Definitive role for natriuretic peptide receptor-C in mediating the vasorelaxant activity of C-type natriuretic peptide and endothelium-derived hyperpolarising factor

Abstract

Objective: C-type natriuretic peptide (CNP) has recently been suggested to represent an endothelium-derived hyperpolarising factor (EDHF) in the mammalian resistance vasculature and, as such, important in the regulation of local blood flow and systemic blood pressure. Additionally, this peptide has been shown to protect against ischaemia-reperfusion injury and inhibits leukocyte and platelet activation. Herein, we use a novel, selective natriuretic peptide receptor-C (NPR-C) antagonist (M372049) to highlight the pivotal contribution of CNP/NPR-C signalling in the EDHF-dependent regulation of vascular tone and investigate the mechanism(s) underlying the release and biological activity of CNP.

Methods: In vitro pharmacological investigation was conducted in rat (Sprague-Dawley) aorta and mesenteric resistance arteries. Relaxant responses to CNP, atrial natriuretic peptide (ANP), the nitric oxide donor spermine-NONOate (SPER-NO) and the endothelium-dependent vasodilator, acetylcholine (ACh) were examined in the absence and presence of M372049 or inhibitor cocktails shown previously to block endothelium-dependent dilatation in the resistance vasculature. RT-PCR was employed to characterize the expression of NPR subtypes in the vessels studied.

Results: M372049 produced concentration-dependent inhibition of the vasorelaxant activity of CNP in rat isolated mesenteric resistance arteries but not aorta; in contrast, M372049 did not affect relaxations to ANP or SPER-NO in either vessel. M372049 or ouabain alone produced small, significant inhibition of EDHF-dependent relaxations in mesenteric arteries and in combination acted synergistically to abolish such responses. A combination of M372049 with established inhibitors of EDHF-dependent relaxation revealed that multiple, distinct pathways coordinate the bioactivity of EDHF in the resistance vasculature, and that CNP/NPR-C signalling represents a major component.

Conclusions: These data substantiate CNP/NPR-C signalling as a fundamental pathway underlying EDHF-dependent regulation of vascular tone in the rat mesenteric resistance vasculature. An increased understanding of the physiological roles of CNP/NPR-C signalling in the vasculature (now facilitated by the identification of a selective NPR-C antagonist) should aid determination of the (patho)physiological importance of EDHF and might provide the rationale for the design of novel therapeutics.

Figure 1

Chemical structure of M372049.

Figure 2

Relaxation of rat isolated mesenteric artery by CNP (0.001-1μM; upper panels), ANP (0.001-1μM; lower left panel) and SPER-NO (0.001-10μM; lower right panel) in the absence and presence of M372049 (0.1-100nM), Ba²⁺ (30μM), ouabain (1mM) or following endothelial denudation. Data is represented as mean±s.e.mean; n≥6; *P<0.05, significantly different to control.

Figure 3

Relaxation of rat isolated mesenteric artery to ACh (0.001-10μM) in the absence and presence of M372049 (100nM), ouabain (1mM) or a combination of M372049 plus ouabain. Data is represented as mean±s.e.mean; n≥6; *P<0.05, significantly different to control.

Figure 4

Relaxation (upper panel) and hyperpolarisation (lower panel) of rat isolated mesenteric artery by ACh (0.01-10μM) in the absence and presence of M372049 (100nM) or M372049 plus ouabain (1mM). Data is represented as mean±s.e.mean; n≥6; *P<0.05, significantly different to control.

Figure 5

Relaxation of rat isolated aorta by CNP (0.001-1μM; upper left panel), ANP (0.001-1μM; upper right panel), ACh (0.001-1μM; lower left panel) and SPER-NO (0.001-10μM; lower right panel) in the absence and presence of M372049 (100nM). Data is represented as mean±s.e.mean; n≥6; *P<0.05, significantly different to control.

Figure 6

Relaxation of rat isolated mesenteric artery by ACh (0.001-10μM) in the absence and presence of TRAM-34 (10μM), apamin (100μM) or both (upper left panel); apamin, M372049 (100nM) or both (upper right panel); apamin, ouabain (1mM) or both (lower left panel); or TRAM-34, M372049/Ba²⁺ (30μM) or both (lower right panel). Data is represented as mean±s.e.mean; n≥6; *P<0.05, significantly different to control.

Figure 7

RT-PCR analysis of NPR subtypes present in isolated rat aorta and mesenteric small vessels. Each receptor subtype (i.e. NPR-A, NPR-B & NPR-C) was found in both vessels. Expected product sizes: NPR-A, 698bp; NPR-B 699bp; NPR-C, 533bp. This is representative of 3 separate experiments.

Figure 8

Schematic representation of the proposed multiple EDHF pathways present in rat mesenteric resistance arteries. One ‘arm’ of the EDHF response is triggered by SK_Ca channels, is dependent on the release of CNP, activation of NPR-C and opening of a Ba²⁺-sensitive GIRK. An additional component is provided by an as yet unidentified mediator that is released following IK_Ca channel activation and is dependent on stimulation of the ouabain-sensitive Na⁺/K⁺-ATPase.