Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

Abstract

The importance of the brain renin angiotensin system (RAS) in blood pressure (BP) regulation and water homeostasis is well accepted. However, it remains unclear whether renin and/or its precursor, prorenin, are expressed in the brain and whether they trigger signaling pathways in specific brain regions. We previously used double transgenic mice expressing enhanced green fluorescent protein controlled by renin promoter and beta‐galactosidase controlled by the human angiotensinogen (AGT) promoter to localize their expression in the brain. We reported that neurons expressing renin are localized in close proximity to AGT‐expressing cells in the rostral ventrolateral medulla (RVLM), a brainstem nucleus involved in sympathetic activity and BP control. Here, we hypothesized that prorenin induces phosphorylation of intracellular cascades such as extracellular signal‐regulated kinases (ERK1/2) and activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPHox) in the brainstem. Neonatal brainstem cells were cultured from C57BL/6J mice at postnatal day 1–3 and maintained for 7–10 days in B27‐supplemented neurobasal medium. Immunofluorescence experiments demonstrated the cultures contain both MAP2+ neurons and GFAP+ astrocytes. Expression of type 1 angiotensin receptor (AT1R), renin, and prorenin receptor (PRR) was confirmed by qPCR. To quantify ERK1/2 phosphorylation, cells were stimulated with recombinant prorenin (rProrenin) at 100 nM for 0, 30, and 60 min. Total and phosphorylated ERK1/2 were detected by Western Blot. The effect of rProrenin on NADPHox activity was measured by lucigenin chemiluminescence assay. Specific activity of NADPHox was expressed as chemiluminescence units per min per microgram of protein. Vehicle and phorbol myristate acetate were used as negative and positive controls, respectively. Incubation with rProrenin increased the ratio of phosphorylated‐to‐total ERK1/2 from 0.65±0.07 A.U. at baseline to 1.01±0.13 A.U. at 30 min (54% increase; p=0.0035; n=7) and 1.29±0.09 A.U. at 60 min (102% increase; p<0.0001; n=7). We generated preliminary data showing that both PRO20, a PRR blocker, and Ro‐31, a protein kinase C inhibitor, abrogated prorenin‐induced ERK1/2 phosphorylation. In an additional experiment, we observed that Angiotensin II (Ang II) at 100 nM induced ERK1/2 phosphorylation from 0.98 A.U. to 1.52 A.U. at 15 min (55 % increase), 1.61 A.U. at 30 min (65% increase), and 1.35 A.U. at 60 min (38% increase). Incubation of cells with 100 nM rProrenin for 6 hours resulted in 47.2±9.5 % induction of NADPHox activity compared to vehicle control (p<0.05, n=5). Finally, fluorescent in situ hybridization (RNAscope) data suggests that PRR is expressed in the RVLM neurons and RVLM‐targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane‐anesthetized mice. We conclude that prorenin in the brainstem induces downstream signaling involving ERK/2 phosphorylation and generation of ROS, which might contribute to BP elevation and sympathoexcitation. Future studies will determine whether prorenin‐elicited signaling in the brainstem requires binding to PRR, generation of Ang II, and/or AT1R.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa