Morphological changes in the adrenergic innervation of pancreatic islets after chemical sympathectomy by use of 6-hydroxydopamine and the influence of the sympatho-adrenal system on insulin secretion were investigated in the mouse and rat.

The intracellular regulation of thyrotropin-stimulated iodide efflux was studied in polarized porcine thyrocytes grown as a continuous, tight monolayer in bicameral culture chambers. From a previous study using this system we know that thyrotropin rapidly increases iodide efflux in the apical but not basal direction of the polarized epithelium. [125I]-iodide efflux in apical direction was stimulated by thyrotropin in a concentration-dependent manner (1–10 U/I), whereas efflux in basal direction was unchanged at any thyrotropin dose. Thyrotropin-induced elevation of intracellular cAMP showed a corresponding concentration dependence. The selective stimulation of apical efflux by thyrotropin was evident also when re-uptake of iodide released in basal direction was blocked by perchlorate. The effect of thyrotropin on apical efflux was mimicked by 8-bromo-cAMP and forskolin, whereas agents known to activate the Ca2+/phosphatidylinositol cascade (epidermal growth factor) and protein kinase C (phorbol ester) or increase cytosolic [Ca2+] (A23187) were inactive. We conclude that the selective stimulation by thyrotropin of apical iodide efflux, corresponding to efflux in luminal direction in intact follicles, occurs via cAMP-regulated iodide channels present in the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.

Rats were treated with daily doses of T4 (20 μg, sc) for 2, 7, 14, and 21 days in order to suppress secretion of TSH and thereby decrease thyroid protein synthesis. Twenty minutes before perfusion fixation of the thyroids, the animals were given either TSH (0.5 IU, iv) or saline. The number of endocytotic structures (pseudopods and colloid droplets) were counted in the light microscope. The membrane surface areas of exocytotic vesicles, apical plasma membrane, and endocytotic structures were estimated by electron microscopic stereology. Light microscopy showed that the number of endocytotic structures induced by TSH decreased with increasing time of T4 treatment. Electron microscopy showed that in all non-TSHtreated groups the follicle cells contained exocytotic vesicles but their number and size and, hence, their membrane surface area decreased progressively during T4 treatment. Administration of TSH induced exocytosis, resulting in a reduction of the surface area of the exocytotic vesicles; in relative terms, this reduction was similar in all groups and exceeded 85%. TSH also induced endocytosis in all groups, but the membrane surface area of the endocytotic structures decreased with increasing time of T4 treatment. The sum of the surface areas of the endocytotic structures and the remaining exocytotic vesicles in the TSHtreated groups was almost identical to the surface area of the exocytotic vesicles in the corresponding non-TSH-injected groups. In view of our previous finding that exocytosis results in addition of exocytotic vesicle membrane to the apical plasma membrane, the present observations suggest that the size of the pool of exocytotic vesicles in the follicle cells determines the size of the endocytotic response to a large dose of TSH.

Rats, pretreated with thyroxine for 2 days, were given one or two iv injections of 500 mU of TSH; in some groups the second TSH dose was replaced by 0.75 micronmol isoproternol. The effects of the thyroid stimulators on the following parameters were studied: the number of exocytotic vesicles in the follicle cells; the incorporation of 125I into thyroid proteins, measured over periods of 5 min; and the thyroidal cAMP contents. At 2 h after TSH administration, a second dose of TSH failed to stimulate iodination while at 8 h the iodination response was "normal". Two hours after TSH the follicle cells contained practically no exocytotic vesicles but at 8 h they had a full supply of vesicles, and this was emptied by the second TSH injection. THE CAMP content was less increased by the second TSH injection than by the first one, but the stimulatory effect of the second TSH dose on cAMP was the same at 2 h and at 8 h; this indicates that the lack of iodination response at 2 h was not simply due to blocking of TSH receptors. Isoproternol, which acts on other receptors than does TSH, cause a similar cAMP increase incontrols and at 2 h and 8 h after TSH, but stimulated iodination only in controls and at 8 h after TSH; this supported the conclusion that the lack of iodination response to a second TSH dose at 2 h was not due to impairment of the adenylate cyclase-cAMP system. These observations taken together strongly indicate that a rapid iodination response to TSH depends on stimulated exocytosis which, in turn, requires a pool of exocytotic vesicles in the follicle cells. Such a coupling between exocytosis and iodination seems appropriate since by exocytosis uniodinated thyroglobulin and membrane, showing peroxidase activity histochemically, are delivered to the site of iodination, the apical cell surface.

Accumulation and subcellular localization of dopamine (DA) in pancreatic B-cells and its effects on insulin secretion were investigated in mice following a single injection of L-3,4-dihydroxyphenyl-alanine (L-DOPA). Electron microscopic autoradiography showed that3H-DA formed from administered3H-DOPA was present over B-cells as well as over other types of islet cells. Pretreatment of the animals with a decarboxylase inhibitor greatly reduced the number of autoradiographic grains. In the B-cells the3H-DA-grains were associated with the secretory granules. The location of the label may suggest an incorporation in the periphery of the β-granule, rather than in the dense core, supposed to contain insulin. Accumulation of DA in the B-cells following L-DOPA administration was found to inhibit partially the insulin secretory response to different insulin secretagogues (glucose, glibenclamide and L-isopropylnoradrenaline (L-IPNA)). Treatment with monoamine oxidase inhibitor + L-DOPA induced an almost total suppression of L-IPNA-stimulated insulin secretion, whereas glucose-induced insulin release was still only partially inhibited. Pretreatment with a decarboxylase inhibitor abolished the effects of L-DOPA. It is suggested that intracellularly accumulated DA in the B-cell exerts an inhibitory action on insulin releasing mechanisms induced by different secretagogues and that this action might involve interference with a calcium translocation process at the level of the secretory granule.

The purpose of this study was to explore the nature of the protein(s) in the exocytotic vesicles in the thyroid follicle cells and to ascertain whether or not thyroglobulin and peroxidase are transported by the same vesicles through the apical region of the cells to the follicle lumen. The study was performed on rats pretreated with thyroxine for 2 days in order to inhibit endocytosis. A fraction of exocytotic vesicles was isolated by centrifugation in continuous and discontinuous sucrose density gradients. The protein content of the vesicles were analysed by electrophoresis in continuous polyacrylamide gradient gels. The vesicles contained (uniodinated) thyroglobulin, 12-S protein and thyralbumin. Parallel histochemical studies in the electron microscope. These observations have important bearings on the mechanisms for thyroglobulin iodination, since it has been demonstrated that iodination does not occur in the exocytotic vesicles but in connection with the opening of the vesicles at the apical cell surface.

The effect of vinblastine in vivo on ultrastructure and insulin releasing capacity of the B-cell was studied in mice. Treatment with vinblastine (1.1 mumole/mouse) resulted in a 75% decrease of the amount of normal microtubules and the appearance of characteristic paracrystals. Basal plasma immunoreactive insulin levels were depressed to about 60% of the control level. The dose-response pattern for insulin release (first phase) following two chemically unrelated insulin secretagogues, the potent sulphonyl-urea derivative, glibenclamide, and the beta-adrenegic agonist L-isopropylnoradrenaline, (L-IPNA), was tested with and without vinblastine pretreatment. The dose-response curves for L-IPNA-induced insulin release in vinblastine-treated and control animals did not deviate significantly from each other, whereas insulin release following glibenclamide was almost totally suppressed by vinblastine except at the lowest dose level. Injection of maximal doses of glibenclamide or L-IPNA did not alter the ultrastructural changes induced by vinblastine in the B-cells. It is suggested that the microtubular system of the B-cell might play a minor role for certain insulin-releasing processes and/or that vinblastine might have other important effects on the insulin secretory machinery.

Studies were performed in mice to investigate the sympathetic innervation of the thyroid and to evaluate its role in the regulation of thyroid hormone secretion. Fluorescence histochemistry and electron microscopic autoradiography were used to study the distribution of adrenergic fibers and terminals in the thyroid and their relation to follicle cells and vessels. Bilateral surgical sympathectomy was performed by extirpation of the superior cervical ganglia, and pharmacological sympathectomy by iv injections of 6-hydroxydopamine. The secretion of thyroid hormone was estimated by daily measurements of the blood radioiodine (BRI) levels. Sympathetic, adrenergic nerve fibers were numerous in the mouse thyroid, and were mainly found in 2 locations: in relation to arterioles, and in interfollicular spaces. The terminals of the latter group had a very close relation to follicles and often were in direct contact with them, only the follicular basement membrane separating the adrenergic terminal and the follicle cell. In addition, interfollicular terminals often had a close relation to capillaries. It is concluded that in mice there is a morphological basis for a direct, nonvascular influence of the sympathetic nervous system on thyroid follicle cells. In addition, sympathetic stimuli may influence thyroid blood flow by effects on both arterioles and capillaries. Both pharmacological and surgical sympathectomy elicited a reduction in BRI levels that persisted for 1 or 2 days, presumably as a reflection of decreased thyroid hormone secretion. In view of this finding, and since a previous study has shown that unilateral sympathetic stimulation causes a large increase in BRI levels, preceded by formation of colloid droplets in the portions of the thyroid supplied by the stimulated nerve, it is suggested that the sympathetic innervation of the thyroid serves as a means for effecting prompt, short-term alterations in the rate of thyroid hormone secretion. (Endocrinology94: 959, 1974)

In normal rats, the apical region of the thyroid follicle cells harbors a heterogeneous population of vesicular elements. Functionally, these are of at least two categories: endocytotic and exocy-totic. Elimination of TSH secretion, either by hypophysectomy or by suppression with daily doses (of L-thyroxine (T4), was followed by marked changes in the population of vesicles: the heterogeneity disappeared, and only one type of vesicles remained. These remaining vesicles had a diameter of 1,000–3,000 A and they were characterized by a very dense content and a bounding membrane of 60 A. During continuous suppression of TSH secretion, the number of such vesicles decreased, but at a slow rate and they were still present at the sixth day. In contrast, colloid droplets vanished within one day. In T4-suppressed animals, the rate of incorporation of leucine-l4C into thyroid proteins, including thyroglobulin, was slowly affected, being reduced by only 15% after 1 day and by 50% after 6 days. In contrast, plasma prot...

The tracheo-bronchial mucosa of the mouse has been found to contain an extensive system of argyrophilic epithelial cells. In the trachea the cells morphologically resemble enterochromaffin cells. Normally, these enterochromaffin-like cells contain no fluorogenic amine, as revealed by the Falck-Hillarp formaldehyde technique. On the other hand the cells have the capacity to take up and decarboxylate 3,4-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan (5-HTP); the amine formed is stored in the cytoplasm in a reserpine-sensitive store. This capacity to produce and store amines under experimental conditions may reflect the presence in the tracheal enterochromaffin-like cells of an amine which can not be demonstrated with available fluorescence histochemical techniques. In the electron microscope the tracheal enterochromaffin-like cells were identified by a positive argyrophil reaction and by their capacity to accumulate radioactivity after administration of 3H-DOPA or 3H-5-HTP as revealed by autoradiography. The radioactive labelling was associated with cytoplasmic electron-dense granules (800–1000 Å), suggesting that the amine formed was stored in these granules. Accordingly, the granules stained argentaffin after DOPA-pre-treatment of the animal. It is suggested that, like similar cells in the gastric mucosa, these argyrophilic enterochromaffin-like cells constitute an endocrine system in which amines are of cytophysiological importance.

The relations of plasma TSH, thyroidal amine-containing mast cells, and thyroid hormone secretion were studied in the rat and the mouse by a combination of bioassay, chemical, histochemical, and electron-microscopical procedures.