RESUMO
Cytochrome P450-derived epoxides of arachidonic acid [i.e., the epoxyeicosatrienoic acids (EETs)] are important lipid signaling molecules involved in the regulation of vascular tone and angiogenesis. Because many actions of 11,12-cis-epoxyeicosatrienoic acid (EET) are dependent on the activation of protein kinase A (PKA), the existence of a cell-surface G(s)-coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12-EET are enantiomer specific and linked to a potential G protein-coupled receptor, we assessed 11,12-EET-induced, PKA-dependent translocation of transient receptor potential (TRP) C6 channels, as well as angiogenesis. In primary cultures of human endothelial cells, (±)-11,12-EET led to the rapid (30 seconds) translocation a TRPC6-V5 fusion protein, an effect reproduced by 11(R),12(S)-EET, but not by 11(S),12(R)-EET or (±)-14,15-EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)-11,12-EET and 11(R),12(S)-EET, whereas 11(S),12(R)-EET and 11,12-dihydroxyeicosatrienoic acid were without effect. The effects of (±)-11,12-EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists, and TRP channel trafficking was also prevented by a PKA inhibitor. The small interfering RNA-mediated downregulation of G(s) in endothelial cells had no significant effect on responses stimulated by vascular endothelial growth or a PKA activator but abolished responses to (±)-11,12-EET. The downregulation of G(q)/11 failed to prevent 11,12-EET-induced TRPC6 channel translocation or the formation of capillary-like structures. Taken together, our results suggest that a G(s)-coupled receptor in the endothelial cell membrane responds to 11(R),12(S)-EET and mediates the PKA-dependent translocation and activation of TRPC6 channels, as well as angiogenesis.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Canais de Cátion TRPC/metabolismo , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/farmacologia , Indutores da Angiogênese/farmacologia , Movimento Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Humanos , Cultura Primária de Células , RNA Interferente Pequeno/farmacologia , Estereoisomerismo , Canal de Cátion TRPC6 , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Activation of the basal forebrain (BF), the primary source of acetylcholine (ACh) in the cortex, broadly increases cortical cerebral blood flow (CBF), a response downstream to ACh release. Although endothelial nitric oxide and cholinoceptive GABA (γ-aminobutyric acid) interneurons have been implicated, little is known about the role of pyramidal cells in this response and their possible interaction with astrocytes. Using c-Fos immunohistochemistry as a marker of neuronal activation and laser-Doppler flowmetry, we measured changes in CBF evoked by BF stimulation following pharmacological blockade of c-Fos-identified excitatory pathways, astroglial metabolism, or vasoactive mediators. Pyramidal cells including those that express cyclooxygenase-2 (COX-2) displayed c-Fos upregulation. Glutamate acting via NMDA, AMPA, and mGlu receptors was involved in the evoked CBF response, NMDA receptors having the highest contribution (~33%). In contrast, nonselective and selective COX-2 inhibition did not affect the evoked CBF response (+0.4% to 6.9%, ns). The metabolic gliotoxins fluorocitrate and fluoroacetate, the cytochrome P450 epoxygenase inhibitor MS-PPOH and the selective epoxyeicosatrienoic acids (EETs) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) all blocked the evoked CBF response by ~50%. Together, the data demonstrate that the hyperemic response to BF stimulation is largely mediated by glutamate released from activated pyramidal cells and by vasoactive EETs, likely originating from activated astrocytes.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Potenciais Evocados/fisiologia , Células Piramidais/metabolismo , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/metabolismo , Acetilcolina/metabolismo , Amidas/farmacologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Velocidade do Fluxo Sanguíneo/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Citratos/toxicidade , Ciclo-Oxigenase 2/metabolismo , Potenciais Evocados/efeitos dos fármacos , Fluoracetatos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células Piramidais/citologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The increase in cerebral blood flow (CBF) during neuronal activation can be only partially attenuated by individual inhibitors of epoxyeicosatrienoic acids (EETs), cyclooxgenase-2, group I metabotropic glutamate receptors (mGluR), neuronal nitric oxide synthase (nNOS), N-methyl-D-aspartate receptors, or adenosine receptors. Some studies that used a high concentration (500 µM) of the cyclooxygenase-1 inhibitor SC-560 have implicated cyclooxygenase-1 in gliovascular coupling in certain model systems in the mouse. Here, we found that increasing the concentration of SC-560 from 25 µM to 500 µM over whisker barrel cortex in anesthetized rats attenuated the CBF response to whisker stimulation. However, exogenous prostaglandin E(2) restored the response in the presence of 500 µM SC-560 but not in the presence of a cyclooxygenase-2 inhibitor, thereby suggesting a limited permissive role for cyclooxygenase-1. Furthermore, inhibition of the CBF response to whisker stimulation by an EET antagonist persisted in the presence of SC-560 or a cyclooxygenase-2 inhibitor, thereby indicating that the EET-dependent component of vasodilation did not require cyclooxygenase-1 or -2 activity. With combined inhibition of cyclooxygenase-1 and -2, mGluR, nNOS, EETs, N-methyl-D-aspartate receptors, and adenosine 2B receptors, the CBF response was reduced by 60%. We postulated that the inability to completely block the CBF response was due to tissue acidosis resulting from impaired clearance of metabolically produced CO2. We tested this idea by increasing the concentration of superfused bicarbonate from 25 to 60 mM and found a markedly reduced CBF response to hypercapnia. However, increasing bicarbonate had no effect on the initial or steady-state CBF response to whisker stimulation with or without combined inhibition. We conclude that the residual response after inhibition of several known vasodilatory mechanisms is not due to acidosis arising from impaired CO2 clearance when the CBF response is reduced. An unidentified mechanism apparently is responsible for the rapid, residual cortical vasodilation during vibrissal stimulation.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Circulação Cerebrovascular/fisiologia , Ciclo-Oxigenase 1/fisiologia , Ciclo-Oxigenase 2/fisiologia , Córtex Somatossensorial/irrigação sanguínea , Córtex Somatossensorial/metabolismo , Vibrissas/fisiologia , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacologia , Acidose/metabolismo , Animais , Bicarbonatos/farmacologia , Circulação Cerebrovascular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Ratos , Ratos Wistar , Córtex Somatossensorial/efeitos dos fármacosRESUMO
Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC-3, DU-145 and LNCaP) cells. 11,12-Epoxyeicosatrienoic acid (11,12-EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration-dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12-EET-induced cell migration. Importantly, synthetic EET antagonists (14,15-epoxyeicosa-5(Z)-enoic acid [14,15-EEZE], 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester [14,15-EEZE-PEG] and 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide [14,15-EEZE-mSI]) inhibited EET-induced cell invasion and migration. 11,12-EET induced cell stretching and myosin-actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15-EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor-mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Carcinoma/metabolismo , Movimento Celular/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Imunofluorescência , Humanos , Masculino , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Epoxyeicosatrienoic acids (EETs) are endothelium-derived metabolites of arachidonic acid. They relax vascular smooth muscle by membrane hyperpolarization. These actions are inhibited by the EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE5ZE). We synthesized 20-(125)iodo-14,15-EE5ZE (20-(125)I-14,15-EE5ZE), a radiolabeled EET antagonist, and characterized its binding to cell membranes. 14,15-EET (10(-9)-10(-5)M) caused a concentration-related relaxation of the preconstricted bovine coronary artery and phosphorylation of p38 in U937 cells that were inhibited by 20-(125)I-14,15-EE5ZE. Specific 20-(125)I-14,15-EE5ZE binding to U937 cell membranes reached equilibrium within 5 min and remained unchanged for 30 min. The binding was saturable and reversible, and it exhibited K(D) and B(max) values of 1.11 +/- 0.13 nM and 1.13 +/- 0.04 pmol/mg protein, respectively. Guanosine 5'-O-(3-thio)triphosphate (10 muM) did not change the binding, indicating antagonist binding of the ligand. Various EETs and EET analogs (10(-10)-10(-5)M) competed for 20-(125)I-14,15-EE5ZE binding with an order of potency of 11,12-EET = 14,15-EET > 8,9-EET = 14,15-EE5ZE > 15-hydroxyeicosatetraenoic acid = 14,15-dihydroxyeicosatrienoic acid. 8,9-Dihydroxyeicosatrienoic acid and 11-hydroxyeicosatetraenoic acid did not compete for binding. The soluble and microsomal epoxide hydrolase inhibitors (1-cyclohexyl-3-dodecyl-urea, elaidamide, and 12-hydroxyl-elaidamide) and cytochrome P450 inhibitors (sulfaphenazole and proadifen) did not compete for the binding. However, two cytochrome P450 inhibitors, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) and miconazole competed for binding with K(i) of 1558 and 315 nM, respectively. Miconazole and MS-PPOH, but not proadifen, inhibited 14,15-EET-induced relaxations. These findings define an EET antagonist's binding site and support the presence of an EET receptor. The inhibition of binding by some cytochrome P450 inhibitors suggests an alternative mechanism of action for these drugs and could lead to new drug candidates that target the EET binding sites.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Membrana Celular/metabolismo , Compostos de Epóxi/farmacologia , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/síntese química , Ácido 8,11,14-Eicosatrienoico/química , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , Sítios de Ligação , Western Blotting , Bovinos , Membrana Celular/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Relação Dose-Resposta a Droga , Epóxido Hidrolases/antagonistas & inibidores , Compostos de Epóxi/síntese química , Compostos de Epóxi/química , Humanos , Radioisótopos do Iodo , Ligantes , Fosforilação , Células U937 , Vasodilatação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Células Cultivadas , Citocromo P-450 CYP2C8 , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Oligonucleotídeos Antissenso/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Fatores de Tempo , TransfecçãoRESUMO
We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ERalpha-knockout (ERalpha-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ERalpha-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ERalpha-KO arteries, N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited FID by approximately 26%, whereas indomethacin inhibited dilations by approximately 50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/cm(2) shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ERalpha-KO mice subjected to L-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ERalpha-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ERbeta in ERalpha-KO arteries. Treatment of ERalpha-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ERalpha-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone.
Assuntos
Ácido 8,11,14-Eicosatrienoico/farmacologia , Aromatase/fisiologia , Artérias/fisiologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Vasodilatação/genética , Vasodilatação/fisiologia , Ácido 8,11,14-Eicosatrienoico/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Western Blotting , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Técnicas In Vitro , Indometacina/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Compostos Organofosforados/farmacologia , Peptídeos/farmacologia , Perfusão , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse MecânicoRESUMO
Epoxyeicosatrienoic acids (EETs) are potent regulators of vascular homeostasis and are bound by cytosolic fatty acid-binding proteins (FABPs) with K(d) values of approximately 0.4 microM. To determine whether FABP binding modulates EET metabolism, we examined the effect of FABPs on the soluble epoxide hydrolase (sEH)-mediated conversion of EETs to dihydroxyeicosatrienoic acids (DHETs). Kinetic analysis of sEH conversion of racemic [(3)H]11,12-EET yielded K(m) = 0.45 +/- 0.08 microM and V(max) = 9.2 +/- 1.4 micromol min(-1) mg(-)(1). Rat heart FABP (H-FABP) and rat liver FABP were potent inhibitors of 11,12-EET and 14,15-EET conversion to DHET. The resultant inhibition curves were best described by a substrate depletion model, with K(d) = 0.17 +/- 0.01 microM for H-FABP binding to 11,12-EET, suggesting that FABP acts by reducing EET availability to sEH. The EET depletion by FABP was antagonized by the co-addition of arachidonic acid, oleic acid, linoleic acid, or 20-hydroxyeicosatetraenoic acid, presumably due to competitive displacement of FABP-bound EET. Collectively, these findings imply that FABP might potentiate the actions of EETs by limiting their conversion to DHET. However, the effectiveness of this process may depend on metabolic conditions that regulate the levels of competing FABP ligands.