Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS) are highly implicated in the development of angiotensin II (AngII)-dependent hypertension mediated in part through the hypothalamic paraventricular nucleus (PVN). This region contains vasopressin and non-vasopressin neurons that are responsive to cardiovascular dysregulation, however it is not known if ROS is generated by one or both cell-types in response to “slow pressor” infusion of AngII. We addressed this question using ROS imaging and electron microscopic dual labeling for vasopressin and p47phox, a cytoplasmic NADPH oxidase subunit requiring mobilization to membranes for the initiation of ROS production. C57BL/6 mice or vasopressin-enhanced green fluorescent protein (VP-eGFP) mice were infused systemically with saline or AngII (600 ng/kg/min; s.c.) for two weeks during which they slowly developed hypertension. Ultrastructural analysis of the PVN demonstrated p47phox immunolabeling in many glial and neuronal profiles, most of which were postsynaptic dendrites. Compared with saline, AngII recipient mice had a significant increase in p47phox immunolabeling on endomembranes just beneath the plasmalemmal surface (+42.1±11.3%; p<0.05) in non-vasopressin dendrites. In contrast, AngII infusion decreased p47phox immunolabeling on the plasma membrane (−35.5±16.5%; p<0.05) in vasopressin dendrites. Isolated non-VP-eGFP neurons from the PVN of AngII-infused mice also showed an increase in baseline ROS production not seen in VP-eGFP neurons. Our results suggest that chronic low dose AngII may offset the homeostatic control of blood pressure by differentially affecting membrane assembly of NADPH oxidase and ROS production in vasopressin and non-vasopressin neurons located within the PVN.

The incidence of hypertension increases after menopause. Similar to humans, “slow-pressor” doses of angiotensin II (AngII) increase blood pressure in young males, but not in young female mice. However, AngII increases blood pressure in aged female mice, paralleling reproductive hormonal changes. These changes could influence receptor trafficking in central cardiovascular circuits and contribute to hypertension. Increased post-synaptic NMDA receptor activity in the hypothalamic paraventricular nucleus (PVN) is crucial for the sympathoexcitation driving AngII hypertension. Estrogen receptors beta (ERβ) are present in PVN neurons. We tested the hypothesis that changes in ovarian hormones with age promote susceptibility to AngII hypertension, and influence NMDA receptor NR1 subunit trafficking in ERβ-containing PVN neurons. Transgenic mice expressing enhanced green fluorescent protein (EGFP) in ERβ-containing cells were implanted with osmotic minipumps delivering AngII (600 ng/kg/min) or saline for 2 weeks. AngII increased blood pressure in 2 month-old males and 18 month-old females, but not in 2 month-old females. By electron microscopy, NR1-silver-intensified immunogold (SIG) was mainly in ERβ-EGFP dendrites. At baseline, NR1-SIG density was greater in 2 month-old females than in 2 month-old males or 18 month-old females. After AngII infusion, NR1-SIG density was decreased in 2 month-old females, but increased in 2 month-old males and 18 month-old females. These findings suggest that, in young female mice, NR1 density is decreased in ERβ-PVN dendrites thus reducing NMDA receptor activity and preventing hypertension. Conversely, in young males and aged females, NR1 density is upregulated in ERβ-PVN dendrites and ultimately leads to the neurohumoral dysfunction driving hypertension.

Adaptive changes in glutamatergic signaling within the hypothalamic paraventricular nucleus (PVN) may play a role in the neurohumoral dysfunction underlying the hypertension induced by "slow-pressor" ANG II infusion. We hypothesized that these adaptive changes alter production of gp91phox NADPH oxidase (NOX)-derived reactive oxygen species (ROS) or nitric oxide (NO), resulting in enhanced glutamatergic signaling in the PVN. Electron microscopic immunolabeling showed colocalization of NOX2 and N-methyl-D-aspartate receptor (NMDAR) NR1 subunits in PVN dendrites, an effect enhanced (+48%, P < 0.05 vs. saline) in mice receiving ANG II (600 ng·kg⁻¹·min⁻¹ sc). Isolated PVN cells or spinally projecting PVN neurons from ANG II-infused mice had increased levels of ROS at baseline (+40 ± 5% and +57.6 ± 7.7%, P < 0.01 vs. saline) and after NMDA (+24 ± 7% and +17 ± 5.5%, P < 0.01 and P < 0.05 vs. saline). In contrast, ANG II infusion suppressed NO production in PVN cells at baseline (-29.1 ± 5.2%, P < 0.05 vs. saline) and after NMDA (-18.9 ± 2%, P < 0.01 vs. saline), an effect counteracted by NOX inhibition. In whole cell recording of unlabeled and spinally labeled PVN neurons in slices, NMDA induced a larger inward current in ANG II than in saline groups (+79 ± 24% and +82.9 ± 6.6%, P < 0.01 vs. saline), which was reversed by the ROS scavenger MnTBAP and the NO donor S-nitroso-N-acetylpenicillamine (P > 0.05 vs. control). These findings suggest that slow-pressor ANG II increases the association of NR1 with NOX2 in dendrites of PVN neurons, resulting in enhanced NOX-derived ROS and reduced NO during glutamatergic activity. The resulting enhancement of NMDAR activity may contribute to the neurohumoral dysfunction underlying the development of slow-pressor ANG II hypertension.