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

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Abstract

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 (Δ = 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.

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