Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase

Superoxide anion (O-2(-.)) production is elevated in sympathetic ganglion neurons and in the vasculature of hypertensive animals; however, it is not known what enzymatic pathway(s) are responsible for O-2(-.) production. To determine the pathway(s) of O-2(-.) production in sympathetic neurons, we examined the presence of mRNA of NADPH oxidase subunits in sympathetic ganglionic neurons and differentiated PC-12 cells. The mRNAs for NADPH oxidase subunits p47(phox), p22(phox), gp91(phox), and NOX1 were present in sympathetic neurons and PC-12 cells, whereas the NOX4 homologue was present in sympathetic neurons but not PC-12 cells. Freshly dissociated celiac ganglion neurons from normal rats and PC-12 cells produced O-2(-.) when treated with the PKC activator PMA; O-2(-.) production increased by 317% and 254%, respectively. The PMA-evoked increases were reduced by pretreatment with the NADPH oxidase inhibitor apocynin. These findings indicate that NADPH oxidase is the primary source of O-2(-.) in sympathetic ganglion neurons. When celiac ganglia from hypertensive rats were incubated with apocynin, O-2(-.) levels were reduced to the same levels as normotensive animals, indicating that NADPH oxidase activity accounted for the elevated O-2(-.) levels in hypertensive animals. To test this latter finding, we compared NADPH oxidase activity in extracts of prevertebral sympathetic ganglia of DOCA-salt hypertensive rats and sham-operated rats. NADPH oxidase activities were 49.9% and 78.6% higher in sympathetic ganglia of DOCA rats compared with normotensive controls when using beta-NADH and beta-NADPH as substrates, respectively. Thus elevated O-2(-.) levels in hypertension may be a result of the increased activity of NADPH oxidase in postganglionic sympathetic neurons.