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1.
Biochim Biophys Acta Gen Subj ; 1865(1): 129767, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33141062

RESUMO

BACKGROUND: The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate. When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity. METHODS: We deciphered the reasons of this disparity in studying the role of key parameters, including. cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O2 consumption rates. RESULTS: Our results show that the O2•- low permeability of the vesicle membrane as well as secondary reactions (O2•- and H2O2 disproportionations) are strong clues to shed light on this inconsistency. CONCLUSION AND GENERAL SIGNIFICANCE: These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.


Assuntos
NADPH Oxidase 2/metabolismo , Ácido Araquidônico/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , NADP/metabolismo , Neutrófilos/metabolismo , Oxirredução , Consumo de Oxigênio
2.
PLoS One ; 15(12): e0240070, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382700

RESUMO

Dietary nitrate lowers blood pressure and improves athletic performance in humans, yet data supporting observations that it may increase cerebral blood flow and improve cognitive performance are mixed. We tested the hypothesis that nitrate and nitrite treatment would improve indicators of learning and cognitive performance in a zebrafish (Danio rerio) model. We utilized targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the extent to which treatment resulted in changes in nitrate or nitrite concentrations in the brain and altered the brain metabolome. Fish were exposed to sodium nitrate (606.9 mg/L), sodium nitrite (19.5 mg/L), or control water for 2-4 weeks and free swim, startle response, and shuttle box assays were performed. Nitrate and nitrite treatment did not change fish weight, length, predator avoidance, or distance and velocity traveled in an unstressed environment. Nitrate- and nitrite-treated fish initially experienced more negative reinforcement and increased time to decision in the shuttle box assay, which is consistent with a decrease in associative learning or executive function however, over multiple trials, all treatment groups demonstrated behaviors associated with learning. Nitrate and nitrite treatment was associated with mild anxiogenic-like behavior but did not alter epinephrine, norepinephrine or dopamine levels. Targeted metabolomics analysis revealed no significant increase in brain nitrate or nitrite concentrations with treatment. Untargeted metabolomics analysis found 47 metabolites whose abundance was significantly altered in the brain with nitrate and nitrite treatment. Overall, the depletion in brain metabolites is plausibly associated with the regulation of neuronal activity including statistically significant reductions in the inhibitory neurotransmitter γ-aminobutyric acid (GABA; 18-19%), and its precursor, glutamine (17-22%). Nitrate treatment caused significant depletion in the brain concentration of fatty acids including linoleic acid (LA) by 50% and arachidonic acid (ARA) by 80%; nitrite treatment caused depletion of LA by ~90% and ARA by 60%, change which could alter the function of dopaminergic neurons and affect behavior. Nitrate and nitrite treatment did not adversely affect multiple parameters of zebrafish health. It is plausible that indirect NO-mediated mechanisms may be responsible for the nitrate and nitrite-mediated effects on the brain metabolome and behavior in zebrafish.


Assuntos
Aprendizagem por Associação/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Função Executiva/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Nitratos/farmacologia , Nitrito de Sódio/farmacologia , Animais , Ansiedade/induzido quimicamente , Ansiedade/psicologia , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/metabolismo , Comportamento Animal/efeitos dos fármacos , Tamanho Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Dopamina/metabolismo , Epinefrina/metabolismo , Feminino , Glutamina/metabolismo , Ácido Linoleico/antagonistas & inibidores , Ácido Linoleico/metabolismo , Masculino , Metaboloma/fisiologia , Norepinefrina/metabolismo , Reflexo de Sobressalto/efeitos dos fármacos , Reforço Psicológico , Peixe-Zebra/metabolismo , Ácido gama-Aminobutírico/metabolismo
3.
Nat Cell Biol ; 22(9): 1049-1055, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32868902

RESUMO

Rapid wound detection by distant leukocytes is essential for antimicrobial defence and post-infection survival1. The reactive oxygen species hydrogen peroxide and the polyunsaturated fatty acid arachidonic acid are among the earliest known mediators of this process2-4. It is unknown whether or how these highly conserved cues collaborate to achieve wound detection over distances of several hundreds of micrometres within a few minutes. To investigate this, we locally applied arachidonic acid and skin-permeable peroxide by micropipette perfusion to unwounded zebrafish tail fins. As in wounds, arachidonic acid rapidly attracted leukocytes through dual oxidase (Duox) and 5-lipoxygenase (Alox5a). Peroxide promoted chemotaxis to arachidonic acid without being chemotactic on its own. Intravital biosensor imaging showed that wound peroxide and arachidonic acid converged on half-millimetre-long lipid peroxidation gradients that promoted leukocyte attraction. Our data suggest that lipid peroxidation functions as a spatial redox relay that enables long-range detection of early wound cues by immune cells, outlining a beneficial role for this otherwise toxic process.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Peroxidação de Lipídeos/fisiologia , Ferimentos e Lesões/metabolismo , Peixe-Zebra/metabolismo , Animais , Ácido Araquidônico/metabolismo , Leucócitos/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
5.
PLoS One ; 15(9): e0235384, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925915

RESUMO

Interstitial cystitis/bladder pain syndrome (IC) is a debilitating condition of chronic pelvic pain with unknown etiology. Recently, we used a genetic approach in a murine model of IC to identify the lipase acyloxyacyl hydrolase (AOAH) as a modulator of pelvic pain. We found that AOAH-deficient mice have elevated pelvic pain responses, and AOAH immunoreactivity was detected along the bladder-brain axis. Lipidomic analyses identified arachidonic acid (AA) and its metabolite PGE2 as significantly elevated in the sacral spinal cord of AOAH-deficient mice, suggesting AA is a substrate for AOAH. Here, we quantified the effects of AOAH on phospholipids containing AA. Spinal cord lipidomics revealed increased AA-containing phosphatidylcholine in AOAH-deficient mice and concomitantly decreased AA-phosphatidylethanolamine, consistent with decreased CoA-independent transferase activity (CoIT). Overexpression of AOAH in cell cultures similarly altered distribution of AA in phospholipid pools, promoted AA incorporation, and resulted in decreased membrane fluidity. Finally, administration of a PGE2 receptor antagonist reduced pelvic pain in AOAH-deficient mice. Together, these findings suggest that AOAH represents a potential CoA-independent AA transferase that modulates CNS pain pathways at the level of phospholipid metabolism.


Assuntos
Ácido Araquidônico/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cistite Intersticial/metabolismo , Dor Pélvica/metabolismo , Fosfolipídeos/metabolismo , Animais , Cistite Intersticial/complicações , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Dor Pélvica/complicações , Bexiga Urinária/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-32750662

RESUMO

Cystic fibrosis (CF) is a recessively inherited fatal disease that is the subject of extensive research and ongoing development of therapeutics targeting the defective protein, cystic fibrosis transmembrane conductance regulator (CFTR). Despite progress, the link between CFTR and clinical symptoms is incomplete. The severe CF phenotypes are associated with a deficiency of linoleic acid, which is the precursor of arachidonic acid. The release of arachidonic acid from membranes via phospholipase A2 is the rate-limiting step for eicosanoid synthesis and is increased in CF, which contributes to the observed inflammation. A potential deficiency of docosahexaenoic acid may lead to decreased levels of specialized pro-resolving mediators. This pathophysiology may contribute to an early and sterile inflammation, mucus production, and to bacterial colonization, which further increases inflammation and potentiates the clinical symptoms. Advances in lipid technology will assist in elucidating the role of lipid metabolism in CF, and stimulate therapeutic modulations of inflammation.


Assuntos
Ácido Araquidônico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Ácidos Docosa-Hexaenoicos/deficiência , Ácido Linoleico/deficiência , Ácido Araquidônico/deficiência , Fibrose Cística/fisiopatologia , Humanos , Inflamação/metabolismo , Ácido Linoleico/metabolismo , Metabolismo dos Lipídeos , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/fisiopatologia , Muco/metabolismo
7.
PLoS One ; 15(7): e0228607, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645009

RESUMO

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.


Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase/química , Ácido Araquidônico/química , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Nanoestruturas/ultraestrutura , Ligação Proteica , Conformação Proteica , Sacarose
8.
Artigo em Inglês | MEDLINE | ID: mdl-32717531

RESUMO

Oxylipins, which are circulating bioactive lipids generated from polyunsaturated fatty acids (PUFAs) by cyclooxygenase, lipooxygenase and cytochrome P450 enzymes, have diverse effects on endothelial cells. Although studies of the effects of oxylipins on endothelial cell function are accumulating, a review that provides a comprehensive compilation of current knowledge and recent advances in the context of vascular homeostasis is lacking. This is the first compilation of the various in vitro, ex vivo and in vivo reports to examine the effects and potential mechanisms of action of oxylipins on endothelial cells. The aggregate data indicate docosahexaenoic acid-derived oxylipins consistently show beneficial effects related to key endothelial cell functions, whereas oxylipins derived from other PUFAs exhibit both positive and negative effects. Furthermore, information is lacking for certain oxylipin classes, such as those derived from α-linolenic acid, which suggests additional studies are required to achieve a full understanding of how oxylipins affect endothelial cells.


Assuntos
Células Endoteliais/metabolismo , Ácidos Graxos Insaturados/metabolismo , Oxilipinas/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidônico/metabolismo , Dieta , Ácido Eicosapentaenoico/metabolismo , Humanos , Ácido Linoleico/metabolismo
9.
Med Hypotheses ; 143: 109843, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32492560

RESUMO

SARS-CoV-2 virus has resulted in a devastating pandemic of COVID-19. Exploring compounds that could offer a breakthrough in treatment is the need of the hour. Re-positioning cheap, freely available and safe drugs is a priority. The paper proposes evidence for the potential use of diethylcarbamazine (DEC) in the treatment of COVID-19. DEC has inhibitory effects on arachidonic acid metabolism to prostaglandins, little known anti-viral effects on animal retroviruses and demonstrated anti-inflammatory actions in animal models of lung inflammation indicating the need to explore this hypothesis further. We believe this is the first time DEC is being proposed to treat COVID-19.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Dietilcarbamazina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , Ácido Araquidônico/metabolismo , Betacoronavirus , Reposicionamento de Medicamentos , Filaricidas/uso terapêutico , Humanos , Inflamação , Pulmão/patologia , Pandemias , Prostaglandinas/metabolismo
10.
Chemosphere ; 257: 126892, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32480082

RESUMO

We used the freshwater insect Hydropsyche sp. to investigate the impact of diets lacking arachidonic acid (ARA) and an environmentally relevant mixture of NSAIDs (Ibuprofen, Ketoprofen, Diclofenac and Naproxen at a nominal concentration of all compounds together 16.75 µg L-1) on their metabolism of ARA and prostaglandins (PGs). The organisms were exposed for 16 days to four different treatments: a reference (FF), a diet lacking ARA (O), to NSAIDs in water (FFN) and to the combination of the two factors (ON). Mortality, biomass and bioconcentration of pharmaceuticals were investigated. The ARA and PGs levels in the organisms were monitored by utilising a targeted metabolomics approach. NSAIDs or dietary constraints did not produce significant differences in biomass or mortality of Hydropsyche sp. among treatments. In organisms exposed to NSAIDs, all pharmaceuticals were detected, except for Ketoprofen. Metabolomic approach determined the presence of PGH2, PGE1 and PGD1. Levels of ARA diminished significantly in those organisms in treatment ON. The levels of PGs responded negatively to the absence of ARA in diet: PGH2 diminished significantly with respect to the reference in treatment O while PGE1 diminished significantly in treatment ON. Regarding the effects of NSAIDs on ARA metabolism, our results suggest that it was sensitive to NSAIDs, but effects were weak and did not imply a general decrease in the PGs. We confirmed that ARA was the main substrate for the synthesis of PGs in Hydropsyche sp, their absence or poor levels of ARA in diet, produced changes in the PG levels.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Invertebrados/fisiologia , Prostaglandinas/metabolismo , Animais , Ácido Araquidônico/metabolismo , Diclofenaco , Dieta , Ibuprofeno , Invertebrados/metabolismo , Cetoprofeno , Metabolismo dos Lipídeos , Naproxeno
11.
Nature ; 580(7804): 524-529, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322056

RESUMO

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis.


Assuntos
Carcinogênese , Neoplasias Colorretais/patologia , Intestinos/patologia , Mesoderma/patologia , Células-Tronco Neoplásicas/patologia , Comunicação Parácrina , Nicho de Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos Ly/metabolismo , Ácido Araquidônico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Organoides/metabolismo , Organoides/patologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Análise de Célula Única
12.
Zhongguo Zhong Yao Za Zhi ; 45(6): 1225-1231, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32281329

RESUMO

Since the outbreak of 2019-nCoV, the epidemic has developed rapidly and the situation is grim. LANCET figured out that the 2019-nCoV is closely related to "cytokine storm". "Cytokine storm" is an excessive immune response of the body to external stimuli such as viruses and bacteria. As the virus attacking the body, it stimulates the secretion of a large number of inflammatory factors: interleukin(IL), interferon(IFN), C-X-C motif chemokine(CXCL) and so on, which lead to cytokine cascade reaction. With the exudation of inflammatory factors, cytokines increase abnormally in tissues and organs, interfering with the immune system, causing excessive immune response of the body, resulting in diffuse damage of lung cells, pulmonary fibrosis, and multiple organ damage, even death. Arachidonic acid(AA) metabolic pathway is principally used to synthesize inflammatory cytokines, such as monocyte chemotactic protein 1(MCP-1), tumor necrosis factor(TNF), IL, IFN, etc., which is closely related to the occurrence, development and regression of inflammation. Therefore, the inhibition of AA metabolism pathway is benefit for inhibiting the release of inflammatory factors in the body and alleviating the "cytokine storm". Based on the pharmacophore models of the targets on AA metabolic pathway, the traditional Chinese medicine database 2009(TCMD 2009) was screened. The potential herbs were ranked by the number of hit molecules, which were scored by pharmacophore fit value. In the end, we obtained the potential active prescriptions on "cytokine storm" according to the potential herbs in the "National novel coronavirus pneumonia diagnosis and treatment plan(trial version sixth)". The results showed that the hit components with the inhibitory effect on AA were magnolignan Ⅰ, lonicerin and physcion-8-O-ß-D-glucopy-ranoside, which mostly extracted from Magnoliae Officinalis Cortex, Zingiberis Rhizoma Recens, Lonicerae Japonicae Flos, Rhei Radix et Rhizoma, Salviae Miltiorrhizae Radix et Rhizoma, Scutellariae Radix, Gardeniae Fructus, Ginseng Radix et Rhizoma, Arctii Fructus, Dryopteridis Crassirhizomatis Rhizoma, Paeoniaeradix Rubra, Dioscoreae Rhizoma. Finally the anti-2019-nCoV prescriptions were analyzed to obtain the potential active prescriptions on AA metabolic pathway, Huoxiang Zhengqi Capsules, Jinhua Qinggan Granules, Lianhua Qingwen Capsules, Qingfei Paidu Decoction, Xuebijing Injection, Reduning Injection and Tanreqing Injection were found that may prevent 2019-nCoV via regulate cytokines. This study intends to provide reference for clinical use of traditional Chinese medicine to resist new coronavirus.


Assuntos
Ácido Araquidônico/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Citocinas/imunologia , Medicamentos de Ervas Chinesas/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Betacoronavirus , Humanos , Medicina Tradicional Chinesa , Redes e Vias Metabólicas , Pandemias
13.
J Med Chem ; 63(9): 4824-4836, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302132

RESUMO

Mammary-tissue-restricted cytochrome P450 4Z1 (CYP4Z1) has garnered interest for its potential role in breast cancer progression. CYP4Z1-dependent metabolism of arachidonic acid preferentially generates 14,15-epoxyeicosatrienoic acid (14,15-EET), a metabolite known to influence cellular proliferation, migration, and angiogenesis. In this study, we developed time-dependent inhibitors of CYP4Z1 designed as fatty acid mimetics linked to the bioactivatable pharmacophore, 1-aminobenzotriazole (ABT). The most potent analogue, 8-[(1H-benzotriazol-1-yl)amino]octanoic acid (7), showed a 60-fold lower shifted-half-maximal inhibitory concentration (IC50) for CYP4Z1 compared to ABT, efficient mechanism-based inactivation of the enzyme evidenced by a KI = 2.2 µM and a kinact = 0.15 min-1, and a partition ratio of 14. Furthermore, 7 exhibited low off-target inhibition of other CYP isozymes. Finally, low micromolar concentrations of 7 inhibited 14,15-EET production in T47D breast cancer cells transfected with CYP4Z1. This first-generation, selective mechanism-based inhibitor (MBI) will be a useful molecular tool to probe the biochemical role of CYP4Z1 and its association with breast cancer.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Família 4 do Citocromo P450/antagonistas & inibidores , Ácidos Graxos/farmacologia , Triazóis/farmacologia , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/metabolismo , Desenho de Fármacos , Ácidos Graxos/síntese química , Ácidos Graxos/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxirredução , Triazóis/síntese química , Triazóis/metabolismo
14.
Am J Physiol Renal Physiol ; 318(6): F1369-F1376, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308018

RESUMO

Cytochrome P-450 (Cyp) epoxygenase-dependent metabolites of arachidonic acid (AA) have been shown to inhibit renal Na+ transport, and inhibition of Cyp-epoxygenase is associated with salt-sensitive hypertension. We used the patch-clamp technique to examine whether Cyp-epoxygenase-dependent AA metabolites inhibited the basolateral 40-pS K+ channel (Kir4.1/Kir5.1) in the distal convoluted tubule (DCT). Application of AA inhibited the basolateral 40-pS K+ channel in the DCT. The inhibitory effect of AA on the 40-pS K+ channel was specific because neither linoleic nor oleic acid was able to mimic the effect of AA on the K+ channel. Inhibition of Cyp-monooxygenase with N-methylsulfonyl-12,12-dibromododec-11-enamide or inhibition of cyclooxygenase with indomethacin failed to abolish the inhibitory effect of AA on the 40-pS K+ channel. However, the inhibition of Cyp-epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide abolished the effect of AA on the 40-pS K+ channel in the DCT. Moreover, addition of either 11,12-epoxyeicosatrienoic acid (EET) or 14,15-EET also inhibited the 40-pS K+ channel in the DCT. Whole cell recording demonstrated that application of AA decreased, whereas N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide treatment increased, Ba2+-sensitive K+ currents in the DCT. Finally, application of 14,15-EET but not AA was able to inhibit the basolateral 40-pS K+ channel in the DCT of Cyp2c44-/- mice. We conclude that Cyp-epoxygenase-dependent AA metabolites inhibit the basolateral Kir4.1/Kir5.1 in the DCT and that Cyp2c44-epoxygenase plays a role in the regulation of the basolateral K+ channel in the mouse DCT.


Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido Araquidônico/farmacologia , Família 2 do Citocromo P450/metabolismo , Túbulos Renais Distais/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacologia , Amidas/farmacologia , Animais , Ácido Araquidônico/metabolismo , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/genética , Inibidores Enzimáticos/farmacologia , Túbulos Renais Distais/metabolismo , Masculino , Potenciais da Membrana , Camundongos da Linhagem 129 , Camundongos Knockout , Bloqueadores dos Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo
15.
PLoS One ; 15(3): e0230780, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214349

RESUMO

Calprotectin is a heterodimeric protein complex with two subunits called S100A8/A9. The protein has an essential role in inflammation process and various human diseases. It has the ability to bind to unsaturated fatty acids including Arachidonic acid, Oleic acid and etc., which could be considered as a major carrier for fatty acids. In this study we aimed to appraise the thermodynamics and structural changes of Calprotectin in presence of Arachidonic acid/Oleic acid) using docking and molecular dynami simulation method. To create the best conformation of Calprotectin-Oleic acid/Arachidonic acid complexes, the docking process was performed. The complexes with the best binding energy were selected as the models for molecular dynamics simulation process. Furthermore, the structural and thermodynamics properties of the complexes were evaluated too. The Root Mean Square Deviation and Root Mean Square Fluctuation results showed that the binding of Arachidonic acid/Oleic acid to Calprotectin can cause the protein structural changes which was confirmed by Define Secondary Structure of Proteins results. Accordingly, the binding free energy results verified that binding of Oleic acid to Calprotectin leads to instability of S100A8/A9 subunits in the protein. Moreover, the electrostatic energy contribution of the complexes (Calprotectin-Oleic acid/Arachidonic acid) was remarkably higher than van der Waals energy. Thus, the outcome of this study confirm that Oleic acid has a stronger interaction with Calprotectin in comparison with Arachidonic acid. Our findings indicated that binding of unsaturated fatty acids to Calprotectin leads to structural changes of the S100A8/A9 subunits which could be beneficial to play a biological role in inflammation process.


Assuntos
Ácido Araquidônico/farmacologia , Complexo Antígeno L1 Leucocitário/química , Complexo Antígeno L1 Leucocitário/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácidos Oleicos/farmacologia , Ácido Araquidônico/metabolismo , Ligação de Hidrogênio , Ácidos Oleicos/metabolismo , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos
16.
Artigo em Inglês | MEDLINE | ID: mdl-32151768

RESUMO

His596 of human ALOX12 has been suggested to interact with the COO--group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed light on the molecular basis of the observed functional alterations we performed in silico substrate docking studies and molecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. The combined theoretical and experimental data suggest that Gln596 may not directly interact with the COO--group of arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15 orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acids in the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interface depends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchange impairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functional importance of Gln596 for ALOX15 catalysis.


Assuntos
Sítio Alostérico , Araquidonato 15-Lipoxigenase/química , Simulação de Acoplamento Molecular , Substituição de Aminoácidos , Animais , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Estabilidade Enzimática , Glutamina/química , Glutamina/genética , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Coelhos , Especificidade por Substrato
17.
Vascul Pharmacol ; 127: 106651, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32044414

RESUMO

Aspirin is a widely used drug with anti-coagulating and anti-inflammatory effects on atherosclerotic vascular disease. The goal of this study was to investigate expression of microRNA (miR) in association with changes in arachidonic acid (AA) metabolism in cardiac and surrounding fat mesenchymal stem cells (MSCs) treated with or without aspirin. Aspirin-targeted endogenous lipid metabolites that impact specific miRNA expression were examined by mass spectrometry. The pattern of miR expression was characterized using a microarray of 1100 miRs. There were a dozen miRs expressed differentially in MSCs from human myocardium and peri-myocardial fat tissue at baseline, including hsa-miR-1307-3p, 765, 4739, 3613-3p, 4281, 6816-5p, 2861, and 146b-5p. After exposure to aspirin, cardiac MSCs expressed an array of of miRs (eg, hsa-miR-4734, 10a-5p, 4267, 3197, and 3182), while generation of their endogenous AA metabolites was depressed. However, in the peri-cardiac adipose tissue-derived MSCs, treatment with the same doses of aspirin caused mild changes in the miR expression levels. In conclusion, MSCs from human myocardium and peri-myocardial fat tissue respond differentially to aspirin treatment by alterations in miR expression and AA metabolism. The study further raises an intriguing issue as to whether the copious amounts of aspirin taken worldwide by patients with cardiovascular disease may have direct impacts on their heart repair processes by regulation of stromal cell miR expression and AA metabolism.


Assuntos
Tecido Adiposo/citologia , Anticoagulantes/farmacologia , Ácido Araquidônico/metabolismo , Aspirina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/metabolismo , Miocárdio/citologia , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Fenótipo , Transcriptoma
18.
Sci Rep ; 10(1): 3300, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094450

RESUMO

Aedes aegypti is the primary vector for transmission of Dengue, Zika and chikungunya viruses. Previously it was shown that Dengue virus infection of the mosquito led to an in increased expression of the odorant binding protein 22 (AeOBP22) within the mosquito salivary gland and that siRNA mediated knockdown of AeOBP22 led to reduced mosquito feeding behaviors. Insect OBPs are implicated in the perception, storage and transport of chemosensory signaling molecules including air-borne odorants and pheromones. AeOBP22 is unusual as it is additionally expressed in multiple tissues, including the antenna, the male reproductive glands and is transferred to females during reproduction, indicating multiple roles in the mosquito life cycle. However, it is unclear what role it plays in these tissues and what ligands it interacts with. Here we present solution and X-ray crystallographic studies that indicate a potential role of AeOBP22 binding to fatty acids, and that the specificity for longer chain fatty acids is regulated by a conformational change in the C-terminal tail that leads to creation of an enlarged binding cavity that enhances binding affinity. This study sheds light onto the native ligands for AeOBP22 and provides insight into its potential functions in different tissues.


Assuntos
Aedes/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Odorantes , Animais , Apoproteínas/química , Ácido Araquidônico/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Soluções , Homologia Estrutural de Proteína
19.
Sci Rep ; 10(1): 3490, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103057

RESUMO

Spatial heterogeneity is a fundamental feature of the tumor microenvironment (TME), and tackling spatial heterogeneity in neoplastic metabolic aberrations is critical for tumor treatment. Genome-scale metabolic network models have been used successfully to simulate cancer metabolic networks. However, most models use bulk gene expression data of entire tumor biopsies, ignoring spatial heterogeneity in the TME. To account for spatial heterogeneity, we performed spatially-resolved metabolic network modeling of the prostate cancer microenvironment. We discovered novel malignant-cell-specific metabolic vulnerabilities targetable by small molecule compounds. We predicted that inhibiting the fatty acid desaturase SCD1 may selectively kill cancer cells based on our discovery of spatial separation of fatty acid synthesis and desaturation. We also uncovered higher prostaglandin metabolic gene expression in the tumor, relative to the surrounding tissue. Therefore, we predicted that inhibiting the prostaglandin transporter SLCO2A1 may selectively kill cancer cells. Importantly, SCD1 and SLCO2A1 have been previously shown to be potently and selectively inhibited by compounds such as CAY10566 and suramin, respectively. We also uncovered cancer-selective metabolic liabilities in central carbon, amino acid, and lipid metabolism. Our novel cancer-specific predictions provide new opportunities to develop selective drug targets for prostate cancer and other cancers where spatial transcriptomics datasets are available.


Assuntos
Redes e Vias Metabólicas/genética , Neoplasias da Próstata/patologia , Ácido Araquidônico/metabolismo , Cisteína/metabolismo , Bases de Dados Factuais , Humanos , Masculino , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Neoplasias da Próstata/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/metabolismo , Ácido Succínico/metabolismo , Suramina/química , Suramina/metabolismo , Microambiente Tumoral
20.
Circ Res ; 126(7): 839-853, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32078445

RESUMO

RATIONALE: High-salt diet is one of the most important risk factors for hypertension. Intestinal flora has been reported to be associated with high salt-induced hypertension (hSIH). However, the detailed roles of intestinal flora in hSIH pathogenesis have not yet been fully elucidated. OBJECTIVE: To reveal the roles and mechanisms of intestinal flora in hSIH development. METHODS AND RESULTS: The abovementioned issues were investigated using various techniques including 16S rRNA gene sequencing, untargeted metabolomics, selective bacterial culture, and fecal microbiota transplantation. We found that high-salt diet induced hypertension in Wistar rats. The fecal microbiota of healthy rats could dramatically lower blood pressure (BP) of hypertensive rats, whereas the fecal microbiota of hSIH rats had opposite effects. The composition, metabolism, and interrelationship of intestinal flora in hSIH rats were considerably reshaped, including the increased corticosterone level and reduced Bacteroides and arachidonic acid levels, which tightly correlated with BP. The serum corticosterone level was also significantly increased in rats with hSIH. Furthermore, the above abnormalities were confirmed in patients with hypertension. The intestinal Bacteroides fragilis could inhibit the production of intestinal-derived corticosterone induced by high-salt diet through its metabolite arachidonic acid. CONCLUSIONS: hSIH could be transferred by fecal microbiota transplantation, indicating the pivotal roles of intestinal flora in hSIH development. High-salt diet reduced the levels of B fragilis and arachidonic acid in the intestine, which increased intestinal-derived corticosterone production and corticosterone levels in serum and intestine, thereby promoting BP elevation. This study revealed a novel mechanism different from inflammation/immunity by which intestinal flora regulated BP, namely intestinal flora could modulate BP by affecting steroid hormone levels. These findings enriched the understanding of the function of intestinal flora and its effects on hypertension.


Assuntos
Pressão Sanguínea/fisiologia , Corticosterona/biossíntese , Microbioma Gastrointestinal/fisiologia , Hipertensão/fisiopatologia , Intestinos/química , Animais , Ácido Araquidônico/metabolismo , Bacteroides fragilis/fisiologia , Corticosterona/sangue , Transplante de Microbiota Fecal , Fezes/microbiologia , Humanos , Hipertensão/etiologia , Hipertensão/microbiologia , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Metabolômica/métodos , Ratos Wistar , Cloreto de Sódio na Dieta/efeitos adversos
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