Ang II-Stimulated Superoxide Production Is Mediated via Phospholipase D in Human Vascular Smooth Muscle Cells

• Published in: Hypertension (Dallas, Tex. 1979) Tex. 1979), 1999-10, Vol.34 (4, Part 2), p.976-982
• Main Authors: Touyz, Rhian M, Schiffrin, Ernesto L
• Format: Article
• Language: English
• Subjects: Angiotensin II - pharmacology; Arterial hypertension. Arterial hypotension; Biological and medical sciences; Blood and lymphatic vessels; Cardiology. Vascular system; Cells, Cultured; Clinical manifestations. Epidemiology. Investigative techniques. Etiology; Enzyme Inhibitors - pharmacology; Humans; Medical sciences; Muscle, Smooth, Vascular - metabolism; Phospholipase D - metabolism; Receptors, Angiotensin - metabolism; Signal Transduction - drug effects; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; Superoxides - metabolism; Vasoconstrictor Agents - pharmacology
• ISSN: 0194-911X; 1524-4563
• DOI: 10.1161/01.hyp.34.4.976

Intracellular signaling events that mediate the long-term effects of Ang II in vascular smooth muscle cells are unclear, but oxidative stress may play an important role. This study examined the ability of Ang II to generate reactive oxygen species and investigated the putative role of phospholipase D (PLD)-dependent signaling pathways for its production in human vascular smooth muscle cells. In addition, we assessed whether redox-sensitive pathways influence Ang II-stimulated cell growth. Primary and low-passage cells (passages 1 to 4) derived from resistance arteries of subcutaneous gluteal biopsies from healthy subjects were studied. Oxidative stress was measured with the fluorescent probe 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (8 μmol/L), and the role of PLD was assessed with the PLD inhibitor D-erythro-sphingosine, dihydro (sphinganine) (10 μmol/L). To determine whether NADH/NADPH oxidase contributes to production of reactive oxygen species, Ang II-stimulated cells were pretreated with the specific flavoprotein inhibitor diphenylene iodinium (DPI) (10 μmol/L). DNA and protein synthesis were determined by [H]thymidine and [H]leucine incorporation, respectively. Ang II increased CM-H2DCFDA fluorescence, and this was inhibited by catalase (350 U/mL), indicating that the fluorescence signal was derived predominantly from H2O2. Ang II dose-dependently increased H2O2 production (Emax=57.6±1.7 nmol/L, pD2=7.7±0.06) and PLD activation (Emax=207±3.3% of control, pD2=7.7±0.5). H2O2 effects were evident within 1 hour, and maximal PLD activation occurred within 40 minutes after stimulation. DPI inhibited (P