Vascular relaxation induced by C-type natriuretic peptide involves the ca2+/NO-synthase/NO pathway

Abstract

Aims: C-type natriuretic peptide (CNP) and nitric oxide (NO) are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3) contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery.

Main methods: Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry.

Key findings: CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas.

Significance: These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.

Figure 1. Relaxation induced by CNP in intact endothelium (e⁺) and denuded endothelium (e⁻) aortic rings after contraction with phenylephrine.

Responses are represented as the percentage of the relaxation induced by CNP on the contraction with phenylephrine (0.1 µmol/L). Data are means ± SEM of at least five experiments. **P<0.01 for pD₂ values were obtained for e⁻ vs. e⁺ rat aortic rings (Student t test).

Figure 2. Representative tracings showing the amplitude of the pre-contraction induced by phenylephrine and the profile of relaxation induced by CNP.

A) In endothelium-denuded aortic rings B) Endothelium-intact aortic rings, C) Endothelium-intact aortic rings after incubation with L-NAME.

Figure 3. Effects of L-NAME and hydroxocobalamin on the relaxation induced by CNP in intact endothelium aortic rings (e⁺).

Concentration-effect curves were constructed in the absence (control) or after incubation with these drugs for 30 minutes, _L-NAME (100 µmol/L) (A) and hydroxocobalamin (Hydroxo, 10 µmol/L) (B). Responses are represented as the percentage of the relaxation induced by CNP on the contraction with phenylephrine (0.1 µmol/L). Data are means ± SEM of five experiments. *P<0.05 for pD₂ values was obtained with _L-NAME or hydroxo vs. intact endothelium rat aortic rings (e⁺) (Student t test).

Figure 4. Phosphorylation of NOS-3 in rat aorta.

Representative blots showing the protein expression of p-NOS-3-Ser¹¹⁷⁷ after stimulation with vehicle (control) and CNP (0.3 µmol/L). Each lane represents the protein obtained from the homogenate of an independent rat aorta. Bars show the optic densitometry of the blots (n = 6–7/group).

Figure 5. Cytosolic Ca²⁺ increase in endothelial cells and cytosolic Ca²⁺ decrease in smooth muscle cells induced by CNP.

A) Aortic rings were preloaded with Fluo-3 AM and then stimulated with CNP (0.3 µmol/L). Serial Ca²⁺ images of Fluo-3 fluorescence in aortic segment were recorded at the times (t) 0, 210, 410 and 610 seconds (s) after addition of CNP. A) image in differential contrast phase-DIC; B) image of Fluo-3 fluorescence and C) merged image in pseudocolor (*black line ECs), (*white line SMCs) and (*yellow line arterial lumen). The effects of CNP were represented in surface plots of the endothelial cells (a–d) and the smooth muscle cells (a1–d1). The arbitrary intensity values from low to high [Ca²⁺] are indicated by pseudocolor values. B) Effect of CNP (0.3 µmol/L) on average fluorescence intensity (%ΔFI) of smooth muscle cells (SMCs) and endothelial cells (ECs) from aortic ring segment preloaded with Fluo-3 AM.

Figure 6. Expression of NPR-C-receptor in rat aorta artery.

A) Representative photomicrography of an aortic ring showing endothelial (black arrows) and adventitial (white arrows) immunohistochemical staining for NPR-C receptor (×100, and inset ×400). B) Representative photomicrography of an aortic ring showing negative control to immunohistochemical staining for NPR-C receptor.