Abstract-The atherogenic effect of the renin-angiotensin system can be explained, in part, by the influence of its effector, angiotensin II (Ang II), on vascular smooth muscle cell (VSMC) growth. There is evidence that reactive oxygen species (ROS) play a role in the atherogenesis and activation of mitogen-activating protein (MAP) kinases, which are involved in proliferation and differentiation. The study was performed to further characterize the role of ROS in Ang II-mediated MAP kinase activation and the regulation of the transcription factor activator protein-1 (AP-1). Rat VSMCs were stimulated with Ang II. The activities of MAP kinases were assessed by Western blot analysis or by immunocomplex kinase assay. AP-1 binding was determined by using an electrophoretic mobility shift assay. Rat VSMCs were treated with Ang II-activated MAP kinases, extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK), p38 MAP kinase (p38 MAPK), and their downstream effector, AP-1. Interestingly, only the activation of ERK1/2, but not JNK or p38 MAPK, was tyrosine kinase, protein kinase C, and MEK1/2 dependent. Ang II also induced the rapid formation of ROS, which could be inhibited by a specific antibody as well as by antisense against the p22phox subunit of the NAD(P)H oxidase. JNK and p38 MAPK, but not ERK, activation was inhibited by an inhibitor of NAD(P)H oxidase. Antisense against p22phox also solely inhibited p38 MAPK but did not affect ERK. The results indicate that in VSMCs, Ang II activates MAP kinases and AP-1 through different pathways; the results further suggest that ROS, generated by p22phox, mediate Ang II-induced JNK and p38 MAPK activation, which may contribute to the pathogenesis of atherosclerosis. Key Words: angiotensin II Ⅲ atherosclerosis Ⅲ reactive oxygen species Ⅲ mitogen-activated protein kinase Ⅲ activator protein-1 A ngiotensin II (Ang II), the main peptide hormone of the renin-angiotensin system, plays an important role in the pathogenesis of cardiovascular diseases, including atherosclerosis, myocardial infarction, and hypertension. 1 Ang II exerts hypertrophic and hyperplastic effects by activating a number of intracellular signal transduction pathways through a 7-transmembrane heterotrimeric G protein-coupled receptor called the Ang II type 1 (AT 1 ) receptor. 2 New data indicate that Ang II plays an important role in the generation of reactive oxygen species (ROS) by activation of NAD(P)H oxidase, a plasma membrane-bound protein. 3,4

Insulin caused a transient increase in H202 accumulation in human fat cell suspensions that was observed only in the presence of an inhibitor of catalase and heme-containing peroxidases, such as azide, and reached peak levels of 30 gM within 5 min. The cells contained a plasma membrane-bound NADPH oxidase, producing 1 mol H202/mol of NADPH oxidation, that was activated on exposure of intact cells to insulin at contrations that are physiologically relevant (0.1-10 nM). The hormone effect was rapid and was due to a selective increase in substrate affinity. The enzyme was magnesium dependent, required a flavine nucleotide for optimal activity, and was most active at pH 5.0-6.5. In contrast to all other hormone-or cytokine-sensitive NADPH oxidases that have been characterized in sufficient detail, the human fat cell oxidase retained its hormone responsiveness after cell disruption, and only Mn2', but no ATP, was required for a ligand-induced activation in crude plasma membranes. The results demonstrate that insulin utilizes tyrosine kinase-independent pathways for receptor signaling and strongly support the view that H202 contributes to the intracellular propagation of the insulin signal. (J. Clin. Invest.

Human fat cells possess a multireceptor-linked H 2 O 2 -generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H 2 O 2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H 2 O 2 generation by Mn 2؉ and insulin was blocked by GDP␤S (guanosine 5-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-G␣ i1-2 or the corresponding peptide.Consistently, manganese could be replaced by micromolar concentrations of GTP␥S (guanosine 5-O-(3-thiotriphosphate)), which increased NADPH-dependent H 2 O 2 generation by 20 -40%. Insulin shifted the dose response curve for GTP␥S to the left (>10-fold) and increased the maximal response. In the presence of 10 M GTP␥S, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor.The insulin receptor and G i were co-adsorbed on anti-G␣ i and anti-insulin receptor ␤-subunit (anti-IR␤) affinity columns. Partially purified insulin receptor preparations contained G␣ s , G␣ i2 , and G␤␥ (but no G␣ i1 or G␣ i3 ). The functional nature of the insulin receptor-G i2 complex was made evident by insulin's ability to modulate labeling of G i by bacterial toxins. Insulin action was mimicked by activated G␣ i , but not by G␣ o or G␤␥, indicating that insulin's signal was transduced via G␣ i2 . Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.

3T3 L1-cells, which undergo adipose conversion in vitro, possess a stimulus-sensitive H2O2-generating system in their plasma membrane, and its properties are virtually identical with those of the insulin-sensitive human fat-cell oxidase [Krieger-Brauer and Kather (1992) J. Clin. Invest. 89, 1006-1013]. Insulin and insulin-like growth factor I were found to be active stimulators of NADPH-dependent H2O2 generation. Surprisingly, the acidic (a) and basic (b) isoforms of fibroblast growth factor (FGF) as well as the AA and BB homodimers of platelet-derived growth factor (PDGF) had antagonistic effects on NADPH-dependent H2O2 generation in plasma membranes which were parallelled by corresponding changes in H2O2 accumulation in intact cells. bFGF and PDGF BB (which inhibit NADPH-dependent H2O2 generation) prevented the adipose conversion of 3T3 L1-preadipocytes, and this effect could be reversed by exogenously supplied H2O2. Conversely, aFGF and PDGF AA, which stimulated H2O2 generation, accelerated adipocyte conversion in the presence of insulin and were adipogenic in themselves. Consistently, expression of the adipocyte phenotype induced by insulin, dexamethasone and isobutylmethylxanthine was enhanced in the presence of exogenous hypoxanthine/xanthine oxidase, whereas antioxidants, such as N-acetylcysteine or ascorbate, suppressed the process of differentiation. It is concluded that the H2O2 produced in response to hormones and cytokines may contribute to the development and maintenance of the differentiated state.