high concentrations of TSH and measurements of cAMP as indicator of activity of secretory processes should be interpreted very cautiously; the prolonged lag in thyroid hormone secretion observed after stimulation with low concentrations of TSH is accompanied by a corresponding lag in activation of the cAMP system. This pattern suggest that the duration of late secretory processes such as thyroglobulin pinocytosis and hydrolysis is independent of the degree of stimulation and not involved in the variations in secretion latency.     

Is there a double pathway for the secretion of thyroglobulin: a "short circuit" weakly glycosyl-iodinated and a "long circuit" strongly glycosyl-iodinated?

Intact rat thyroid lobes incubated in vitro release recently synthesized thyroglobulin (Tg) into the media at a faster rate than they release thyroglobulin stored in follicular structures. Differential release of this Tg fraction cannot be explained by morphological alterations in thyroid architecture during incubation. This rapidly excreted fraction exhibits a low density on rubidium chloride gradients characteristic of poorly sialylated and poorly iodinated thyroglobulin, comigrating on rubidium chloride gradients with thyroglobulin isolated from tunicamycin treated glands. This poorly sialylated and poorly iodinated thyroglobulin is itself unaffected in its density or release into the media by tunicamycin treatment. Tg isolated from the media of tunicamycin treated glands has nearly the same low iodine and low sialic acid content as rat serum thyroglobulin and does not incorporate radiolabelled glucosamine. This fraction thus appear to duplicate properties of low glycosylated-low iodinated thyroglobulin released from thyroid cells in organisms that have no follicular structures and no follicular storage process as well as from thyroid tissue in patients with thyroid disease states, particularly thyroid tumors. Thus it is proposed a "short loop" pathway of low-glycosylated low-iodinated thyroglobulin directly into circulation, that bypasses and is not stored in the follicular lumen, the "long loop".