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

J Hypertens. 2001 Jul;19(7):1245-54. doi: 10.1097/00004872-200107000-00009.


Objective: We tested the hypothesis that increased responsiveness of phospholipase D (PLD) to angiotensin II (Ang II) is associated with increased oxidative stress and exaggerated growth responses in vascular smooth muscle cells (VSMC) from untreated essential hypertensive patients.

Design: VSMCs from peripheral resistance arteries of normotensive and hypertensive subjects were studied. Production of reactive oxygen species (ROS) was measured with the fluoroprobe 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). PLD and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase were assessed with the inhibitors, dihydro-D-erythro-sphingosine (sphinganine) and diphenylene iodinium (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-H2DCFDA fluorescence signal, derived predominantly from H2O2. Ang II-induced generation of DPI-inhibitable ROS was significantly enhanced in cells from hypertensives compared with normotensives (Emax = 72 +/- 2 versus 56.9 +/- 1.8 fluorescence units, P< 0.01). PLD inhibition attenuated Ang II-induced ROS generation, with greater effects in the hypertensive group than the normotensive group (delta = 42 +/- 3.3 versus 21 +/- 2 units). PKC inhibition partially decreased Ang II-elicited signals. Ang II-stimulated PLD activity and DNA and protein synthesis were significantly greater in cells from hypertensives than normotensives. These effects were normalized by DPI and sphinganine.

Conclusions: Our results suggest that in essential hypertension enhanced oxidative stress and augmented growth-promoting actions of Ang II are associated with increased activation of PLD-dependent pathways. These processes may contribute to vascular remodeling in hypertension.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Angiotensin II / pharmacology*
  • Arteries / metabolism*
  • Arteries / pathology
  • Cell Division / physiology
  • Humans
  • Hypertension / metabolism*
  • Hypertension / pathology
  • Middle Aged
  • Muscle, Smooth, Vascular / metabolism*
  • Muscle, Smooth, Vascular / pathology
  • NADH, NADPH Oxidoreductases / metabolism
  • NADH, NADPH Oxidoreductases / physiology
  • NADPH Oxidases
  • Phospholipase D / metabolism
  • Phospholipase D / physiology
  • Reactive Oxygen Species / metabolism
  • Superoxides / metabolism*
  • Vascular Resistance*


  • Reactive Oxygen Species
  • Superoxides
  • Angiotensin II
  • NADH, NADPH Oxidoreductases
  • NADPH Oxidases
  • Phospholipase D