Mechanisms underlying prorenin actions on hypothalamic neurons implicated in cardiometabolic control

Abstract

Background: Hypertension and obesity are highly interrelated diseases, being critical components of the metabolic syndrome. Despite the growing prevalence of this syndrome in the world population, efficient therapies are still missing. Thus, identification of novel targets and therapies are warranted. An enhanced activity of the hypothalamic renin-angiotensin system (RAS), including the recently discovered prorenin (PR) and its receptor (PRR), has been implicated as a common mechanism underlying aberrant sympatho-humoral activation that contributes to both metabolic and cardiovascular dysregulation in the metabolic syndrome. Still, the identification of precise neuronal targets, cellular mechanisms and signaling pathways underlying PR/PRR actions in cardiovascular- and metabolic related hypothalamic nuclei remain unknown.

Methods and results: Using a multidisciplinary approach including patch-clamp electrophysiology, live calcium imaging and immunohistochemistry, we aimed to elucidate cellular mechanisms underlying PR/PRR actions within the hypothalamic supraoptic (SON) and paraventricular nucleus (PVN), key brain areas previously involved in cardiometabolic regulation. We show for the first time that PRR is expressed in magnocellular neurosecretory cells (MNCs), and to a lesser extent, in presympathetic PVN neurons (PVNPS). Moreover, we show that while PRR activation efficiently stimulates the firing activity of both MNCs and PVNPS neurons, these effects involved AngII-independent and AngII-dependent mechanisms, respectively. In both cases however, PR excitatory effects involved an increase in intracellular Ca(2+) levels and a Ca(2+)-dependent inhibition of a voltage-gated K(+) current.

Conclusions: We identified novel neuronal targets and cellular mechanisms underlying PR/PRR actions in critical hypothalamic neurons involved in cardiometabolic regulation. This fundamental mechanistic information regarding central PR/PRR actions is essential for the development of novel RAS-based therapeutic targets for the treatment of cardiometabolic disorders in obesity and hypertension.

Keywords: Angiotensin; PVN; Potassium; Prorenin receptor; SON; Sympathetic.

Figure 1

PR increases the firing activity of SON/PVN MNCs and PVN_PSneurons. A, Representative example of a patched eGFP-VP neuron, showing that focal application of PR (2.5 nM, 5 s) increased its firing activity (B). C, Summary data showing mean firing frequencies (Hz) before and after PR application in MNCs (n = 12). D, Representative example of a patched PVN_PS neuron, showing that focal application of PR (2.5 nM, 5 s) increased its firing activity (E). F, Summary data showing mean firing frequencies (Hz) before and after PR application in PVN_PS neurons (n = 12). **p < 0.01. Scale bars: 10 μm.

Figure 2

PR effects involve distinct AngII-independent and dependent signaling mechanisms in MNCs and PVN_PSneurons. A, Mean Δ frequency (Hz, n = 12 and 5 in PR and PR + PRO20) (A1) and sample trace (A2) showing that bath application of the PRR-antagonist PRO20 (250 nM, 10 min), prevented PR-evoked excitation in MNCs. B, Summary data (n = 12 and 6 in PR and PR + LOS) (B1) and representative trace (B2) showing that the AT₁-R blocker losartan (50 μM, 20 min), blocked PR-evoked excitation in PVN_PS neurons. **p < 0.01.

Figure 3

PR effects on MNCs and PVN_PSneurons involve an increase in [Ca²⁺]i and are abolished by the Ca²⁺chelator BAPTA. A, Representative example of an eGFP-VP neuron loaded with the Ca²⁺ indicator Fluo-5F (50 μM) (A1). Pseudocolor images showing PR-evoked Ca²⁺ changes over time in that neuron are shown below (A2–A4). B, Sample traces showing simultaneous membrane potential (B1) and somatic (B2) and dendritic (B3) Δ[Ca²⁺]i measurements in the eGFP-VP neuron shown in A, in response to focally-applied PR (5 s, arrow). Arrowheads correspond to the images shown in A2–A4. Note that PR evoked a slow increase in Δ[Ca²⁺]i along with membrane depolarization, which preceded onset of firing and an abrupt action potential-mediated increase in Δ[Ca²⁺]i (asterisks). C, Summary data showing mean PR-evoked peak Ca²⁺ changes (F/F0) prior to action potential firing in MNCs (p < 0.01, n = 8) and PVN_PS neurons (p < 0.05, n = 5). D, Summary data showing that chelation of intracellular Ca²⁺ with BAPTA (10 mM) in the patch pipette blunted PR excitatory effect in both MNCs (p > 0.2, n = 19) and PVN_PS neurons (p > 0.5, n = 5). Scale bars: 20 μm.

Figure 4

PR inhibits voltage-dependent K⁺currents in a Ca²⁺-dependent manner. A, Representative example of currents elicited by voltage ramps (−100 mV to +40 mV, 5s) before (black) and after (red) PR application (2.5 nM, 5 s) in an eGFP-VP neuron. Note the decreased magnitude of the outward component in PR. The isolated PR-sensitive current is shown in the inset. B, Representative example showing that the PR-mediated inhibition of K⁺ currents was abolished in an eGFP-VP neuron dialyzed with BAPTA (10 mM). Note in the inset that the PR-sensitive inward current was absent, while a minor PR-sensitive outward current was unveiled. C and D, Summary data showing the mean PR-sensitive peak current amplitude in MNCs (C) and PVN_PS neurons (D) in control conditions (n = 18 and 12, respectively) and in cells dialyzed with BAPTA (n = 12 and 8, respectively). **p < 0.01.

Figure 5

Prorenin receptor (PRR) immunoreactivity in identified MNCs and PVN_PSneurons in the SON and PVN. A, PRR immunoreactivity in the PVN (green, antibody generated in Dr. Feng's laboratory, A1), in which oxytocin (OT) MNCs (red) and PVN_PS neurons (blue) were identified (A2). In A3, images in A1 and A2 were superimposed, and the areas contained within the white squares are reimaged at higher magnification and displayed in D–E. B, PRR immunoreactivity in the PVN (green, Abcam antibody, B1), in which vasopressin (VP) MNCs (red) and PVN_PS neurons (blue) were identified (B2). In B3, images in B1 and B2 were superimposed. C, PRR immunoreactivity in the SON (green, antibody generated in Dr. Feng's laboratory, C1), in which both OT and VP neurons were identified (red, C2). In C3, images in C1 and C2 were superimposed. F, Summary data showing mean PRR immunoreactivity intensity in identified VP neurons in the SON and PVN, and PVN_PS neurons in the PVN (n = 153, 116 and 117 respectively, from 3 rats). ***p < 0.0001 vs. all other groups. 3V: third ventricle; dc; dorsal cap; lm: lateral magnocellular subnucleus, ot: optic tract. Scale bars in A–C: 50 μm and D–E: 20 μm. Vertical and horizontal arrows in A2 point dorsally and medially, respectively.