C-type natriuretic peptide (CNP) in the paraventricular nucleus-mediated renal sympatho-inhibition

Abstract

Volume reflex produces sympatho-inhibition that is mediated by the hypothalamic paraventricular nucleus (PVN). However, the mechanisms for the sympatho-inhibitory role of the PVN and the neurochemical factors involved remain to be identified. In this study, we proposed C-type natriuretic peptide (CNP) as a potential mediator of this sympatho-inhibition within the PVN. Microinjection of CNP (1.0 μg) into the PVN significantly decreased renal sympathetic nerve activity (RSNA) (-25.8% ± 1.8% vs. -3.6% ± 1.5%), mean arterial pressure (-15.0 ± 1.9 vs. -0.1 ± 0.9 mmHg) and heart rate (-23.6 ± 3.5 vs. -0.3 ± 0.9 beats/min) compared with microinjection of vehicle. Picoinjection of CNP significantly decreased the basal discharge of extracellular single-unit recordings in 5/6 (83%) rostral ventrolateral medulla (RVLM)-projecting PVN neurons and in 6/13 (46%) of the neurons that were not antidromically activated from the RVLM. We also observed that natriuretic peptide receptor type C (NPR-C) was present on the RVLM projecting PVN neurons detected by dual-labeling with retrograde tracer. Prior NPR-C siRNA microinjection into the PVN significantly blunted the decrease in RSNA to CNP microinjections into the PVN. Volume expansion-mediated reduction in RSNA was significantly blunted by prior administration of NPR-C siRNA into the PVN. These results suggest a potential role for CNP within the PVN in regulating RSNA, specifically under physiological conditions of alterations in fluid balance.

Keywords: C-type natriuretic peptide; central nervous system; paraventricular nucleus; renal sympathetic nerve activity; volume reflex.

FIGURE 1

(A) Segments of original recordings demonstrating the representative responses of heart rate (HR), mean arterial pressure (MAP), integrated (Int) renal sympathetic nerve activity (RSNA), and raw RSNA to microinjections of C-type natriuretic peptide (CNP) (1.0 μg) or artificial cerebrospinal fluid (aCSF) (arrows) into the paraventricular nucleus (PVN). Bar = 1 min. (B) HR, MAP, and RSNA responses to the low dose of CNP (0.5 μg), high dose of CNP (1.0 μg), B-type natriuretic peptide (BNP) (1.0 μg) and aCSF microinjection into the PVN, n = 5–9. Values are presented as mean ± SE. *p < 0.05 compared with aCSF.

FIGURE 2

(A) Segments of original recordings demonstrating the representative responses of Int.RSNA, RSNA, integrated lumbar sympathetic nerve activity (int.LSNA), and LSNA to microinjections of CNP (1.0 μg) into the PVN. Bar = 1 min. (B) Cumulative data of change in RSNA and LSNA in response to two doses of CNP (0.5 and 1.0 μg) (n = 6) microinjected into the PVN. Values are presented as mean ± SE. *p < 0.05 compared with LSNA.

FIGURE 3

(A) Segments of original recordings from an individual PVN-RVLM neurons after picoinjection of CNP (right), or aCSF (left) in a rat. (B) Spike discriminator demonstrating a single-unit discharge. (C) Antidromic stimulation evoked action potential with constant latency (32 ms, a, b, and d), for the same neuron as in c, RVLM stimulation evoked an antidromic spike that was canceled (arrow) when the interval between spontaneous action potential and stimulation was reduced to <31 ms, and high frequency (333 Hz, 3 ms) following test (e). All segments (a through e) represent 2 superimposed sweeps.

FIGURE 4

(A) Percent changes in discharge after picoinjection of CNP or aCSF on PVN-RVLM neurons (n = 5) or no-evoked neurons (n = 13), which did not evoke antidromic spikes by antidromic stimulation of the RVLM with constant latency. (B) Percent of PVN-RVLM neurons and no-evoked neurons response to CNP picoinjection. Values are presented as mean ± SE. *p < 0.05 vs. aCSF injected rats.

FIGURE 5

Representative low-magnification (A) and high-magnification (B) confocal images showing co-localization of immunoreactivity of retrogradely labeled PVN-RVLM neurons (A1 and B1, green) and NPR-C receptors (A2 and B2, red). Note that some retrogradely labeled neurons and NPR-C immunoreactivity are co-localized in the PVN (A3 and B3, white arrow). Scale bar, 100 μm (A) and 25 μm (B). All images are single confocal optical sections. 3V, third ventricle.

FIGURE 6

Representative western blots and mean values of NPR-C protein expression with NPR-C siRNA (siRNAa, siRNAb, siRNAc) and scramble RNA (scRNA) in neuronal NG108 cells. Values are presented as mean ± SE, n = 3. *p < 0.05 compared with the control group.

FIGURE 7

Change in HR (A), MAP (B) and RSNA (C) in response to CNP (1.0 μg) (n = 6) microinjected into the PVN in two groups of rats that had prior microinjection of NPRC siRNAb or scRNA. Values are presented as mean ± SE. *p < 0.05 compared with scRNA group.

FIGURE 8

Infusion volume and central venous pressure (CVP)-mediated changes in RSNA in rats that had prior microinjections of NPR-C siRNAb or scRNA bilaterally within the PVN. Values are presented as mean ± SE. *p < 0.05 compared with scRNA group.

FIGURE 9

(A) The microinjection sites of CNP in the PVN to record RSNA or LSNA. (B) Approximate locations of the evoked and non-evoked PVN neurons stimulated by CNP. The distance (in mm) posterior to bregma is shown for each section. (C) Histological photo showing the microinjection site in the PVN of one rat. Arrowhead shows a marked injection site. Bar = 200 μm. AH, anterior hypothalamic nucleus; f, fornix; 3V, third ventricle; OX, optic tract; SO, supraoptic nucleus.

FIGURE 10

A schematic diagram is showing that activation of afferents from volume receptors in the low pressure side of the circulation due to volume expansion causes an increase in CNP in the PVN resulting in specific inhibition of preautonomic glutamatergic neurons (NR1 receptors) innervating the kidneys (RSNA) but not LSNA innervating other general vasculature. Inhibition of RSNA would cause a decrease in renin release, reduced renal vasoconstriction and reduced sodium retention by renal tubules. CNP in the PVN inhibits pre-autonomic neurons that project to the kidneys by acting on the NPR-C. (Templates used from BioRender).