RESUMEN
The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an anti-fibrotic lipid mediator that induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death are still unresolved. Interestingly, the inducible isoform of cyclooxygenase, COX-2, can metabolize 2-AG to pro-apoptotic prostaglandin glycerol esters (PG-GEs). We analyzed the roles of COX-2 and endocannabinoid-derived PG-GEs in the differential susceptibility of primary activated HSCs and hepatocytes toward 2-AG-induced cell death. HSCs displayed significant COX-2 expression in contrast to hepatocytes. Similar to 2-AG, treatment of HSCs with PGD2-GE dose-dependently induced cell death independently from cannabinoid receptors that was accompanied by PARP- and caspase 3-cleavage. In contrast to 2-AG, PGD2-GE failed to induce significant ROS formation in HSCs, and depletion of membrane cholesterol did not rescue HSCs from PGD2-GE-induced apoptosis. These findings indicate differential engagement of initial intracellular signaling pathways by 2-AG and its COX-2-derived metabolite PGD2-GE, but similar final cell death pathways. Other PG-GEs, such as PGE2-or PGF2α-GE did not induce apoptosis in HSCs. Primary rat hepatocytes were mainly resistant against 2-AG- and PGD2-GE-induced apoptosis. HSCs, but not hepatocytes were able to metabolize 2-AG to PGD2-GE. As a proof of principle, HSCs from COX-2(-/-) mice lacked PDG2-GE production after 2-AG treatment. Accordingly, COX-2(-/-) HSCs were resistant against 2-AG-induced apoptosis. In conclusion, the divergent expression of COX-2 in HSCs and hepatocytes contributes to the different susceptibility of these cell types towards 2-AG-induced cell death due to the generation of pro-apoptotic PGD2-GE by COX-2 in HSCs. Modulation of COX-2-driven metabolization of 2-AG may provide a novel physiological concept allowing the specific targeting of HSCs in liver fibrosis.
Asunto(s)
Apoptosis/fisiología , Ácidos Araquidónicos/administración & dosificación , Ciclooxigenasa 2/metabolismo , Endocannabinoides/administración & dosificación , Glicéridos/administración & dosificación , Células Estrelladas Hepáticas/fisiología , Hepatocitos/fisiología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endocannabinoides/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de OxígenoRESUMEN
BACKGROUND: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. OBJECTIVES: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. METHODS: S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. RESULTS: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 µM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. CONCLUSIONS: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.
Asunto(s)
Plaquetas/metabolismo , Lisofosfolípidos/sangre , Esfingosina/análogos & derivados , Tromboxanos/biosíntesis , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Células Cultivadas , Cromatografía en Capa Delgada , Humanos , Receptores de Tromboxanos/agonistas , Esfingosina/sangre , Trombina/farmacologíaRESUMEN
We used an experimental gingivitis study design to compare crevicular fluid concentrations of Migration Inhibitory Factor (MIF) and Prostaglandin E(2) (PGE(2)) in younger (18 to 30 yrs) and older (46 to 77 yrs) healthy adults. PGE(2) increased after 1 wk in younger participants, whereas it decreased in older individuals after 1 wk of plaque accumulation. A significant interaction between age and time was observed for PGE(2) (p = 0.04). High concentrations of MIF were identified in both age groups at baseline. MIF increased in the younger participants, whereas in the older individuals a decrease over time was observed. MIF concentration was positively correlated with plaque index and gingival index in the older age group. Total counts of bacteria, Parvimonas micra and Prevotella intermedia, were significantly correlated with MIF concentration in older participants. In conclusion, MIF and PGE(2) production in response to bacterial accumulation seems to be modified by age.
Asunto(s)
Dinoprostona/metabolismo , Líquido del Surco Gingival/química , Gingivitis/inmunología , Gingivitis/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Técnicas de Tipificación Bacteriana , Placa Dental/microbiología , Dinoprostona/análisis , Femenino , Gingivitis/microbiología , Humanos , Inmunidad Celular/fisiología , Factores Inhibidores de la Migración de Macrófagos/análisis , Masculino , Persona de Mediana Edad , Peptostreptococcus/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Prevotella intermedia/aislamiento & purificación , Adulto JovenRESUMEN
BACKGROUND: Selective inhibitors of cyclooxygenase-2 (COX-2) called coxibs, are effective anti-inflammatory and analgesic drugs. Recently, these drugs were associated with an increased risk for myocardial infarction and atherothrombotic events. The hypothesis of thromboxane-prostacyclin imbalance has been preferred to explain these unwanted effects. METHODS: We studied the effects of 14 days intake of rofecoxib (25 mg q.d.), celecoxib (200 mg b.i.d.), naproxen (500 mg b.i.d.) and placebo in a randomized, blinded, placebo-controlled study in young healthy volunteers (median age 25-30 years, each group n = 10). We assessed prostanoid metabolite excretion (PGE-M, TXB(2), 6-keto-PGF(1alpha), 11-dehydro-TXB(2), 2,3-dinor-TXB(2), and dinor-6-keto-PGF(1alpha)), the expression of platelet activation markers (CD62P, PAC-1, fibrinogen), platelet-leukocyte formation, the endogenous thrombin potential, platelet cAMP content and plasma thrombomodulin level. RESULTS: Naproxen suppressed biosynthesis of PGE-M, prostacyclin metabolites and thromboxane metabolites and thrombomodulin levels. In contrast, both coxibs had an inhibitory effect only on PGE-M, 6-keto-PGF(1alpha), and on dinor-6-keto-PGF(1alpha), whereas TXB(2), 2,3-dinor-TXB(2) and 11-dehydro-TXB(2) excretion were unaffected. None of the coxibs exerted significant effects on the expression of platelet activation markers, cAMP generation, platelet-leukocyte formation, or on thrombomodulin plasma levels. Interestingly, platelet TXB(2) release during aggregation was enhanced after coxib treatment following arachidonic acid or collagen stimulation. CONCLUSION: In young healthy volunteers coxibs inhibit systemic PGE(2) and PGI(2) synthesis. Platelet function and expression of platelet aggregation markers are not affected; however, coxibs can stimulate TXB(2) release from activated platelets. Combined decrease in vasodilatory PGE(2) and PGI(2) together with increased TXA(2) in proaggregatory conditions may contribute to coxib side effects.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/sangre , Lactonas/farmacología , Naproxeno/farmacología , Activación Plaquetaria/efectos de los fármacos , Prostaglandinas/sangre , Pirazoles/farmacología , Sulfonamidas/farmacología , Sulfonas/farmacología , Adulto , Antígenos de Neoplasias/sangre , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Plaquetas/metabolismo , Celecoxib , Adhesión Celular/efectos de los fármacos , AMP Cíclico/sangre , Inhibidores de la Ciclooxigenasa 2/efectos adversos , Inhibidores de la Ciclooxigenasa 2/sangre , Método Doble Ciego , Fibrinógeno/metabolismo , Humanos , Lactonas/efectos adversos , Lactonas/sangre , Leucocitos/efectos de los fármacos , Masculino , Naproxeno/efectos adversos , Naproxeno/sangre , Selectina-P/sangre , Fosfolipasas A/sangre , Agregación Plaquetaria/efectos de los fármacos , Glicoproteína IIb de Membrana Plaquetaria/sangre , Prostaglandinas/orina , Pirazoles/efectos adversos , Pirazoles/sangre , Valores de Referencia , Sulfonamidas/efectos adversos , Sulfonamidas/sangre , Sulfonas/efectos adversos , Sulfonas/sangre , Trombina/metabolismo , Trombomodulina/sangre , Tromboxanos/sangre , Tromboxanos/orina , Factores de TiempoRESUMEN
BACKGROUND AND OBJECTIVE: The aim of this study was to identify the expression of cyclooxygenase-1, cyclooxygenase-2, cyclooxygenase-3, and microsomal prostaglandin E synthase-1 in young and elderly subjects. MATERIAL AND METHODS: Periodontally healthy subjects were divided into young (18-30 years, n = 7) and elderly (46-77 years, n = 7). A gingival biopsy was taken at baseline. After experimental gingivitis, clinical examination was repeated and a second biopsy was taken. The expression of cyclooxygenase-1, cyclooxygenase-2, cyclooxygenase-3, and microsomal prostaglandin E synthase-1 was analyzed by means of immunohistochemistry. RESULTS: In both healthy age groups, cyclooxygenase-1 and microsomal prostaglandin E synthase-1 were expressed in epithelial cells, endothelial cells and fibroblast-like connective tissue cells. Cyclooxygenase-1 was found in Langerhans' cells of the epithelium. Cyclooxygenase-2 expression was observed in cells exhibiting the morphology of epithelial mitosis cells, and the expression of cyclooxygenase-2 in periodontally healthy elderly subjects was significantly lower (p < or = 0.05). Following experimental gingivitis, cyclooxygenase-1 and microsomal prostaglandin E synthase-1 expression did not change. However, the expression of cyclooxygenase-2 was significantly increased in both age groups (p < or = 0.05). Cyclooxygenase-3 was not detected in any group investigated. CONCLUSION: Cyclooxygenase-1 and microsomal prostaglandin E synthase-1 were expressed constitutively in gingival tissue, and expression was unaffected by age or inflammation states. In contrast, the expression of cyclooxygenase-2 was weaker in elderly subjects. In the course of experimental gingivitis, cyclooxygenase-2 was induced in both age groups.
Asunto(s)
Encía/enzimología , Gingivitis/enzimología , Prostaglandinas/biosíntesis , Adolescente , Adulto , Factores de Edad , Anciano , Biopsia/métodos , Ciclooxigenasa 1/análisis , Ciclooxigenasa 2/análisis , Métodos Epidemiológicos , Femenino , Encía/citología , Gingivitis/etiología , Humanos , Oxidorreductasas Intramoleculares/análisis , Masculino , Persona de Mediana Edad , Prostaglandina-E Sintasas , Prostaglandina-Endoperóxido Sintasas/análisisRESUMEN
Hyperprostaglandin E syndrome/antenatal Bartter syndrome is characterized by NaCl wasting and volume depletion, juxtaglomerula hypertrophy, hyperreninism and secondary hyperaldosteronism. Primary causes are mutations in the gene for Na-K-2Cl-cotransporter, NKCC2, or for potassium channel, ROMK, responsible for medullary NaCl malabsorption. Most intriguing aspect of the syndrome is the association with a massively increased renal prostaglandin production which contributes substantially to the clinical picture of the patients. Therefore the term hyperprostaglandin E syndrome has been introduced. It is unclear how prostaglandins aggravate the NaCl transport deficiency. Aspects to prostaglandin synthesis and receptor-mediated function within the kidney in patients suffering from hyperprostaglandin E syndrome/antenatal Bartter syndrome will be discussed.
Asunto(s)
Síndrome de Bartter/fisiopatología , Prostaglandina-Endoperóxido Sintasas/fisiología , Receptores de Prostaglandina E/fisiología , Diuresis , Humanos , Riñón/metabolismo , Prostaglandinas E/biosíntesisRESUMEN
Hyaluronic acid (HA) is a prominent constituent of the extracellular matrix of atherosclerotic vascular lesions in humans known to modulate vascular smooth muscle phenotype. The regulation of HA synthesis by vasodilatory prostaglandins was analyzed in human arterial smooth muscle cells (SMCs). The prostacyclin analogue, iloprost (100 nmol/L), markedly increased pericellular formation of HA coats and HA secretion into the cell culture medium in human arterial SMCs (8.7+/-1.6-fold). Expression of HA synthase 2 (HAS2) was determined by semiquantitative RT-PCR and found to be strongly upregulated at concentrations of iloprost between 1 and 100 nmol/L after 3 hours. Furthermore, endogenous cyclooxygenase-2 (COX2) activity was required for basal expression of HAS2 mRNA in SMCs in vitro. Total HA secretion in response to iloprost was markedly decreased by RNA interference (RNAi), specific for HAS2. In addition, siRNA targeting HAS2 strongly increased the spreading of human SMCs compared with mock-transfected cells. HAS2 mRNA levels were also stimulated by a selective prostacyclin receptor (IP) agonist, cicaprost (10 nmol/L), prostaglandin E(2) (10 nmol/L), and the EP(2) receptor agonist, butaprost (1 micromol/L). Induction of HAS2 mRNA and HA synthesis by prostaglandins was mimicked by stable cAMP analogues and forskolin. In human atherectomy specimens from the internal carotid artery, HA deposits and COX2 expression colocalized frequently. In addition, strong EP(2) receptor expression was detected in SMCs in HA-rich areas. Therefore, upregulation of HAS2 expression via EP(2) and IP receptors might contribute to the accumulation of HA during human atherosclerosis, thereby mediating proatherosclerotic functions of COX2.
Asunto(s)
Alprostadil/análogos & derivados , Arteriosclerosis/metabolismo , Epoprostenol/análogos & derivados , Matriz Extracelular/metabolismo , Glucuronosiltransferasa/biosíntesis , Iloprost/farmacología , Músculo Liso Vascular/efectos de los fármacos , Receptores de Prostaglandina E/efectos de los fármacos , Sulfonamidas , Vasodilatadores/farmacología , 6-Cetoprostaglandina F1 alfa/biosíntesis , Acetofenonas/farmacología , Alprostadil/farmacología , Arteriosclerosis/patología , Becaplermina , Benzopiranos/farmacología , Bucladesina/farmacología , Enfermedades de las Arterias Carótidas/patología , Arteria Carótida Interna/patología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colforsina/farmacología , AMP Cíclico/fisiología , Ciclooxigenasa 2 , Inducción Enzimática/efectos de los fármacos , Epoprostenol/farmacología , Glucuronosiltransferasa/genética , Humanos , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/metabolismo , Indoles/farmacología , Isoenzimas/fisiología , Isoquinolinas/farmacología , Macrófagos/metabolismo , Maleimidas/farmacología , Proteínas de la Membrana , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Toxina del Pertussis/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Prostaglandina-Endoperóxido Sintasas/fisiología , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Subtipo EP2 de Receptores de Prostaglandina ERESUMEN
Thromboxane A(2) (TXA(2)) is an important mediator for platelet aggregation and blood vessel constriction. TXA(2) receptor (TP receptor) is expressed in different cell types including smooth muscle cells, endothelial cells and platelets. Expression level of TP receptor may modulate the action of TXA(2) on target cells. In megakaryoblastic MEG-01 cells, a cell line representing a model for platelet precursor cells, addition of phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) caused an increase in transcriptional activity of TP receptor gene promoter. Within 20 h a rise in expression of TP receptor mRNA and protein was observed. The effect of TPA was concentration-dependent and was blocked by specific inhibitors of protein kinase C. Flow cytometry analysis indicated that the increase in TP receptor expression appeared to be one of the earliest events in the course of TPA-induced maturation of MEG-01 cells. Stimulation of the protein kinase A pathway by incubation with forskolin or IBMX caused a decrease in transcriptional activity. Promoter deletion experiments indicated that the responsive elements for protein kinase A and C are located upstream and downstream, respectively, of -700 bp of the TP receptor gene. These experiments indicate that the expression of the human thromboxane receptor is differently regulated in platelet precursor cells by the protein kinase A and C pathway.
Asunto(s)
Regulación de la Expresión Génica , Megacariocitos/fisiología , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Plaquetas/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Genes Reporteros , Humanos , Megacariocitos/citología , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Transcripción GenéticaRESUMEN
We examined the direct epithelial effects of the major product of arachidonic acid metabolism in the kidney, prostaglandin E(2) (PGE(2)), on ion transport and signal transduction in the hormone-sensitive Madin-Darby canine kidney (MDCK) C7 subclone as a model of renal collecting duct principal cells. MDCK C7 cells were grown on microporous permeable filter supports and mounted in Ussing-type chambers. Reverse transcriptase (RT)-PCR and sequencing were used to determine E-prostanoid (EP) receptor expression. Basolateral and, about 14-fold less potent, apical addition of PGE(2) increased short-circuit current (I(sc)) in a concentration-dependent manner. This ion transport was biphasic with a rapid peak not detectable under chloride-free conditions. The remaining, stably elevated current was unaffected by furosemide, hydrochlorothiazide, ethylisopropanol amiloride, and 5-nitro-2-(3-phenyl-propyl-amino)benzoic acid (NPPB). In contrast, apical amiloride (10 microM) significantly decreased I(sc), indicating sodium reabsorption. The effect of PGE(2) was attenuated in the presence of vasopressin. Agonists acting by cAMP elevation like dibutyryl-cAMP and theophylline also induced an amiloride-sensitive ion transport with similar kinetics as PGE(2). Moreover, PGE(2) rapidly increased intracellular cAMP levels. RT-PCR demonstrated mRNA expression of the epithelial sodium channel (ENaC), and of the EP2 receptor in MDCK C7 cells. Accordingly, EP2 receptor agonist butaprost mimicked PGE(2) epithelial action. In conclusion, PGE(2) induces amiloride-sensitive sodium reabsorption in MDCK C7 monolayers. This ion transport is most likely mediated by EP2 receptor activation leading to increased intracellular cAMP levels. Therefore, PGE(2) might also contribute to Na(+) reabsorption in the mammalian collecting duct.
Asunto(s)
Alprostadil/análogos & derivados , Dinoprostona/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales Colectores/metabolismo , Sodio/metabolismo , Alprostadil/farmacología , Animales , Bucladesina/farmacología , Línea Celular , AMP Cíclico/metabolismo , Perros , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio , Túbulos Renales Colectores/citología , Técnicas de Placa-Clamp , Prostaglandinas E Sintéticas/farmacología , Receptores de Prostaglandina E/metabolismo , Sistemas de Mensajero Secundario/fisiología , Canales de Sodio/genética , Canales de Sodio/metabolismo , Teofilina/farmacologíaRESUMEN
Thromboxane is an important modulator of hemostasis and smooth muscle tonus and signals via G-protein-coupled thromboxane receptor. Previously, we characterized the TP receptor gene and suggested the presence of three promoter regions within the gene. The aim of the present study was to examine the regulation of transcriptional gene expression. By primer extension experiments the major transcription initiation site was shown to be a doublet at -160/165 bp upstream of the ATG codon in human megakaryoblastic MEG-01 cells, endothelial ECV 304 cells and in human myometrium smooth muscle cells. In the erythroleukemic HEL 1 cells transcription initiation site was identified at -10 bp. Transcriptional activity of the three 5'flanking regions of TP receptor gene representing the putative promoter regions was evaluated by transfection of MEG-01 cells with chimeric constructs containing luciferase gene-encoding sequence. Promoter region I displayed highest transcriptional activity and RT-PCR analysis confirmed the transcription of TP receptor mRNA driven by promoter I. Although, weak transcriptional activity was also observed regarding promoter region II, we were unable to amplify cDNA fragments representing promoter II-driven mRNA synthesis. Considering promoter region III, transcriptional activity was barely detectable. Various deletions of the 3.9 kb promoter I region revealed a size-dependent transcriptional activity. Further, for full activity a 'core' promoter corresponding to the region from -160/165 to -588 bp appeared to be necessary for full transcriptional activity of promoter 1.
Asunto(s)
Región de Flanqueo 5'/genética , Regiones Promotoras Genéticas/genética , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Línea Celular , Clonación Molecular , Regulación de la Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: It is well established that the diuretic- and renin-stimulated effects of loop diuretics can be attenuated by nonselective cyclooxygenase inhibitors. Since it is yet unclear which of the isoforms of cyclooxygenases, COX-1 and COX-2, is relevant in this context, our study aimed to determine the effects of selective COX-2 inhibition on the renal effects of the loop diuretic furosemide, as well as the diuretic hydrochlorothiazide, which acts on the distal tubule. METHOD: Male Sprague-Dawley rats were treated with furosemide (12 mg/day subcutaneously by osmotic pump) or hydrochlorothiazide (30 mg/kg body weight/day orally by gavage). In addition, parallel groups received rofecoxib (1 to 10 mg/kg body weight/day) for selective inhibition of COX-2. Controls were treated with vehicle. RESULTS: Induction of COX-2 mRNA expression due to furosemide was paralleled by increased renal excretion of prostanoids. Also, hydrochlorothiazide led to a rise in prostanoid excretion. Rofecoxib blunted the diuretic-induced increase in prostanoid excretion, thus confirming an effective blockade of COX-2. Moreover, the COX-2 inhibitor rofecoxib dose-dependently attenuated diuresis and saluresis, as well as the stimulation of the renin system induced by furosemide. Furthermore, rofecoxib completely reversed diuresis and saluresis and prevented the increase of plasma renin activity induced by hydrochlorothiazide. CONCLUSIONS: These findings suggest that COX-2-derived prostanoids are of major relevance in modulating the renal effects of diuretics. COX-2 inhibitors might be valuable drugs to treat salt and water wasting during Bartter and Gitelman diseases.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Diuréticos/farmacología , Isoenzimas/fisiología , Lactonas/farmacología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Isoenzimas/genética , Masculino , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Renina/genética , Cloruro de Sodio/metabolismo , SulfonasRESUMEN
The aim of this study was to examine the effects of highly selective inhibition of cyclooxygenase 2 (COX-2) with rofecoxib on the renin system during long-term stimulation and after short-term stimulation. Six healthy male volunteers received, in a randomized crossover design, a low-sodium diet for days 1 through 9 with or without 25 mg rofecoxib twice daily on days 5 through 9 and, in addition, 20 mg of furosemide intravenously on day 8. Plasma renin activity increased 2 to 3 times over baseline with a low-sodium diet and 5 times over baseline 30 minutes after intravenous furosemide; it was still elevated nearly 5 times on day 9. These effects were completely blocked by rofecoxib. Plasma aldosterone and urinary aldosterone concentrations basically reflected the findings with plasma renin activity. Urinary sodium excretion decreased during a low-sodium diet and increased after intravenous furosemide without being significantly affected by rofecoxib. We have concluded that low-sodium and furosemide-stimulated renin and aldosterone secretion is completely blocked in healthy volunteers during COX-2 inhibition with rofecoxib, suggesting that intact COX-2 is of major importance for stimulation of the renin system under these conditions in man.
Asunto(s)
Dieta Hiposódica , Diuréticos/farmacología , Furosemida/farmacología , Isoenzimas/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Renina/sangre , Adulto , Aldosterona/metabolismo , Creatinina/sangre , Estudios Cruzados , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/orina , Humanos , Lactonas/farmacología , Masculino , Proteínas de la Membrana , Sodio/orina , SulfonasRESUMEN
Patients with hyperprostaglandin E syndrome/antenatal Bartter syndrome typically have renal salt wasting, hypercalciuria with nephrocalcinosis, and secondary hyperaldosteronism. Antenatally, these patients have fetal polyuria, leading to polyhydramnios and premature birth. Hyperprostaglandin E syndrome/antenatal Bartter syndrome is accompanied by a pathologically elevated synthesis of prostaglandin E(2), thought to be responsible for aggravation of clinical symptoms such as salt and water loss, vomiting, diarrhea, and failure to thrive. In this study administration of the cyclooxygenase-2 (COX-2) specific inhibitor nimesulide to patients with hyperprostaglandin E syndrome/antenatal Bartter syndrome blocked renal prostaglandin E(2) formation and relieved the key parameters hyperprostaglandinuria, secondary hyperaldosteronism, and hypercalciuria. Partial suppression of serum thromboxane B(2) synthesis resulting from platelet COX-1 activity and complete inhibition of urinary 6-keto-prostaglandin F(1alpha), reflecting endothelial COX-2 activity, indicate preferential inhibition of COX-2 by nimesulide. Amelioration of the clinical symptoms by use of nimesulide indicates that COX-2 may play an important pathogenetic role in hyperprostaglandin E syndrome/antenatal Bartter syndrome. Moreover, on the basis of our data we postulate that COX-2-derived prostaglandin E(2) is an important mediator for stimulation of the renin-angiotensin-aldosterone system in the kidney.
Asunto(s)
Síndrome de Bartter/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas E/sangre , Sulfonamidas/uso terapéutico , Tromboxano B2/análogos & derivados , 6-Cetoprostaglandina F1 alfa/orina , Adolescente , Síndrome de Bartter/sangre , Síndrome de Bartter/fisiopatología , Síndrome de Bartter/orina , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Niño , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/orina , Humanos , Indometacina/uso terapéutico , Riñón/efectos de los fármacos , Riñón/metabolismo , Proteínas de la Membrana , Prostaglandinas E/orina , Tromboxano B2/análisis , Tromboxano B2/biosíntesis , Tromboxano B2/orinaRESUMEN
This study aimed to assess the impact of cyclooxygenase-2 (COX-2) on the secretion and expression of renin in the kidney cortex. For this purpose renocortical COX-2 expression was moderately stimulated by a low-salt diet or strongly stimulated (increase in mRNA about fivefold) by the combination of a low-salt diet and the angiotensin-I-converting enzyme inhibitor ramipril in male Sprague-Dawley rats. None of these manoeuvres changed medullary COX-2 expression or cortical or medullary COX-1 expression. Treatment with low salt plus ramipril but not with low salt alone led to a three- to fourfold increase of the urinary output of all major prostanoids. The selective COX-2 inhibitor rofecoxib (10 mg/kg per day) markedly lowered basal urinary prostanoid excretion and blunted the stimulation of prostanoid excretion during treatment with low salt plus ramipril. The stimulation of renin secretion by the low-salt diet but not by low salt plus ramipril was attenuated by rofecoxib. The low-salt diet led to a moderate increase of renin gene expression, and additional treatment with ramipril caused a 15-fold increase of renin mRNA. However, no effect of rofecoxib on renin gene expression was observed in any group. These findings suggest that stimulation of COX-2 in the renal cortex leads to the increased formation of all major prostanoids. COX-2-derived prostanoids may play a role in the regulation of renin secretion but not in renin gene expression during the intake of a low-salt diet. However, no major relevance of COX-2-derived prostanoids to renin secretion or renin gene expression during ramipril treatment or a combination of ramipril and a low-salt diet was found.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Expresión Génica/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Corteza Renal/metabolismo , Prostaglandinas/orina , Renina/genética , 6-Cetoprostaglandina F1 alfa/orina , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dieta Hiposódica , Dinoprostona/orina , Isoenzimas/genética , Médula Renal/enzimología , Lactonas/farmacología , Masculino , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/genética , Ramipril/farmacología , Ratas , Ratas Sprague-Dawley , Renina/sangre , Renina/metabolismo , SulfonasRESUMEN
Isoprostanes comprise a group of free radical-catalyzed products of arachidonic acid. However, there is recent evidence pointing towards an enzyme-dependent formation of isoprostanes. With the use of isolated rat glomeruli we addressed the mechanisms of isoprostane generation. Synthesis of prostanoids and isoprostanes, including 8-epi-PGF(2alpha), was studied under conditions favouring radical formation. Cultured glomeruli formed different prostanoids including 8-epi-PGF(2alpha). Upon LPS challenge cyclo-oxygenase (COX)-2 expression was enhanced, and this was paralleled by a 2 - 9-fold increase in prostanoid formation, including isoprostanes. Addition of COX-isoform unselective inhibitors (diclofenac, indomethacin) or a selective inhibitor (NS-398) suppressed the synthesis of prostanoids, 8-epi-PGF(2alpha) and total isoprostane fraction; however, inhibition of the latter was less pronounced. Antioxidants such as butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), or dimethylurea exhibited an only minimal inhibitory effect on 8-epi-PGF(2alpha) synthesis. Moreover, ROS-generating drugs (menadione, methylviologen) or NADPH-driven radical formation were unable to cause the generation of significant amounts of 8-epi-PGF(2alpha) by rat glomeruli. In contrast, the total isoprostane fraction could be increased by menadione addition. These data provide further evidence for a radical-independent, but COX-dependent formation of 8-epi-PGF(2alpha) in renal tissue. Regarding the other isoprostanes, both radicals and COX enzymes contribute to their formation. Based on our data we assume that elevated release of vasoactive 8-epi-PGF(2alpha) has to be expected under conditions when the prostanoid system in the kidney is stimulated, e.g. under inflammatory conditions. Regarding renal oxidative injuries, the usefulness of 8-epi-PGF(2alpha) as a representative marker molecule of oxidative stress has to be questioned.
Asunto(s)
Dinoprost/metabolismo , Radicales Libres/metabolismo , Glomérulos Renales/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Ácido Araquidónico/química , Ácido Araquidónico/farmacología , Western Blotting , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Diclofenaco/farmacología , Dinoprost/análogos & derivados , Relación Dosis-Respuesta a Droga , Epoprostenol/genética , Epoprostenol/metabolismo , Ésteres , F2-Isoprostanos , Femenino , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/enzimología , Masculino , Proteínas de la Membrana , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitrobencenos/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sulfonamidas/farmacología , Tromboxano-A Sintasa/genética , Tromboxano-A Sintasa/metabolismoRESUMEN
BACKGROUND: Bartter-like syndrome (BLS) is a heterogeneous set of congenital tubular disorders that is associated with significant renal salt and water loss. The syndrome is also marked by increased urinary prostaglandin E2 (PGE2) excretion. In rodents, salt and volume depletion are associated with increased renal macula densa cyclooxygenase-2 (COX-2) expression. The expression of COX-2 in human macula densa has not been demonstrated. The present studies examined whether COX-2 can be detected in macula densa from children with salt-wasting BLS versus control tissues. METHODS: The intrarenal distribution of COX-2 protein and mRNA was analyzed by immunohistochemistry and in situ hybridization in 12 patients with clinically and/or genetically confirmed BLS. Renal tissue rejected for transplantation, from six adult patients not affected by BLS, was also examined. RESULTS: The expression of COX-2 immunoreactive protein was observed in cells of the macula densa in 8 out 11 patients with BLS. In situ hybridization confirmed the expression of COX-2 mRNA in the macula densa in 6 out of 10 cases. COX-2 protein was also detected in the macula densa in a patient with congestive heart failure. The expression of COX-2 immunoreactive protein was not observed in cells associated with the macula densa in kidneys from patients without disorders associated with hyper-reninemia. CONCLUSION: These studies demonstrate that COX-2 may be detected in the macula densa of humans. Since macula densa COX-2 was detected in cases of BLS, renal COX-2 expression may be linked to volume and renin status in humans, as well as in animals.
Asunto(s)
Síndrome de Bartter/metabolismo , Isoenzimas/genética , Aparato Yuxtaglomerular/enzimología , Prostaglandina-Endoperóxido Sintasas/genética , Síndrome de Bartter/patología , Niño , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Hibridación in Situ , Isoenzimas/análisis , Aparato Yuxtaglomerular/patología , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/análisis , ARN Mensajero/análisis , Renina/metabolismo , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Four prostaglandin E2 receptor subtypes designated EP1, EP2, EP3, and EP4 have been shown to mediate a variety of effects of prostaglandin E2 (PGE2) on glomerular hemodynamics, tubular salt and water reabsorption, and on blood vessels in the human kidney. Despite the important role of renal PGE2, the localization of PGE2 receptor proteins in the human kidney is unknown. The present study used antipeptide antibodies to the EP1 to EP4 receptor proteins for immunolocalization in human kidney tissue. Immunoblot studies using these antibodies demonstrated distinct bands in membrane fraction from human kidney. By means of immunohistochemistry, expression of the human EP1 receptor subtype protein in renal tissue was detected mainly in connecting segments, cortical and medullary collecting ducts, and in the media of arteries and afferent and efferent arterioles. The human EP2 receptor subtype protein was detectable only in the media of arteries and arterioles. The human EP3 receptor subtype protein was strongly expressed in glomeruli, Tamm-Horsfall negative late distal convoluted tubules, connecting segments, cortical and medullary collecting ducts, as well as in the media and the endothelial cells of arteries and arterioles. Staining of the human EP4 receptor subtype protein was observed in glomeruli and in the media of arteries. However, no signal of either receptor subtype was detected in the thick ascending limb, the macula densa, or in adjacent juxtaglomerular cells. These results support the concept that PGE2 modulates specific functions in different anatomical structures of the human kidney.
Asunto(s)
Riñón/metabolismo , Receptores de Prostaglandina E/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Western Blotting , Humanos , Inmunohistoquímica , Riñón/anatomía & histología , Riñón/irrigación sanguínea , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Conejos , Receptores de Prostaglandina E/química , Receptores de Prostaglandina E/inmunología , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Distribución TisularRESUMEN
1. In this study we describe that activin A, a transforming growth factor (TGF) beta-like polypeptide affects the expression of inflammatory response genes and their products. 2. In rat bone marrow derived macrophages 15 nM activin A caused the stimulation of prostaglandin (PG) E2 and thromboxane (TX) A2 formation, production of nitrite as a marker for nitric oxide (NO) and the release of the cytokines tumour necrosis factor (TNF) alpha and interleukin (IL) -1beta. As shown by mRNA analysis induction of cyclo-oxygenase-2 and inducible nitric oxide synthase by activin A gave rise to the enhanced release of prostanoids and NO. 3. Costimulation of bone marrow derived macrophages with 15 nM activin A and 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA) potentiated the synthesis of prostanoids in a synergistic manner. With respect to NO formation the effect of activin A and TPA was additive. 4. In contrast to the nitrite production activin A induced PGE2 synthesis was susceptible to tyrosine kinase inhibition by genistein and tyrphostin 46 (IC50 was 10 and 20 microM, respectively). This observed inhibition was caused by the selective suppression of activin A induced cyclo-oxygenase-2 mRNA expression. Further, the release of TNFalpha in the presence of activin A was potentiated by tyrosine kinase inhibition. 5. In summary, we report that activin A exerts proinflammatory activity which results in the formation of prostanoids, NO and cytokines in rat bone marrow derived macrophages. Tyrosine kinase dependent and independent signalling pathways are involved leading to the increased synthesis of these metabolites. Based upon these results, we speculate that activin A may be considered as a possible component of inflammatory processes affecting at least the haematopoietic system.
Asunto(s)
Células de la Médula Ósea/metabolismo , Citocinas/biosíntesis , Dinoprostona/biosíntesis , Inhibinas/farmacología , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Tromboxano A2/biosíntesis , Activinas , Animales , Ciclooxigenasa 2 , Isoenzimas/fisiología , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Prostaglandina-Endoperóxido Sintasas/fisiología , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: Prostacyclin is an important mediator of renal hemodynamics. Furthermore, recent studies argue for a role of this arachidonic acid metabolite in the regulation of salt and water handling in the distal nephron. To gain insight into the network of prostacyclin signal transduction, we analyzed the intrarenal distribution of the prostacyclin receptor (IP receptor) in adult human kidney. METHODS: Specific polyclonal antibodies against a synthetic peptide of the human IP receptor were generated. By means of immunohistology the localization of IP receptor protein was studied. The mRNA expression for IP receptor was analyzed by in situ hybridization using specific cRNA probes. RESULTS: In human kidney sections both IP receptor-immunoreactive protein and mRNA were expressed in smooth muscle cells and endothelial cells. Expression of the IP receptor was observed in glomerular cells, namely mesangial cells, endothelial cells, and podocytes. Both mRNA and protein expression for IP receptor was observable in Tamm-Horsfall-negative distal tubules and collecting ducts. CONCLUSIONS: The vascular expression of the IP receptor is consistent with the known vasodilatory effect of prostacyclin in vascular beds. Glomerular expression argues for a role of this autacoid in the regulation of glomerular hemodynamics. The tubular distribution might point towards the involvement of prostacyclin in renal salt and water handling.
Asunto(s)
Riñón/metabolismo , Receptores de Prostaglandina/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Riñón/irrigación sanguínea , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , ARN Mensajero/metabolismo , Receptores de Epoprostenol , Receptores de Prostaglandina/genética , Distribución TisularRESUMEN
OBJECTIVE: To compare the expression of the cyclooxygenase (COX) isoforms, COX-1 and COX-2, in synovial tissue samples between patients with inflammatory arthritis (i.e., rheumatoid arthritis [RA], ankylosing spondylitis [AS], or psoriatic arthritis [PsA]) and patients with osteoarthritis (OA). METHODS: Paraffin-embedded sections of synovial tissue from patients with OA (n = 18), RA (n = 35), AS (n = 9), and PsA (n = 16) were immunostained for COX-1 and COX-2. Staining intensity was quantified videodensitometrically from specific synovial cell areas. In addition, samples of OA and RA synovial tissue were analyzed for levels of COX-1 and COX-2 messenger RNA (mRNA) using reverse transcriptase-polymerase chain reaction. RESULTS: Strong COX-2 immunostaining was observed in synovial blood vessel endothelium, synovial lining cells, chondrocytes, and subsynovial fibroblast-like cells in patients with inflammatory arthritides. In the blood vessels, the mean (+/-SD) optical density (MOD) of staining was elevated, especially in AS samples (2.73 +/- 0.63), but also in PsA (1.99 +/- 0.66) and RA samples (1.54 +/- 0.73), in comparison with OA synovial tissue (0.84 +/- 0.30; P < 0.01 versus other groups). COX-1 staining was almost exclusively localized in synovial lining cells, with no significant differences in the MOD between the diseases. COX-2 mRNA expression was higher in RA than in OA samples (P < 0.05). CONCLUSION: The expression of COX-2, but not the expression of COX-1, was found to be elevated in a disease-related pattern in the synovial tissue from patients with RA, AS, or PsA in comparison with OA samples, and was especially high in AS synovial tissue. These results may improve our understanding of the pathogenesis of different arthritic diseases, and may have implications for the use of selective COX-2 inhibitors in the treatment of inflammatory joint symptoms.