Increased generation of superoxide by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients: role of phospholipase D-dependent NAD(P)H oxidase-sensitive pathways

Objective We tested the hypothesis that increased responsiveness of phospho lipase D (PLD) to angiotensin II (Ang II) is associated with increased oxid ative stress and exaggerated growth responses in vascular smooth muscle cel ls (VSMC) from untreated essential hypertensive patients, Design VSMCs from peripheral resistance arteries of normotensive and hypert ensive subjects were studied. Production of reactive oxygen species (ROS) w as measured with the fluoroprobe 5-(and 6)-chloromethyl-2 ' ,7 ' -dichlored ihydrofluorescein diacetate (CM-H-2 DCFDA). PLD and reduced nicotinamide ad enine dinucleotide (phosphate) (NAD(P)H) oxidase were assessed with the inh ibitors, dihydro-D-erythro-sphingosine (sphinganine) and diphenylene iodini um (DPI), respectively, and protein kinase C (PKC) effects were determined using chelerythrine chloride and calphostin C, PLD activity was measured by the transphosphatidylation assay. Results Ang II increased the CM-H(2)DCFDA fluorescence signal, derived pred ominantly from H2O2 Ang Ii-induced generation of DPI-inhibitable ROS was si gnificantly enhanced in cells from hypertensives compared with normotensive s (E-max = 72 +/- 2 versus 56.9 +/- 1.8 fluorescence units, P < 0.01). PLD inhibition attenuated Ang Ii-induced ROS generation, with greater effects i n the hypertensive group than the normotensive group (Delta = 42 +/- 3.3 ve rsus 21 +/- 2 units). PKC inhibition partially decreased Ang Ii-elicited si gnals. Ang Ii-stimulated PLD activity and DNA and protein synthesis were si gnificantly greater in cells from hypertensives than normotensives. These e ffects were normalized by DPI and sphinganine. Conclusions Our results suggest that in essential hypertension enhanced oxi dative stress and augmented growth-promoting actions of Ang II are associat ed with increased activation of PLD-dependent pathways. These processes may contribute to vascular remodeling in hypertension. (C) 2001 Lippincott Wil liams & Wilkins.