RESUMO
Emerging evidence indicates that reactive oxygen species are important regulators of vascular function. Although NAD(P)H oxidases have been implicated as major sources of superoxide in the vessel wall, the molecular identity of these proteins remains unclear. We recently cloned nox1 (formerly mox-1), a member of a new family of gp91(phox) homologues, and showed that it is expressed in proliferating vascular smooth muscle cells (VSMCs). In this study, we examined the expression of three nox family members, nox1, nox4, and gp91(phox), in VSMCs, their regulation by angiotensin II (Ang II), and their role in redox-sensitive signaling. We found that both nox1 and nox4 are expressed to a much higher degree than gp91(phox) in VSMCS: Although serum, platelet-derived growth factor (PDGF), and Ang II downregulated nox4, they markedly upregulated nox1, suggesting that this enzyme may account for the delayed phase of superoxide production in these cells. Furthermore, an adenovirus expressing antisense nox1 mRNA completely inhibited the early phase of superoxide production induced by Ang II or PDGF and significantly decreased activation of the redox-sensitive signaling molecules p38 mitogen-activated protein kinase and Akt by Ang II. In contrast, redox-independent pathways induced by PDGF or Ang II were unaffected. These data support a role for nox1 in redox signaling in VSMCs and provide insight into the molecular identity of the VSMC NAD(P)H oxidase and its potentially critical role in vascular disease.
Assuntos
Glicoproteínas de Membrana/genética , Músculo Liso Vascular/metabolismo , Animais , Northern Blotting , Linhagem Celular , Células Cultivadas , DNA Antissenso/genética , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Maleimidas/farmacologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oxirredução , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Análise de Sequência de DNA , Transdução de Sinais , Superóxidos/metabolismo , Fatores de TempoRESUMO
Recently, heme oxygenase-1 (HO-1) has been shown to be present in vascular smooth muscle cells. In the present study, we examined the effect of angiotensin II (Ang II) on HO-1 in rat vascular smooth muscle cells. After treatment with 100 nmol/L Ang II, HO-1 mRNA levels were decreased, with a nadir at 2 hours (39+/-9% of the control level, P<.01). This downregulation was completely blocked by the Ang II type I receptor antagonist losartan. Western blot analysis showed that HO-1 protein is also significantly downregulated, with a nadir at 4 hours (52+/-6% of the control level, P<.01). Heme oxygenase activity was also significantly decreased at 4 hours (control, 0.35+/-0.86 nmol bilirubin/mg per hour; Ang II, 0.10+/-0.06). This downregulation was observed in serum-starved cells to a similar extent as in serum-supplemented cells. Inhibitors of protein kinase C, lipoxygenase, cyclooxygenase, cytochrome P450 monooxygenase, and phospholipase A2 did not block this downregulation. However, this effect was not observed in the absence of calcium and presence of EGTA (2 mmol/L). Furthermore, a 2-hour incubation with calcium ionophore or arginine vasopressin decreased HO-1 mRNA levels, suggesting that an increase of intracellular calcium mediates the downregulation. In conclusion, Ang II decreases HO-1 mRNA in a calcium-dependent manner in vascular smooth muscle cells, which may provide a novel mechanism for the modulation of vascular tone and oxidative stress.
Assuntos
Angiotensina II/fisiologia , Antagonistas de Receptores de Angiotensina , Heme Oxigenase (Desciclizante)/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Vasoconstritores/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Compostos de Bifenilo/farmacologia , Células Cultivadas , Regulação para Baixo , Imidazóis/farmacologia , Losartan , Masculino , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Tetrazóis/farmacologiaRESUMO
Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.