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

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 r eactive oxygen species and investigated the putative role of phospholipase D (PLD)-dependent signaling pathways for its production in human vascular s mooth muscle cells. In addition, we assessed whether redox-sensitive pathwa ys influence Ang II-stimulated cell growth. Primary and low-passage cells ( passages 1 to 4) derived from resistance arteries of subcutaneous gluteal b iopsies from healthy subjects were studied. Oxidative stress was measured w ith the fluorescent probe: 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluo rescein diacetate (CM-H(2)DCFDA) (8 mu mol/L). and the rule of PLD was asse ssed with the PLD inhibitor D-erythro-sphingosine, dihydro (sphinganine) (1 0 mu mol/L). To determine whether NADH/NADPH oxidase contributes to product ion of reactive oxygen species, Ang II-stimulated cells were pretreated wit h the specific flavoprotein inhibitor diphenylene iodinium (DPI) (10 mu mol /L). DNA and protein synthesis were determined by [H-3]thymidine and [H-3]l eucine incorporation, respectively. Ang II increased CM-H(2)DCFDA fluoresce nce, and this was inhibited by catalase (350 U/mL), indicating that the flu orescence signal was derived predominantly from H2O2. Ang II dose-dependent ly increased H2O2 production (E-max=57.6+/-1.7 nmol/L, pD(2)=7.7+/-0.06) an d PLD activation E-max=207+/-3.3% of control, pD(2)=7.7+/-0.5). H2O2 effect s were evident within 1 hour, and maximal PLD activation occurred within 40 minutes after stimulation. DPI inhibited (P<0.01) Ang II-stimulated respon ses. PLD inhibition significantly attenuated (P<0.05) Ang II-elicited H2O2 production (E-max=29+/-5 nmol/L). DPT and sphinganine inhibited Ang II-indu ced DNA and protein synthesis, These data indicate that in vascular smooth muscle cells from human peripheral resistance arteries, Ang II increases H2 O2 generation via PLD-dependent, NADH/NADPH oxidase-sensitive pathways. The se cascades may function as secund messengers in long-term Ang II-mediated growth-signaling events.