Yoshifusa Shimizu

Epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium in cultured porcine thyroid cells

The initial signal for thyroid cell proliferation is unknown. This is the first report to show that epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium ([Ca2+]i) in the thyroid gland. In cultured porcine thyroid cells, 10 nM EGF produces a breakdown of phosphatidylinositol and stimulates inositol phosphate production. Ten nM EGF increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the EGF-induced [Ca2+]i response occurs immediately, reaches a maximum within several seconds, and then slowly declines. EGF stimulates production of inositol phosphates, which seem to increase [Ca2+]i. Inositol phosphate production and an increase in [Ca2+]i after EGF-stimulation may function as an initial signal for thyroid cell proliferation.

Analyses of treadmill locomotion in adult spinal dogs

The locomotion of the hindlimbs of two adult female spinal dogs, who were able to walk steadily on their hindlimbs 10 months after transection of the spinal cord (T10), and of two normal dogs was analyzed on a treadmill by means of high-speed cinematography and electromyography. With increase in walking speed, the duration of the step cycle was shortened by reduction of the duration of the stance phase, and the stride length was extended mainly by elongation of the transfer distance during the swing phase in both normal and spinal dogs. The patterns of muscle discharges of the hindlimbs in spinal dogs were similar to those in normal dogs. With increase in walking speed, reductions in the burst duration of the extensors were observed in both normal and spinal dogs. These results indicate that spinal dogs can adjust their locomotion speed in the same manner as normal dogs; this supports the theory that a central pattern generator regulating the locomotive activities of the hindlimbs exists in the spinal cord below the transection site.

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1α production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6‐keto PGF1α an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura‐2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6‐keto PGF1α production; the maximum effects are obtained after 2–4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.

Epidermal growth factor (GGF) and tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.

Trophic effects of sympathetic ganglia on normal and dystrophic chicken skeletal muscles in tissue culture

Abstract Neurotrophic effects of sympathetic ganglia on normal and dystrophic skeletal muscles were studied in tissue culture. In isolated cultures of muscle, no prominent differences were observed in spontaneous contractions and the formation of striations between muscles from normal (NM) and muscles from dystrophic chickens (DM). But DM degenerated faster than NM and almost disappeared after 3 weeks. On the contrary, NM were maintained 4 weeks and degenerated gradually afterward. In isolated cultures of sympathetic ganglia, no differences were observed between sympathetic ganglia from normal (NS) and sympathetic ganglia from dystrophic chickens (DS). Many catecholamine-positive nerve fibers radiated from the explants of NS and DS, and they were maintained more than 6 weeks. In cocultures of muscle and sympathetic ganglion, either DM or NM muscle fibers survived for a longer time than those in isolated cultures of muscle. In combinations of DM and NS or DM and DS, however, they began to degenerate after 3 weeks. In cocultures of muscle and sympathetic ganglion, a bridge was formed between both explants. In long-term culture, only some nerve fibers displayed catecholamine fluorescence in these bridges, although many nerve fibers were stained by the silver impregnation method. Some of the nerve fibers terminated on the muscle fibers, accompanied by acetylcholinesterase activity. Prominent differences were not observed histochemically in the cholinergic terminals in cocultures of the various combinations.

Immunoreactive luteinizing hormone (ir-LH) cells in the lung and stomach of chick embryos

Abstract.Luteinizing hormone (LH) immunoreactivity was detected in the lung and stomach of chick embryos by the immunoperoxidase staining technique using specific antiserum to chicken LH. Immunoreactive LH (ir-LH) cells first appeared in the primordial cells of the epithelial layer of lung bud and foregut as well as of Rathke’s pouch in the 3-day-old embryo, Hamburger and Hamilton stage 21. Ir-LH cells increased in number with advancing age of embryos in the lung, stomach, and pituitary gland. In the lung of 7-day-old embryos, stage 31, the ir-LH cells were distributed in the epithelium of primary, secondary, and tertiary bronchi, and their shapes were pseudostratified columnar, simple columnar, and simple cuboidal, depending on their sites in the intrapulmonary airway. Ir-LH cells were more numerous in the median part than in the lateral part of the lung, and the population in the epithelial layer of entobronchi of the secondary bronchi was 4 times higher than that in ectobronchi and laterobronchi of the secondary bronchi and in the primary bronchi. The immunoreactive products were found, either in the entire cell or in the apical part, facing the lumina of bronchi. In the stomach, ir-LH cells were found in the epithelial layer of gastric glands. No ir-LH cells were observed in interstitial regions, which consisted of mesenchymal cells and blood vessels, in the lung and stomach tissues. With advancing age, ir-LH cells changed their shapes to flat or squamous, coincident with the formation of parabronchi. Other pituitary hormones were not observed immunohistochemically in either the lung or stomach before hatching. Preabsorption of the antiserum against avian LH with the purified chicken LH or the extract of pituitaries from 10-day-old embryos completely destroyed the immunoreactivity to the cells in the lung and the pituitary. A single band of the immunoreaction products, whose molecular weight was around 25 K daltons, was shown by the immunostaining of nitrocellulose membrane transblotted after sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified pituitary LH, extracts of pituitaries from 10-day-old embryos, and the extracts of lungs from 7-, 10-, and 14-day-old chick embryos. These results demonstrated that ir-LH cells are present in extrapituitary tissues, and may play an important role during the development of chick embryonic lung and stomach.

Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells

Effects of calcium ionophore A23187 and BAY-K-8644, a calcium channel agonist, on cytoplasmic free calcium ([Ca2+]i) and H2O2 generation were studied in cultured porcine thyroid cells. We monitored continuously the effects of A23187 and BAY-K-8644 on [Ca2+]i and H2O2 generation, using the intracellularly trapped fluorescent Ca2+ indicator, fura-2, and homovanillic acid, respectively. A23187 and BAY-K-8644 induce an immediate increase in [Ca2+]i and H2O2 generation. The A23187- and BAY-K-8644-induced [Ca2+]i responses and H2O2 generation occur immediately, reach a maximum within several seconds, and then slowly decline. The minimum doses of A23187 or BAY-K-8644 to increase [Ca2+]i stimulate H2O2 generation. H2O2 generation is regulated by [Ca2+]i.

Augmentation of prostacyclin and depression of PGE2, PGF2α and thromboxane A2 by TSH in cultured porcine thyroid cells: An important role of prostacyclin in maintaining thyroid cell function

The importance of prostaglandins (PCs) in thyroid physiology has long been a controversial subject. In [l-3] 6-ketoprostaglandin Fr, was isolated and found to be an end-metabolite of the unstable compound, prostacyclin [4,.5]. Prostacyclin producing activity has been reported in many tissues and cells [6-81, and biological significance of prostacyclin in maintaining homeostasis has become apparent. Thromboxane Ba, an end-metabolite of thromboxane Aa, has been isolated [9] and this thromboxane A* seems to play some role to maintain homeostasis. However, prostacyclin and thromboxane Aa producing activities in the thyroid have not been reported and their biological significance needs to be studied. To evaluate the role of endogenous PGs in the regulation of thyroid function, we have estimated prostaglandin Ea (PGE,), prostaglandin Fzor (PGFa,), 6-ketoprostaglandin Fr, and thromboxane Bz (TXBa) and correlations of these PGs and thyroid functions were studied using cultured porcine thyroid cells. Here, we show that in the absence of TSH, the cells are unable to take up iodide or organify it but product; PGE2, PGFzo, and TXBz and that in the presence of TSH, the cells are able to take up iodide and organify it but preferentially produce 6-ketoprostaglandin Fr,, an end-metabolite of prostacyclin.

An important role of prostacyclin in porcine thyroid cells in culture Stimulation and refractoriness of cyclic AMP synthesis and iodine metabolism

The discovery of prostacyclin [l] opened a new area of prostaglandin research. PGIz is a potent stimulator of the adenylate cyclase-cyclic AMP (CAMP) system [2,3] and is considered to be an important regulator of cell metabolism. We have shown that cultured porcine thyroid cells produce PGIz [4]. This raised a possibility that PGIz plays an important role in the thyroid physiology. To test this hypothesis, we first studied the effects of PGIz on CAMP synthesis and iodine metabolism, using cultured porcine thyroid cells. We then attempted to study whether pre-exposure to PGEr or PGEz could make the thyroid cells refractory to further stimulation of PGIa on CAMP synthesis and iodine metabolism. The results show that PGIz stimulates CAMP synthesis and iodine metabolism and that preexposure to PGEr or PGEz induces refractoriness to further PGI*-stimulation.

Interrelationship between insulin-like growth factor I-induced activation of the Na+H+-antiporter and intracellular Ca2+-mobilization in thyroid cells

Insulin-like growth factor I (IGF-I) increased cytoplamic pH (pHi) and cytoplasmic Ca2+ [( Ca2+]i) in cultured porcine thyroid cells. Inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of external Na(+)-concentrations attenuates the increases in pHi and [Ca2+]i. The [Ca2+]i response to IGF-I is a pHi-dependent process. IGF-I activates Na+/H(+)-antiporter and alkalinizes thyroid cells. The resulting increase in pHi facilitates the [Ca2+]i response by adjusting the pHi closer to the pHi-optimum of the intracellular Ca(2+)-mobilizing system. One of the biological functions of IGF-I-induced activation of the Na+/H(+)-antiporter is to shift the pHi to an optimal value for the [Ca2+]i response.