Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.
 ...
PMID:Adenosine 3',5'-monophosphate and thyroid hormone control of uncoupling protein messenger ribonucleic acid in freshly dispersed brown adipocytes. 137 9

We have detected the presence of nuclear 3,5,3'-triiodothyronine (T3) receptors in primary cultures of chick embryo hepatocytes and neurons. Hepatocytes were isolated from livers of embryos of 12, 16 and 19 days by treatment with 0.2% collagenase and hyaluronidase. They were plated at a density of 3-4 x 10(5)/35-mm petri dish in Ham's F-10 medium containing fetal calf serum, tryptose phosphate, and antibiotics. Cells were used for the binding assay at Day 3 of culture. Neurons from 8-day-old embryo brains were cultured in a serum-free medium at a density of 1.2 x 10(6) cells/35-mm petri dish and used for the binding assay after 7 days of culture. Biological activity of hepatocytes was determined by measuring insulin binding, inositol phosphate formation, and 5'-monodeiodinase activity. Neurons or glial cells in culture were identified by immunostaining with anti-neurofilaments and anti-glial fibrillary acidic protein antisera. Binding assay was performed with isolated nuclei and 0.4 M NaCl nuclear extracts. With the latter preparation, the Scatchard analysis showed, in both cells, a single, high-affinity, low-capacity T3 receptor. In the hepatocytes of 12-, 16-, and 19-day-old embryos association constants (Ka) were, respectively, 0.93 +/- 0.02, 0.74 +/- 0.03, and 0.56 +/- 0.04 nM-1, whereas the maximal binding capacities (MBC) were 2.26 +/- 0.2, 2.72 +/- 0.33, and 1.83 +/- 0.19 fmol/microgram DNA (mean +/- SE, n = 3). In neurons Ka was 1.25 +/- 0.53 nM-1 and MBC 0.59 +/- 0.14 fmol/microgram DNA (n = 3). The receptor had a sedimentation coefficient of 3.4 S, an estimated Mr of 59 kDa, and the following relative affinity for thyroid hormone analogues: TRIAC greater than L-T3 greater than L-T4. These data indicate that cultured hepatocytes and neurons of chick embryo contained T3 receptors with properties similar to those described in intact tissues from this and other species. Only the MBC of neurons was 50% lower than that observed in whole brain of embryo, but was comparable to values observed in cultured neurons from other species.
 ...
PMID:Characterization of 3,5,3'-triiodothyronine receptors in primary cultures of hepatocytes and neurons from chick embryo. 160 Dec 52

We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.
 ...
PMID:Physiological de novo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3',5'-monophosphate alone is sufficient for thyroid hormone formation. 215 8

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.
 ...
PMID:Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini. 248 94

The plasma clearance rate of high density lipoprotein is reduced in the hypothyroid rat. Because the liver is an important site of HDL-cholesterol catabolism, the present study was undertaken to investigate whether thyroid hormone deficiency affects binding of HDL to liver cells. Male Wistar rats were made hypothyroid by feeding propylthiouracil (0.1% w/w). Liver cells were isolated by in situ perfusion of the liver with a buffered collagenase solution. 125I-labelled rat HDL binding to isolated liver cells was carried out at low temperature on ice. For both control and hypothyroid rat liver cells, 125I-HDL binding was significantly inhibited by excess unlabelled rat HDL and also by human HDL3 and human LDL but was unaffected by the addition of 10 mM EDTA. From Scatchard analysis of dose-response studies, hypothyroid cells displayed a lower HDL binding capacity (P less than 0.01) and a higher binding affinity (P less than 0.025) compared to control cells. These results suggest that thyroid hormone affects the expression of the HDL binding site in liver cells which may contribute to the reduced HDL clearance in the hypothyroid animal.
 ...
PMID:Hypothyroidism reduces HDL binding to rat liver cells. 280 42

Although Ismail-Beigi and Edelman demonstrated in 1971 that thyroid hormones control the activity of Na-K-ATPase in the mammalian kidney, the actual site of this regulation inside the organ was not located. We therefore decided to study the relationship between thyroid hormones and Na-K-ATPase activity in individual nephron segments obtained by microdissection of collagenase-treated rabbit kidneys. For this purpose, the changes in the activity and number of catalytic sites of Na-K-ATPase in response to thyroidectomy or triiodothyronine administration were examined. Eight to 12 days after thyroidectomy, Na-K-ATPase activity had dropped by 40 to 80% in the convoluted and straight portions of the proximal tubules, and in the cortical and outer medullary collecting tubules, but not in the thick ascending limbs of Henle's loops or distal convoluted tubules. The apparent number of catalytic sites for Na-K-ATPase, as measured by specific binding of 3H-ouabain, decreased in parallel with Na-K-ATPase activity, and therefore this enzyme's specific activity was not altered. Fourty eight hours after injection of thyroidectomized animals with a single dose of either 100 or 500 micrograms/kg triiodothyronine, Na-K-ATPase activity in target segments was restored to the level measured in control animals. These effects of thyroid hormone were specific for Na-K-ATPase, since the activity of adenylate cyclase, another marker of the basolateral membrane, was not altered by thyroidectomy. The results obtained indicate that triiodothyronine controls Na-K-ATPase activity in specific nephron segments, by altering the number of this enzyme's catalytic sites.
 ...
PMID:Sites of thyroid hormone action on Na-K-ATPase along the rabbit nephron. 299 94

The relationship of structural polarity to functional activities was examined in cultured human thyroid follicles, which were isolated from the thyroid gland of patients with Graves' disease by collagenase treatment. Structural polarity was examined morphologically by electron microscopy, while the functional response to bovine TSH was examined by measuring intracellular cAMP accumulation and T3 release. In freshly isolated thyroid follicles, structural polarity was normal and TSH induced significant cAMP accumulation but no significant release of T3. After culture for 5 days the structural polarity of thyroid follicles became inverted in the absence of thyroid stimulators, but normal polarity was retained in the presence of TSH or dibutyryl cAMP [Bu)2 cAMP). The response to TSH of cAMP accumulation increased markedly after culture in either the presence or absence of TSH, suggesting that cAMP accumulation in response to TSH is not related to structural polarity. In contrast, thyroid follicles cultured without thyroid stimulators showed no significant T3 release in response to TSH, whereas those cultured with TSH or (Bu)2 cAMP showed significant T3 release in response to TSH. These data indicate that in cultured human thyroid follicles, the responses to TSH of cAMP accumulation and T3 release are not always correlated. Among many other explanations, the results were at least compatible with the idea that normal structural polarity is necessary for thyroid hormone release in response to TSH.
 ...
PMID:Relation of structural polarity to responses of cyclic 3', 5'-adenosine monophosphate accumulation and thyroid hormone release in cultured human thyroid follicles. 608 98

Studies were carried out on the effect of triiodothyronine (T3) on the oxygen consumption of dispersed rat liver cells incubated for 2 hr at 37 degrees C. Thyroidectomized SD-NIH rats were kept on a low iodine diet with calcium chloride in the drinking water for 4 weeks or longer to assure hypothyroidism, verified by low serum thyroxine and T3 concentrations. Liver cells were obtained by portal vein perfusion with oxygenated collagenase-enriched Krebs-Ringer-bicarbonate buffer, after the method of Berry and Friend. Cell viability was evaluated by morphology, by trypan blue exclusion, and by biochemical parameters prior to 2-hr incubations with or without added hormone. The oxygen consumption of cell suspensions was measured with the Clark oxygen electrode after the 2-hr incubations at 37 degrees C with oxygenation of the flasks and alanine (5-10 mM) as substrate. In 31 experiments the oxygen consumption (QO2) was enhanced to 121% of control values with T3 in the medium at 3.3 nM ("physiological" level) and with an even greater effect (138% of control values) with 300-1000 nM T3 ("hyperthyroid" level). Cycloheximide at 100 microM was used to inhibit new protein synthesis by incubated hepatocytes. In 18 parallel experiments with cycloheximide blockade, no alteration of the stimulatory effect of T3 was evident. The results signify that incubated liver cells show an early response to thyroid hormone by extranuclear pathways that do not require new protein synthesis.
 ...
PMID:Rapid thyroid hormone action in vitro in the absence of new protein synthesis. 653 49

The skin of an anuran tadpole undergoes region-dependent metamorphic changes: the body (head and body trunk) transforms into the adult type, while the tail falls into programmed cell death. The present study was undertaken to investigate the regional specificity of metamorphosis at a molecular level, focusing on genes of collagen and collagenase that are known to be activated in their synthesis at metamorphosis. A cDNA probe utilized for collagen was Hf677 (a clone of human type I collagen alpha 1 chain). A probe for collagenase gene was cloned in the present study from a cDNA library of bullfrog tadpole skin, characterized and named Tc1. Tc1 contained the consensus sequence of zinc-metalloproteinases and showed a high homology to mammalian collagenases. Using these recombinant DNAs as probes, RNA blot analyses were performed for the body and tail skin of tadpoles that had been in spontaneous metamorphosis, induced to metamorphosis by the injection of thyroid hormone, or had been induced to grow by prolactin treatment. Collagenase gene was activated irrespective of regions of the skin, body or tail at the early metamorphic climax stage, although the extent of activation was region-dependent. In contrast, metamorphic changes of collagen gene expression showed a clear regional dependency. The transcription level in body skin was enhanced at the onset of metamorphosis while that in tail skin was markedly suppressed. Thyroid hormone was shown to be responsible for this region-dependent expression of collagen genes at metamorphosis. Prolactin, a suppressor hormone of amphibian metamorphosis, enhanced the transcription of collagen genes and suppressed that of collagenase.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Regionally and hormonally regulated expression of genes of collagen and collagenase in the anuran larval skin. 798 Oct 43

The activity of type I 5'-deiodinase (5'DI) is known to correlate with thyroid status; it is high in hyperthyroidism and low in hypothyroidism. Recently, it was shown that the increased activity of type I 5'DI in hyperthyroidism is associated with an increase in enzyme contents as well as its mRNA in liver. However, it remains unknown whether thyroid hormone directly regulates the expression of 5'DI mRNA in hepatocytes. In this study, the direct actions of thyroid hormone as well as rT3 and dexamethasone on type I 5'DI mRNA were investigated using primary cultures of rat hepatocytes. Hepatocytes were prepared from euthyroid rats by collagenase perfusion and plated either on collagen-coated dishes for conventional monolayer cultures or on positively charged dishes for spheroid cultures. After hormonal treatments, the levels of mRNAs for type I 5'DI and albumin were determined by Northern blotting. In spheroid cultures, T3 increased type I 5'DI mRNA in a dose- and time-dependent manner, whereas the albumin mRNA level was not altered. A lesser effect was observed in hepatocytes cultured as monolayers. The T3-induced increase in 5'DI mRNA was not inhibited by pretreatment with cycloheximide, indicating that the effect of thyroid hormone on 5'DI mRNA is direct, not requiring de novo protein synthesis. rT3 did not affect the levels in type I 5'DI mRNA increased by T3. On the other hand, dexamethasone alone increased 5'DI mRNA and, when added together with T3, had a synergistic effect. In contrast to T3, dexamethasone increased albumin mRNA. Dexamethasone-induced increases in mRNAs for 5'DI and albumin were inhibited by pretreatment of cycloheximide. The present study indicated that T3 increases 5'DI mRNA through a direct action on its gene, whereas the effect of dexamethasone requires de novo synthesis of a protein factor(s).
 ...
PMID:Effects of thyroid and glucocorticoid hormones on the level of messenger ribonucleic acid for iodothyronine type I 5'-deiodinase in rat primary hepatocyte cultures grown as spheroids. 824 26


1 2 Next >>