C-Type Natriuretic Peptide Induces Anti-contractile Effect Dependent on Nitric Oxide, Oxidative Stress, and NPR-B Activation in Sepsis

Abstract

Aims: To evaluate the role of nitric oxide, reactive oxygen species (ROS), and natriuretic peptide receptor-B activation in C-type natriuretic peptide-anti-contractile effect on Phenylephrine-induced contraction in aorta isolated from septic rats.

Methods and results: Cecal ligation and puncture (CLP) surgery was used to induce sepsis in male rats. Vascular reactivity was conducted in rat aorta and resistance mesenteric artery (RMA). Measurement of survival rate, mean arterial pressure (MAP), plasma nitric oxide, specific protein expression, and localization were evaluated. Septic rats had a survival rate about 37% at 4 h after the surgery, and these rats presented hypotension compared to control-operated (Sham) rats. Phenylephrine-induced contraction was decreased in sepsis. C-type natriuretic peptide (CNP) induced anti-contractile effect in aortas. Plasma nitric oxide was increased in sepsis. Nitric oxide-synthase but not natriuretic peptide receptor-B expression was increased in septic rat aortas. C-type natriuretic peptide-anti-contractile effect was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation. Natriuretic peptide receptor-C, protein kinase-Cα mRNA, and basal nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were lower in septic rats. Phenylephrine and CNP enhanced ROS production. However, stimulated ROS production was low in sepsis.

Conclusion: CNP induced anti-contractile effect on Phenylephrine contraction in aortas from Sham and septic rats that was dependent on nitric oxide-synthase, ROS, and natriuretic peptide receptor-B activation.

Keywords: C-type natriuretic peptide; natriuretic peptide receptor B; nitric oxide; phenylephrine-induced contraction; reactive oxygen species; sepsis.

Figure 1

Percentage of survival analysis (A) of control-operated (Sham n = 20) and CLP (n = 30) rats. Mean arterial pressure (B) (MAP, mmHg) before (0 h) or after (from 1 to 4 h) surgeries in Sham (n = 6) or CLP (n = 6) rats. Cumulative concentration-effect curves to phenylephrine (PE) in the absence or in the presence of CNP (10 nmol.L⁻¹; Sham n = 8; CLP n = 8) in aorta (C) or resistance mesenteric artery (RMA, Sham n = 6; CLP n = 7) (D) with endothelium, from Sham or CLP rats. Data are represented as mean ± S.E.M. In (B): ^*different from 0 h; ^#different from Sham respective time; ^adifferent from CLP 1, 2, or 3 h. In (C): ^*different from Sham CO; ^#different from CLP CO; ^adifferent from Sham CNP. In (D): ^*different from Sham CO; ^**different from Sham CNP. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons (P < 0.05).

Figure 2

NPR-C staining in vascular layers in Sham or CLP rat aortas. Photomicrograph of immunofluorescence of NPR-C staining in Sham (n = 6) or CLP (n = 6) rat aortas. DIC represents dichroic contrast phase, DAPI represents nuclei staining, α-actin from α-actin smooth muscle-FITC staining. Bar represents 50 μm. L, M, and Adv represent lumen, media layer, and adventitia, respectively. Each experimental n represents samples isolated from different animals.

Figure 3

Protein expression of NOS3 (A, Sham n = 4; CLP n = 4), NOS2 (B, Sham n = 4; CLP n = 4) and NPR-B (C, Sham n = 4; CLP n = 6) on aorta homogenate of Sham or CLP rats. Measurement (D) of fluorescence intensity (FI) emitted by anti-NPR-C antibody in Sham (n = 6) or CLP (n = 6) rat aortas. Measurement of plasma nitrite (E, Sham n = 4; CLP n = 4) and nitrate (F, Sham n = 7; CLP n = 7). Western blotting data were normalized by β-actin, and presented as mean ± S.E.M. ^*different from Sham. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons; Student t-test to compare two different groups (P < 0.05).

Figure 4

Cumulative concentration-effect curves to phenylephrine (PE) in the absence (CO) or presence of CNP (10 nmol.L⁻¹); non-selective NOS-inhibitor L-NAME (100 μmol.L⁻¹) or CNP; and L-NAME in Sham (A) (n = 11) or CLP rat aorta (B) (n = 12). pD₂ values (C) of PE curves of Sham or CLP rat aorta. Maximum effect values (D) (g.g⁻¹) of PE curves of Sham or CLP rat aorta. Data are presented as mean ± S.E.M. ^*different from Sham CO; ^#different from CLP CO; ^adifferent from Sham CNP; ^cdifferent from CLP CNP. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons (P < 0.05).

Figure 5

Cumulative concentration-effect curves to phenylephrine (PE) in the absence (CO) or presence of CNP (10 nmol.L⁻¹); selective NOS1-inhibitor N^ω-propyl-L-arginine (N^ω-propyl, 50 nmol.L⁻¹) or CNP + N^ω-propyl in Sham (A) (n = 9) or CLP rat aorta (B) (n = 5). Cumulative concentration-effect curves to PE in the absence (CO) or presence of CNP (10 nmol.L⁻¹); selective NOS2-inhibitor 1400 W (100 nmol.L⁻¹) or CNP + 1400 W in Sham (C) (n = 8) or CLP rat aorta (D) (n = 6). Data are presented as mean ± S.E.M. ^*different from Sham CO; ^#different from CLP CO; ^adifferent from Sham CNP; ^bdifferent from Sham + 1400 W; ^cdifferent from CLP CNP. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons (P < 0.05).

Figure 6

Cumulative concentration-effect curves to phenylephrine (PE) in the absence (CO) or presence of CNP (10 nmol.L⁻¹); ${\text{O}}_2^{–}$ scavenger Tiron (100 μmol.L⁻¹) or CNP + Tiron in Sham (A) (n = 6) or CLP rat aorta (B) (n = 13). Cumulative concentration-effect curves to PE in the absence (CO) or presence of CNP (10 nmol.L⁻¹); intracellular H₂O₂ breakdown agent PEG-catalase (250 U.mL⁻¹) or CNP + PEG-catalase in Sham (C) (n = 5) or CLP rat aorta (D) (n = 11). Data are presented as mean ± S.E.M. ^*different from Sham CO; ^#different from CLP CO; ^adifferent from Sham CNP; ^bdifferent from Sham Tiron; ^cdifferent from CLP CNP. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons (P < 0.05).

Figure 7

Cumulative concentration-effect curves to phenylephrine (PE) in the absence (CO) or presence of CNP (10 nmol.L⁻¹); NPR-A/B antagonist Anantin (Ana) in lower concentration (0.1 μmol.L⁻¹) or CNP + Ana in Sham (A) (n = 8) or CLP rat aorta (B) (n = 13). Cumulative concentration-effect curves to PE in absence or in the presence of Ana in higher concentration (1 μmol.L⁻¹) in Sham (n = 5) or CLP rat aorta (n = 11) (C). Data are presented as mean ± S.E.M. ^*different from Sham CO; ^#different from CLP CO; ^adifferent from Sham CNP; ^cdifferent from CLP CNP; ^edifferent from Sham Ana. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons (P < 0.05).

Figure 8

Endogenous CNP staining in vascular layers. Photomicrograph of immunofluorescence of CNP staining in Sham (n = 4) or CLP (n = 6) rat aortas. DIC represents dichroic contrast phase, DAPI represents nuclei staining, α-actin from α-actin smooth muscle-FITC staining. Bar represents 50 μm. L, M, and Adv represent lumen, media layer, and adventitia, respectively. Each experimental n represents samples isolated from different animals.

Figure 9

Measurement of FI emitted by anti-CNP antibody (A) in Sham (n = 4) or CLP (n = 6) rat aortas. Protein expression of Nox1 (B) and NADPH-dependent reactive oxygen species (ROS) production (C) in aorta homogenate from Sham (n = 4) or CLP (n = 4) rats. ROS production (relative luminescence units.mg⁻¹.min⁻¹) was evaluated in the absence (basal, Sham n = 5; CLP n = 3) or in the presence of PE (10 μmol.L⁻¹, Sham n = 8; CLP n = 5) or CNP (10 nmol.L⁻¹) + PE (10 μmol.L⁻¹, Sham n = 6; CLP n = 5). Negative control was evaluated in the presence of non-selective inhibitor of flavoproteins DPI (10 μmol.L⁻¹, n = 3) in Sham aorta. Relative PKCα mRNA expression (D) in aorta homogenate from Sham (n = 4) or CLP (n = 4) rats. Western blotting data were normalized by β-actin. Quantitative PCR analysis was normalized by GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA expression. Data are presented as mean ± S.E.M. In (B): ^*different from Sham endothelium; ^#different from CLP endothelium; ^**different from VSM from respective groups. In (C): ^*different from Sham. In (D): ^*different from Sham basal; ^#different from CLP basal; ^adifferent from Sham DPI; ^**different from Sham PE; ^##different from CLP PE; ^cdifferent from Sham PE CNP. In (E): ^*different from Sham. Each experimental n represents samples isolated from different animals. Two-way Anova, Bonferroni correction to evaluate interaction factor; One-way Anova, pos-hoc Newman-Keuls to three or more comparisons; Student t-test to compare two different groups (P < 0.05).