Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension

Abstract

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension.

Keywords: (pro)renin receptor; central nervous system; hypertension.

Figure 1

Binding of fluorescently labeled PRO20 to mouse and human brain tissues. A and B, Representative images show the binding of PRO20-fluorescein isothiocyanate (PRO20-FITC; 5 nmol/L; green) in the mouse paraventricular nucleus of hypothalamus (PVN), with or without coincubation with unlabeled mouse prorenin (1 μmol/L). Cell nuclei are indicated by blue DAPI (4′,6-diamidino-2-phenylindole) staining. C, Fluorescence in the mouse PVN was quantified for a series of concentrations (1, 2, 5, 10, and 20 nmol/L) of PRO20-FITC (n=5–6/concentration). D and E, Representative images show the binding of PRO20-FITC (1 nmol/L) in the human PVN, with or without coincubation with unlabeled human prorenin (1 μmol/L). F, Fluorescence in the human PVN was quantified for a series of concentrations (0.2, 0.5, 1, 2, and 5 nmol/L) of PRO20-FITC (n=5–6/concentration). Relative fluorescence unit (RFU) was calculated by subtracting the background from the total fluorescence density in each image quantified by Image J (Version 1.48).

Figure 2

PRO20 prevents human prorenin–induced calcium influx in SH-SY5Y cells. A, Representative traces of changes in fluorescence (ΔRFU) compared with time for SH-SY5Y cells loaded with the calcium-indicator Fluo-4-AM in response to application of ATP (1 μmol/L), angiotensin II (Ang II; 10 nmol/L), or Ang II + losartan (1 μmol/L). The arrow indicates drug injection time. B, Summary data for experiments shown in A (n=7–11/group; *P<0.05 vs control; #P<0.05 vs Ang II). C, Representative traces of changes in fluorescence induced by human prorenin (4 nmol/L), prorenin + captopril (1 μmol/L), or prorenin + losartan (1 μmol/L). D, Summary data for experiments shown in C (n=7–11/group; *P<0.05 vs control, #P<0.05 vs prorenin). E, Representative traces of changes in fluorescence induced by human prorenin + scrambled peptide (1 μmol/L), and prorenin with or without PRO20 (0.12 and 1.2 μmol/L). F, PRO20 inhibited prorenin (4 nmol/L)-induced calcium influx in a concentration-dependent manner.

Figure 3

PRO20 attenuates pressor responses induced by intracerebroventricular infusion of prorenin. A, Real-time blood pressure (BP) trace recorded before and during intracerebroventricular infusion of mouse prorenin (2.4 μmol/L) + scrambled peptide (100 μmol/L). Arrows indicate the beginning of the 10-minute intracerebroventricular infusion. B, Real-time BP trace recorded before and during intracerebroventricular infusion of mouse prorenin (2.4 μmol/L) + PRO20 (100 μmol/L). Arrows indicate the beginning of the 10-minute intracerebroventricular infusion. C, Changes in mean arterial pressure (ΔMAP) induced by intracerebroventricular infusion of mouse prorenin (2.4 μmol/L) or prorenin + PRO20 (1.8, 5.3, 16, 48, and 144 μmol/L; *P<0.05 vs prorenin). D, PRO20 inhibited mouse prorenin (2.4 μmol/L)-induced increases in MAP in a dose-dependent manner (n=5/concentration).

Figure 4

Intracerebroventricular infusion of PRO20 lowers blood pressure (BP) in mice with established angiotensin (Ang II)-dependent and deoxycorticosterone acetate (DOCA)-salt–induced hypertension. Real-time BP trace recorded before, during, and after intracerebroventricular infusion of (A) scrambled peptide (1 mmol/L) in renin–angiotensinogen (RA) double-transgenic mice, (B) scrambled peptide (1 mmol/L) in DOCA-salt hypertensive mice, (C) PRO20 (1 mmol/L) in RA mice, and (D) PRO20 (1 mmol/L) in DOCA-salt hypertensive mice. Arrows indicate the beginning of the 10-minute intracerebroventricular infusion. E, Summary data showing changes in mean arterial pressure (ΔMAP) in response to a 10-minute intracerebroventricular infusion of scrambled peptide (1 mmol/L), PRO20 (1 mmol/L), or losartan (3 mmol/L) in hypertensive RA mice (*P<0.05 vs RA + scrambled; n=4). F, ΔMAP during intracerebroventricular infusion of scrambled peptide (1 mmol/L), PRO20 (1 mmol/L), or losartan (3 mmol/L) in DOCA-salt hypertensive mice (*P<0.05 vs DOCA + scrambled; n=4 in the SHAM group, n=5 in the DOCA group).

Figure 5

Chronic intracerebroventricular infusion of PRO20 attenuates the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Daily mean arterial pressure (MAP; A) and heart rate (HR; B) after chronic intracerebroventricular (ICV) infusion of artificial cerebrospinal fluid (aCSF; n=9) or PRO20 (n=9), and subcutaneous (SC) infusion of PRO20 (n=5) at baseline and after DOCA-salt treatment for 21 days. C, Maximum changes in HR in response to intraperitoneal propranolol (5 mg/kg; cardiac sympathetic tone). D, Maximum changes in MAP in response to intraperitoneal chlorisondamine (6 mg/kg; vasomotor sympathetic tone). E, Maximum changes in HR in response to intraperitoneal methylatropine (1 mg/kg; cardiac parasympathetic tone). F, Spontaneous baroreflex sensitivity (SBRS) in mice with intracerebroventricular-administered aCSF or PRO20 at baseline and 21 days after DOCA-salt treatment. G, Intrinsic HR recorded after injection of propranolol (5 mg/kg, IP) or methylatropine (1 mg/kg, IP). H, Ang II levels in the cerebral cortex (Cor), hypothalamus (Hypo), and brain stem (BS) of mice in SHAM, DOCA-salt + intracerebroventricular aCSF, and DOCA-salt + intracerebroventricular PRO20 groups (*P<0.05 vs baseline aCSF intracerebroventricular and #P<0.05 vs DOCA + aCSF intracerebroventricular in C, D, E, and F; *P<0.05 vs SHAM and #P<0.05 vs DOCA + aCSF intracerebroventricular in H).

Figure 6

Phosphorylated extracellular signal-regulated protein kinase (ERK) 1 and 2 (P-ERK-1/2) and total ERK (T-ERK-1/2) in Neuro-2A cells. Representative Western blots showing P-ERK-1/2 (upper blot) and T-ERK-1/2 (lower blot) following the indicated treatments. Quantification of the blots indicates that prorenin increased ERK-1/2 phosphorylation. PRO20 or prorenin receptor knockdown (PRR-KO) prevented prorenin-induced ERK-1/2 phosphorylation; however, handle region peptide (HRP) had no effect (n=4/group; *P<0.05 vs losartan treatment; #P<0.05 vs losartan + prorenin treatment).

Figure 7

Proposed pathways for angiotensinergic neurons in the regulation of sympathetic activity. In presympathetic neurons, prorenin (PR) binds intracellular (pro)renin receptor (PRR), leading to intracellular formation of angiotensin II (Ang II), which is subsequently secreted into the extracellular space. Alternatively, extracellular prorenin binds to the PRR on the neuronal membrane and metabolizes extracellular angiotensinogen (AGT) secreted by astrocytes or neurons to generate Ang I. Angiotensin-converting enzyme (ACE), located on the external surface of cell membranes or in the interstitial fluid, converts Ang I to Ang II. The intracellular Ang II can be transported to axon terminals to act as a neurotransmitter. Extracellular Ang II binds to the angiotensin II type 1 receptor (AT₁R) to modulate neuronal activity and neurotransmitter release at the synapse. PVN indicates paraventricular nucleus of hypothalamus; and RVLM, rostral ventrolateral medulla nucleus.