Activation of the (pro)renin receptor in the paraventricular nucleus increases sympathetic outflow in anesthetized rats

Abstract

Previous studies have indicated that hyperactivity of brain prorenin receptors (PRR) is implicated in neurogenic hypertension. However, the role of brain PRR in regulating arterial blood pressure (ABP) is not well understood. Here, we test the hypothesis that PRR activation in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA). In anaesthetized adult Sprague-Dawley (SD) rats, bilateral PVN microinjection of human prorenin (2 pmol/side) significantly increased splanchnic SNA (SSNA; 71 ± 15%, n = 7). Preinjection of either prorenin handle region peptide, the PRR binding blocker (PRRB), or tiron (2 nmol/side), the scavenger of reactive oxygen species (ROS), significantly attenuated the increase in SSNA (PRRB: 32 ± 5% vs. control, n = 6; tiron: 8 ± 10% vs. control, n = 5; P < 0.05) evoked by prorenin injection. We further investigated the effects of PRR activation on ROS production as well as downstream gene expression using cultured hypothalamus neurons from newborn SD rats. Incubation of brain neurons with human prorenin (100 nM) dramatically enhanced ROS production and induced a time-dependent increase in mRNA levels of inducible nitric oxide synthase (iNOS), NAPDH oxidase 2 subunit cybb, and FOS-like antigen 1 (fosl1), a marker for neuronal activation and a component of transcription factor activator protein-1 (AP-1). The maximum mRNA increase in these genes occurred 6 h following incubation (iNOS: 201-fold; cybb: 2 -fold; Ffosl1: 11-fold). The increases in iNOS and cybb mRNA were not attenuated by the AT1 receptor antagonist losartan but abolished by the AP-1 blocker curcumin. Our results suggest that PVN PRR activation induces sympathoexcitation possibly through stimulation of an ANG II-independent, ROS-AP-1-iNOS signaling pathway.

Keywords: (pro)renin receptor; paraventricular nucleus; reactive oxygen species; sympathetic nerve activity.

Fig. 1.

The paraventricular nucleus (PVN) prorenin receptor (PRR) is primarily expressed in neurons. Representative confocal images showing coimmunofluorescence staining of PRR (red) and neuronal nuclear (NeuN; green), a marker for neuronal nuclei (top), or PRR (red) and glial fibrillary acidic protein (GFAP; green), a marker for astrocyte (bottom). Left: low-magnification images. Right: high-magnification images. Cell nuclei were stained with DAPI (blue).

Fig. 2.

Activation of PVN PRR increases sympathetic nerve activity (SNA). A: representative traces showing splanchnic sympathetic nerve activity (SSNA), renal sympathetic nerve activity (RSNA), and mean arterial blood pressure (BP; MAP) responses to bilateral microinjections of 0.9% saline (vehicle control) followed by bilateral microinjections of prorenin (2 pmol/side) into the PVN (left) and bilateral microinjections of prorenin handle region peptide, a prorenin receptor blocker (PRRB; 200 pmol/side), followed by bilateral microinjections of prorenin (2 pmol/side) into the PVN (right). Each injection (100 nl) of saline, PRRB, and prorenin (arrowhead) was completed over a period of ∼1 min. The interval between saline or PRRB and prorenin microinjection was ∼10 min. B, left: 2.5-s specimen traces of SSNA (top) and RSNA (bottom) before injection of saline into the PVN and after microinjection of prorenin into the PVN. B, right: 2.5-s specimen traces of SSNA (top) and RSNA (bottom) before injection of PRRB into the PVN and after microinjection of prorenin into the PVN. C: summary data showing changes in SSNA and MAP in response to bilateral microinjections of saline (n = 7): saline + prorenin (n = 7), PRRB + prorenin (n = 6), and tiron + prorenin (n = 5). *P < 0.05 compared with all other 3 groups.

Fig. 3.

PRR activation in brain neurons increases reactive oxygen species (ROS) production. Representative micrographs of dihydroethidium (DHE) staining showing ROS production. Top: control neurons. Bottom: neurons incubated with prorenin (100 nM) for 1 h. Left: DHE staining showing ROS (red). Middle: DAPI staining showing cell nuclei (blue). Right: merged micrographs.

Fig. 4.

PRR activation in brain neurons increases the mRNA levels of FOS-like antigen 1 (fosl1), inducible nitric oxide synthase (iNOS), and the NAPDH oxidase 2 subunit cybb. Brain neurons were incubated in primary cultures from the hypothalamus of new born Sprague-Dawley rats containing the PVN with prorenin (100 nM) for different time courses, the neurons were then collected, RNA was purified, and real-time PCR was performed using specific primers and probes specific for iNOS, fosl1, and cybb. The mRNA level in the control sample was assigned to be arbitrary unit (a.u) 1. *P ≤ 0.05 vs. control; n = 6 each group.

Fig. 5.

PRR-mediated increases in target genes are attenuated by the activator protein-1 (AP-1) inhibitor curcumin. Cultured brain neurons were incubated with 100 nM of human prorenin with or without curcumin (50 μmol/l), an AP-1 inhibitor, or losartan (2 μmol/l), an antagonist of ANG II type I receptor (AT₁-R), for 6 h, and the neurons were then subjected to real-time PCR to assay mRNA levels of fosl1, iNOS, and cybb. The mRNA level in the control sample was assigned to be arbitrary unit 1. *P ≤ 0.05 vs. control; ^#P ≤ 0.05 vs. prorenin + curcumin, n = 6 each group.