Increased free radical production in hypertension due to increased expression of the NADPH oxidase subunit p22(phox) in lymphoblast cell lines.
OBJECTIVE
To confirm increased production of reactive oxygen species (ROS) in hypertension, to demonstrate the source of ROS and to analyse NADPH oxidase subcomponent expression in hypertension.


METHODS
A lymphoblast model was used, as this has previously been used in the study of hypertension and of NADPH oxidase. Chemiluminescence (CL) was chosen to assay ROS production, as it is simple and sensitive.


METHODS
Lymphocytes from 12 hypertensive patients (HT), and 12 age- and sex-matched normotensive (NT) subjects, were immortalized. Luminol, isoluminol and Cypridina luciferin analogue (CLA) CL were used to assay ROS production. NADPH oxidase subunits were measured by Western blot analysis.


RESULTS
Stimulation with 50 micromol/l arachidonic acid (AA) resulted in increased ROS production in HT cell lines with luminol, CLA and isoluminol CL. Stimulation with 500 nmol/l 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a detectable increase in HT ROS production with luminol and with CLA, whereas there was no significant difference with isoluminol. The ROS production was abolished by diphenyleneiodonium chloride (DPI) but not by rotenone, indicating that a non-mitochondrial flavoprotein such as NADPH oxidase is the source of ROS. Analysis of NADPH oxidase subcomponents revealed an increase in p22(phox) in HT subjects.


CONCLUSIONS
We have shown there is increased ROS production in lymphoblasts derived from hypertensive subjects, probably originating from NADPH oxidase. As the ROS production persists in transformed cells, this suggests a genetic predisposition to increased ROS production. Increased expression of p22(phox) in HT lymphoblasts may account for some of the increased ROS.
Pettit
Andrew I
AI
Royal United Hospital Bath, Bath, UK.
Wong
Richard K M
RK
Lee
Virginia
V
Jennings
Sonja
S
Quinn
Pauline A
PA
Ng
Leong L
LL
D016718:Arachidonic Acid / Q000494:pharmacology; D002460:Cell Line; D005260:Female; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006973:Hypertension / Q000201:enzymology* / Q000378:metabolism; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D026901:Membrane Transport Proteins; D008875:Middle Aged; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005609:Free Radicals; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D016718:Arachidonic Acid; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase; D013755:Tetradecanoylphorbol Acetate