Membrane Trafficking of NADPH Oxidase p47^phoxin Paraventricular Hypothalamic Neurons Parallels Local Free Radical Production in Angiotensin II Slow-Pressor Hypertension

Abstract

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.

Angiotensin II slow-pressor hypertension enhances NMDA currents and NOX2-dependent superoxide production in hypothalamic paraventricular neurons

“…This phenomenon is likely to reflect NOX2 trafficking to membrane sites of NMDA receptor activation, since there was no observed increase in mRNA expression. Dendritic mobilization of NOX2 in PVN dendrites of mice showing ANG II-induced slow pressor hypertension is consistent with our recent demonstration of NOX p47 phox subunit trafficking in PVN dendrites in this hypertension model (12). These observations, collectively, suggest that ANG II slowpressor hypertension leads to a compartmental redistribution of NMDA receptors and associated signaling molecules in PVN neurons, which may be critical for the enhanced ROS production and glutamate currents observed in these cells.…”

“…As described elsewhere (9), wild-type mice were anesthetized intraperitoneally (ketamine ϩ xylazine) and implanted subcutaneously with 14-day osmotic minipumps (ALZET; Durect, Cupertino, CA) loaded with saline or ANG II (600 ng·kg Ϫ1 ·min Ϫ1 ), a concentration widely used by us and others (9,10,12,33,50). A Hatteras MC-4000 tail-cuff blood pressure system (Cary, NC) was used to document the increase in systolic blood pressure produced by ANG II.…”

Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons

“…It is also possible that the increase in ROS production upon exposure to BMP-7 is a local increase within certain cellular compartments but not an overall increase in ROS throughout the cell. Localized ROS production has been observed in paraventricular hypothalamic neurons and in rat sympathetic neurons (Coleman et al, 2013; Natera-naranjo et al, 2012). A localized increase in ROS would be consistent with our observation that there was no toxicity associated with BMP-7 treatment.…”

Sex differences in NMDA GluN1 plasticity in rostral ventrolateral medulla neurons containing corticotropin-releasing factor type 1 receptor following slow-pressor angiotensin II hypertension

“…Systolic blood pressure (SBP) was measured in awake mice (N = 3/group) prior to implanting pumps and on days 2, 9–10 and 13 post-implant using tail cuff plethysmography (Model MC4000, Hatteras Instruments, Cary, NC) as previously described (Coleman et al, 2010). Tail-cuff plethysmography provides a reliable non-invasive method to compare SBP measurements between groups (Capone et al, 2012; Coleman et al, 2013; Wang et al, 2013). The limitations of using tail-cuff phethysmography have been discussed previously (Marques-Lopes et al, 2014).…”

“…A guinea pig polyclonal antiserum raised against arginine vasopressin (AVP; #t-5048, Peninsula Laboratories, San Carlos, CA) has been shown to recognize AVP without oxytocin cross-reactivity (Coleman et al, 2013). No immunolabeling is seen using this antiserum in Brattleboro rats, which do not express AVP (Drouyer et al, 2010).…”