C-type natriuretic peptide and natriuretic peptide receptor B signalling inhibits cardiac sympathetic neurotransmission and autonomic function

Abstract

Aims: B-type natriuretic peptide (BNP)-natriuretic peptide receptor A (NPR-A) receptor signalling inhibits cardiac sympathetic neurotransmission, although C-type natriuretic peptide (CNP) is the predominant neuropeptide of the nervous system with expression in the heart and vasculature. We hypothesized that CNP acts similarly to BNP, and that transgenic rats (TGRs) with neuron-specific overexpression of a dominant negative NPR-B receptor would develop heightened sympathetic drive.

Methods and results: Mean arterial pressure and heart rate (HR) were significantly (P < 0.05) elevated in freely moving TGRs (n = 9) compared with Sprague Dawley (SD) controls (n = 10). TGR had impaired left ventricular systolic function and spectral analysis of HR variability suggested a shift towards sympathoexcitation. Immunohistochemistry demonstrated co-staining of NPR-B with tyrosine hydroxylase in stellate ganglia neurons. In SD rats, CNP (250 nM, n = 8) significantly reduced the tachycardia during right stellate ganglion stimulation (1-7 Hz) in vitro whereas the response to bath-applied norepinephrine (NE, 1 μM, n = 6) remained intact. CNP (250 nM, n = 8) significantly reduced the release of ³H-NE in isolated atria and this was prevented by the NPR-B antagonist P19 (250 nM, n = 6). The neuronal Ca²⁺ current (n = 6) and intracellular Ca²⁺ transient (n = 9, using fura-2AM) were also reduced by CNP in isolated stellate neurons. Treatment of the TGR (n = 9) with the sympatholytic clonidine (125 µg/kg per day) significantly reduced mean arterial pressure and HR to levels observed in the SD (n = 9).

Conclusion: C-type natriuretic peptide reduces cardiac sympathetic neurotransmission via a reduction in neuronal calcium signalling and NE release through the NPR-B receptor. Situations impairing CNP-NPR-B signalling lead to hypertension, tachycardia, and impaired left ventricular systolic function secondary to sympatho-excitation.

Keywords: Calcium; Hypertension; Natriuretic peptides; Norepinephrine; Sympathetic.

Figure 1

Telemetric measurements and powerspectral analysis in conscious SD wildtype (WT, n = 9) and pNSE-NPR-BΔKC transgenic (TG, n = 10) rats. (A) MAP, SBP, and diastolic blood pressure (DBP) as well as HR in SD and TG rats. (B–D) Cumulative spectral moduli of HRV and SBP in the LF and HF bands in control and NSE-NPR-BΔKC TG rats. (E, F) Spontaneous BRS-LF and calculated as the gain of the LF-SBP. (G, H) Overall HRV was assessed as the standard deviation of the NN interval (SDNN) along with the standard deviation of systolic blood pressure (SD-SBP) (*P < 0.05, **P < 0.01 transgene vs. SD wild-type).

Figure 2

(A) Representative raw data traces showing the effect of 250 nM CNP (grey) compared with control (black) on the HR response to stimulation of the right stellate ganglion at 1–7 Hz in isolated atria-stellate preparations from the SD rat in vitro. (B) Group mean data showing the effect of CNP at 50 (n = 7), 100 (n = 8), 250 (n = 8), and 500 (n = 6) on the tachycardia to right stellate stimulation at 1–7 Hz (**P < 0.01 control vs. CNP).

Figure 3

(A) Representative raw data trace showing ³H efflux in counts per minute over time and the effect of 250 nM CNP on the evoked release of ³H-NE (S2) in response to field stimulation (5 Hz) compared with control (S1) in right atria from the SD rat. (B) Group data (including mean and standard error) as a dot plot showing a significantly reduced release of ³H-NE (percentage increase from baseline) in response to field stimulation following addition of 250 nM CNP (n = 8 atria). The action of CNP is lost in the presence of the NPR-B antagonist P19 (250 nM, n = 6 atria). (C) Raw data trace showing increase in fluorescence (au) over time, of a stellate sympathetic neuron in a solution containing the NE reuptake transporter (NET) assay. The control slope is represented in black and the slope after addition of 250 nM CNP in black with grey fill. The fluorescence increase is blocked by the NET inhibitor desipramine (DMI, 1 μM, in grey). (D) Group data (including mean and standard error) as a dot plot showing no change in NET activity between CNP 250 nM and control. DMI blocks fluorescence uptake in both groups. (Control, n = 19 neurons; CNP n = 12 neurons). *P < 0.05 control vs. CNP, ***P < 0.001 vs. control or CNP.

Figure 4

(A) Immunohistochemistry showing tyrosine hydroxylase (TH) staining with texas red in cultured sympathetic neurons of the right stellate ganglia from SD rats also staining positive for the NPR-B in green (fluorescein) Nuclear staining is shown with DAPI in blue and co-localization is demonstrated by overlap of staining in yellow (×20 magnification). (B) Representative whole cell calcium current density traces with (grey S2) or without (black) 250 nM CNP. Current evoked by test pulses from a holding potential of − 90 to − 10 mV. (C) Mean current density–voltage relations in the presence or absence of 250 nM CNP (n = 6). (*P < 0.05, **P < 0.01 control vs. CNP). (D) Representative raw data trace of calcium transients induced by 50 mM KCl (30 s) in a single stellate sympathetic neuron in control (black S1) and in the presence of 100 or 250 nM CNP (grey). (E) Group data (including mean and standard error) as a dot plot showing a significant reduction in KCl-evoked calcium transients in isolated stellate neurons from the SD rat after 100 nM (n = 6) and 250 nM (n = 9) CNP. *P < 0.05, **P < 0.01 vs. control.

Figure 5

Telemetric measurement in freely moving conscious NSE-NPR-BΔKC TGR (TG) and SD wild type (WT) following clonidine treatment. Clonidine significantly reduced MAP (A), and HR (B). After a washout phase, parameters were indistinguishable from baseline values (n = 9 per group. *P < 0.05 vs. baseline).