Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human thyroid cells were transfected with SV40 DNA using the calcium phosphate co-precipitation technique. The transfected cells grew rapidly and could be passaged readily. TSH and the immunoglobulin fraction from a patient with hyperthyroid Graves' disease stimulated adenylate cyclase activity in the transfected cells. These cells could potentially be used in clinical assays of patients' immunoglobulins without concern for species incompatibilities. On immunoblot analysis, extracts from SV40-transfected human thyroid cells, from sheep thyroid cells and from rat FRTL5 cells reacted with the immunoglobulin fraction from the patient with hyperthyroid Graves' disease but not with the immunoglobulin fraction from a pool of serum from normal subjects. Proteins of molecular weights 77,000, 70,000 and 54,000 were identified by the patient's immunoglobulin fraction in the three thyroid-derived cell types.
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PMID:Human thyroid cells transfected with SV40 DNA retain the TSH receptor. 217 Feb 63

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin-beta gene. 217 92

This study was carried out to clarify the way in which thyrotropin (TSH) and forskolin regulate the adenylylcyclase complex in thyroid follicle cells. We examined the effects of chronic treatment of pig thyroid follicles with TSH or forskolin on the state of G proteins by (a) assaying adenylylcyclase activity, (b) analyzing the ADP-ribosylation of stimulatory G protein (Gs) by cholera toxin, and (c) quantifying the Gs subunits by Western blotting with antipeptide antibodies. Chronic exposure (18 h) of thyroid follicles to a low concentration of TSH (0.01-0.1 milliunit/ml) enhanced the subsequent response of adenylylcyclase to TSH. Higher concentration of TSH (1 milliunit/ml) induced a homologous desensitization of this response. In cells pretreated with forskolin, the TSH-stimulated adenylylcyclase activity was higher than in control cells. The forskolin-or guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p)-stimulated adenylylcyclase activity was always significantly increased after chronic treatment of cells with TSH or forskolin. Treatment of cultured thyroid follicle membranes with [32P]NAD and cholera toxin resulted in labeling of the Gs alpha (45-52-kDa) component. Culturing follicles with TSH (0.001-1 milliunit/ml) or forskolin (0.01-10 microM) greatly affected the cholera toxin-mediated ADP-ribosylation of the Gs alpha subunit. Gs alpha labeling increased progressively to level off at 1 milliunit/ml TSH or 1 microM forskolin (150-200%). Gs alpha immunoreactivity was increased in parallel (200-300%). The immunoreactivity of G beta subunits in cells cultured with TSH or forskolin was also increased compared with control cells. Cycloheximide abolished the effects of TSH and forskolin on the follicles, suggesting that new protein synthesis is required. These results indicate that Gs protein subunits are up-regulated by TSH and forskolin and suggest that their synthesis in thyroid cells is mediated, at least in part, by a cyclic AMP-dependent mechanism.
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PMID:Cyclic AMP regulation of Gs protein. Thyrotropin and forskolin increase the quantity of stimulatory guanine nucleotide-binding proteins in cultured thyroid follicles. 217 58

Amiodarone is an antiarrhythmic drug that often induces thyroid disorders. Its effects on several aspects of thyroid function were studied using cultured dog thyroid cells. Within 5-60 min of incubation of cell membranes with amiodarone, there were profound changes in adenylate cyclase activity and TSH receptor binding. Amiodarone specifically decreased TSH-stimulated adenylate cyclase activity, but not the basal or forskolin-stimulated activities, while it increased the binding of 125I-labeled TSH to its receptors. Significant effects were seen with 5-10 microM amiodarone, with maximal effects at 50-100 microM, when TSH-stimulated adenylate cyclase activity was completely blocked and the labeled TSH binding increased 4- to 5-fold over control. These effects of amiodarone were reversible, since membranes exposed to 50 microM amiodarone for 1 h exhibited normal binding and cyclase activities, when amiodarone was removed by washing before the assay. The above effects of amiodarone were also observed when cells, instead of membranes, were treated with the drug, although the magnitude of changes was less than in membranes. Lower concentrations of amiodarone (10-25 microM) caused significant inhibition of iodide organification, without affecting iodide uptake, while higher concentrations (50-100 microM) inhibited organification by nearly 75% and uptake by about 20%. Amiodarone (10-100 microM) also inhibited [3H]2-deoxy-glucose uptake and the increase in intracellular calcium concentration in response to TSH and carbachol. In contrast to membranes, treatment of cells with amiodarone caused persistent inhibition of TSH-stimulated cAMP formation and iodide organification even 24-48 h after removal of the drug. However, amiodarone had no effect on cell viability, as judged by trypan blue exclusion and ability to remain attached to the culture dishes. These results suggest that amiodarone has specific inhibitory effects on agonist-stimulated functions in thyroid cells, possibly by interfering with TSH-receptor interactions and also at the level of cholinergic receptors.
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PMID:Amiodarone effects on thyrotropin receptors and responses stimulated by thyrotropin and carbachol in cultured dog thyroid cells. 217 39

The effect of thyrotropin (TSH) on cyclic AMP accumulation, phosphatidylinositol bisphosphate (PIP2) hydrolysis and [Ca2+]i rise has been studied in CHO cells stably transfected with human TSH receptor (hTSHR) cDNA. In human thyroid slices, TSH activates these two intracellular cascades with a higher affinity for the adenylate cyclase activation (from 0.1 to 1 mU/ml TSH) than for phospholipase C activation (from 1 to 10 mU/ml TSH). The CHO cells transfected with the recently cloned cDNA of human TSH receptor respond in the same way to TSH. They respond between 0.1 and 1 mU/ml TSH for cyclic AMP accumulation and between 1 and 10 mU/ml TSH for inositol monophosphate (IP1) increase. In these same cells, TSH 10 mU/ml, but not forskolin (10 microM), or dibutyryl cyclic AMP (100 microM), clearly enhances intracellular calcium concentration [( Ca2+]i). Our results demonstrate unequivocally that a single transcription unit has the potential to encode receptor molecules coupled to both cascades.
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PMID:Thyrotropin activates both the cyclic AMP and the PIP2 cascades in CHO cells expressing the human cDNA of TSH receptor. 217 5

TSH, the major signalling factor for the thyroid follicles, controls thyrocyte function in concert with other modulators of cell growth, differentiation and structural organization of the follicular-endothelial network. Most of the TSH effects are mediated by TSH binding to the TSH receptor which stimulates adenylate cyclase catalyzed cAMP production. TSH is involved in the regulation of thyroidal uptake of small molecules and nutrients, intracellular transport of thyrocyte specific proteins, and in most of the steps of thyroid hormone synthesis, storage and release. These cellular events require the fine tuned regulation of metabolic reactions, morphological differentiation and cell proliferation. Thyrocytes also express a highly active Type I iodothyronine 5' deiodinase which is controlled by TSH stimulated cAMP production. The thyrocyte specific 5' deiodinase isozyme has marked influence on the amount of T3 secreted by the thyroid. This 5' deiodinase isozyme shows most of the characteristics of the type I 5' deiodinase found in liver and kidney and is also blocked by PTU, other 5' deiodinase inhibitors, and iodinated X-ray contrast agents such as iopanoic acid, which are occasionally used in thyrotoxicosis to inhibit thyroidal T3-production by this enzyme. In contrast to the liver and kidney type I 5' deiodinase T4 and T3 are not able to induce this enzyme in thyrocytes. However, TSH stimulated cAMP production increases the thyroidal isozyme activity in contrast to the liver and kidney enzyme. This review summarizes the data on the various experimental models used up to date to characterize the thyroidal type I 5' deiodinase in various species including man.
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PMID:Thyrotropin (TSH) action on thyroid hormone deiodination and secretion: one aspect of thyrotropin regulation of thyroid cell biology. 221 Jun 28

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.
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PMID:Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells. 222 79

Gangliosides (GM1, GT1b, GD3) were incorporated in bovine thyroid plasma membranes using the nonspecific lipid transfer protein from beef liver. The transfer of GT1b or GD3 in the presence of 16 units of transfer protein was twice as high as that of GM1. However, taking into account the spontaneous exchange (approximately 8% for GT1b or GD3 and 1% for GM1) the transfer protein seemed to be more effective for GM1. Incorporation of these gangliosides in bovine thyroid plasma membranes caused a concentration dependent inhibition of the TSH-stimulated adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was not significantly affected by ganglioside modification of the plasma membranes, indicating that the gangliosides do not act at the level of the catalyst of adenylate cyclase. Binding experiments on the other hand revealed that TSH binding to bovine thyroid plasma membranes was inhibited with the same order of efficacy (GT1b greater than GD3 greater than GM1) and to the same extent as their inhibitory effect on TSH stimulation. Therefore, this indicates that the ganglioside induced drop in TSH binding might be an important factor in the decrease in TSH-stimulated adenylate cyclase activity. Incorporation of GT1b or GD3 (approximately 11 nmol) in bovine thyroid plasma membranes, however, also induced a substantial decrease in cholera toxin-stimulated adenylate cyclase activity (approximately 30%) and to a lesser degree a decrease in NaF-stimulated activity (approximately 17%), whereas GM1 incorporation did not significantly affect these stimulated activities. These latter inhibitory effects were paralleled by changes in fluorescence steady-state anisotropy: GT1b modification of the plasma membranes provoked a slight increase in TMA-DPH anisotropy, whereas the anisotropy of DPH was substantially enhanced after incorporation of GD3 or GT1b. These results suggest that gangliosides might also interfere with the coupling between the alpha-subunit of the stimulatory GTP-binding regulatory protein and the catalyst of the adenylate cyclase system by affecting the membrane fluidity.
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PMID:Modification of the adenylate cyclase activity of bovine thyroid plasma membranes by manipulating the ganglioside composition with a nonspecific lipid transfer protein. 233 19

Iodide inhibits cyclic AMP accumulation in the thyroid by a process which is prevented by inhibition of iodide uptake and of thyroid peroxidase. By a similar process, it also exerts other independent effects such as the enhancement of iodinated protein release. Iodide inhibited the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin and forskolin. The action of iodide was not relieved by phosphodiesterase inhibitors and was not additive with the effect of norepinephrine or adenosine. Iodide did not decrease the cellular level of ATP. The data are compatible with an inhibition of adenylate cyclase beyond the level of the receptor, presumably at the level of the catalytic unit or its interaction with the positive transducing unit NS. The effect of iodide required TSH for its expression but not for its installation. It was decreased under all conditions in which iodide organification was decreased: decreased iodide or increased methimazole concentration, absence of calcium in the medium, etc. However, the relation between iodide binding to proteins and effect was not linear. The effect was not relieved by washing in the absence of iodide and in the presence of perchlorate, but it was partly reversible in the presence of methimazole propylthiouracyl or thiourea. It was not relieved by cooling to 20 degrees C and cytochalasin b, which block stimulated thyroglobulin hydrolysis and iodothyronine release, nor by actinomycin D, cycloheximide, puromycin, mepacrine or indomethacin. The data suggest that iodide binds to a saturable cell component by a reaction which is reversible only in the presence of thiol-containing drugs.
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PMID:Further characterization of the iodide inhibitory effect on the cyclic AMP system in dog thyroid slices. 240 38

We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 microM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating 'uncoupling' of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of the adenylate cyclase by epinephrine (10(-6) M-10(-4) M in the presence of 10(-4) M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.
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PMID:Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line. 241 13


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