Ang II-stimulated superoxide production is mediated via phospholipase D in human vascular smooth muscle cells

Abstract

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 genera e 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 he 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 [3H]thymidine and [3H]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 (E(max)=57.6±1.7) nmol/L, pD2=7.7±0.06) and PLD activation (E(max)=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<0.01) Ang II-stimulated responses. PLD inhibition significantly attenuated (P<0.05) Ang II-elicited H2O2 production (E(max)=29±5 nmol/L). DPI and sphinganine inhibited Ang II- induced DNA and protein synthesis. These data indicate that in vascular smooth muscle cells from human peripheral resistance arteries, Ang II increases H2O2 generation via PLD-dependent, NADH/NADPH oxidase-sensitive pathways. These cascades may function as second messengers in long-term Ang II-mediated growth-signaling events.