Abstract-Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle cell growth, an important feature of hypertension and atherosclerosis. Many of these effects are dependent on the production of reactive oxygen species (ROS). Ang II induces phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules. Here, we provide novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. Pretreatment of vascular smooth muscle cells with the antioxidants diphenylene iodonium, Tiron, N-acetylcysteine, and ebselen significantly inhibited (Ϸ80% to 90%) tyrosine phosphorylation of the EGF-R by Ang II but not by EGF. Of the 5 autophosphorylation sites on the EGF-R, Ang II mainly phosphorylated Tyr1068 and Tyr1173 in a redox-sensitive manner. The Src family kinase inhibitor PP1, overexpression of kinase-inactive c-Src, or chelation of intracellular Ca 2ϩ attenuated EGF-R transactivation. Although antioxidants had no effects on the Ca 2ϩ mobilization or phosphorylation of Ca 2ϩ -dependent tyrosine kinase Pyk2, they inhibited c-Src activation by Ang II, suggesting that c-Src is 1 signaling molecule that links ROS and EGF-R phosphorylation. Furthermore, Ang II-induced tyrosine phosphorylation of the autophosphorylation site and the SH2 domain of c-Src was redox sensitive. These findings emphasize the importance of ROS in specific Ang II-stimulated growth-related signaling pathways and suggest that redox-sensitive EGF-R transactivation may be a potential target for antioxidant therapy in vascular disease. A ngiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle growth, a cardinal feature of hypertension and atherosclerosis. Ang II exerts its effects via activation of a complex set of enzymatic cascades exhibiting considerable cross talk. Recently, attention has focused on the role of reactive oxygen species (ROS) in transducing growthrelated molecular signals and cardiovascular disease. Ang II rapidly increases superoxide and hydrogen peroxide (H 2 O 2 ) production in vascular smooth muscle cells (VSMCs) via activation of a p22phox-based NAD(P)H oxidase. 1-4 These ROS are required for activation of a specific set of downstream kinases, including p38 mitogen-activated protein kinase (p38 MAPK) and Akt. 4,5 Importantly, both of these pathways are required for the hypertrophic response by Ang II. 4,5 The most proximal mechanisms coupling these responses to the Ang II type 1 (AT 1 ) receptor remain unclear.

The regulation of intracellular calcium concentration in single smooth muscle cells was investigated by simultaneously monitoring electrical events at the surface membrane and calcium concentration in the cytosol. Cytosolic calcium concentration rose rapidly during an action potential or during a voltage-clamp pulse that elicited calcium current; a train of voltage-clamp pulses caused further increases in the calcium concentration up to a limit of approximately 1 microM. The decline of the calcium concentration back to resting levels occurred at rates that varied with the calcium concentration in an apparently saturable manner. Moreover, the rate of decline at any given calcium concentration was enhanced after a higher, more prolonged increase of calcium. The process responsible for this enhancement persisted for many seconds after the calcium concentration returned to resting levels. Thus, the magnitude and duration of a calcium transient appear to regulate the subsequent calcium removal.

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The role of Ca24 in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2I] simultaneously in individual single smooth-muscle cells. [Ca24] was measured with fura-2 and a high timeresolution dual-wavelength digital microfluorimeter, and force was measured with an ultrasensitive force transducer attached to a probe around which was tied one end of the cell. Both [Ca2+1 and force increase after maximal electrical stimulus, with [Ca24] Calcium is considered the key regulator of contraction of smooth muscle (1, 2), although details of this regulation remain somewhat unclear. The relationship between force and [Ca2 '] has been studied in skinned smooth-muscle strips (3-11) and in smooth muscles that were loaded with aequorin by temporarily permeabilizing the tissue (12). The sensitivity of force to [Ca24] found in chemically permeabilized (skinned) preparations by various investigators varies by almost two orders of magnitude, and several studies showed that the sensitivity can be altered by calmodulin addition (5,6,13) per se, accounts for the several hundred-msec delay between stimulation and onset of active-force production characteristic of smooth muscle. Finally, our results show that the contractile process exhibits decreasing sensitivity to calcium during slow contraction-relaxation cycles. METHODSSmooth muscle cells were isolated from the stomach of the toad Bufo marinus as described (14). Briefly, the procedure involves incubating thin slices of stomach wall first in a dilute solution of collagenase and trypsin and then in a solution of collagenase alone. Cells released from the tissue in this latter solution are collected by mechanically separating the tissue from the cell suspension. Soybean trypsin inhibitor was added to all cell suspensions to suppress any remaining trypsin activity.Cells were loaded with the calcium-sensitive dye fura-2 (15) by incubating them in a solution of the acetoxymethyl ester form ofthe dye, fura-2/AM (Molecular Probes, Eugene, OR). Cells were incubated in this solution at 31°C for 60 min and then returned to room temperature. The amount of fura-2/AM added to the cell suspension was adjusted according to cell density at the ratio of 1.25 x 10-9 mol of fura-2/AM per 106 cells. These loading conditions were found to produce intracellular fura-2 concentrations of 30-100 ,M as estimated by comparing total fluorescence to that from droplets of fura-2 solutions in oil.A dilute suspension of cells that had been loaded with fura-2 was placed on a cover-slip chamber on the stage of an inverted microscope, and the cells were allowed to settle. A single cell was then attached at both ends to two special glass microprobes, each of which had a single anionic-exchangeresin particle cemented to its tip. After attachment of cell to probe, a small section of the muscle cell was wrapped at each end by micromanipulation around the attached probe. The procedure was basically that described by Warshaw and Fay (16). One probe was attached to a micr...

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT 1 receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by ␤-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca 2؉ increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosinephosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.Angiotensin II (Ang II) 1 is a highly pluripotential hormone in vascular smooth muscle cells (VSMCs) and stimulates multiple signaling pathways, including Src family kinases, as well as mitogen-activated protein kinases (MAPKs) and Akt/protein kinase B (PKB), that mediate VSMC hypertrophy and growth via AT 1 receptors (AT 1 Rs). Increasing evidence suggests that transmodulation of the epidermal growth factor receptor (EGF-R), which serves as a scaffold for various signaling molecules, plays an essential role in organizing Ang II-mediated tyrosine kinase signaling pathways. We and others (1, 2) have demonstrated that EGF-R transactivation by Ang II is mediated through Ca 2ϩ , c-Src, and NADPH oxidase-derived reactive oxygen species, leading to activation of downstream signaling such as extracellular signal regulated kinase (ERK) and Akt/ PKB in VSMCs.Relatively little is known of the mechanisms controlling the spatial and temporal organization of AT 1 R signaling in VSMCs or of how specificity is achieved. We showed originally that Ang II signaling in VSMC is biphasic and that internalization or sequestration of the agonist-occupied receptor into a "signaling domain" ...