Nox2, Ca2+, and protein kinase C play a role in angiotensin II -: Induced free radical production in nucleus tractus solitarius

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca2+ currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca2+ or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-( 4[ didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II ( 3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC50=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis ( benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca2+ currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca2+ attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca2+ currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca2+ currents that underlie the actions of Ang II on central autonomic regulation.