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

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.


INTRODUCTION
One of the most important clinical characteristics of sepsis and septic shock is the vascular hyporesponsivity to contractile agonists (Donaldson and Myers, 1996;Vromen et al., 1996;Strunk et al., 2001). It represents an important condition for patient survival. Different therapeutic strategies aimed to improve vital organ function (Leone and Martin, 2008). The identification of new intracellular signaling related to sepsis progression might contribute to the development of therapeutic strategies to reduce sepsis-associated mortality.
Sepsis cardiovascular dysfunction involves excessive nitric oxide (NO) production (Strunk et al., 2001) by NO-synthases (NOS) (Thomas et al., 2008). The endothelial NOS (NOS3) plays main role on organ blood flow distribution and is related to microvascular permeability regulation and cell interaction. Sand et al. (2015) showed that mesenteric blood flow decreases in a time-dependent manner and in parallel with the development of metabolic acidosis and organ dysfunction after induction of sepsis. Inducible NOS (NOS2) is constitutively expressed on few quantities and its protein expression is enhanced after inflammatory stimuli in different cells (Strunk et al., 2001). Sustained NO production following NOS2 induction is associated with sepsis hyporesponsivity (Donaldson and Myers, 1996;Vromen et al., 1996;Strunk et al., 2001). Furthermore, the role of neuronal NOS (or NOS1) seems to contribute to the hyporesponsivity of contractile adrenergic agonists in sepsis (Nardi et al., 2014). The inhibition of soluble guanylyl cyclase (sGC), enzyme which is the main target to NO, is able to recover mean arterial pressure (MAP) and enhance cardiac contractility in septic patients (Fernandes et al., 2009).
Regarding to endothelial cells, Li et al. (2016) showed that lipopolysaccharide (LPS) treatment of human umbilical vein endothelial cells (HUVEC) increases oxidative stress, malondialdehyde levels, superoxide dismutase 2 (SOD2) protein expression and phosphorylation of c-Jun N-terminal kinases (JNK), and decreases SOD1 expression. Moreover, Zhou et al. (2012) observed that intravenous bolus injection of ascorbate, 30 min prior to CLP surgery or 3 h after CLP surgery, attenuated NOS activity and prevented vascular permeability increases.
The C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family with high conserved amino acid sequence (Inoue et al., 2003). It can be synthesized, stored, and released from endothelial cells. CNP is present in LPS-, cytokines-, and growth factors-stimulated endothelial cells (Suga et al., 1993). However, CNP remains low on the plasma concentration in healthy subjects (Stingo et al., 1992). CNP is an endothelium-derived vasodilator agent (Suga et al., 1992), it induces vasodilation in an endothelium-dependent manner (Amin et al., 1996;Andrade et al., 2014) and it can hyperpolarize plasma membrane of vascular smooth muscle (VSM) cell through natriuretic peptide receptor (NPR)-C activation (Chauhan et al., 2003;Garcha and Hughes, 2006). Natriuretic peptides, i.e., atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and CNP are able to induce intravascular shedding of glycocalyx and increase vascular permeability (Jacob et al., 2013). Furthermore, the authors suggest that this condition might be present in the pathophysiology of sepsis.
In VSM cells stimulated with LPS or tumor necrosis factorα (TNF-α), natriuretic peptides increase nitrite formation while the NOS inhibitor reduces CNP responses, suggesting the involvement of NO on CNP effects (Marumo et al., 1995). NPR-A/B antagonist infusion attenuated the hypotension in a sepsis model (Hinder et al., 1997). According to Panayiotou et al. (2010), knockout mice to NPR-A exposed to LPS have low systemic hypotension, and lower levels of NO metabolites, cGMP levels and NOS2 expression compared to wild-type septic animals. Therefore, the contribution of natriuretic peptide system to the vascular dysfunction seems to be evident on sepsis. However, little is known about the role of CNP in this dysfunction and if it could act as a modulator of α 1 -adrenergic vascular contraction on animals septic vessels. The hypothesis of the present work was that CNP induces anti-contractile effect on phenylephrine (PE) contraction in septic vessels, which effect would be dependent of NO and reactive oxygen species (ROS). This study aimed to evaluate the contribution of intracellular NO and ROS, and the role of activation of NPR-B in the CNP-anticontractile effect on PE-contraction in aortas isolated from rats submitted to CLP surgery to induce sepsis.

Animals
All the procedures were performed in accordance with the standards and policies of the Ethics Committee on Animal Care and Use of the University of São Paulo (#144/2011). Male rats (200 g) were anesthetized with Tribromoethanol (0.25 g.Kg −1 , i.p.) before cathetherization surgery on femoral artery. On the next day, the MAP was measured for basal values (before surgery), using pressure transducer. Then, the rats were anesthetized with Tribromoethanol (0.25 g.Kg −1 , i.p.) and randomly submitted to CLP surgery to induce sepsis, with 12 punctures using a 16 gauge-needle on anti-mesenteric border of the cecum (Wichterman et al., 1980;Araújo et al., 2011), or the control group was submitted to a medial laparotomy only (Sham). Survival and MAP were evaluated for 24 h after surgeries. Animals were maintained on standard rat chow and water.

Functional Studies by Vascular Reactivity
Sham and CLP rats were killed by decapitation under anesthesia (inhaled Isoflurane) and thoracic aorta and resistance mesenteric artery (RMA) were used to measure the isometric tension.

Vascular Reactivity on RMA
RMA were isolated and cut into rings (2 mm) using a dissection magnifier in cold Krebs solution for small arteries. Only the second branch of RMA with 200-350 µm of internal circumference were used. A tungsten wire (40 µm) was passed into the vessel and tied in myograph to resistance vessels and another was connected to isometric force transducer. Cumulative concentration-effect curves were constructed to PE (0.1 nmol.L −1 −100 µmol.L −1 ) in intact endothelium RMA in the absence or in the presence of CNP (10 nmol.L −1 , 30 min). The area under the curve (AUC) of PE contraction was evaluated.

Measurement of Plasma NO Metabolites
Cardiac blood samples were collected from Sham or CLP rats in heparin-containing tubes. Plasma aliquots of Sham or CLP rats were analyzed for their nitrite content using an ozone-based reductive chemiluminescence assay (Pinheiro et al., 2012). The plasma NOx concentration was determined by using the Griess reaction (Pinheiro et al., 2012).

Confocal Microscopy Analysis
Aorta was isolated from Sham or CLP rats and frozen on cryoprotection liquid. Sheets were prepared with 10 µm of thickness and immunofluorescence was performed to evaluate endogenous CNP (1:100) or NPR-C (1:100) expression. The secondary antibody mouse anti-goat Alexafluor 647 (1:1000) or sheep anti-rabbit Alexafluor 647 (1:1000) was incubated for 1 h, at room temperature. Fluoroshield R with DAPI was applied overnight at 4 • C. Images were acquired using confocal microscopy.

Lucigenin Chemiluminescence Analysis
Vascular nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production was assessed in intactendothelium aorta from Sham or CLP rats, previously stimulated with PE (0.1 µmol.L −1 ) in the absence or in the presence of CNP (10 nmol.L −1 , 30 min) or not stimulated (basal). After that, aortas were collected and frozen on liquid nitrogen. The luminescence was measured using Single Tube Luminometer Berthold FB12, at 37 • C. Data were presented as relative luminescence units (RLU).mg −1 .min −1 .

Quantitative Polymerase Chain Reaction (qPCR) Analysis
Total ribonucleic acid (RNA) was extracted from aorta homogenates isolated from Sham or CLP rats with TRIzol R reagent according to manufacturer's instruction. The cDNA was synthesized with 1 µg of RNA using anchored random hexadeoxynucleotide in the reaction conditions for transcriptor First-strand cDNA Synthesis Kit. qPCR was performed using SYBR R FAST qPCR kit Master Mix Universal with 50 nmol.L −1 of primers for PKCα (protein kinase-Cα) or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The samples were incubated for 2 min at 50 • C and 10 min at 95 • C followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min.

Drugs and Solutions
Drugs and solutions are presented on Supplemental Material.

Statistical Analysis
Results are presented as mean ± S.E.M. Each experimental n represents samples isolated from different animals. Comparisons between groups were conducted by the Student's t-test, Oneway analysis of variance followed by Newman-Keuls, or Twoway analysis of variance using Bonferroni correction for multiple comparisons, as appropriate. The level of statistical significance was defined as P < 0.05.

NPR-C was Decreased in CLP Compared to Sham Aortas
As shown in the photomicrographs (Figure 2) and in the measurement of fluorescence intensity (FI) emitted by anti-NPR-C antibody, the NPR-C expression was lower in CLP (4.35 ± 0.46 U, n = 6) compared to Sham aortas (7.81 ± 0.80 U, n = 6).

NPR-C was Lower and NPR-B was Not
Changed in CLP in Relation to Sham Aortas NPR-B protein expression was not different between Sham (0.79 ± 0.07 U, n = 4) and CLP (0.92 ± 0.06 U, n = 6), whereas NPR-C expression was lower in CLP (4.35 ± 0.46 U, n = 6) aortas as compared to Sham (7.81 ± 0.80 U, n = 6), as shown in Figure 3.

CNP-Induced Anti-contractile Effect was Dependent on ROS in Rat Aortas
The superoxide (O − 2 ) scavenger Tiron reduced the potency to PE in Sham (6.70 ± 0.15) but not in CLP (6.69 ± 0.20) aortas (Figure 6). However, ME to PE was enhanced on CLP (1034 ± 70 g.g −1 ) aortas. Moreover, CNP-anti-contractile effect on PE 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. . 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). pD 2 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; a different from Sham CNP; c different 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).

Endogenous Expression of CNP was Lower in Endothelium and Higher in VSM Layer in CLP Than in Sham Rat Aortas
The endothelial expression of endogenous CNP, as shown in the photomicrographs (Figure 8) and the measurement of FI of anti-CNP antibody (Figure 9), was lower in CLP (6.50 ± 0.66 U, n = 6) than in Sham aortas (9.78 ± 0.50 U, n = 4). However, CNP expression on VSM was higher in CLP (9.72 ± 0.60 U, n = 6) compared to Sham aortas (7.32 ± 0.11 U, n = 4). It was not different in the adventitia isolated from Sham (13.35 ± 1.00 U, n = 4) and CLP aortas (15.02 ± 1.20 U, n = 6). It can be noted that endogenous CNP is higher in the adventitia than in the endothelium which is higher than in the VSM in Sham. CNP is higher in adventitia than in the VSM that is higher than in the endothelium in CLP aortas.

DISCUSSION
The main findings of this work were: (i) CNP induced anticontractile effect on PE contraction in rat aortas but not in rat RMA, (ii) this CNP anti-contractile effect was dependent on NOS activity, ROS production and NPR-B activation in Sham and CLP aortas, (iii) endogenous CNP was reduced on endothelium and increased on the VSM cells in CLP aortas, (iv) NPR-C expression was low on VSM layer of CLP aortas, (v) basal NPR-B activity was able to modulate PE-induced vasoconstriction, and (vi) basal NADPH-dependent ROS production was low in CLP aortas, and PE and CNP increased ROS production. It is the first report that CNP can modulate PE contraction, which effect was higher in CLP than Sham aortas. CLP experimental sepsis model represents clinical conditions observed on patients with intestinal puncture, developing severe sepsis within few hours or some days (Lundblad et al., 1995). Moreover, it was observed that CLP group had a pronounced systemic hypotension and low survival rate, corroborating with findings from Tyml et al. (1998), Liaw et al. (2005), and Tsao et al. (2010). However, we have shown that Sham rats presented lower hypotensive effect than CLP group. According to Jong et al. (2002) general anesthetics can change cardiovascular parameters. Therefore, in our study, the decreased MAP observed on Sham rats could be due to tribromoethanol effect. Besides systemic hypotension showed on CLP rats, it was noted that PE-induced contraction on aorta and RMA was lower in CLP than in Sham rats.
In accordance to Vromen et al. (1996) and Wang et al. (2004), in CLP rat aortas there is low vasoconstriction induced by norepinephrine. Silva-Santos et al. (2002) noted LPS-exposure of aortas during 12 h, at isolated organ bath, reduced contraction to PE. Usually, such reduced contraction to different agonists have been associated with larger NO production by NOS1 and/or NOS2 (Vromen et al., 1996;Silva-Santos et al., 2002;Wang et al., 2004;Enkhbaatar et al., 2009;Tsao et al., 2010;Nardi et al., 2014), oxidative stress (Tsao et al., 2010), and greater inflammatory mediators production (Panayiotou et al., 2010). Enkhbaatar et al. (2009) showed that specific NOS1 inhibitor, called as ZK234238, attenuated hypotension and the fall in systemic vascular resistance, inhibited the increase in plasma nitrate/nitrite, and attenuated inflammatory markers such as myeloperoxidase activity, IL (interleukin)-6 mRNA, and reactive 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).
nitrogen species. Furthermore, Nardi et al. (2014) demonstrated that the treatment with 7-nitroindazole, another NOS1 inhibitor, reverted the hyporesponsivity to PE and norepinephrine, and reduced the production of cGMP stimulated by PE in CLP rat aortas. Our results are in agreement with the others and it was noted raise on plasma NO levels, as nitrite and nitrate, and raise on protein expression of NOS2 and NOS3 in CLP rat aortas. Moreover, the non-selective NOS inhibitor L-NAME, or the selective NOS1 inhibitor N ω -propyl, or the selective NOS2 inhibitor 1400 W, increased PE contraction in both groups; but PE-induced maximum contraction was still lower in CLP aortas, suggesting additional mechanisms which are responsible for PE hyporresponsivity.
CNP-induced anti-contractile effect was greater in CLP than in Sham aortas. However, we did not observe CNP-induced modulation of PE contraction in RMA from both groups, using a single concentration of CNP in these studies. Villar et al. (2007) showed CNP-induced relaxation in RMA isolated from rats that was dependent on NPR-C activation. Besides, this group demonstrated all the three NPRs in rat aorta and RMA. We cannot exclude the possibility that higher CNP concentration could induce the anti-contractile effect on these resistance vessels, since the contraction elicited by PE needed higher concentration in RMA than in aorta. Since this CNP anti-contractile effect was observed on aortas at low concentration (10 nmol.L −1 ), we decided to continue to investigate this vessel. CNP anticontractile effect was completely reversed by NOS inhibitors showing CNP could activate NOS and NO production could be responsible for CNP anti-contractile effect in aortas. In the same way, Murthy et al. (1998) had demonstrated that a selective agonist of NPR-C, called cANP(4-23), can increase NOS activity via Gi protein and calcium influx. Costa et al. (2007) reported that CNP increased NOS activity in aorta homogenate and our research group (Andrade et al., 2014) has described endothelium-dependent relaxation induced by CNP in rat aortas that seems to lead to NPR-C activation, but with no alteration in phosphorylation status of Ser 1177 of NOS3. As described by Anand-Srivastava et al. (1996), the activation of cytosolic domain of NPR-C leads to adenylyl cyclase inhibition via Pertussis toxin sensitive-G protein. Furthermore, Anand-Srivastava (2005) reviewed NPR-C intracellular signaling with inositol-trisphosphate (IP 3 ) and diacylglycerol (DAG) formation, calcium mobilization, potassium channel, NOS, and PKC activation.
Although Nox1 protein expression was higher in CLP aortas, the basal activity of NADPH-dependent ROS production was low in CLP aortas, suggesting another source of ROS in CLP aortas. Moreover, Brandes et al. (1999) determined after LPS stimulus, that there are increased NADPH oxidase and xanthine oxidase expression in rat aortas, leading to O − 2 , H 2 O 2 production and after reaction with NO to form peroxynitrite (ONOO − ).
Since NADPH oxidase and xanthine oxidase produce O − 2 and H 2 O 2 , these results suggest the contribution of O − 2 to the contractile effect to PE in Sham and CLP rat aortas. However, when intracellular H 2 O 2 was removed, there were no changes on PE-induced contraction in both groups. It is possible that ROS production in VSM cells could compensate the removal of endothelial ROS on PE-induced contraction.
NADPH-dependent ROS production was increased after PE alone or CNP and PE stimuli, suggesting that CNP can positively modulate ROS production in rat aortas. In relation to the CNP anti-contractile effect in rat aortas, it seems dependent on H 2 O 2 , possibly through O − 2 dismutation by SOD. As presented by Anand-Srivastava (2005), CNP could stimulate PKC which in turn activates NADPH oxidase (Brandes et al., 2014). Thus, NADPH-dependent ROS production increased by CNP and PE could be due to NPR-C activation dependent on PKC. Jao et al. (2001) reported low protein expression of PKCα in rat model of sepsis. The reduced effect of PE or CNP and PE on NADPH-dependent ROS production could be due to reduced PKC expression and/or PKC deficient activity in CLP aortas. In fact, PKCα mRNA expression was lower in CLP aortas compared to Sham. Furthermore, protein expression of NPR-C on VSM cells from CLP aortas was lower compared to Sham. It might suggest a reduction on protein expression or activity of the NPR-C-PKCα-NADPH oxidase axis in CLP aortas. Weber et al. (1991) identified Anantin as the first competitive antagonist of NPR associated with cGMP formation, i.e., NPR-A and NPR-B receptors. Then, Anantin has been used as non-selective NPR-A/B antagonist. As CNP is the endogenous agonist of NPR-B (Koller and Goeddel, 1992) and has low affinity for NPR-A, Anantin could be considered a NPR-B antagonist for CNP effects. We demonstrated that CNP anticontractile effect was dependent on NPR-B activation, since Anantin reversed this effect. Madhani et al. (2003) showed that CNP relaxation was inhibited by NPR-B antagonist in mice aortas. Rautureau et al. (2010) reported that CNP increased cGMP production independently of sGC in endothelial cells that was accompanied by NO production. Simon et al. (2009) showed CNP hyperpolarizes the endothelial cell membrane in a cGMP-dependent protein kinase (GK) manner. Activation of NPR-B by CNP was evident in Sham and CLP aortas. However, basal NPR-B activity without exogenous CNP was enough to modulate PE-induced vasoconstriction. Anantin at low concentration was able to increase PE maximum contraction only in CLP aortas. The greater effect of Anantin on high concentration suggests an important contribution of endogenous CNP for regulation of vascular tone. Besides, protein expression of NPR-B was not altered in aortas from Sham and CLP rats.
According to Hinder et al. (1997) and Stubbe et al. (2004) described that NPR-A/B antagonist preventing hypotension due to sepsis. This is the first mention that endogenous CNP or NPR-B intrinsic activity can contribute to modulation of vascular tone in aortas from Sham and CLP rats. Moreover, endogenous CNP production can be stimulated by several factors, such as LPS, TNF-α, IL-1α, and IL-1β, acetylcholine and bradykinin (Suga et al., 1993;Anand-Srivastava, 2005). Sepsis is a condition in which plasma levels of CNP are high (Hama et al., 1994). We demonstrated that endogenous CNP on vascular endothelium was lower in CLP than in Sham aortas. However, the peptide expression on VSM cells was greater in CLP than Sham aortas. It suggests a possible release of CNP from the endothelium to VSM in CLP aortas. Although the protein expression of NPR-B was similar in both groups, NPR-B activity might be different in CLP aortas.
Our results showed for the first time the CNP induced anticontractile effect on PE-induced contraction that was greater in CLP than Sham aortas. This effect is dependent on NOS activity, ROS production and NPR-B activation. O − 2 and H 2 O 2 production seems to be part of the same signaling pathway, and PE alone or CNP and PE increase ROS production. In CLP aortas, CNP could be released from the endothelial to VSM cells. NPR-B intrinsic activity or endogenous CNP could modulate the vascular tone. Furthermore, the lower protein expression of NPR-C on VSM cells of CLP aortas and lower relative PKCα mRNA expression could contribute to the reduction of CNPinduced NADPH-dependent ROS production in CLP aortas. If the activation of NOS and NPR-B belongs to a common intracellular signaling remains to be investigated. Moreover, it is evident the contribution of CNP system on vascular hyporeactivity to α 1 -adrenergic activation in CLP aorta, and it might represents a potential target for future pharmacological studies.