Biomedical subjects

N Emoto

Publications and source records attributed to N Emoto.

At least 37 records · Page 2Linked to original sources

PubMed1997

Oncostatin M: a new potent inhibitor of iodine metabolism inhibits thyroid peroxidase gene expression but not DNA synthesis in porcine thyroid cells in culture.

The functions of thyroid cells are regulated by a number of cytokines and growth factors in addition to TSH. Recent studies have revealed that several cytokines including interleukin (IL)-6 are involved in thyroid dysfunction. Oncostatin M (OSM) is a glycoprotein belonging to the same family of cytokines as IL-6, to which it is related by sequence and structural homology and the use of the signal-transducing receptor component gp130. We, therefore, studied the effect of OSM on iodide uptake and DNA synthesis by porcine thyroid cells in culture. OSM increased c-fos and c-jun mRNA levels but did not stimulate DNA synthesis. OSM inhibited iodide uptake stimulated by TSH; while IL-6 also inhibited iodide uptake, it was only about one-tenth as potent. IL-6 had about the same potency as OSM when it was added with soluble IL-6 receptor. OSM had no effect on cAMP production but inhibited iodide uptake stimulated by 8-bromo-cAMP and forskolin. These findings suggest that OSM exerts its inhibitory effects at the post-cAMP production step(s). OSM also inhibited thyroid peroxidase mRNA levels but had little effect on thyroglobulin mRNA levels. Investigations of the signal transduction system showed that gp130 and leukemia inhibitory factor (LIF) receptor beta subunit mRNA were detectable in porcine thyroid cells by reverse transcription (RT)-polymerase chain reaction (PCR). Together with the report that serum OSM and IL-6 concentrations are elevated to the same levels in patients with sepsis, these results suggest that OSM may contribute to the thyroid dysfunction in this condition.

Animals↗

PubMed1997

Localization of endothelin-converting enzyme-1 in human kidney.

The distribution of endothelin-converting enzyme-1 (ECE-1) mRNA and protein was investigated in human kidney excised because of renal tumors. ECE-1 immunoreactivity was detected by immunohistochemistry throughout the different areas of the kidney in the vascular and tubular structures. In the cortex, ECE-1 immunostaining was present in the endothelial surface of arcuate and interlobular arteries and in arterioles. Weak specific immunoreactivity was present over some proximal and distal tubules. Few endothelial glomerular cells contained ECE-1 protein. In the medulla, ECE-1 immunoreactivity was observed in the vasa recta bundles and capillaries. ECE-1 immunostaining was also detected in the outer and inner medullary collecting ducts and thin limbs of Henle's loops. Immunohistochemical detection of the von Willebrand factor on adjacent sections confirmed the endothelial nature of the vascular cells that exhibited ECE-1 immunostaining. The distribution patterns of ECE-1 mRNA, investigated by in situ hybridization, appeared similar to that obtained by immunohistochemistry in the cortical and medullary vasculature and in different portions of the nephron. Northern blot and densitometric analyses demonstrated that ECE-1 mRNA levels were quantitatively similar in both the renal cortex and medulla. These results demonstrate that vascular endothelial and tubular epithelial cells in the cortex and medulla of the human kidney synthesize ECE-1, which, in turn, may play an important role in regulating endothelin production in physiological and pathological conditions.

Adult↗

PubMed1997

Microinjection of rat GH but not human IGF-I into a defined area of the hypothalamus inhibits endogenous GH secretion in rats.

It has been surmised that GH exerts feedback action on the hypothalamus and thereby regulates its own secretion. Our previous studies suggested that GH acts on somatostatin neurons in the hypothalamic periventricular nucleus (PeV) and neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC). However, there remains uncertainty whether GH acts directly or indirectly through the generation of IGFs on the hypothalamus to regulate its own secretion. To examine this, rat GH (rGH) or human IGF-I was injected directly into a defined area of the hypothalamus, and the blood GH profile was observed in conscious male rats. In the rats given 0.5 microgram rGH into the ARC or PeV bilaterally, GH secretion was inhibited, and the inhibition lasted for 12 h. During the period of inhibition, the duration and amplitude of GH pulses were significantly decreased and the episodic secretion of GH appeared irregularly compared with the vehicle-injected control rats. In control rats given the vehicle or those given rGH into the lateral hypothalamus, the blood GH profile did not change and pulsatile GH secretion was produced every 3 h. When 0.1 microgram IGF-I was injected into the ARC or PeV bilaterally, the blood GH secretory pattern was not affected. Together with the results of our previous studies showing that c-fos gene expression was induced by systemic administration of GH and that GH receptor mRNA was contained in somatostatin neurons in the PeV and NPY neurons in the ARC, the data of the present study indicate that GH, but not IGF-I, acts on the cells in the ARC and the PeV or in their vicinity to inhibit its own secretion, presumably by activating the somatostatin and NPY neurons.

Animals↗

PubMed1997

Cushing's syndrome due to bilateral adrenocortical adenomas with different pathological features.

A 48-year-old woman with Cushing's syndrome due to bilateral adrenocortical adenomas is reported. The patient presented with a typical Cushingoid appearance. The serum cortisol level was elevated with loss of the diurnal rhythm and the plasma adrenocorticotropic hormone (ACTH) level was undetectable. Dynamic testing showed no suppression of urinary 17-OHCS by high-dose dexamethasone and no stimulation by metyrapone. An abdominal computed tomography (CT) scan showed bilateral adrenal tumors. Bilateral adrenalectomy was performed. The right adrenal gland contained a tumor that was encapsulated and consisted mainly of compact cells. The surrounding cortex was atrophic. The left adrenal gland contained an encapsulated tumor composed predominantly of clear cells. There were numerous small adrenocortical nodules in the surrounding cortex. Immunohistochemical analysis of steroidogenic enzymes (P450scc, 3beta-HSD, P450c21, P450c17 and P450c11) was performed. Immunoreactivity of all the enzymes was intense in the compact cells of the right adrenocortical adenoma, while the adjacent non-neoplastic cortex was negative for the enzymes. In the left adrenal tumor, the immunoreactivity of 3beta-HSD was intense, while that of P450c17 was weak. In the adrenocortical nodules, 3beta-HSD activity was sporadically observed. G protein genes encoding Gs alpha and Gi2 were examined for activating mutations at codons 201 and 227 (Gs alpha) and codons 179 and 205 (Gi2 alpha) in the bilateral adrenal tumors, but no mutations were found. The bilateral adenomas of this patient showed marked differences in microscopic and immunohistochemical studies, suggesting that the capacity of steroidogenesis differs between the right and left tumors.

Adrenal Cortex Neoplasms↗

PubMed1996

Increased pituitary growth hormone-releasing factor (GRF) receptor messenger ribonucleic acid expression in food-deprived rats.

In prolonged food-deprived rats, growth hormone (GH) secretion in response to exogenous GH-releasing factor (GRF) is enhanced both in vivo and in vitro. We tested the hypothesis that, in fasted rats, GRF receptors in the pituitary may be up-regulated. The expression of mRNA for the GRF receptor in the pituitary and GRF binding to the pituitary membrane were examined in adult male Wistar rats deprived of food for 72 h. The level of GRF receptor mRNA in the pituitary was significantly increased after 48 h food deprivation and was more than 3 times the level in control rats after 72 h food deprivation. GRF binding to the pituitary was significantly increased after 72 h food deprivation. The results of the present study provide evidence regarding changes in the GH axis in fasted rats, involving increased pituitary responsiveness to GRF and an increase in the pituitary membrane GRF receptor concentration.

Animals↗

PubMed1996

Co-expression of endothelin-1 and endothelin-converting enzyme-1 in patients with chronic rhinitis.

Several studies have suggested an important role for endothelin in the pathophysiology of the upper and lower respiratory tract. In the present study, we have investigated the localization of endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1) messenger RNAs(mRNAs) and immunoreactivities in the human nasal mucosa by immunohistochemistry and in situ hybridization. Inferior turbinates were obtained from 32 patients at surgery. Histopathologic examination of nasal biopsy specimens revealed that 17 patients had chronic inflammation and 15 patients had normal mucosa. Immunostaining for ET-1 and ECE-1 was seen in the surface epithelium, endothelial cells, submucosal glands, and infrequently in vascular smooth muscle cells, in all subjects. There was significantly more staining for ET-1 and ECE-1 in nasal glands and inflammatory cells in patients with chronic inflammation than in those with normal nasal mucosa. In patients with chronic inflammation, strong immunoreactivity for ECE-1 was seen over inflammatory cells. In situ hybridization showed abundant expression of ET-1 and ECE-1 mRNAs over the surface epithelium, glands, vessels, and inflammatory cells. Both immunohistochemistry and in situ hybridization demostrated the expression of ET-1 and ECE-1 immunoreactivities and mRNAs in similar sites, and also independently. The present findings demonstrate the co-expression of ET-1 and its converting enzyme in the human nasal mucosa and suggest a possible role for the endothelin system in the pathogenesis of chronic rhinitis.

Adult↗

PubMed1995

Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum.

Endothelins (ET) are a family of potent vasoactive peptides that are produced from biologically inactive intermediates, termed big endothelins, via a proteolytic processing at Trp21-Val/Ile22. We recently cloned and characterized a membrane-bound metalloprotease that catalyzes this proteolytic activation, endothelin-converting enzyme-1 (ECE-1) (Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M. (1994) Cell 78, 473-485). This enzyme was shown to function in the secretory pathway as well as on the cell surface. Here we report molecular cloning of another novel enzyme, ECE-2, that produces mature ET-1 from big ET-1 both in vitro and in transfected cells. The cDNA sequence predicts that bovine ECE-2 is a metalloprotease structurally related to ECE-1, neutral endopeptidase 24.11, and human Kell blood group protein. The deduced amino acid sequence of ECE-2 is most similar to ECE-1, with an overall identity of 59%. ECE-2 resembles ECE-1 in that it is inhibited in vitro by phosphoramidon and FR901533 but not by thiorphan or captopril, and it converts big ET-1 more efficiently than big ET-2 or big ET-3. However, ECE-2 also exhibits the following striking differences from ECE-1. (i) The sensitivity of ECE-2 to phosphoramidon is 250-fold higher as compared with ECE-1, while FR901533 inhibits both enzymes at similar concentrations. (ii) ECE-2 has an acidic pH optimum at pH 5.5, which is in sharp contrast to the neutral pH optimum of ECE-1. ECE-2 has a narrow pH profile and is virtually inactive at neutral pH. Chinese hamster ovary (CHO) cells, which lack detectable levels of endogenous ECE activity, secrete mature ET-1 into the medium when doubly transfected with ECE-2 and prepro-ET-1 cDNAs. However, ECE-2-transfected CHO cells do not efficiently produce mature ET-1 when present with an exogenous source of big ET-1 through coculture with prepro-ET-1-transfected CHO cells. These findings suggest that ECE-2 acts as an intracellular enzyme responsible for the conversion of endogenously synthesized big ET-1 at the trans-Golgi network, where the vesicular fluid is acidified.

Amino Acid Sequence↗

PubMed1995

Degradation of cell surface heparan sulfates decreases the high affinity binding of basic FGF to endothelial cells, but not to FRTL-5 rat thyroid cells.

The role of cell surface heparan sulfate proteoglycans in the effect of basic fibroblast growth factor (bFGF) on FRTL-5 rat thyroid cells was investigated and compared with that of endothelial cells. FRTL-5 cells were incubated for 2 h with heparitinase (0.5-5.0 mU/mL), which specifically degrades heparan sulfate proteolgycans, and then stimulated by bFGF. The mitogenic effect of bFGF was estimated by measuring [3H]thymidine incorporation. Although cell surface heparan sulfates have been believed to be necessary for bFGF binding to its high affinity receptors, the heparitinase treatment had no significant effect on the DNA synthesis of FRTL-5 cells stimulated by bFGF. The binding study revealed that heparitinase treatment decreased low affinity bindings of [125I]bFGF to FRTL-5 cells by only 50% and did not attenuate the high affinity binding, while the same treatment abolished the high and low affinity binding to bovine pulmonary artery endothelial (CPAE) cells. Analysis of trypsin accessible cell surface 35SO4-labeled materials by Q-sepharose anion-exchange column chromatography showed that heparan sulfate proteoglycans, peaked at 0.55 M NaCl elution, disappeared from the surface of FRTL-5 cells after treatment with 2.0 mU/mL of heparitinase, indicating that the heparitinase resistant low-affinity binding sites are not heparan sulfates. These results demonstrate that cell surface heparan sulfates are not required for the high affinity binding of bFGF to FRTL-5 rat thyroid cells, while proteoglycans are necessary for binding to endothelial cells, and suggest that the mechanism of the action of bFGF is different in rat thyroid cells compared with endothelial cells.

Animals↗

PubMed1995

Effects of transforming growth factor alpha (TGF-alpha) on DNA synthesis and thyrotropin-induced iodine metabolism in cultured porcine thyroid cells.

Transforming growth factor alpha (TGF-alpha) is a potent mitogen that is similar structurally to epidermal growth factor (EGF). As EGF is a potent growth stimulator and an inhibitor of iodine metabolism in cultured thyroid cells of several species, we studied whether TGF-alpha has similar effects using porcine thyroid cells in culture. Recombinant human TGF-alpha dose-dependently stimulated DNA synthesis of thyroid cells, with maximal stimulation (eight- to ninefold above basal) occurring at 2 nmol/l. The potency was approximately 50% that of mouse EGF and correlated with the ability to compete with EGF for receptor binding, suggesting that the action of TGF-alpha is mediated by interaction with EGF receptors. When thyroid cells were cultured for 3 days with thyrotropin (TSH) in the presence of TGF-alpha, TSH-induced iodide uptake was inhibited in a dose-dependent manner. The potency of TGF-alpha again was approximately 50% that of EGF. Transforming growth factor alpha did not inhibit TSH-stimulated cAMP production. Moreover, iodide uptake stimulated by either forskolin or 8-bromo-cAMP also was inhibited by TGF-alpha. Thus, we conclude that TGF-alpha inhibits TSH-induced iodine metabolism largely by acting at the steps distal to cAMP production. Northern blot analysis revealed expression of TGF-alpha mRNA in porcine thyroid cells. These observations suggest that TGF-alpha acts as an autocrine modulator of growth and differentiated functions in porcine thyroid cells.

Animals↗

PubMed1994

Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.

Defects in the gene encoding the endothelin-B receptor produce aganglionic megacolon and pigmentary disorders in mice and humans. We report that a targeted disruption of the mouse endothelin-3 ligand (EDN3) gene produces a similar recessive phenotype of megacolon and coat color spotting. A natural recessive mutation that results in the same developmental defects in mice, lethal spotting (ls), failed to complement the targeted EDN3 allele. The ls mice carry a point mutation of the EDN3 gene, which replaces the Arg residue at the C-terminus of the inactive intermediate big EDN3 with a Trp residue. This mutation prevents the proteolytic activation of big EDN3 by ECE-1. These findings indicate that interaction of EDN3 with the endothelin-B receptor is essential in the development of neural crest-derived cell lineages. We postulate that defects in the human EDN3 gene may cause Hirschsprung's disease.

Amino Acid Sequence↗

PubMed1994

ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1.

Endothelin-1 (ET-1), a 21-residue vasoactive peptide, is produced in vascular endothelial cells from the 38-residue inactive intermediate big endothelin-1 via a specific cleavage at Trp-21-Val-22. The protease that catalyzes the conversion, endothelin-converting enzyme (ECE), constitutes a potential regulatory site for the production of the active peptide. We report the identification of ECE-1, a novel membrane-bound neutral metalloprotease that is expressed abundantly in endothelial cells in vivo and is structurally related to neutral endopeptidase 24.11 and Kell blood group protein. When transfected into cultured cells that normally secrete only big ET-1, the ECE-1 cDNA conferred the ability to secrete mature ET-1. In transfected cells, ECE-1 processes endogenously synthesized big ET-1 as well as exogenously supplied big ET-1, which interacts with ECE-1 on the cell surface. ECE-1 may provide a target for pharmacological intervention to alter ET-1 production.

Amino Acid Sequence↗

PubMed1994

Mechanism of inhibitory actions of minocycline and doxycycline on ascitic fluid production induced by mouse fibrosarcoma cells.

Semisynthetic tetracyclines (TCNs) are used for the management of malignant pleural effusions as sclerosing agents. However, their precise mechanism of actions are uncertain. In the present study, the mechanism of inhibitory effects of minocycline (MINO) and doxycycline (DOXY), on the accumulation of ascitic fluid induced by mouse fibrosarcoma (Meth-A) cells were investigated using male mice. Meth-A cells inoculated intraperitoneally elicited 2.5-4 ml of bloody ascites 10 days after implantation. The production of ascitic fluid was suppressed in a dose-related manner by daily intraperitoneal injections of MINO or DOXY, whereas vehicle (normal saline with 0.01N HCl) did not exert a significant effect. The inhibitory activity of these two substances was quite similar; one mg/mouse of MINO or DOXY inhibited the accumulation of fluid by 87% and 84%, respectively. The survival rate of Meth-A-bearing mice treated with MINO or DOXY was higher than that of the controls. Macroscopic examination of the peritoneal cavity did not reveal any obvious effects, such as adhesions, in mice treated with either MINO or DOXY. In vitro studies showed that MINO and DOXY suppressed Meth-A cell growth with IC50s of 5 microM and 8 microM, respectively. Maximal suppression (95%) was achieved at MINO and DOXY concentrations of 25 microM. The above observations suggest that MINO and DOXY inhibit the accumulation of ascites by a direct effect on Meth-A cell growth. Therefore, it appears that TCNs injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.

Animals↗

PubMed1994

Basic fibroblast growth factor (FGF-2) in renal cell carcinoma, which is indistinguishable from that in normal kidney, is involved in renal cell carcinoma growth.

To investigate the role of basic fibroblast growth factor (FGF) in renal cell carcinoma growth, we have analyzed the expression of mRNA of basic FGF. In 7 of 15 cases, basic FGF mRNA level in renal cell carcinoma tissues was higher than that in corresponding normal tissues. However, the tumor-to-normal ratios of expression levels are chiefly less than 2.0 and, in 5 cases, are even less than 1.0. Furthermore, there was no correlation between the ratio and the clinical stage. In protein analysis, we could not find any difference between basic FGF extracted from renal cell carcinomas and that from normal kidney tissues in bioactivity, immunoreactivity, molecular weight and affinity to heparin. On the other hand, anti-basic FGF monoclonal antibody inhibited the growth of a renal cell carcinoma cell line, VMRC-RCW, and this inhibition was reversed by an extraphysiological amount of exogenous basic FGF (100 ng./ml.). These results suggest that basic FGF itself may have no pivotal role in renal cell carcinoma etiology but is involved in the growth of renal cell carcinomas in an autocrine manner.

Animals↗

PubMed1994

Increased activity of insulin-like growth factor-binding protein in human thyroid papillary cancer tissue.

It has been shown that both insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) are produced by thyroid cells in culture and that the cells respond to IGF-I with increased DNA synthesis, suggesting an autocrine/paracrine role of IGF-I in the regulation of thyroid cell growth. We investigated the tissue contents of immunoreactive IGF-I (irIGF-I) and IGFBPs in human papillary carcinoma and compared them with those of normal thyroid tissue. When irIGF-I was measured after separation of the IGFBPs by gel-filtration, its content in carcinoma tissue was not different from that in adjacent normal tissue (566 +/- 58 vs. 424 +/- 75 pg/mg protein, N = 10). Nor was there any difference in the abundance of IGF-I mRNA expression determined by slot blot analysis. On the other hand, IGFBP activity measured in terms of 125I-IGF-I binding was significantly higher in cancer extracts. Western ligand blot analysis of IGFBPs revealed several species (24-42 kDa) of IGFBPs. The IGF-I-binding activity of 38-41 kDa species (corresponding to IGFBP-3) was not different between extracts of cancer tissue and those of normal tissue, whereas that of 28-32 kDa species was significantly higher in cancer tissue extracts. Since IGFBPs have been reported to modulate cellular responses to IGF-I, the present data suggest that higher IGFBP activity in cancer tissue is involved in regulating growth of thyroid papillary carcinoma cells.

Adenoma↗

PubMed1994

Progressively increased serum 1,25-dihydroxyvitamin D2 concentration in a hypoparathyroid patient with protracted hypercalcemia due to vitamin D2 intoxication.

A 76-year-old female patient who had been taking vitamin D2 100,000 U/day for more than 14 years due to hypoparathyroidism following total throidectomy was admitted because of protracted hypercalcemia. On admission, the levels of serum vitamin D2 (99.8 ng/ml) and 25-OHD2 (356 ng/ml) were very high, and 1,25-(OH)2D2 was low (4.0-18.7 pg/ml). Serum D3' 25-OHD3 and 1,25-(OH)2D3 were below the normal range. Despite intensive hydration with saline, intravenous hyperalimentation with phosphate- and calcium-free nutrients, and administration of glucocorticoid and calcitonin, the hypercalcemia persisted, accompanied by hypoproteinemia, edema, pleural effusion and congestive heart failure. The serum D2 and 25-OHD2 concentrations remained high and were accompanied by a gradual increase in 1,25-(OH)2D2 (121 pg/ml), which further increased after the administration of bisphosphonate (pamidronate) to 183 pg/ml. Seventeen months later, serum calcium and 1,25-(OH)2D2 were normalized but serum D2 and 25-OHD2 remained high. The serum 24,25-(OH)2D2/25-OHD2 ratio was relatively constant throughout her clinical course, whereas the low serum 1,25-(OH)2D2/25-OHD2 ratio at admission gradually increased during admission, suggesting that the increase in serum 1,25-(OH)2D2 is due to increased production rather than decreased degradation. The administration of pamidronate further increased serum 1,25-(OH)2D2. These features of the clinical course demonstrate that the 1,25-dihydroxyvitamin D concentration in hypercalcemic patients with protracted vitamin D intoxication may be decreased, normal or increased. Possible factors responsible for a protracted increase in serum 1,25-(OH)2D2 are body weight loss, hypoproteinemia, and phosphate depletion. In addition, some bisphosphonates would certainly promote PTH-independent production of 1,25-(OH)2D2.

Aged↗

PubMed1994

Interaction of endothelin-1 with porcine thyroid cells in culture: a possible autocrine factor regulating iodine metabolism.

Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a Kd of 0.45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Inhibition of human pancreatic cancer cell (MIA PaCa-2) growth by cholera toxin and 8-chloro-cAMP in vitro.

The effects of cholera toxin (CT) and 8-chloro-cAMP (8-Cl-cAMP) on cell growth were investigated using two human pancreatic carcinoma cell lines (MIA PaCa-2, Panc-1). CT, which catalyses the ADP ribosylation of Gs, suppresses the proliferation of MIA PaCa-2(PC) cells. CT at the low dose of 0.1 pg ml-1 was inhibitory of PC cell growth, and the maximum suppression (70%) was achieved at a CT concentration of 100 pg ml-1. This phenomenon was reversible. The production of cAMP by CT (100 pg ml-1) in PC cells was enhanced 320-fold compared with the control. In addition, cAMP analogues (8-Cl-cAMP, 8-Br-cAMP) and forskolin decreased the growth rate of PC cells in a dose-dependent manner. These results support the view that CT suppresses PC cell growth by stimulating cAMP production. Conversely, Panc-1 cells were far less sensitive to CT in cell growth and cAMP production. 8-Cl-cAMP was also less effective on Panc-1 cell growth. The binding of an insulin-like growth factor (IGF)-I and transforming growth factor (TGF)-alpha, which has been shown to stimulate PC cell growth in an autocrine manner, to PC cells was not modified in cells treated with CT or 8-Cl-cAMP. The results suggest that the inhibitory actions of these substances do not occur at the level of the receptor for IGF-I or EGF/TGF-alpha. We have previously shown that phorbol esters, which decrease the binding of TGF-alpha to PC cells, has an anti-proliferative activity on these tumour cells. Inhibited cell growth by maximum suppressive dose of CT or 8-Cl-cAMP was further inhibited by TPA. In addition, an oncogene product of K-ras which is commonly activated in pancreatic cancer, was increased by CT and 8-Cl-cAMP. It is concluded that CT and 8-Cl-cAMP inhibit PC cell growth, presumably in a similar manner, and their mechanism(s) of action may be different from that of TPA. The anti-proliferative effect of CT or 8-Cl-cAMP was enhanced by TPA, implying that the combination of these substances results in increased inhibition of the PC cell growth.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Iodine regulation of endothelin-1 gene expression in cultured porcine thyroid cells: possible involvement in autoregulation of the thyroid.

We studied the regulation of endothelin (ET)-1 gene expression in porcine thyroid cells in culture. First, we demonstrated prepro-ET-1 mRNA in porcine thyroid cells. The level of the mRNA was increased by phorbol 12-myristate 13-acetate (TPA), a protein kinase C stimulator, but was decreased by TSH (1 mU/mL). However, transforming growth factor-beta and interleukin-1 beta had no effect. The amount of immunoreactive (ir)-ET-1 secreted from the cells was also increased by TPA and was decreased by TSH. Next, we studied the effect of iodide, as iodide has various effects on thyroid cells. NaI (100 microM) increased the prepro-ET-1 mRNA level. The effect of NaI was attenuated by 1 mM methimazole (MMl). The amount of ir-ET-1 released from the cells was also increased by the NaI treatment and the increase was also attenuated by MMl. These observations indicate that ET-1 gene expression is induced by organified iodine compounds in thyroid cells in a manner very similar to the inhibitory actions of iodide on thyroid cell function. The protein synthesis inhibitor, cycloheximide, superinduced prepro-ET-1 mRNA within 4 h, but NaI did not. The difference between cycloheximide and NaI suggests that the iodine effect on the gene expression is not due to nonspecific inhibition of protein synthesis. Together with our previous findings that porcine thyroid cells have ET-1 receptors and that ET-1 modulates iodine metabolism, we speculate that ET-1 produced by thyroid cells is involved in thyroid autoregulation including thyroid blood flow.

Animals↗