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1.
Eur Respir J ; 25(1): 88-95, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15640328

ABSTRACT

The correlation of clinical and epidemiological data suggests that intrauterine infection/inflammation can promote foetal lung injury. The aim of this study was: 1) to characterise the early inflammatory response elicited in infected foetal lungs, in terms of nitric oxide-derived oxidative stress and programmed cell death; and 2) to investigate the effects of antibiotic therapy on these parameters. A previously described rabbit experimental model of materno-foetal infection was used. Animals were divided into three groups: controls; Escherichia coli infected (12 h); and E. Coli infected (12 h) and treated (24 h gentamicin+ceftriaxone). Foetal lungs were examined in terms of histology, nitric oxide synthase (NOS) activity, immunohistochemical detection of 3-nitrotyrosine, and detection of apoptotic cells by the TUNEL assay and Hoechst staining. In the infected group, a moderate inflammatory response was observed, associated with a significant increase in inducible NOS activity, the formation of 3-nitrotyrosine residues in epithelial and immune cells, the down-regulation of constitutive NOS activity and clusters of apoptotic cells, as compared with the control group. Early antibiotic therapy, initiated at 12 h post-inoculation, elicited a significant decrease in the infection-induced nitrosative stress. Levels of 3-nitrotyrosine and of apoptotic cells were decreased in the infected-and-treated group compared with the infected group, mainly by the re-expression of constitutive NOS and of the basal level of inducible NOS. Altogether, these findings indicate that early antibiotic therapy can curb the inflammatory reaction and help avert antenatal lung injury, which is known to be involved in the onset of bronchopulmonary dysplasia.


Subject(s)
Anti-Bacterial Agents/pharmacology , Nitric Oxide Synthase/metabolism , Oxidative Stress/physiology , Pneumonia, Bacterial/drug therapy , Pregnancy, Animal , Analysis of Variance , Animals , Apoptosis/drug effects , Apoptosis/physiology , Disease Models, Animal , Female , Fetus/drug effects , Fetus/pathology , Immunohistochemistry , In Situ Nick-End Labeling , Lung/drug effects , Lung/pathology , Nitric Oxide Synthase/drug effects , Oxidative Stress/drug effects , Pneumonia, Bacterial/pathology , Pregnancy , Pregnancy Complications, Infectious , Probability , Rabbits , Reference Values , Sensitivity and Specificity
2.
Br J Cancer ; 90(12): 2364-9, 2004 Jun 14.
Article in English | MEDLINE | ID: mdl-15150612

ABSTRACT

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.


Subject(s)
Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Guanylate Cyclase/biosynthesis , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide/metabolism , Down-Regulation , Humans , Immunohistochemistry , Inflammation , Neoplasm Staging , Nitric Oxide Synthase Type I , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction
3.
Biochem J ; 344 Pt 3: 643-8, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10585850

ABSTRACT

This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e. anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power. Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation. The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl. 16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by (14)CO(2) production from 2-deoxy[1-(14)C]glucose. Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.


Subject(s)
Carbohydrate Metabolism , Nitric Oxide/pharmacology , Adenosine Triphosphate/analysis , Cell Survival/drug effects , Culture Media , Deoxyglucose/metabolism , Epithelial Cells , Galactose/metabolism , Glucose/metabolism , Glutathione/analogs & derivatives , Glutathione/metabolism , Glutathione/pharmacology , HT29 Cells , Humans , Lactic Acid/metabolism , Nitroso Compounds/metabolism , Nitroso Compounds/pharmacology , Oligomycins/pharmacology , Pyruvic Acid/metabolism , S-Nitrosoglutathione , Tyrosine/analogs & derivatives , Tyrosine/metabolism
4.
Gut ; 45(2): 246-51, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10403737

ABSTRACT

BACKGROUND: Interleukin (IL) 1beta converting enzyme (now known as caspase 1) is able to process pro-IL-1beta into its active form and is involved in proapoptotic signalling. AIMS: To characterise IL-1 and caspase 1 expression in human colonic epithelial cells. METHODS: Intracellular IL-1 content (IL-1alpha and IL-1beta) was measured by ELISA in freshly isolated human normal colonocytes. Caspase 1 expression was determined both at the mRNA level using in situ hybridisation and reverse transcription polymerase chain reaction, and at the protein level by immunoblotting experiments using antibodies specific for the proform of caspase 1 and for its cleavage forms. RESULTS: Low amounts of IL-1beta were found in nearly all preparations (92%), and IL-1alpha was detected in only 45% of human colonocyte preparations. The normal colonic epithelium strongly expressed caspase 1, both at the mRNA level and at the protein level in its latent form. In contrast, caspase 1 was not expressed in colon cancer (primary colonic adenocarcinomas and cancer cell lines). CONCLUSIONS: The demonstration that the human colonic epithelial barrier is able to express caspase 1 and its substrate IL-1beta reinforces the concept that, under certain conditions, the epithelium could trigger an inflammatory reaction. In addition, the finding that caspase 1 was downregulated in colonic adenocarcinomas supports the concept that disrupted apoptosis pathways may be involved in tumour formation and/or may confer resistance to treatment.


Subject(s)
Caspase 1/metabolism , Colon/metabolism , Colonic Neoplasms/metabolism , Interleukin-1/metabolism , Neoplasm Proteins/metabolism , Adult , Aged , Aged, 80 and over , Blotting, Western , Colon/physiopathology , Colonic Neoplasms/physiopathology , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Female , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/physiopathology , Male , Middle Aged , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods
5.
Eur J Clin Invest ; 29(1): 48-55, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10092989

ABSTRACT

BACKGROUND: Colonic epithelial cells may behave as antigen-presenting cells. Interleukin 10 (IL-10) is known to play a major role in the intestinal immune system; however, it remains to be determined whether human intestinal epithelial cells express IL-10 receptor, and whether this cytokine modulates their expression of antigen presentation-associated molecules. METHODS: The binding of biotinylated IL-10 was studied in SW 1116, HT-29 and T84 human colonic epithelial cell lines and freshly isolated normal colonic epithelial cells. Reverse transcription-polymerase chain reaction was also performed to detect IL-10 receptor mRNA. The effect of IL-10 on antigen presentation associated molecules was assessed by flow cytometry. RESULTS: Biotinylated IL-10 bound to SW 1116, HT-29, T84, and normal colonic epithelial cells. IL-10 receptor mRNA was detected in SW 1116 and normal epithelial cells. SW 1116 and HT-29 cells expressed MHC class I and ICAM-1, but not CD80, and SW 1116 constitutively expressed HLA-DR. Interferon-gamma up-regulated HLA-DR and ICAM-1 expression on both cells, whereas lipopolysaccharide increased ICAM-1 expression only on SW 1116. IL-10 failed to modulate these antigens, even after stimulation by lipopolysaccharide or interferon-gamma. Moreover, these molecules decreased IL-10 binding in both lines. CONCLUSION: The presence of IL-10 receptor on intestinal epithelial cells suggest that IL-10 may play a role in mucosal physiology, however its effect on the immune response remains to be determined.


Subject(s)
Antigen Presentation , Colon/immunology , Interleukin-10/immunology , Intestinal Mucosa/immunology , Receptors, Interleukin/isolation & purification , Adenocarcinoma/immunology , B7-1 Antigen/immunology , Cell Separation , Colon/cytology , Down-Regulation , Epithelial Cells/cytology , Epithelial Cells/immunology , HLA-DR Antigens/immunology , Histocompatibility Antigens Class I/immunology , Humans , Intercellular Adhesion Molecule-1/immunology , Interferon-gamma/immunology , Intestinal Mucosa/cytology , Lipopolysaccharides/immunology , Major Histocompatibility Complex , Receptors, Interleukin-10 , Tumor Cells, Cultured
6.
J Biol Chem ; 274(10): 6107-13, 1999 Mar 05.
Article in English | MEDLINE | ID: mdl-10037693

ABSTRACT

In human cardiac myocytes, we have previously identified a functional beta3-adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, beta3-adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the electrophysiological effects of beta3-adrenoceptor stimulation in non-cystic fibrosis tissues. We therefore investigated the control of CFTR activity by human beta3-adrenoceptors in a recombinant system: A549 human cells were intranuclearly injected with plasmids encoding CFTR and beta3-adrenoceptors. CFTR activity was functionally assayed using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe and the patch-clamp technique. Injection of CFTR-cDNA alone led to the expression of a functional CFTR protein activated by cAMP or cGMP. Co-expression of CFTR (but not of mutated DeltaF508-CFTR) with high levels of beta3-adrenoceptor produced an increased halide permeability under base-line conditions that was not further sensitive to cAMP or beta3-adrenoceptor stimulation. Patch-clamp experiments confirmed that CFTR channels were permanently activated in cells co-expressing CFTR and a high level of beta3-adrenoceptor. Permanent CFTR activation was not associated with elevated intracellular cAMP or cGMP levels. When the expression level of beta3-adrenoceptor was lowered, CFTR was not activated under base-line conditions but became sensitive to beta3-adrenoceptor stimulation (isoproterenol plus nadolol, SR 58611, or CGP 12177). This later effect was not prevented by protein kinase A inhibitors. Our results provide molecular evidence that CFTR but not mutated DeltaF508-CFTR is regulated by beta3-adrenoceptors expression through a protein kinase A-independent pathway.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Receptors, Adrenergic, beta/metabolism , Signal Transduction , Animals , COS Cells , Cell Line , Humans , Mutation , Myocardium/metabolism , Receptors, Adrenergic, beta/genetics , Receptors, Adrenergic, beta-3
7.
Biochem J ; 331 ( Pt 3): 713-7, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9560296

ABSTRACT

In this work, we addressed the issue of whether exogenous NO and ONOO- (peroxynitrite) are able to alter growth, viability and/or differentiation of normal epithelial cells using cultured normal human keratinocytes as a model. 3-Morpholino-sydnonimine (SIN-1), a donor of both NO and O2(-)., leading to the production of ONOO-, dose-dependently inhibited growth of human keratinocytes without loss of viability. This inhibitory effect was lowered when SIN-1 was transformed into a pure NO donor by scavenging O2(-). with superoxide dismutase/catalase. Finally, scavenging NO release from SIN-1 with carboxy-1H-imidazol-1-yloxy,2-(4-carboxyp henyl)-4,5-dihydro-4,4,5,5 -tetramethyl-3-oxide (PTIO) resulted in a loss of the inhibitory effect of SIN-1. Together these findings suggest that both ONOO- and NO exert a growth inhibitory effect on human keratinocytes without cytotoxicity. Further support for this conclusion came from the treatment of human keratinocytes with the NO. donor propanamine 3-(2-hydroxy-2-nitroso-1-propyl hydrazino) or with authentic peroxynitrite. Moreover, only SIN-1 or peroxynitrite, and not NO, was able to trigger the expression of markers of terminal differentiation in human keratinocytes. From a physiological perspective, the ability of peroxynitrite, a known genotoxic and potentially carcinogenic agent, to direct proliferating keratinocytes towards terminal differentiation may be crucial to protect the genomic stability of this barrier epithelium.


Subject(s)
Cell Differentiation/drug effects , Cell Division/drug effects , Keratinocytes/drug effects , Nitrates/pharmacology , Nitric Oxide/pharmacology , Catalase/metabolism , Cyclic N-Oxides/pharmacology , Fluorescent Antibody Technique , Free Radical Scavengers/metabolism , Humans , Hydrazines/metabolism , Imidazoles/pharmacology , Keratinocytes/cytology , Molsidomine/analogs & derivatives , Molsidomine/metabolism , Nitric Oxide/metabolism , Superoxide Dismutase/metabolism , Thymidine/metabolism
8.
Gastroenterology ; 112(6): 1887-94, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9178681

ABSTRACT

BACKGROUND & AIMS: Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function. The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells. METHODS: Two model systems were used: the HT29-CI.16E colonic goblet cell line and primary cultures of human normal colonocytes. RESULTS: Toxin A exerted a rapid and dose-related inhibition of stimulated mucin exocytosis without altering baseline (constitutive) mucin exocytosis from HT29-CI.16E cells. Toxin A was also able to induce the secretion of IL-8 from both HT29-CI.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation. CONCLUSIONS: The results show that while toxin A is able to down-regulate stimulated mucin exocytosis, it is able to up-regulate the secretion of an important chemoattractant chemokine, IL-8. These modifications illustrate the ability of colonocytes to recruit inflammatory and immune cells that will eventually bring about major mucosal damage.


Subject(s)
Bacterial Toxins , Colon/drug effects , Enterotoxins/toxicity , Cell Line , Colon/cytology , Dose-Response Relationship, Drug , Humans , Time Factors
9.
Br J Pharmacol ; 121(2): 187-92, 1997 May.
Article in English | MEDLINE | ID: mdl-9154326

ABSTRACT

1. The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to produce interleukin-1 (IL-1), the cellular mechanisms involved in IL-1 release, and the intracellular signalling pathways involved in IL-1 up-regulation during inflammatory stress. 2. This study was based on the human colonic epithelial cell line HT29-Cl.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with various proinflammatory cytokines (interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), IL-1 beta) alone or in combination. 3. IL-1 production, restricted to IL-1 alpha, was induced by the combination of IFN gamma/TNF alpha. When IL-1 beta was added to IFN gamma/TNF alpha, it led to an additional production of IL-1 alpha. IL-1 alpha release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL-1 production, and was not accounted for by a secretory mechanism. 4. Both IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO2-/NO3- by use of the Griess reagent, quantitation of cells scoring positive with an anti-iNOS antibody and detection of mRNAs coding for iNOS by RT-PCR. The use of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, led to the demonstration of two distinct signalling pathways in IL-1 production by HT29-Cl.16E cells, one dependent on NO (L-NMMA-sensitive) under treatment with IFN gamma/TNF alpha/IL-1 beta, and the other independent of NO (L-NMMA-insensitive) under treatment with IFN gamma/TNF alpha. 5. Moreover, we examined whether a redox-based mechanism could be responsible for the apparent discrepancy between NO production and NO implication in IL-1 production under IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta treatments. Experiments with cysteine, which acts as a powerful reductant, suggest that the nitrosonium character of NO is involved in the NO-dependent pathway in IL-1 production.


Subject(s)
Colon/metabolism , Interleukin-1/biosynthesis , Nitric Oxide/biosynthesis , Stress, Physiological/metabolism , Cells, Cultured/metabolism , Epithelium/metabolism , Humans , Nitric Oxide/physiology
10.
Biochem J ; 323 ( Pt 2): 521-4, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9163347

ABSTRACT

The aim of this work was to investigate the role of nitric oxide (NO) on the macromolecular exocytotic function of human epithelial cells. We tested the effects of two NO-generating drugs, i.e. 1-hexanamine 6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl (MAHMA NONOate) and sodium nitroprusside (SNP), on mucin exocytosis from the human colonic epithelial HT29-Cl.16E cell line. Our results show that MAHMA NONOate and SNP elicited a rapid mucin exocytotic response through a cGMP-dependent and a cGMP-independent pathway respectively. Indeed, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a newly available specific inhibitor of soluble guanylate cyclase, inhibited both cGMP accumulation and subsequent mucin exocytosis evoked by MAHMA NONOate. By contrast, SNP did not alter intracellular cGMP levels, and SNP-mediated mucin exocytosis was not inhibited by ODQ. As expected from two NO donors acting through distinct pathways, the combined action of MAHMA NONOate and SNP led to an additive effect on mucin exocytosis. SNP was likely to act through S-nitrosylation of a cellular target, because cysteine, a reductive thiol that provides decoy targets for SNP through the formation of nitrosocysteine, abolished the early stimulatory effect of SNP on mucin exocytosis. Finally, the fact that in the presence of cysteine SNP was able to trigger a late, ODQ-inhibitable, mucin exocytotic response demonstrates the ability of NO to shift its intracellular signalling pathway depending on the changes of the redox state of the milieu.


Subject(s)
Cyclic GMP/metabolism , Exocytosis , Mucins/metabolism , Nitric Oxide/metabolism , 1-Methyl-3-isobutylxanthine/metabolism , Cysteine/pharmacology , Enzyme Inhibitors/pharmacology , Epithelium/metabolism , Exocytosis/drug effects , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , Humans , Hydrazines/pharmacology , Nitroprusside , Oxadiazoles/pharmacology , Quinoxalines/pharmacology , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism
12.
Gut ; 38(2): 240-2, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8801204

ABSTRACT

The stable differentiated human colonic epithelial cell line, HT29-C1.16E, was used to study the effects of interleukin-1 (IL-1) on mucin exocytosis. The main findings include: (a) IL-1 stimulated a rapid release of mucin from filter grown HT29-C1.16E cells, this effect being dose related; (b) this secretory effect was abolished in the presence of the blocking monoclonal antibody M4 specific for IL-1 receptors type I, showing that IL-1 receptors type I mediated IL-1 action; (c) experiments based on chamber cultures showed that these receptors were located on the basolateral membranes of HT29-C1.16E cells; (d) finally, mRNA for IL-1 receptors type I were detected by reverse transcriptase-polymerase chain reaction in these cells. To extend these findings to the in vivo situation, the rapid stimulatory effect of IL-1 on mucin exocytosis may contribute to the wash out of noxious agents during mucosal inflammation.


Subject(s)
Colon/chemistry , Interleukin-1/pharmacology , Mucins/drug effects , Receptors, Interleukin-1/drug effects , Antibodies, Monoclonal/drug effects , Cell Line , Colon/cytology , Exocytosis/drug effects , HT29 Cells , Humans , Mucins/metabolism , Receptors, Interleukin-1/analysis
13.
Biochem J ; 313 ( Pt 1): 35-8, 1996 Jan 01.
Article in English | MEDLINE | ID: mdl-8546706

ABSTRACT

We evaluated the effects of two NO donors, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), characterized by alternative redox states, i.e. nitrosonium ion (NO+) and nitric oxide (NO.) respectively, on intracellular interleukin-1 (IL-1) production, by a human colonic epithelial cell line (HT29-Cl.16E). SNP was able to induce intracellular IL-1 alpha production up to 10 h incubation, in a dose-dependent manner. Several experiments provide evidence that the NO+ redox form, and not the free radical NO., is implicated in the IL-1 alpha production: (i) SIN-1, devoid of any NO+ character, led to a very weak IL-1 production as compared with SNP; (ii) the reductive action of a thiol such as cysteine on NO+ led to a dose-dependent increase in NO, concentration, measured as NO2-/NO3- accumulation, and to large decrease in IL-1 production. Dibutyryl cGMP had no effect on IL-1 production, this finding supporting the concept that a cGMP-independent pathway is involved in the intracellular signalling of NO+. Together these results point out that NO, depending on its redox form, is able to modulate IL-1 production in cultured colonic epithelial cells.


Subject(s)
HT29 Cells/drug effects , HT29 Cells/metabolism , Interleukin-1/biosynthesis , Molsidomine/analogs & derivatives , Nitric Oxide/pharmacology , Nitroprusside/pharmacology , Vasodilator Agents/pharmacology , Cell Line , Epithelium/metabolism , Free Radicals/metabolism , Free Radicals/pharmacology , Humans , Kinetics , Molsidomine/metabolism , Molsidomine/pharmacology , Nitric Oxide/metabolism , Nitroprusside/metabolism , Oxidation-Reduction , Vasodilator Agents/metabolism
15.
Cell Signal ; 7(4): 319-23, 1995 May.
Article in English | MEDLINE | ID: mdl-8527300

ABSTRACT

Regulation by unsaturated fatty acids of glucocorticoid-sensitive gene transcription was studied in HeLa cells transiently transfected with a mouse mammary tumour virus-luciferase reporter gene. Arachidonic acid and docosahexaenoic acid by themselves had no effect on basal levels of luciferase expression. However, they were able to enhance dexamethasone-induced transcription by 1.4-2.3 times (25-42 times the control levels) in a dose-dependent manner (ED50: 18 and 8 microM) for arachidonic and docosahexaenoic acid, respectively. The glucocorticoid antagonist RU486 effectively antagonized the dexamethasone response as well as the synergistic effect observed in the presence of arachidonic and docosahexaenoic acids, suggesting that the glucocorticoid receptor was an intermediate in the fatty acid synergism of the dexamethasone response. These studies show that fatty acids may be playing a role in modulating the intracellular steroid hormone signalling pathway to co-regulate a glucocorticoid-sensitive promoter.


Subject(s)
Arachidonic Acid/pharmacology , Dexamethasone/pharmacology , Docosahexaenoic Acids/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Glucocorticoids/pharmacology , Luciferases/biosynthesis , Animals , Dexamethasone/antagonists & inhibitors , Dose-Response Relationship, Drug , Drug Synergism , Glucocorticoids/antagonists & inhibitors , HeLa Cells/enzymology , HeLa Cells/virology , Hormone Antagonists/pharmacology , Humans , Mammary Tumor Virus, Mouse/physiology , Mice , Mifepristone/pharmacology , Receptors, Glucocorticoid/physiology , Transcription, Genetic/drug effects , Transfection
16.
Steroids ; 59(1): 46-54, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8140602

ABSTRACT

Stimulating lipase activity with heparin (200 IU/kg b.w.) increased the plasma free fatty acid (FFA) concentration of immature rats (15 days). The effect of this elevated FFA concentration on glucocorticoid binding to corticosteroid binding globulin (CBG), and liver cytosol glucocorticoid receptor (GR), was analyzed. The plasma FFA concentration increased 2-fold, 10 minutes (P < 0.001), 20 minutes (P < 0.01), and 60 minutes (P < 0.01) post-heparin. The corticosterone (B) and progesterone concentrations were unchanged 60 minutes post-injection. The binding activity of immature rat CBG for B dropped 50% (P < 0.001) 60 minutes post-heparin injection, decreased B binding and increased plasma FFA were correlated (r = -0.8). The decreased B binding resulted from a 2-fold decrease in the apparent number of CBG binding sites; the affinity constant (Ka) remained unchanged. The liver cytosol endogenous FFA content of immature rats was also increased 2-fold, 60 minutes after heparin-induced lipolysis. The increased cytosol FFA, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor. The decrease resulted from a significantly lower apparent Ka for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between Ka (r = -0.93) and n (r = -0.90) and the increased liver cytosol FFA content. Thus the higher plasma FFA induced in vivo by lipase activation or a standard FFA mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR.


Subject(s)
Fatty Acids, Nonesterified/pharmacology , Glucocorticoids/metabolism , Liver/metabolism , Receptors, Glucocorticoid/metabolism , Transcortin/metabolism , Animals , Corticosterone/blood , Cytosol/metabolism , Dexamethasone/metabolism , Fatty Acids, Nonesterified/blood , Fatty Acids, Nonesterified/metabolism , Heparin/pharmacology , Kinetics , Lipase/blood , Lipids/blood , Lipolysis/drug effects , Male , Progesterone/blood , Rats , Rats, Wistar
17.
Acta Endocrinol (Copenh) ; 129(4): 348-55, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8237254

ABSTRACT

Polyunsaturated fatty acids have been shown to decrease the binding of [3H]dexamethasone to rat liver glucocorticoid receptors by mixed non-competitive inhibition, suggesting that these fatty acids interact at a site on the receptor different from the hormone binding site. The present study was undertaken to localize the site of interaction of polyunsaturated fatty acids on the receptor by comparing the differential effects of docosahexaenoic acid (a 22-carbon polyunsaturated fatty acid of the series n-3) on antagonist (RU486) and agonist binding, by covalent cross-linking of the hsp 90 and other proteins to the receptor to attempt to mask the site of interaction, by limited trypsinization to cleave the site and by using antibodies against specific epitopes to prevent fatty acid access by steric hindrance. Binding [3H]RU486 was not inhibited by docosahexaenoic acid at a concentration (60 mumol/l) that increases the dissociation constant of [3H]dexamethasone eightfold. Covalent stabilization of the hetero-oligomeric glucocorticoid receptor structure did not keep the fatty acid from inhibiting [3H]dexamethasone binding. The binding to the receptor of monoclonal and polyclonal antibodies against different domains of the receptor did not sterically hinder the fatty acid interaction with the receptor. After limited trypsinization of the receptor, the fatty acid still increased the dissociation rate constant of [3H]dexamethasone binding, indicating that the site of interaction of polyunsaturated fatty acids is on a fragment of the receptor containing the hormone-binding domain and some sequences C-terminal of the DNA-binding domain.


Subject(s)
Fatty Acids, Unsaturated/metabolism , Liver/metabolism , Receptors, Glucocorticoid/metabolism , Animals , Antibodies, Monoclonal , Cytosol/metabolism , Dexamethasone/metabolism , Docosahexaenoic Acids/pharmacology , Male , Mifepristone/metabolism , Rats , Rats, Sprague-Dawley , Tissue Distribution
18.
AIDS Res Hum Retroviruses ; 9(8): 755-60, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8217344

ABSTRACT

Liposoluble extracts of serum from healthy men and AIDS patients (stages IVC1 and IVD by CDC criteria) inhibited the incorporation of [3H]thymidine into isolated rat thymocytes, but AIDS extracts were less inhibitory, requiring 1.8 times more cortisol in the AIDS extracts than in the healthy extracts to inhibit [3H]thymidine incorporation by 50%. Although the total serum extracts from AIDS patients contained 1.7 times more cortisol than the extracts from healthy controls, the AIDS extracts decreased the binding affinity (Ka) of [3H]dexamethasone to rat thymus glucocorticoid receptors by 50% less than the healthy control extracts. The present study seems to indicate that a substance(s) can be extracted from the serum of AIDS patients that attenuates the inhibitory effect of cortisol on thymocyte proliferation and interferes with the binding of cortisol to the glucocorticoid receptor.


Subject(s)
Acquired Immunodeficiency Syndrome/blood , Dexamethasone/pharmacology , Thymus Gland/drug effects , Animals , Cells, Cultured , Cytosol/metabolism , Dexamethasone/antagonists & inhibitors , Dexamethasone/metabolism , Fatty Acids, Nonesterified/blood , Humans , Hydrocortisone/blood , Male , Rats , Rats, Sprague-Dawley , Receptors, Glucocorticoid/metabolism , Solubility , Thymidine , Thymus Gland/cytology
19.
Cell Signal ; 5(2): 181-6, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8388702

ABSTRACT

Activity of the enzyme phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) was demonstrated in MCF-7 human breast cancer cell homogenate. The addition of 10(-9) M 17 beta-estradiol to the culture medium elicited in the cells two types of responses depending on the period of exposure. Enzyme activity was rapidly activated at 15 s of incubation. After 5 min, PIP2-PLC activity was inhibited, and this effect continued at least until 24 h of exposure to the hormone. When 17 beta-estradiol was added in vitro to the total homogenate of untreated cells, enzyme activity was stimulated in a dose-dependent manner. These findings indicate that 17 beta-estradiol induces early and long-term modifications of the phosphoinositide signal pathway in intact MCF-7 cells as well as in vitro. The rapidity of the early effect suggests a non-genomic action of estradiol.


Subject(s)
Estradiol/pharmacology , Phosphoric Diester Hydrolases/metabolism , Signal Transduction/drug effects , Cell Division , Enzyme Activation/drug effects , Humans , Phosphoinositide Phospholipase C , Time Factors , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology
20.
AIDS Res Hum Retroviruses ; 8(9): 1693-7, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1457214

ABSTRACT

The total liposoluble extract of sera from AIDS patients, IVC1 and IVD stages, containing cortisol and free fatty acids (FFA) inhibited [3H]dexamethasone binding to a lesser extent than did the same quantity of total liposoluble extract of sera from healthy men. FFA isolated from extracts of AIDS sera by Sephadex LH20 chromatography had less effect on [3H]dexamethasone binding to rat liver glucocorticoid receptor than those extracted from sera of healthy men. These results suggest the presence in sera of AIDS patients of a liposoluble substance which could be limiting the inhibitory effect of FFA on [3H]dexamethasone binding to glucocorticoid receptor by inducing a conformational change in glucocorticoid receptor that could alter the biological action of glucocorticoids. The pathological consequence could be the apparent contradiction of high cortisolemia and clinical symptoms of adrenal insufficiency that have been observed in AIDS patients.


Subject(s)
Acquired Immunodeficiency Syndrome/blood , Cytosol/metabolism , Liver/metabolism , Receptors, Glucocorticoid/metabolism , Adult , Animals , Dexamethasone/metabolism , Fatty Acids, Nonesterified/metabolism , Humans , Hydrocortisone/metabolism , Lipid Metabolism , Male , Rats , Rats, Wistar , Solubility
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