Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension

Abstract

Accumulating evidence supports that the brain renin-angiotensin system (RAS), including prorenin (PR) and its receptor (PRR), two newly discovered RAS players, contribute to sympathoexcitation in salt-sensitive hypertension. Still, whether PR also contributed to elevated circulating levels of neurohormones such as vasopressin (VP) during salt-sensitive hypertension, and if so, what are the precise underlying mechanisms, remains to be determined. To address these questions, we obtained patch-clamp recordings from hypothalamic magnocellular neurosecretory neurons (MNNs) that synthesize the neurohormones oxytocin and VP in acute hypothalamic slices obtained from sham and deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats. We found that focal application of PR markedly increased membrane excitability and firing responses in MNNs of DOCA-salt, compared with sham rats. This effect included a shorter latency to spike initiation and increased numbers of spikes in response to depolarizing stimuli and was mediated by a more robust inhibition of A-type K⁺ channels in DOCA-salt compared with sham rats. On the other hand, the afterhyperpolarizing potential mediated by the activation of Ca²⁺-dependent K⁺ channel was not affected by PR. mRNA expression of PRR, VP, and the Kv4.3 K⁺ channel subunit in the supraoptic nucleus of DOCA-salt hypertensive rats was increased compared with sham rats. Finally, we report a significant decrease of plasma VP levels in neuron-selective PRR knockdown mice treated with DOCA-salt, compared with wild-type DOCA-salt-treated mice. Together, these results support that activation of PRR contributes to increased excitability and firing discharge of MNNs and increased plasma levels of VP in DOCA-salt hypertension.NEW & NOTEWORTHY Our studies support that prorenin (PR) and its receptor (PRR) within the hypothalamus contribute to elevated plasma vasopressin levels in deoxycorticosterone acetate-salt hypertension, in part because of an exacerbated effect of PR on magnocellular neurosecretory neuron excitability; Moreover, our study implicates A-type K+ channels as key underlying molecular targets mediating these effects. Thus, PR/PRR stands as a novel therapeutic target for the treatment of neurohumoral activation in salt-sensitive hypertension.

Keywords: K current; hypothalamus; prorenin receptor; vasopressin.

Fig. 1.

Prorenin (PR) enhanced the firing discharge of SON neurons from DOCA-salt-treated (but not sham) rats by reducing the magnitude of the transient outward rectification. A1: representative voltage traces from an SON neuron in a DOCA-salt-treated rat in response to depolarizing current steps of increasing magnitude (0–140 pA, Δ = 10 pA, 400 ms) before (black) and after (red) PR (2.5 nM, 5 s) from a hyperpolarized holding V_m (−80 mV). A2: two depolarizing sweeps that evoked the first action potential (AP) before (70 pA) and after (40 pA) PR are shown superimposed. Note the shorter duration and diminished magnitude of TOR in the presence of PR (~62 ms and 3.2 mV) compared with control (~223 ms and 11.7 mV). A3: two depolarizing sweeps evoked using 70 pA both before and after PR are displayed superimposed, to show better the markedly decreased TOR after PR, resulting also in an increased number of action potentials evoked using the same depolarizing magnitude. A4: same as A3 but in response to a stronger depolarizing pulse (120 pA). B: summary data of the mean delay onset to first spike in SON neurons from sham- and DOCA-salt-treated rats before (open bars) and after (black bars) PR. C: summary data of the mean voltage difference between the onset of the depolarization and the occurrence of the first spike (arrows) in sham- and DOCA-salt-treated rats before (open bars) and after (closed bars) PR. D: summary data of the mean magnitude of the injected current needed to evoke the first action potential in sham- and DOCA-salt-treated rats before (open bars) and after (closed bars) PR. E: summary data of the mean number of action potentials evoked in response to a depolarizing pulse (120 pA) in sham- and DOCA-salt-treated rats before (open bars) and after (closed bars) PR. **P < 0.01, ***P < 0.001, and ****P < 0.001 versus respective pre-PR. (2-way ANOVA-RM, n = 10/group.) All measurements were obtained from male rats. DOCA, deoxycorticosterone acetate; SON, supraoptic nuclei; TOR, transient outward rectification.

Fig. 2.

Prorenin (PR) inhibits the A-type K⁺ current (I_A) in SON neurons of DOCA-salt-treated rats. Representative samples of I_A triggered by a depolarizing set (−75 to −30 mV, 400 ms) before and after PR (2.5 nM, 5 s) in a DOCA-salt-treated rat (A). Note the diminished I_A magnitude after PR. Inset: full depolarizing protocol used to evoke I_A at different voltage steps. Mean plots of I_A peak amplitude versus command potential in sham (B) and DOCA-salt-treated (C) rats, before (black) and after (red) PR. Mean plots of normalized I_A peak amplitude versus command potential in sham- (D) and DOCA-salt-treated (E) rats, before (black) and after (red) PR. *P < 0.05 and **P < 0.01 versus respective pre-PR. (3-way ANOVA, n = 10/group). All measurements were obtained from male rats. DOCA, deoxycorticosterone acetate; SON, supraoptic nuclei.

Fig. 3.

Prorenin (PR) did not alter the magnitude of the afterhyperpolarization (AHP) in SON neurons from sham- or DOCA-salt-treated rats. Representative example of bursts of action potentials (APs) triggered by depolarizing voltage steps (75 pA, 1 s) in a sham rat before and after PR (2.5 nM, 5s) (A). *Note the slow, long lasting AHP following the bursts, which was not affected by PR. Summary data of the mean AHP peak amplitude as a function of depolarizing voltage commands of increasing magnitude (5–75 pA, Δ5 pA) in sham (B) and DOCA-salt treated (C) rats before (black) and after (red) PR. Summary data of the mean number of evoked APs as a function of depolarizing voltage commands of increasing magnitude (5–75 pA, Δ5 pA) in sham (D) and DOCA-salt-treated (E) rats before (black) and after (red) PR. n = 10/group. All measurements were obtained from male rats. DOCA, deoxycorticosterone acetate.

Fig. 4.

Changes in the expression of voltage-gated K⁺ channel subunits, prorenin receptor (PRR) and vasopressin (VP) mRNA in DOCA-salt treated rats. Summary data of normalized mRNA expression for VP (A), PRR (B), and the voltage-gated K⁺ channel subunits Kv4.2, Kv4.3, and Kv1.2 (C) in SON punches obtained from sham- and DOCA-salt-treated rats. Note the increased expression of Kv4.3, PRR, and VP in DOCA-salt-treated compared with sham rats. *P < 0.05 versus respective sham (n = 5 and 7 in sham and DOCA-salt treated, respectively). Insets: representative samples of PCR amplification plots (y-axis: ΔRn; x-axis: cycle). All measurements were obtained from male rats. SON, supraoptic nuclei.

Fig. 5.

PRR expression in neurons contributes to elevated plasma VP levels in DOCA-salt-treated mice. Summary data of mean plasma VP levels in wild-type (WT) and neuron filament promoter PRR knockdown (Nefh-PRRKO) mice following sham or DOCA-salt treatment. ***P < 0.01 or ****P < 0.001 vs. respective sham; ##P < 0.01 vs. WT + DOCA, one-way ANOVA (n = 3 mice/group). All measurements were obtained from male mice. DOCA, deoxycorticosterone acetate; PRR, prorenin receptor; VP, vasopressin.