RESUMEN
BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing skin disorder with pruritic skin symptoms. We previously reported that dihomo-γ-linolenic acid (DGLA) prevented the development of AD in NC/Tnd mice, though the mechanism remained unclear. OBJECTIVE: We attempted to investigate the mechanism of preventive effect of DGLA on AD development in NC/Tnd mice. METHODS: The clinical outcomes of NC/Tnd mice that were given diets containing DGLA, arachidonic acid, or eicosapentaenoic acid were compared. Lipid mediator contents in the skin in each group were also quantified. In addition, release of lipid mediators from RBL-2H3 mast cells treated with either DGLA or prostaglandin D1 (PGD1) was measured. Furthermore, effect of PGD1 on gene expression of thymic stromal lymphopoietin (TSLP) in PAM212 keratinocyte cells was determined. RESULTS: Only DGLA containing diet suppressed the development of dermatitis in vivo. By quantifying the 20-carbon fatty acid-derived eicosanoids in the skin, the application of DGLA was found to upregulate PGD1, which correlated with a better outcome in NC/Tnd mice. Moreover, we confirmed that mast cells produced PGD1 after DGLA exposure, thereby exerting a suppressive effect on immunoglobulin E-mediated degranulation. PGD1 also suppressed gene expression of TSLP in keratinocytes. CONCLUSION: These results suggest that oral administration of DGLA causes preventive effects on AD development in NC/Tnd mice by regulating the PGD1 supply.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/uso terapéutico , Degranulación de la Célula , Dermatitis Atópica/prevención & control , Mastocitos/fisiología , Prostaglandinas D/biosíntesis , Ácido 8,11,14-Eicosatrienoico/farmacología , Administración Cutánea , Animales , Ácido Araquidónico/uso terapéutico , Citocinas/genética , Suplementos Dietéticos , Ácido Eicosapentaenoico/uso terapéutico , Expresión Génica/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Prostaglandina D2/administración & dosificación , ARN Mensajero/metabolismo , Regulación hacia Arriba , Linfopoyetina del Estroma TímicoRESUMEN
Cyclooxygenase (COX) is the key enzyme in the conversion of arachidonic acid to prostanoids, lipid mediators involved in several physiological and pathological processes. Two COX isoenzymes have been characterized, COX-1 and COX-2, that differ in terms of regulatory mechanisms of expression, tissue distribution, substrate specificity, and preferential coupling to upstream and downstream enzymes. Both isoforms play fundamental roles in atherothrombosis; however, whereas the function of COX-1 in this setting is well established, the role of COX-2 remains unclear. Indeed, the intracellular pathways regulating COX-2 induction appear numerous and complicated, varying between cell types and cellular stimulus. In recent years a long series of studies has been performed with the aim of clarifying the role of COX-2 in atherothrombosis, with the major finding that the COX-2 expression pattern in arterial vessels may be associated with either protective or plaque-destabilizing phenotypes according to the downstream synthase that couples with COX-2. In this review we summarize the role of COX-2 as well as the different downstream synthases in atherosclerosis and atherothrombosis. Finally, we briefly review the controversial vascular effects on prostanoid inhibition by COX-2 inhibitors.
Asunto(s)
Aterosclerosis/enzimología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Ácido Araquidónico/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Humanos , Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Oxidorreductasas Intramoleculares/biosíntesis , Prostaglandinas D/biosíntesis , Tromboxano-A Sintasa/biosíntesisRESUMEN
Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.
Asunto(s)
Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Clostridioides difficile/patogenicidad , Enterotoxinas/inmunología , Mastocitos/inmunología , Mastocitos/microbiología , Degranulación de la Célula , Línea Celular , Citocinas/metabolismo , Citoesqueleto/metabolismo , Glucosa/metabolismo , Hexosaminidasas/metabolismo , Humanos , Interleucina-8/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Prostaglandinas D/biosíntesis , Prostaglandinas E/biosíntesis , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Despite the magnitude of the obesity epidemic, the mechanisms that contribute to increases in fat mass and to differences in fat depots are still poorly understood. Prostanoids have been proposed as potent adipogenic hormones, e.g. metabolites of prostaglandin J2 (PGJ2) bind and activate PPARgamma. We hypothesize that an altered expression of enzymes in PGJ2 synthesis may represent a novel pathogenic mechanism in human obesity. We characterized adipose depot-specific expression of enzymes in PGJ2 synthesis, prostaglandin transporter and PPARgamma isoforms. Paired omental and subcutaneous adipose tissue samples were obtained from 26 women undergoing elective abdominal surgery and gene expression examined in whole tissue and cultured preadipocytes using an Affymetrix cDNA microarray technique and validated with quantitative real-time PCR. All enzymes involved in prostaglandin synthesis were expressed in both adipose tissues. Expression of prostaglandin synthase-1 (PGHS1), prostaglandin D synthase (PTGDS), human prostaglandin transporter (hPGT) and PPARgamma2 was higher in OM adipose tissue compared to SC, whereas 17beta-hydroxysteroid dehydrogenase 5 (AKR1C3) showed predominance in SC adipose tissue. In SC adipose tissue, PGHS1 mRNA expression increased with BMI. The differential, depot-specific expression of key enzymes involved in transport, synthesis and metabolism of prostaglandins may have an important impact upon fat cell biology and may help to explain some of the observed depot-specific differences. In addition, the positive correlation between PGHS1 and BMI offers the novel hypothesis that the regulation of PG synthesis may have a role in determining fat distribution in human obesity.
Asunto(s)
Adipogénesis/fisiología , Tejido Adiposo/metabolismo , Prostaglandinas/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Abdomen , Adipocitos/citología , Adulto , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Distribución de la Grasa Corporal , Índice de Masa Corporal , Tamaño de la Célula , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas , Persona de Mediana Edad , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Epiplón/metabolismo , Transportadores de Anión Orgánico/metabolismo , PPAR gamma/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Prostaglandina D2/análogos & derivados , Prostaglandina D2/biosíntesis , Prostaglandina D2/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas D/biosíntesis , Prostaglandinas D/metabolismo , Tejido Subcutáneo/patologíaAsunto(s)
Diferenciación Celular/efectos de los fármacos , Megacariocitos/enzimología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandinas D/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Inducción Enzimática , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Leucemia Megacarioblástica Aguda , Lipocalinas , Células Tumorales CultivadasAsunto(s)
Isoenzimas/biosíntesis , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandinas D/biosíntesis , Ácido Araquidónico/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Ciclooxigenasa 1 , Inducción Enzimática/efectos de los fármacos , Glutatión/metabolismo , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas , Megacariocitos/citología , Proteínas de la Membrana , Acetato de Tetradecanoilforbol/farmacologíaAsunto(s)
Lipooxigenasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas D/biosíntesis , Prostaglandinas F/biosíntesis , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Secuencia de Bases , Evolución Biológica , Epóxido Hidrolasas/metabolismo , Humanos , Lipooxigenasa/genética , Datos de Secuencia Molecular , Prostaglandina-Endoperóxido Sintasas/genéticaRESUMEN
Incubation of [1-14C] arachidonicacid (AA) with homogenates of bovine gallbladder mucosa in the presence or absence of reduced glutathione (GSH) generated large amounts of products that cochromatographed with PGE2 and 6-keto-PGF1 alpha and small amounts of products comigrating with PGF2 alpha, TxB2 and PGD2. In the presence of GSH PGE2-like material was predominantly produced while in the absence of GSH 6-keto-PGF1 alpha-like material was a major product. Identification of PGE2 was performed by its chemical conversion with NaBH4 or KOH treatments and by gas chromatography-mass spectrometry. Identification of 6-keto-PGF1 alpha was confirmed by gas chromatography-mass spectrometry. Gallbladder muscle produced predominantly 6-keto-PGF1 alpha in the presence or absence of GSH. The total amount of cyclooxygenase products was approximately 2 times higher in the mucosa than in the muscle. These results indicate that bovine gallbladder mucosa contains higher levels of fatty acid cyclooxygenase, and PGE2 isomerase and PGI2 synthetase in a proportion different from that in the muscle.
Asunto(s)
Ácido Araquidónico/biosíntesis , Vesícula Biliar/metabolismo , Animales , Bovinos , Epoprostenol/biosíntesis , Glutatión/fisiología , Membrana Mucosa/metabolismo , Prostaglandinas D/biosíntesis , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Tromboxano B2/biosíntesisRESUMEN
1. A sensitive and specific negative ion chemical ionization mass spectrometric assay for the major urinary metabolite of PGD2 has been developed employing a chemically synthesized [18(0)4]-labelled internal standard. 2. The finding that increased urinary excretion of this metabolite occurs in a number of clinical situations suggests that the assay may prove to be a valuable tool to explore the role of PGD2 in the pathophysiology of human disease.
Asunto(s)
Espectrometría de Masas/métodos , Prostaglandina D2/metabolismo , Prostaglandinas D/análisis , Asma/metabolismo , Líquido del Lavado Bronquioalveolar/química , Histamina/metabolismo , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/orina , Técnicas de Dilución del Indicador , Mastocitosis/orina , Niacina/farmacología , Niacina/uso terapéutico , Prostaglandinas D/biosíntesisRESUMEN
We describe the enzymological regulation of the formation of prostaglandin (PG) D2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2 (prostacyclin), and thromboxane (Tx) A2 from arachidonic acid. We discuss the three major steps in prostanoid formation: (a) arachidonate mobilization from monophosphatidylinositol involving phospholipase C, diglyceride lipase, and monoglyceride lipase and from phosphatidylcholine involving phospholipase A2; (b) formation of prostaglandin endoperoxides (PGG2 and PGH2) catalyzed by the cyclooxygenase and peroxidase activities of PGH synthase; and (c) synthesis of PGD2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2, and TxA2 from PGH2. We also include information on the roles of aspirin and other nonsteroidal anti-inflammatory drugs, dexamethasone and other anti-inflammatory steroids, platelet-derived growth factor (PDGF), and interleukin-1 in prostaglandin metabolism.
Asunto(s)
Prostaglandinas D/biosíntesis , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Tromboxanos/biosíntesis , Secuencia de Aminoácidos , Animales , Ácidos Araquidónicos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Endoperóxidos de Prostaglandina/biosíntesis , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
The effect of ultraviolet light-B (UVB) irradiation on the activity of prostaglandin (PG) D synthetase was investigated in adult rat skin. Rats were irradiated with 500 mJ/cm2 of UVB, and PGD synthetase activity was determined in 100,000 g supernatant of the homogenate of rat skin in the presence of glutathione (GSH) before and 3, 6, 12, and 24 h after irradiation. The PGD synthetase activity was decreased time dependently, and within 24 h after UVB irradiation it had dropped to 50% of the control level before irradiation. In contrast, the synthesizing activities of PGE2 and PGF2 alpha were unaffected by UVB irradiation. The reduction of PGD synthetase activity after UVB irradiation was much more prominent in the epidermis than in the dermis, which was separated by heat treatment (55 degrees C, 30 sec). Immunohistochemical studies, using anti-(rat spleen PGD synthetase) antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, decreased in the basal layer of the epidermis 24 h after UVB irradiation. These results, together with the reduction of ATPase positive cells in the epidermis after UVB irradiation, suggest that the decrease of PGD synthetase activity in rat skin by UVB irradiation is, at least in part, due to the reduced Langerhans cell population in the basal layer of the epidermis.
Asunto(s)
Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas D/biosíntesis , Piel/enzimología , Rayos Ultravioleta , Adenosina Trifosfatasas/metabolismo , Animales , Inmunohistoquímica , Células de Langerhans/enzimología , Masculino , Ratas , Ratas Endogámicas , Piel/citología , Piel/efectos de la radiaciónRESUMEN
Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).
Asunto(s)
Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Prostaglandinas D/biosíntesis , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Animales , Encéfalo/enzimología , Citosol/enzimología , Dinoprost , Dinoprostona , Glutatión/metabolismo , Concentración de Iones de Hidrógeno , Técnicas de Inmunoadsorción , Hígado/enzimología , Masculino , Prostaglandina D2 , Endoperóxidos de Prostaglandinas Sintéticos/metabolismo , Prostaglandina H2 , Prostaglandinas H/metabolismo , Ratas , Ratas Endogámicas , Testículo/enzimologíaRESUMEN
Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.
Asunto(s)
Mucosa Intestinal/patología , Leucotrieno B4/biosíntesis , Mastocitos/metabolismo , Prostaglandinas D/biosíntesis , SRS-A/biosíntesis , Animales , Anticuerpos Antiidiotipos/inmunología , Antígenos/inmunología , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Liberación de Histamina , Inmunoglobulina E/inmunología , Infecciones por Nematodos/patología , Nippostrongylus , Cavidad Peritoneal/patología , Prostaglandina D2 , Ratas , Ratas Endogámicas LewRESUMEN
The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied in vitro. After incubation of the whole membraneous cochlea with [3H]-arachidonic acid (AA), syntheses of PGF2 alpha, 6-keto PGF1 alpha, PGE2, thromboxane (TX) P2 and PGD2 were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10(-5) M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37 degrees C for 0-15 min, PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 90-120 pg/cochlea; PGE2, 370 pg/cochlea), reached to the maximum level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 170-200 pg/cochlea; PGE2, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB2 was lower than the detection limit by RIA (less than 50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE2 was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD2, PGF2 alpha and 6-keto PGF1 alpha were about 1/3 of those of PGE2. In the LW, the amounts of PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were about the same level (70-100 pg/LW).
Asunto(s)
Cóclea/metabolismo , Gerbillinae/metabolismo , Prostaglandinas/biosíntesis , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Cóclea/efectos de los fármacos , Nervio Coclear/metabolismo , Dinoprost , Dinoprostona , Femenino , Indometacina/farmacología , Masculino , Órgano Espiral/metabolismo , Prostaglandina D2 , Prostaglandinas D/biosíntesis , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Radioinmunoensayo , Tromboxano B2/biosíntesisRESUMEN
When the amounts of primary prostaglandins formed from endogenous arachidonic acid were determined in homogenates of various tissues of adult rats, prostaglandin D2 was the major prostaglandin found in most tissues. It was formed actively in the spleen (3100 ng/g tissue/5 min at 25 degrees C), intestine (2600), bone marrow (2400), lung (1100), and stomach (630); moderately in the epididymis, skin, thymus, and brain (140-340); and weakly in other tissues (less than 100). Addition of exogenous arachidonic acid (1 mM) accelerated the formation of prostaglandin D2 in all tissues as follows: spleen (15,000); bone marrow, intestine, thymus, liver, and lung (1600-5200); stomach, adrenal gland, epididymis, brain, salivary gland, skin, spinal cord, and seminal vesicle (380-1000); and other tissues (80-310). The activity of prostaglandin D synthetase (prostaglandin-H2 D-isomerase) was detected in 100,000g supernatants of almost all tissues. As judged by glutathione requirement for the reaction, inhibition of the activity by 1-chloro-2,4-dinitrobenzene, and immunotitration or immunoabsorption analyses with specific antibodies, the enzyme in the epididymis, brain, and spinal cord (1.8-9.2 nmol/min/mg protein) was glutathione-independent prostaglandin D synthetase (Y. Urade, N. Fujimoto, and O. Hayaishi (1985) J. Biol. Chem. 260, 12410-12415). The enzyme in the spleen, thymus, bone marrow, intestine, skin, and stomach (2.0-57.1) was glutathione-requiring prostaglandin D synthetase (Y. Urade, N. Fujimoto, M. Ujihara, and O. Hayaishi (1987) J. Biol. Chem. 262, 3820-3825). The activity in the kidney and testis (3.7-4.5) was catalyzed by glutathione S-transferase. The activity in the liver, lung, adrenal gland, salivary gland, heart, pancreas, and muscle (0.6-5.1) was due to both the glutathione-requiring synthetase and the transferase.
Asunto(s)
Oxidorreductasas Intramoleculares , Isomerasas/metabolismo , Prostaglandinas D/biosíntesis , Animales , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , Médula Ósea/metabolismo , Dinoprost , Dinoprostona , Glutatión/farmacología , Glutatión Transferasa/metabolismo , Inmunoensayo , Técnicas de Inmunoadsorción , Mucosa Intestinal/metabolismo , Lipocalinas , Masculino , Prostaglandina D2 , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Ratas , Ratas Endogámicas , Bazo/metabolismo , Timo/metabolismo , Distribución TisularRESUMEN
Fetal calf serum is able to activate arachidonic acid release from phospholipids in NRK 49F cells. We showed that this phenomenon can be potentiated by adding oxysterols to the culture medium. The oxysterol effect was dose-dependent and was not observed in the absence of fetal calf serum. Greater amounts of prostaglandin E2 and prostaglandin F2 alpha were released into the medium in the presence of oxysterols without apparent modification of the cyclooxygenase activity. The most effective oxysterols, in descending order, were the following: calcitriol greater than 7 alpha-hydroxycholesterol greater than 7 beta-hydroxycholesterol greater than 25-hydroxycholesterol. Cholesterol and 7-ketocholesterol were unable to activate phospholipase activity. The mechanism of this activation by oxysterols is still unknown.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Sangre , Hidroxicolesteroles/farmacología , Prostaglandinas/biosíntesis , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animales , Ácido Araquidónico , Radioisótopos de Carbono , Bovinos , Línea Celular , Cromatografía en Capa Delgada , Dinoprost , Dinoprostona , Sangre Fetal , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Cinética , Fosfolípidos/metabolismo , Prostaglandina D2 , Prostaglandinas A/biosíntesis , Prostaglandinas D/biosíntesis , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesisRESUMEN
Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.
Asunto(s)
Mastocitos/citología , Piel/citología , Calcio/fisiología , Separación Celular , Complemento C5/farmacología , Complemento C5a , Liberación de Histamina , Humanos , Inmunoglobulina E/fisiología , Recién Nacido , Pulmón/citología , Mastocitos/fisiología , N-Formilmetionina/farmacología , Prostaglandina D2 , Prostaglandinas D/biosíntesis , SRS-A/biosíntesis , Sustancia P/fisiologíaRESUMEN
The formation of metabolites of arachidonic acid by the cyclo-oxygenase and lipoxygenase pathways were determined in human lung tissue, obtained from surgery. In this measurement the chopped tissue was incubated with the calcium ionophore A23187. Formation of metabolites from [1-14C] arachidonic acid was also determined. The metabolites were extracted, separated by HPLC and identified by measurement of the absorption spectrum at 280 nm, radioactivity, biological activity and by radioimmunoassay. 6-keto-prostaglandin F1 alpha (6-ketoPGF1 alpha), the metabolite of prostacyclin, is the cyclo-oxygenase product present in the highest amount (400 +/- 49 ng g-1), followed by PGD2 (162 +/- 59 ng g-1) thromboxane B2 (102 +/- 32 ng g-1) PGE2 (104 +/- 46 ng g-1) and PGF2 alpha (58 +/- 26 ng g-1). The amounts of the lipoxygenase products are: leukotriene B4 (LTB4), 163 +/- 100 ng g-1; LTC4, 63 +/- 31 ng g-1 and LTE4 121 +/- 34 ng g-1. From [1-14C] arachidonic acid higher amounts of the cyclooxygenase than of the lipoxygenase products were formed, with the exception of PGE2. The effects of several of these substances on the contraction of human small airway smooth muscle were measured. The contractions, induced by equivalent amounts of LTC4 and a synthetic analogue of thromboxane T X A2 were approximately one hundred times those induced by PGD2, PGF2 alpha and histamine. These results suggest that thromboxane A2 and LTC4 are the most important arachidonic acid metabolites that induce bronchoconstriction in the human lung.