RESUMO
In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.
Assuntos
Bradicinina/análogos & derivados , Isoenzimas/fisiologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Receptores da Bradicinina/fisiologia , Transdução de Sinais/fisiologia , Veias Umbilicais/enzimologia , Anti-Inflamatórios não Esteroides/farmacologia , Bradicinina/farmacologia , Ciclo-Oxigenase 2 , Relação Dose-Resposta a Droga , Humanos , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/fisiologia , Proteínas de Membrana , Técnicas de Cultura de Órgãos , Receptor B1 da Bradicinina , Receptor B2 da Bradicinina , Receptores da Bradicinina/agonistas , Transdução de Sinais/efeitos dos fármacos , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
Bradykinin (BK) B1 receptors are not normally expressed in physiological conditions but could be induced in immunopathological states. Molecular approaches have confirmed that BK B1 receptor gene is transcriptionally induced in injured tissues. In these situations, the cytokine network and other proinflammatory mediators are close linked to BK B1 receptor expression. In this article, we describe the functional characterization of the BK B1 receptor up-regulation process in the isolated human umbilical vein and the pharmacological tools employed to demonstrate the de novo synthesis of these receptors. BK B1 receptors are up-regulated in a time- and protein synthesis-dependent process. Furthermore, in this tissue we have demonstrated the close link between the BK B1 receptor sensitization and proinflammatory cytokines, such as interleukin-1 beta and tumor necrosis factor-alpha. We also discuss the possible relationship between nuclear factor-kappa B and BK B1 receptor induction in human umbilical vein.
Assuntos
Receptores da Bradicinina/biossíntese , Veias Umbilicais/metabolismo , Animais , Humanos , Técnicas In Vitro , NF-kappa B/metabolismo , RNA Mensageiro/genética , Receptor B1 da Bradicinina , Receptores da Bradicinina/genética , Regulação para CimaRESUMO
Previous reports have provided evidence to support the view that the de novo synthesis of bradykinin (BK) B(1) receptor is involved in the induction of vascular responses in human umbilical vein (HUV). In the present study, we evaluated different pharmacological tools to further analyze this up-regulation process in HUV. Concentration-response curves to des-Arg(9)-BK, a selective BK B(1) receptor agonist, were performed after 5 h of incubation. Tumor necrosis factor-alpha potentiated BK B(1) receptor responses at 5 h without modifying the maximal response to des-Arg(9)-BK. Pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-kappaB activation, produced a concentration-dependent decrease of the BK B(1) receptor sensitization. When tissues were continuously exposed to actinomycin D, a transcription inhibitor, or cycloheximide, a protein synthesis inhibitor, concentration-response curves to des-Arg(9)-BK were markedly diminished. On the other hand, transitory exposure to cycloheximide allowed the full recovery of BK B(1) receptor-sensitized responses at 5 h. Finally, continuous incubation with the N-linked glycosylation inhibitor, tunicamycin, almost completely abolished des-Arg(9)-BK-mediated responses. In summary, this sensitization process is potentiated by tumor necrosis factor-alpha and is selectively inhibited by pyrrolidine dithiocarbamate, suggesting that BK B(1) receptor up-regulation in HUV involves nuclear factor-kappaB activation. The effects of actinomycin D and tunicamycin provide evidence that the de novo synthesis of a transmembrane glycoprotein has an obligatory role in the BK B(1) up-regulation. The reversion of the cycloheximide effect on BK B(1) response indicates that the time necessary for synthesis, trafficking, and functional membrane expression of this receptor would be less than 1 h.
