RESUMEN
Electrospray/tandem mass spectrometry was used to quantify lipid remodeling in mouse liver and plasma during inhibition of polyunsaturated fatty acid synthesis by the delta6 fatty acid desaturase inhibitor, SC-26196. SC-26196 caused increases in linoleic acid and corresponding decreases in arachidonic acid and docosahexaenoic acid in select molecular species of phosphatidylcholine, phosphatidylethanolamine, and cholesterol esters but not in phosphatidylserine, phosphatidylinositol, or triglycerides. For linoleic acid-, arachidonic acid-, and docosahexaenoic acid-containing phospholipid species, this difference was, in part, determined by the fatty acid at the sn-1 position, namely, palmitic or stearic acid. An understanding of phospholipid remodeling mediated by delta6 desaturase inhibition should aid in clarifying the contribution of arachidonic acid derived via de novo synthesis or obtained directly in the diet during inflammatory responses.
Asunto(s)
Ácido Graso Desaturasas/antagonistas & inhibidores , Metabolismo de los Lípidos , Lípidos/química , Hígado/metabolismo , Animales , Ácido Araquidónico/metabolismo , Sangre/efectos de los fármacos , Sangre/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Ácido Linoleico/metabolismo , Linoleoil-CoA Desaturasa , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Piperazinas/farmacología , Espectrometría de Masa por Ionización de Electrospray/métodosAsunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Arteriosclerosis/tratamiento farmacológico , Aspirina/uso terapéutico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Isoenzimas/antagonistas & inhibidores , Sulindac/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/inmunología , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Aspirina/inmunología , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/patología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/metabolismo , Humanos , Isoenzimas/metabolismo , Proteínas de la Membrana , Ratones , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sulindac/inmunologíaAsunto(s)
Compuestos de Anilina/farmacología , Antiinflamatorios no Esteroideos/farmacología , Grasas Insaturadas en la Dieta/farmacología , Edema/prevención & control , Ácido Graso Desaturasas/antagonistas & inhibidores , Piperazinas/farmacología , Animales , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Sarcoma de Mastocitos , Ratones , Ácido Oléico/farmacología , Células Tumorales CultivadasAsunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Prostaglandinas/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/metabolismo , Celecoxib , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Humanos , Isoenzimas/metabolismo , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/metabolismo , Pirazoles , Sulfonamidas/farmacologíaRESUMEN
Decreased synthesis of arachidonic acid by inhibition of the Delta6 or Delta5 desaturase was evaluated as a means to mitigate inflammation. Using quantitative in vitro and in vivo radioassays, novel compounds representing five classes of Delta5 desaturase inhibitors and one class of Delta6 desaturase inhibitor were identified. The Delta6 desaturase inhibitor, SC-26196, had pharmacokinetic and pharmacodynamic profiles in mice that allowed for the evaluation of the pharmacological effects of chronic inhibition of desaturase activity. SC-26196 decreased edema to the same extent as indomethacin or essential fatty acid deficiency in the carrageenan paw edema model in the mouse. The antiinflammatory properties of SC-26196 were consistent with its mechanism of action as a Delta6 desaturase inhibitor: 1) A correlation existed between inhibition of liver Delta6 desaturase activity and decreases in edema. 2) The onset of the decrease in edema was time dependent. 3) Selective reduction of arachidonic acid occurred dose dependently in liver, plasma and peritoneal cells. 4) In the presence of SC-26196, controlled refeeding of arachidonic acid, but not oleic acid, reversed the changes resulting from desaturase inhibition. The Delta6 desaturase may be a target for development of antiinflammatory drugs whose mechanism of action is unique.
Asunto(s)
Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Ácido Graso Desaturasas/antagonistas & inhibidores , Animales , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Edema/tratamiento farmacológico , Ácidos Grasos Esenciales/deficiencia , Femenino , Ácido Linoleico/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Peroxidasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The anti-inflammatory properties of essential fatty acid deficiency or n-3 polyunsaturated fatty acid supplementation have been attributed to a reduced content of arachidonic acid (AA; 20:4 n-6). An alternative, logical approach to depleting AA would be to decrease endogenous synthesis of AA by selectively inhibiting the delta5 and/or the delta6 fatty acid desaturase. High-throughput radioassays were developed for quantifying delta5, delta6, and delta9 desaturase activities in vitro and in vivo. CP-24879 (p-isopentoxyaniline), an aniline derivative, was identified as a mixed delta5/delta6 desaturase inhibitor during the screening of chemical and natural product libraries. In mouse mastocytoma ABMC-7 cells cultured chronically with CP-24879, there was a concentration-dependent inhibition of desaturase activity that correlated with the degree of depletion of AA and decreased production of leukotriene C4 (LTC4). Production of LTC4 was restored by stimulating the cells in the presence of exogenous AA, indicating that endogenous AA was limiting as substrate. In the livers of mice treated chronically with the maximally tolerated dose of CP-24879 (3 mg/kg, t.i.d.), combined delta5/delta6 desaturase activities were inhibited approximately 80% and AA was depleted nearly 50%. These results suggest that delta5 and/or delta6 desaturase inhibitors have the potential to manifest an anti-inflammatory response by decreasing the level of AA and the ensuing production of eicosanoids.
Asunto(s)
Compuestos de Anilina/síntesis química , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Ácido Graso Desaturasas/metabolismo , Compuestos de Anilina/farmacocinética , Compuestos de Anilina/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Ácido Araquidónico/metabolismo , Células Cultivadas , Dieta , Inhibidores Enzimáticos/farmacocinética , Ácidos Grasos/metabolismo , Técnicas In Vitro , Indicadores y Reactivos , Leucotrieno C4/biosíntesis , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Non-steroidal anti-inflammatory drugs are efficacious treatments for rheumatoid arthritis and osteoarthritis. However, an adverse effect of treatment with non-steroidal anti-inflammatory drugs is acute renal failure, particularly in a subset of patients that are in a state of effective volume depletion. The frequency of this side-effect in the general treated population is not known, but is probably less than 1% per year. Non-steroidal anti-inflammatory drugs act by inhibiting the synthesis of prostaglandins, which are important mediators of renal function. In the volume-depleted state prostaglandins may counter the vasoconstriction associated with the activation of the renin-angiotensin system. Cyclooxygenase is the rate-limiting enzyme involved in the synthesis of prostaglandins. Cyclooxygenase exists in two forms: a constitutive form (cyclooxygenase-1) and an inducible form (cyclooxygenase-2), which is associated with inflammation. Non-steroidal anti-inflammatory drugs are non-specific inhibitors of both forms of cyclooxygenase. New data are emerging regarding the role of cyclooxygenase-2 in the control of renal function. In normal rat and dog kidney, cyclooxygenase-2 is sparsely expressed in the macula densa, but expression is upregulated when animals are volume depleted. This review explores the possible role of cyclooxygenase-2 in the maintenance of normal renal function in volume depleted states.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Antiinflamatorios no Esteroideos/efectos adversos , Isoenzimas/fisiología , Riñón/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Perros , Humanos , Riñón/enzimología , RatasAsunto(s)
Isoenzimas/fisiología , Peroxidasas/fisiología , Prostaglandina-Endoperóxido Sintasas/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Artritis/tratamiento farmacológico , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Humanos , Técnicas In Vitro , Isoenzimas/efectos de los fármacos , Proteínas de la Membrana , Antagonistas de Prostaglandina/farmacología , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Prostaglandinas/biosíntesisRESUMEN
1. Bradykinin (BK, 1 microgram) caused a small (2 fold at 6 h) increase in prostaglandin E2 (PGE2) in the normal rabbit kidney, perfused ex vivo. This was exaggerated (6 fold at 6 h) in the hydronephrotic kidney (HNK). The exaggerated release of PGE2 was attenuated by cycloheximide, an inhibitor of protein synthesis or by dexamethasone, a steroid known to inhibit the induction of cyclo-oxygenase (COX-2). BK (1 microgram) when injected at 6 h of perfusion increased the release of PGE2 from 90 +/- 33 pg ml-1 min-1 to 3069 +/- 946 pg ml-1 min-1. This was reduced to 200 +/- 30 pg ml-1 min-1 in kidneys infused with cycloheximide (1 microM) and to 250 +/- 40 pg ml-1 min-1 in kidneys infused with dexamethasone (n = 8). 2. When tested on human and murine recombinant COX-1 and COX-2 enzymes, DuP-697 was at least 50 fold more selective for COX-2 than for COX-1. 3. DuP-697 reduced the exaggerated release of PGE2 elicited by BK in the HNK (e.g., at 6 h of perfusion BK-evoked PGE2 release decreased from 3069 +/- 946 pg ml-1 min-1 to 187 +/- 22 pg ml-1 min-1 after perfusion with 1 microM DUP-697, n = 8). 4. Cycloheximide, dexamethasone or DuP-697 at doses used to inhibit completely the exaggerated release of PGE2 in the hydronephrotic kidney, failed to inhibit the release of PGE2 elicited by the injection of BK (1 microgram) in the normal contralateral kidney. 5. Indomethacin (1 microM), a non-selective COX-1 and COX-2 inhibitor, completely inhibited PGE2 release in the normal contralateral as well as in the hydronephrotic kidney. 6. We suggest that renal prostaglandin production in the normal kidney is driven by the activity of constitutive COX-1 while at sites of inflammation, such as the hydronephrotic kidney, there is induction of COX-2 that can be blocked selectively by anti-inflammatory glucocorticoids or selective COX-2 inhibitors.
Asunto(s)
Dinoprostona/biosíntesis , Hidronefrosis/metabolismo , Isoenzimas/metabolismo , Riñón/efectos de los fármacos , Riñón/enzimología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Antiinflamatorios/farmacología , Antiinflamatorios no Esteroideos/farmacología , Cicloheximida/farmacología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Masculino , Inhibidores de la Síntesis de la Proteína/farmacología , Conejos , Proteínas Recombinantes/farmacología , Tiofenos/farmacologíaRESUMEN
We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/farmacología , Óxido Nítrico/fisiología , Prostaglandinas/fisiología , Animales , Dexametasona/farmacología , Hemodinámica/efectos de los fármacos , Masculino , Neutrófilos/fisiología , Óxido Nítrico Sintasa , Nitrobencenos/farmacología , Ratas , Ratas Endogámicas Lew , Sulfonamidas/farmacologíaRESUMEN
1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L-arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively. 3. The intravenous infusion of the NO donors sodium nitroprusside (SNP) or glyceryl trinitrate (GTN)increased prostaglandin production in normal animals (for instance urinary PGE2 excretion was increased from 96 +/- 10 to 576 +/- 12 pg min-1 and 400 +/- 24 pg min-1 in the presence of GTN or SNP respectively).4. Proteinuria was measured in order to evaluate the roles of NO and PG in renal damage associated with the in vivo injection of LPS. Interestingly, dexamethasone and the NOS inhibitors attenuated proteinuria in the LPS-treated rats. The COX inhibitors had no effect. It therefore appears that NO and not PG contributes to the LPS-induced renal damage; these findings support the potential use of NOS inhibitors in the treatment of renal inflammation.5. This study demonstrates the regulatory contribution of NO on the in vivo production of prostanoids and suggests that in inflammatory diseases that are driven by both NO and the prostaglandins, NOS inhibitors may act to reduce inflammation by the dual inhibition of cytotoxic NO and pro-inflammatory PG.
Asunto(s)
Óxido Nítrico/fisiología , Prostaglandinas/biosíntesis , 6-Cetoprostaglandina F1 alfa/sangre , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Dexametasona/farmacología , Endotoxinas/farmacología , Inducción Enzimática , Escherichia coli , Estudios de Evaluación como Asunto , Hemodinámica/efectos de los fármacos , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico Sintasa , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Ratas , Ratas Sprague-DawleyAsunto(s)
Aminoácido Oxidorreductasas/biosíntesis , Hidronefrosis/fisiopatología , Inflamación/fisiopatología , Óxido Nítrico/fisiología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Bradiquinina/farmacología , Dinoprostona/metabolismo , Inducción Enzimática , Escherichia coli , Guanidinas/farmacología , Indometacina/farmacología , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa , Conejos , Ratas , omega-N-MetilargininaAsunto(s)
Antiinflamatorios no Esteroideos/farmacología , Aspirina , Inhibidores de la Ciclooxigenasa/farmacología , Analgesia , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Antiinflamatorios no Esteroideos/toxicidad , Artritis Experimental/tratamiento farmacológico , Carragenina , Inhibidores de la Ciclooxigenasa/uso terapéutico , Inhibidores de la Ciclooxigenasa/toxicidad , Diseño de Fármacos , Edema/inducido químicamente , Edema/tratamiento farmacológico , Humanos , Inflamación , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
Acute inflammatory stimuli elevate both the production of prostaglandins and the synthesis and activity of prostaglandin synthase/cyclooxygenase enzyme (COX) in murine peritoneal macrophages. Adrenalectomy also elevates prostaglandin production, COX synthesis and COX activity in these cells. We have utilized cDNA probes and antisera specific for the products of the prostaglandin synthase/cyclooxygenase-1 (COX-1) and TIS10/prostaglandin synthase-2/cyclooxygenase-2 (COX-2) genes to demonstrate that adrenalectomy causes elevation of mRNA and protein from the COX-2 gene, but not from the COX-1 gene, in peritoneal macrophages. Dexamethasone replacement suppressed the elevation of COX-2 mRNA message, COX-2 protein and the increased COX enzyme activity observed in adrenalectomized animals. In contrast, both COX-1 message and COX-1 protein levels were unaffected either by adrenalectomy or by dexamethasone administration. Thus, under normal physiological conditions, tonic glucocorticoid inhibition appears to play a major role in the in vivo regulation of the COX-2 gene. These data are consistent with COX-1 being the constitutive, housekeeping enzyme in macrophages in normal physiological conditions and with the enhanced prostaglandin synthesis seen after an inflammatory stimulus resulting from the rapid induction and activity of COX-2.
Asunto(s)
Dexametasona/farmacología , Regulación Enzimológica de la Expresión Génica , Isoenzimas/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Adrenalectomía , Secuencia de Aminoácidos , Animales , Células Cultivadas , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos Peritoneales/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/metabolismoRESUMEN
The interaction between nitric oxide (NO) and cyclooxygenase (COX) was studied in a rabbit model of renal inflammation, the ureteral obstructed hydronephrotic kidney (HNK). Ex vivo perfusion of the HNK but not the control kidney (e.g., unobstructed contralateral kidney, CLK), led to a time-dependent release of nitrite (NO2-), a breakdown product of NO. Stimulation of the HNK with bradykinin (BK) evoked a time-dependent increase in prostaglandin E2 (PGE2) production. NG-monomethyl-L-arginine (L-NMMA), which blocks the activity of both constitutive and inducible nitric oxide synthase (cNOS and iNOS), aminoguanidine, a recently described selective iNOS inhibitor, dexamethasone, or cycloheximide abolished the release of NO2- and attenuated the exaggerated BK-induced PGE2 production. This supports the existence of iNOS and COX-2 in the HNK. In the CLK, BK elicited release of both NO2- and PGE2 but this did not augment with time. L-NMMA but not aminoguanidine, dexamethasone, or cycloheximide attenuated NO2- and PGE2 release indicative of the presence of constitutive but not inducible NOS or COX. The current study suggests that the endogenous release of NO from cNOS in the CLK activates a constitutive COX resulting in optimal PGE2 release by BK. In addition, in the HNK, NO release from iNOS activates the induced COX resulting in markedly increased release of proinflammatory prostaglandin. The broader implication of this study is that the cyclooxygenase isozymes are potential receptor targets for nitric oxide.
Asunto(s)
Dinoprostona/biosíntesis , Hidronefrosis/metabolismo , Riñón/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Ácidos Cólicos/farmacología , Modelos Animales de Enfermedad , Técnicas In Vitro , Indometacina/farmacología , Riñón/efectos de los fármacos , Masculino , Modelos Biológicos , Óxido Nítrico Sintasa , Perfusión , Prostaglandina-Endoperóxido Sintasas/metabolismo , Conejos , Uréter/cirugía , omega-N-MetilargininaRESUMEN
We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 microgram/ml, 18 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NO2- and PGE2 was blocked by the NOS inhibitors NG-monomethyl-L-arginine or aminoguanidine. The effects of NG-monomethyl-L-arginine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg. PGE2 production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1 beta. These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1 beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 microM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-1 and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.
Asunto(s)
Óxido Nítrico/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/farmacología , Línea Celular , GMP Cíclico/metabolismo , Dinoprostona/biosíntesis , Activación Enzimática/efectos de los fármacos , Guanidinas/farmacología , Humanos , Indometacina/farmacología , Ratones , Nitratos/metabolismo , Óxido Nítrico Sintasa , Proteínas Recombinantes/metabolismo , omega-N-MetilargininaRESUMEN
Intraperitoneal infection with Listeria monocytogenes (LM) results in activation of the peritoneal macrophage population which displays increased surface expression of major histocompatibility (MHC) Class II (Ia) antigen and markedly suppressed prostaglandin (PG) synthesis. We demonstrate here that this decrease in PG production is also seen after treatment by mitogen (Con A) and endotoxin (LPS), and can be explained by reduced cyclooxygenase activity in these cell populations. We show that, whereas Ia expression was augmented at all doses of LM and Con A tested, it displayed a biphasic response to LPS in vivo: increase at the lowest dose and inhibition at higher doses. In order to identify possible endogenous mediators of these responses, we used highly purified preparations of recombinant murine (rMu) cytokines and neutralizing cytokine specific monoclonal antibodies (MAbs) to examine whether interferon-gamma (IFN-gamma) and/or tumor necrosis factor (TNF) down-regulate macrophage cyclooxygenase activity in vivo. We found that IFN-gamma induced Ia expression but had no effect on PG secretion. In contrast, TNF-alpha suppressed PG synthesis and inhibited Ia surface expression. Similarly, in our model of Con A-induced peritoneal macrophage activation, pretreatment of animals with a neutralizing MAb to rMuIFN-gamma completely blocked the induction of Ia positive macrophages by Con A but did not affect Con A-dependent suppression of PG synthesis. Pretreatment with MAb to TNF had no effect on Con A-induced Ia levels, but significantly inhibited suppressed PG synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Citocinas/farmacología , Macrófagos/enzimología , Macrófagos/inmunología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Anticuerpos Monoclonales , Concanavalina A/farmacología , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Lipopolisacáridos/farmacología , Listeriosis/inmunología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Prostaglandinas/biosíntesisRESUMEN
Atriopeptin (AP) is synthesized and stored in the mammalian atria as a 126 amino acid prohormone (AP126). Upon secretion, the prohormone undergoes site specific proteolysis within the atria to yield the carboxyl terminal 28 amino acid hormone (AP28). The atrial cell responsible for AP126 bioactivation has not yet been determined. Primary neonatal rat atrial cell cultures were generated with and without depletion of nonmyocytic cells. The molecular form of AP detected in the conditioned media of mixed cultures was determined to be AP126. Addition of dexamethasone to these cultures resulted in the appearance of a peptide that co-migrated with AP28. In contrast, no AP126 processing was detected in the conditioned media of myocyte enriched cultures when grown in the presence of dexamethasone. Readdition of nonmyocytic atrial cells to myocyte enriched cultures successfully reconstituted the steroid induced AP126 processing. Incubation of recombinant AP126argarg with nonmyocytic atrial cell cultures resulted in the generation of AP28argarg. We conclude that a nonmyocytic atrial cell is responsible for AP126 processing in vitro.
Asunto(s)
Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Corazón/fisiología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/biosíntesis , Células Cultivadas , Dexametasona/farmacología , Corazón/efectos de los fármacos , Atrios Cardíacos , Ventrículos Cardíacos , Inmunohistoquímica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMEN
We recently demonstrated the synthesis and secretion of an atriopeptin (AP)-like prohormone in rat neonatal and adult cortical kidney cell cultures. However, these cultures contained proximal as well as distal tubular epithelial cells; thus characterization of the peptide synthetic cell was not possible. Also, by immunohistochemical techniques, we localized this AP-like prohormone to the distal cortical nephron in adult rat kidney. In this study, we examined further details of the kidney cortical cell type that expresses and secretes this AP-like peptide in adult renal cortical cell cultures, its regulation by adenylate cyclase via adenosine 3',5'-cyclic monophosphate (cAMP) generation, and its ability to stimulate guanylate cyclase. Tubular fragments were derived from cortical tissue of adult Sprague-Dawley rats and separated into four fractions on Percoll density gradient. Cell cultures generated from fraction 3 secreted 5- to 10-fold the amount of this renal peptide compared with fractions 2 and 4. Further cell culture characterization was performed by agonist-stimulated cAMP formation, kallikrein localization, and prostaglandin E2 formation. From these analyses, it was determined that tissue band 3 was enriched for distal cortical connecting tubules. To further evaluate whether mammalian distal nephron synthesizes an AP-like protein, we determined that two immortalized mouse cell lines, derived from either the distal convoluted tubule or cortical collecting tubule, synthesized a radiolabeled AP after being pulsed with [35S]-methionine.(ABSTRACT TRUNCATED AT 250 WORDS)