Fractures of the shaft of the humerus. An epidemiological study of 401 fractures.

We studied the epidemiology of 401 fractures of the shaft of the humerus in 397 patients aged 16 years or older. The incidence was 14.5 per 100,000 per year with a gradually increasing age-specific incidence from the fifth decade, reaching almost 60 per 100, 000 per year in the ninth decade. Most were closed fractures in elderly patients which had been sustained as the result of a simple fall. The age distribution in women was characterised by a peak in the eighth decade while that in men was more even. Simple fractures were by far the most common and most were located in the middle or proximal shaft. The incidence of palsy of the radial nerve was 8% and fractures in the middle and distal shaft were most likely to be responsible. Only 2% of the fractures were open and 8% were pathological. These figures are representative of a population with a low incidence of high-energy and penetrating trauma, which probably reflects the situation in most European countries.

Glutathione peroxidase degrades intracellular hydrogen peroxide and thereby inhibits intracellular protein iodination in thyroid epithelium.

Protein iodination in the thyroid is largely confined to the surface of the epithelium. Intracellular iodine binding is insignificant. We have tested our hypothesis that the key mechanism in the control of intracellular iodination is the control of the intracellular availability of H2O2. The sites of iodination were identified by locating bound radioiodine in electron microscopic autoradiographs, produced from porcine thyroid epithelium grown on filter in Transwell bicameral culture chambers. Autoradiographs obtained after standard incubations with 125I for 15 min to 3 h were all characterized by concentrations of autoradiographic grains along the external surface of the plasma membrane and very few grains over the cytoplasm. The presence of 10 microM H2O2 in the incubation medium resulted in a drastically changed labeling pattern now showing a dissemination of grains over the entire cytoplasm. Epithelia with elevated GSH peroxidase activity produced autoradiographs showing the same restriction of grains to the cell surface as controls; this pattern was the same in the absence and presence of H2O2 (up to 10 microM). Cultures with subnormal GSH peroxidase activity presented cytoplasmic labeling both in the absence and presence of H2O2. In conclusion, iodine binding in filter-cultured thyroid epithelium under normal conditions is an extracellular process located at the cell surface. When H2O2 is available intracellularly, iodination takes place in the cytoplasm, evidently catalyzed by intracellular thyroperoxidase. Normally, this iodination is prevented by cytosolic GSH peroxidase that effectively degrades H2O2 and thus controls intracellular iodination. The observations should be applicable to the thyroid in vivo.

Hydrogen peroxide degradation and glutathione peroxidase activity in cultures of thyroid cells.

The degradation rate of H2O2, added to the incubation medium, and glutathione (GSH) peroxidase activity were measured in cultures of FRTL-5 cells and porcine thyroid cells. The H2O2 degradation rate increased proportionally to the H2O2 concentration and was in FRTL-5 cells, cultured with TSH, approximately 50 nmol/min and mg DNA at 0.01 mM H2O2 and approximately 3 x 10(4) nmol/min and mg DNA at 10 mM H2O2. The GSH peroxidase activity in the same cells was equivalent to an H2O2 degradation of approximately 400 nmol/min and mg DNA. The involvement of enzymes in H2O2 degradation was studied by inhibiting catalase with aminotriazole (ATZ) and reducing GSH peroxidase by omitting glucose in the incubation medium. At 0.1 mM H2O2, ATZ or glucose omission alone did not measurably reduce H2O2 degradation but did so when combined. At 10 mM H2O2 ATZ caused a clear inhibition whereas glucose omission had no additive effect. These observations indicate that GSH peroxidase was involved in H2O2 degradation only at low H2O2 concentrations. The GSH peroxidase activity decreased by reduction of the selenite supply and increased after replenishment. The recovery of the enzyme activity required the presence of TSH in FRTL-5 cells but not in porcine thyrocytes.

Effect of P1-purinergic agonist on thyrotropin stimulation of H2O2 generation in FRTL-5 and porcine thyroid cells.

Our previous studies have shown that the generation of H2O2 in FRTL-5 thyroid cells is regulated via both the adenylate cyclase/cyclic adenosine monophosphate (cAMP) and Ca2+/phosphatidylinositol pathway: thyrotropin (TSH) stimulates H2O2 generation through both pathways, via the former at a low concentration and via the latter at a high concentration. In porcine thyrocytes in primary culture H2O2 generation is stimulated only via the Ca2+/phosphatidylinositol route. In the present study we explored the effect of a P1-purinergic agonist (phenylisopropyladenosine, PIA) on stimulations induced by TSH and by adenosine triphosphate (ATP), an activator of the Ca2+/phosphatidylinositol cascade via the P2-purinergic receptor. In FRTL-5 cells, PIA potentiated H2O2 generation stimulated by TSH at 10 U/l (but not at 1 U/l, Ca2+ mobilization induced by TSH and Ca2+ mobilization induced by ATP at 1 mumol/l (but not 10 mumol/l). Phenylisopropyladenosine strongly inhibited TSH-induced cAMP accumulation in FRTL-5 cells. In pig thyrocytes, PIA had no effect on H2O2 generation stimulated by TSH or ATP and no effect on ATP-stimulated Ca2+ mobilization. Also, PIA did not inhibit TSH-stimulated cAMP accumulation in pig thyrocytes, and by itself had no effect on H2O2 generation or Ca2+ mobilization. Thus, in FRTL-5 cells, but not in porcine thyrocytes, PIA modulates TSH-stimulated H2O2 generation by enhancing the Ca2+/phosphatidylinositol route and inhibiting the adenylate cyclase/cAMP route of the TSH signal. The net result of this modulation apparently depends on the balance between inhibition of the cAMP route and enhancement of the Ca2+ route. This may explain the lack of potentiation observed by 1 U/l TSH.

Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid cells.

Hydrogen peroxide acts as electron acceptor in the oxidative reactions (iodination and coupling) by which the thyroid hormones are formed. Regulation of the generation of H2O2 was studied in monolayer cultures of the FRTL-5 rat thyroid cell line and in primary monolayer cultures of porcine thyroid cells. Both cell types were grown in a medium containing either a six-hormone mixture, including TSH (6H), or a five-hormone mixture without TSH (5H) for 1 to several days before the experiment. The production of H2O2 was measured with the homovanillic acid fluorescence assay and expressed as picomoles of H2O2 formed per min/microgram DNA. In FRTL-5 cells grown in 6H medium, only a weak and varying stimulation of H2O2 production was induced by TSH at high concentration (greater than 10 mU/ml), and no stimulation was seen by TSH at low concentration or by 8-bromo-cAMP, whereas forskolin had a good stimulatory effect. In FRTL-5 cells grown in 5H medium for 1-3 days, all three substances were potent stimulators. In porcine thyrocytes examined in the same way, none of the three presumptive stimulators had any effect in 6H cultures, and only TSH (at high concentration) had a weak effect in 5H medium. ATP, a stimulator of the Ca2+/phosphatidylinositol cascade via a P2-purinergic receptor, had no effect on H2O2 generation in FRTL-5 cells in 6H medium. Phorbol myristate acetate (PMA), a direct activator of protein kinase-C, induced a weak stimulation in these cells. In FRTL-5 cells in 5H medium, both ATP and PMA evoked a strong, and similar, enhancement of H2O2 production. In porcine cells in 6H medium, ATP evoked a moderate and PMA a strong stimulation; the effects in 5H were similar to the corresponding effects in 6H medium. The observations are interpreted to show that in FRTL-5 cells the regulation of H2O2 generation uses both the cAMP cascade and the Ca2+/phosphatidylinositol cascade, whereas in porcine thyrocytes the cAMP route is unimportant. In FRTL-5 cells the Ca2+/phosphatidylinositol cascade may be influenced by the cAMP system.

Polarized efflux of iodide in porcine thyrocytes occurs via a cAMP-regulated iodide channel in the apical plasma membrane.

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/l), 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.

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across 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.

Accelerated exocytosis and H2O2 generation in isolated thyroid follicles enhance protein iodination.

Open pig thyroid follicles in which the apical surface of the follicle cells is in direct contact with the incubation medium were used to study the effect of stimulated exocytosis and stimulated H2O2 generation on the iodination of protein in the incubation medium. In previous studies on this system of follicles we have shown (1) that the apical surface of the follicle cells is a major site of protein iodination and (2), that H2O2 is produced at the apical cell surface. In the present study we confirmed the previous finding that H2O2 generation is greatly stimulated by the Ca2+ ionophore A23187. We further found that TSH at a high concentration (greater than 10 mU/ml) and in the presence of Ca2+ stimulated H2O2 generation; TSH had no such effect on follicles incubated in Ca2+-free medium after pretreatment with EGTA. Forskolin did not stimulate H2O2 generation. Exocytosis of thyroglobulin was stimulated by TSH at a low concentration (0.1 mU/ml), and this stimulation was not dependent on Ca2+. Exocytosis was also stimulated by forskolin but not by A23187. Iodination of protein, including thyroglobulin, in the incubation medium was stimulated by A23187, TSH and forskolin. These observations suggest that stimulation of iodination in association with the apical surface of the follicle cells can be achieved separately by an increased rate of H2O2 generation and increased rate of exocytosis. Generation of H2O2 is Ca2+-dependent, whereas exocytosis is mediated by the adenylate cyclase-cAMP system; TSH at a high concentration can stimulate both these processes.

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles.

The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4 degrees C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37 degrees C for 5-180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabeled. Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.

Localization of the incorporation of 3H-galactose and 3H-sialic acid into thyroglobulin in relation to the block of intracellular transport induced by monensin. Studies with isolated porcine thyroid follicles.

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 degrees 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 degrees C for 1 h in the absence or presence of 1 microM 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. 1987b) 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of monensin on thyroglobulin secretion. Studies with isolated follicles from pig thyroids.

The effect of monensin on the secretion of thyroglobulin was studied in open follicles isolated from pig thyroid tissue; in this system, thyroglobulin is secreted into the incubation medium. When monensin was present during a 4-h chase incubation after pulse-labelling with 3H-leucine, the secretion of labelled thyroglobulin was reduced by about 85%; in electron-microscopic autoradiographs of rat thyroid lobes labelled and chase-incubated under similar conditions the relative number of grains over follicle lumina was strongly reduced when monensin was present during the chase. These observations are in agreement with the consensus that monensin arrests transport of secretory proteins in the Golgi complex. In other experiments, pulse-labelled follicles were chase-incubated for 1.5 h whereby labelled thyroglobulin was transported from the RER to exocytic vesicles. Monensin present during a subsequent chase of 0.5 h caused only a moderate decrease of labelled thyroglobulin secretion. TSH present during the second chase-stimulated secretion in both control and monensin-exposed follicles. TSH also caused a drastic reduction of exocytic vesicles in rat thyroid lobes, and the number of vesicles remaining in the cells was the same in controls and lobes exposed to the ionophore. The observations are interpreted to show that monensin does not inhibit the basal or TSH-stimulated transport of thyroglobulin from the site of monensin-induced arrest in the Golgi complex to the apical cell surface or the exocytosis of thyroglobulin.

Effect of cooling on intracellular transport and secretion of thyroglobulin.

The effect of cooling to 20 degrees 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 degrees 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 degrees and 20 degrees C; within this temperature range the activation energy for secretion, calculated from Arrhenius plots, was found to be 37 kcal/mol. Below 20 degrees C the secretion was scarcely measurable. It is suggested that the three transport blocks at 20 degrees C result mainly from inhibition of membrane fission and fusion due to phase transition in membrane lipids.

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells.

Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.

Localization of iodine binding in the thyroid gland in vitro.

Protein-bound radioiodine was localized by electron microscopic autoradiography in follicles and cells isolated from pig and rat thyroid tissue after incubation with 125I- for 1-10 min. Labeled proteins were analyzed by gel electrophoresis and immunoprecipitation. In closed follicles, with colloid-filled follicle lumina, the autoradiographic labeling was concentrated over the lumina and no labeling occurred over the cells. In open follicles, without colloid content, autoradiographic grains were invariably found along the apical cell surface and in some cells over intracellular lumina. Isolated cells with preserved polarity showed some labeling associated with remaining microvilli whereas isolated cells with lost polarity showed no grains at the cell surface. In isolated cells, particularly those with lost polarity, most grains were located over intracellular lumina (which were common in such cells) and some grains over vesicular elements associated with these lumina. About 80% of the labeled soluble proteins and 40% of the labeled proteins solubilized by sodium dodecyl sulfate were thyroglobulin. It is concluded that the site of iodination in follicles is the same in vitro as in vivo, viz. the apical surface of the follicle cells. When thyroid cells are removed from the follicle and lose their polarity, intracellular lumina become the major site of iodination. This shift in iodination sites is thought to be due to retention of Golgi-derived secretory vesicles in nonpolarized cells, leading to coalescence of vesicles with the formation of intracellular lumina and activation of membrane-bound enzymes catalyzing iodination.

Generation of H2O2 in isolated porcine thyroid follicles.

Generation of H2O2, an essential component in thyroid hormone synthesis, was studied by biochemical and cytochemical methods. Both parts of the study were performed on isolated open pig thyroid follicles in which the cells have preserved polarity and both the apical and basal cell surfaces are exposed to the incubation medium. The biochemical studies, performed with the scopoletin fluorescence assay, showed that H2O2 was released from the follicles into the medium at a rate of 0.5-1.0 nmol min-1 mg-1 DNA under basal conditions. The H2O2 release rate was promptly increased about 10 times by addition of the ionophore A23187 to Ca2+-containing medium. TSH caused an acute but weaker stimulation of H2O2 release, whereas (Bu)2cAMP was without effect, indicating that the TSH action was linked to Ca2+. Both basal and stimulated H2O2 release were strongly inhibited by p-chloromercuribenzene sulfonate. The cytochemical study, performed with the cerium technique, confirmed our previous observations on rat thyroid follicles. Reaction product was found on the apical cell surface but never on the basal cell surface or intracellularly. The apical reaction was enhanced by NADH and NADPH as well as by A23187 in Ca2+-containing medium. The apical reaction was strongly inhibited by p-chloromercuribenzene sulfonate. The observations indicate that the H2O2 released from the open follicles is generated on the apical plasma membrane of the follicle cells, possibly by NAD(P)H oxidase in this membrane. Furthermore, Ca2+ seems to be an important factor in the regulation of this H2O2 generation and, through that, in the regulation of iodination.

Effect of tunicamycin on thyroglobulin secretion.

Secretion of thyroglobulin was studied by incubating pig thyroid follicles, isolated by collagenase digestion and opened up by trypsinization. When followed over periods of 4 h, the secretion of [14C]leucine-labeled thyroglobulin into the medium was reduced by 60-95% in the presence of 1 microgram/ml and 5 micrograms/ml of tunicamycin. These concentrations of the antibiotic reduced incorporation of [3H]mannose into the follicle proteins by 70-80% but did not significantly influence the incorporation of [14C]leucine. Rat thyroid lobes were labeled with [3H]leucine for 20 min and chase-incubated for 0-4 h. In electron microscopic autoradiographs obtained immediately after labeling, the label was restricted to the follicle cells and concentrated over the endoplasmic reticulum both in controls and in specimens exposed to tunicamycin (5 micrograms/ml). After 4 h chase most radioactivity was located in the follicle lumen in controls whereas in tunicamycin-exposed lobes almost all labeled material was retained in the follicle cells. It is concluded that tunicamycin suppresses thyroglobulin secretion and that this is not due to inhibited protein synthesis.

Iodination of thyroglobulin. An intracellular or extracellular process?

The location in the thyroid follicle of the iodination of thyroglobulin has been a matter of debate for several decades. This problem is not a question of mere academic interest. Knowledge of the locus--or loci--of iodination is necessary for a full understanding of the mechanisms involved in thyroid-hormone synthesis and release. In the discussion about this problem 3 fundamentally different views have been--and still are--advocated. The first view implies that the site of iodination is the follicle lumen, the second that iodination is an intracellular process restricted to some organelle(s) in the follicle cells and the third that iodination occurs at the interface between the follicle cells and the follicle lumen. Below I will survey the major observations on which these different opinions are based and discuss the validity of the interpretations.

Site of iodination in hyperplastic thyroid glands deduced from autoradiographs.

We have tried to ascertain the site of iodination in the chronically stimulated, hyperplastic thyroid gland of rats. Rats were fed propylthiouracil in a commercial rat diet for 10 days. Then the diet was changed to a low iodine diet for 5 days. To label the gland, 10 mCi of 125I-iodide was injected into the left heart ventricle. Ten seconds later the animal was perfused through the left ventricle with a fixative solution containing a goitrogen to block further iodination, and stable iodide to help extract uncombined radioiodide. Electron microscopic autoradiographs prepared from the fixed thyroids show strong labeling over the lumen of the follicle and no consistent labeling of any other site or organelle. We conclude that the site of iodination in the chronically stimulated, hyperplastic thyroid is the follicular lumen, i.e. the same as that in the normal gland.