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Author’s Accepted Manuscript MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH 2, regulates the depressor effects induced by endokinin A/B Jing Zhang, Chunbo He, Xiong Pi, Yan Wang, Lanxia Zhou, Shouliang Dong www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(16)30672-0 http://dx.doi.org/10.1016/j.ejphar.2016.10.028 EJP70894

To appear in: European Journal of Pharmacology Received date: 21 April 2016 Revised date: 21 October 2016 Accepted date: 21 October 2016 Cite this article as: Jing Zhang, Chunbo He, Xiong Pi, Yan Wang, Lanxia Zhou and Shouliang Dong, MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH2, regulates the depressor effects induced by endokinin A/B, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.10.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH2, regulates the depressor effects induced by endokinin A/B Jing Zhang a, Chunbo He a, Xiong Pi a, Yan Wang a, Lanxia Zhou b, c, * Shouliang Dong a, d, * a

Institute of Biochemistry and Molecular Biology, School of Life Sciences, 222 Tianshui South Road, Lanzhou 730000, China b

The Core Laboratory of the First Affiliated Hospital, Lanzhou University, 1 Donggang West Road, Lanzhou 730000, China c

Key Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou 730000, P. R. China d

Key Laboratory of Preclinical Study for New Drugs of Gansu Province, 222 Tianshui South Road, Lanzhou 730000, China [email protected] [email protected] *

Corresponding author. Prof. Shouliang Dong, Institute of Biochemistry and Molecular Biology, School of Life Sciences, 222 Tianshui South Road, Lanzhou 730000, China. Tel(Fax): 0086 931 8912428. *

Corresponding author. Prof. Lanxia Zhou, Address: The Core Laboratory of the First Affiliated Hospital, Lanzhou University, 1 Donggang West Road, Lanzhou 730000, China. Tel(Fax): 0086 931 8620394 Abstract

The interactions of the chimeric peptide MCRT (YPFPFRTic-NH2), based on morphiceptin and neuropeptide FF derivative PFRTic-NH2, on the effects of endokinin A/B (EKA/B) on mean arterial blood pressure of the urethane-anaesthetized rat have been investigated in the absence and presence of tachykinin receptor antagonists, naloxone and NO synthase inhibitors. While MCRT produced dose

dependent decreases in mean arterial pressure, in its presence only a small but statistically insignificant decreases in the magnitude and the time course of the depressor effect of EKA/B (10 nmol/kg) were observed. MCRT had little influence on the depressor effect of EKA/B (1 nmol/kg), but strongly potentiated that of EKA/B (100 nmol/kg). The tachykinin NK1 receptor antagonist SR140333B (1 mg/kg) and the NK3 antagonist SR142891 (2.79 mg/kg) both reduced the hypotensive effects of EKA/B and MCRT alone and blocked those of the two peptides in combination. The NK2 antagonist GR159897 (4 mg/kg) partially blocked the depressor effects of EKA/B and MCRT alone. Naloxone (2 mg/kg) completely blocked the depressor effect of MCTR, but partially blocked that of EKA/B. The NO synthase inhibitor L-NAME

(50 mg/kg) partially blocked the depressor effects of EKA/B, MCRT, and

EKA/B + MCRT. These results could help to better understand the role of tachykinin receptors, opioid receptors and neuropeptide FF receptors in cardiovascular system.

Key words: endokinin A/B; MCRT; co-injection; depressor effect

1. Introduction

The mammalian tachykinin peptide family consists of substance P (SP) and neurokinins A (NKA) and B (NKB), which all share a common C terminal region, FXGLM-NH2 (Pennefather et al., 2004). Their biological effects were caused primarily through three neurokinin receptors designated NK1, NK2 and NK3 receptors

(Maggi, 1995; Pennefather et al., 2004). Tachykinin gene 1 (TAC1) encodes SP and NKA, while TAC3 encodes NKB (Kotani et al., 1986; Nawa et al., 1983; Nawa et al., 1984; Page et al., 2001). In 2000, TAC4 was cloned and found to encode hemokinin-1 (HK-1) and endokinins A, B, C, and D (EKA – EKD) (Kurtz et al., 2002; Page et al., 2003; Zhang et al., 2000).

Endokinin A/B (EKA/B, GKASQFFGLM-NH2), the decapeptide sharing the common C-terminal domain of EKA and EKB, is a preferred ligand of NK1 receptor, and it also binds to NK2 and NK3 receptors (Page et al., 2003). It displayed identical hemodynamic effects to SP in rats, causing short-lived falls in mean arterial blood pressure (MAP) associated with tachycardia, mesenteric vasoconstriction, and marked hindquarter vasodilatation (Page et al., 2003). Mechanism studies indicated that tachykinin receptors, opioid receptors, nitric oxide (NO) pathway and soluble guanylyl cyclase were involved in the cardiovascular regulation (Abdelrahman et al., 2005; Jin et al., 2011).

The morphological co-expression of SP and endomorphins in the central nervous system as well as the co-localization of mu opioid receptor and NK1 receptor indicating the existence of possible functional coactions (Aicher et al., 2000; Greenwell et al., 2007; Luo et al., 2014; Wu et al., 2015). In fact, endomorphin-2 (EM-2) was found to inhibit inflammatory pain through suppression of SP release, while SP protected gastric mucosal through increasing the level of EM-2 (Brancati et al., 2013; Wu et al., 2015). When co-injected, the antinociceptive effect of

endomorphin-1 (EM-1) was enhanced by high dose of EKA/B, but abolished by low dose (Yang et al., 2010). Furthermore, EM-1 slightly attenuated the depressor effects of EKA/B (0.1, 1, 10 nmol/kg), but enhanced that of EKA/B (100 nmol/kg) (Jin et al., 2011).

MCRT (YPFPFRTic-NH2), a chimeric peptide based on morphiceptin and PFRTic-NH2, was designed and synthesized originally in our group (Li et al., 2013; Li et al., 2012). Morphiceptin is an analogue of EM-2, and PFRTic-NH2 is a derivative of neuropeptide FF (NPFF) (Chang et al., 1981; Tan et al., 1999). NPFF has been characterized as a modulator of opioid function mainly via its two receptors: NPFF1 and NPFF2 (Lake et al., 1991; Malin et al., 1990; Zajac, 2001). In our previous studies, MCRT decreased MAP significantly stronger than its parent peptides by intravenous (i.v.) or intracerebroventricular injection (Li et al., 2013). Mechanism studies indicated that besides opioid receptors and NO pathway, NPFF receptors could participate in the regulation (Li et al., 2013).

In this study, we attempted to co-inject EKA/B and MCRT to investigate their interactions on the depressor effects at peripheral level. SR140333B, an antagonist of the NK1 receptor, GR159897, an antagonist of the NK2 receptor, SR142801, an antagonist of the NK3 receptor, naloxone, an antagonist of the classical opioid receptor, and L-NAME, an inhibitor of the NO synthase were used in the studies to explore the mechanism. In general, our work may help to better understand the role of tachykinin receptors, opioid receptors and NPFF receptors in cardiovascular system.

2. Materials and methods

The experimental protocols used in the present study were approved by the Ethics Committee of Animal Experiments of Lanzhou University. Every effort was made to minimize the numbers and any suffering of the animals used in the following experiments.

2.1 Animals

Male Wistar rats (Animal Center of Lanzhou University, Lanzhou, China) weighted 200 – 300 g were used. They were housed in a room maintained at 22 – 23°C with an alternating 12 h light/dark cycle. Food and water were available ad libitum.

2.2 Peptides and other compounds

EKA/B and MCRT were synthesized in our laboratory by the solid-phase peptide synthesis and purified by preparative reverse-phase high-performance liquid chromatography (RP-HPLC), and further analyzed by analytical RP-HPLC and characterized by electrospray ionization mass spectrum (ESI-MS). SR140333B or (S)1-(2-(3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl)ethyl)-4phenyl-1-azoniabicyclo(2.2.2)octane chloride and SR142801 or (S)-(+)-N[10]-4-phenylpiperidin-4-yl-N-methylacetamide are generous gifts from Sanofi-Aventis (France). GR159897 or 5-Fluoro-3-(2-(4-methoxy-4-(((R)-phenylsulfinyl)methyl)-1-piperidinyl)ethyl)-1H-ind ole was purchased from Tocris Bioscience (Bristol, UK). Naloxone hydrochloride

dihydrate was purchased from Sigma (Shanghai, China). L-NAME or Nω-nitroL-arginine

methylester hydrochloride was purchased from Sigma-Aldrich (St. Louis,

MO, USA). EKA/B, MCRT, naloxone and L-NAME were dissolved in normal saline. SR140333B, GR159897 and SR142801 were dissolved in 30% dimethyl sulphoxide (DMSO), further diluted with normal saline. Control trials were performed in the presence of corresponding concentration of DMSO to rule out any possible nonspecific action of solvent on MAP. All compounds were stored at − 20◦C. The aliquots were thawed and used on the day of the experiment. During the entire experiment, the drug solutions were kept in crushed ice.

2.3 Surgical procedure

Rats were anesthetized with ethyl carbamate (1.5 g/kg) via intraperitoneal injection; supplemental doses of ethyl carbamate were given as needed to maintain a uniform level of anesthesia. The trachea was incised to facilitate spontaneous respiration as well as to get rid of mucus. A polyethylene catheter was inserted into the external jugular vein for i.v. injection of drugs. The depressor effects were measured from the right common carotid artery via an arterial cannula connected to a PT-100 pressure transducer (Chengdu TSL Technology Co., Ltd., Chengdu, China) and recorded on a BL-420F recorder system (Taimeng, Chengdu Technology & Market. Corp. Ltd., Chengdu, China). Both venous and arterial catheters were filled with saline and heparin in saline (0.2 g/ml) respectively.

2.4 Drug administration

At least 30 min after surgery, when the blood pressure was stable, drug administration was commenced. Each drug was injected by i.v. in a constant volume of 0.1 ml delivered in bolus within 30 s, and the venous catheter was flushed with an additional 0.1 ml saline. A time interval of 30 – 35 min was allowed between each injection for recovery of blood pressure to basal level. The basal value of blood pressure was range from 85 to 115 mmHg, while there was one exception that the value after L-NAME (50 mg/kg) injection was within the range from 140 to 170 mmHg. Each of EKA/B, MCRT and EKA/B + MCRT alone was conducted in a separate rat from lower to higher doses. EKA/B + MCRT (10 + 50 nmol/kg) were tested individually in independent rats as a control, and then we investigated the effect of each antagonist on the depressor effects induced by EKA/B + MCRT (10 + 50 nmol/kg) in each of the rats. The experimental design for EKA/B (10 nmol/kg) and MCRT (400 nmol/kg) was the same as that of EKA/B + MCRT (10 + 50 nmol/kg). In general, studies for each of the peptides alone and for each of the peptides plus antagonist were conducted in separate groups of rats. SR140333B (1 mg/kg), GR159897 (4 mg/kg), SR142801 (2.79 mg/kg), naloxone (2 mg/kg) and L-NAME (50 mg/kg) were injected through the jugular vein 10 min before the second drug administration.

2.5 Statistics

The hypotensive effects were calculated as the mmHg changes from the baseline and the duration was expressed as the half of the recovery time (time taken to attain 50% recovery of the depressor effect). All data were recorded as means ± standard error of

the mean of n experiments. When the variances were homogeneous among the groups, these were analyzed by a one-way analysis of variance (ANOVA) followed by the Dunnett’s test (two-sided) for post hoc comparisons on all time course studies. On the other hand, when the variances were heterogeneous among the groups, these were analyzed by ANOVA followed by Dunnett’s T3 test for post hoc comparisons for all time course studies. A value of P < 0.05 was selected as indicative of a significant difference.

3. Results

3.1 The effects of different doses of MCRT on the depressor effects induced by EKA/B

Our previous studies revealed that MCRT (i.v.) dose-dependently decreased MAP (Fig. 1A) (Li et al., 2013). The effects of EKA/B produced a U-shaped curve and the maximal effect was caused by 10 nmol/kg (Fig. 2A) (Jin et al., 2011). Thus, MCRT (50, 100, 200, 400 nmol/kg) were co-injected with EKA/B (10 nmol/kg) to study the effects of MCRT on the depressor effects induced by EKA/B. A typical record showing the depressor effect induced by EKA/B + MCRT (10 + 50 nmol/kg) could be seen in Fig. S1. The decrease in MAP induced by co-injection of EKA/B + MCRT at 10 + 50, 10 + 100, 10 + 200 and 10 + 400 nmol/kg were – 18.62 ± 1.33, – 18.05 ± 1.50, – 17.85 ± 1.30, and – 19.33 ± 1.50 mmHg respectively (Fig. 1A). Compared with EKA/B (10 nmol/kg), the decrease in MAP induced by co-injection of EKA/B + MCRT were attenuated slightly. However, there was no significant difference between each other. The half of the recovery time induced by co-injection of EKA/B + MCRT was similar to each other too (Fig. 1B). Differently, there was no significant difference compared with EKA/B (10 nmol/kg). Totally speaking, to some extent, MCRT (50, 100, 200, 400 nmol/kg) slightly inhibited the effects of EKA/B (10 nmol/kg) and the effects were similar (Fig. 1).

3.2 The effects of MCRT on the depressor effects induced by different doses of EKA/B

Since MCRT (50, 100, 200, 400 nmol/kg) had a similar inhibiting effect on EKA/B (10 nmol/kg), MCRT (50 nmol/kg) was chosen to be co-injected with EKA/B (1, 10, 100 nmol/kg) to further investigate the effects of MCRT on the depressor effects induced by EKA/B. When EKA/B (1 nmol/kg) was co-injected with MCRT (50 nmol/kg), the decrease in MAP was similar to that of EKA/B (1 nmol/kg, Fig. 2A). When EKA/B (10 nmol/kg) was co-injected with MCRT (50 nmol/kg), the decrease in MAP was slightly attenuated compared with that of EKA/B (10 nmol/kg, Fig. 2A). When EKA/B (100 nmol/kg) was co-injected with MCRT (50 nmol/kg), the decrease in MAP was strongly enhanced compared with that of EKA/B (100 nmol/kg, Fig. 2A). The half of the recovery time induced by co-injection of EKA/B + MCRT at 100 + 50 nmol/kg was significantly greater than the lower doses too (Fig. 2B). In a word, the effects of co-injection of EKA/B (1, 10, 100 nmol/kg) and MCRT (50 nmol/kg) showed a particular curvilinear trend (Fig. 2).

3.3 Effects of SR140333B, GR159897, SR142801, naloxone and L-NAME on the depressor effects induced by EKA/B, MCRT and EKA/B + MCRT

SR140333B for NK1 receptor, GR159897 for NK2 receptor, and SR142801 for NK3 receptor were dissolved in 30% DMSO and administrated 10 min before the injection of EKA/B (10 nmol/kg), MCRT (400 nmol/kg), and EKA/B + MCRT (10 + 50 nmol/kg). Compared with 30% DMSO, MAP was decreased slightly after pretreatment with these antagonists. As seen in Fig. 3, SR140333B fully blocked the depressor effects of EKA/B and EKA/B + MCRT, but only partially abolished the

depressor effect of MCRT. GR159897 was found to partially abolish the depressor effects of EKA/B and MCRT, but had no significant influence on that of EKA/B + MCRT (Fig. 3). SR142801 was found to partially abolish the depressor effects of EKA/B and MCRT too. Interestingly, SR142801 completely antagonized the depressor effect of EKA/B + MCRT, indicating that SR142801 potentiated the restraining level of MCRT on EKA/B (10 nmol/kg, Fig. 3).

Naloxone, the classical opioid receptors antagonist and L-NAME, the NO synthase inhibitor, were also injected 10 min before the peptides. Naloxone was not found to alter MAP. As shown in Fig. 4, naloxone fully blocked the depressor effect of MCRT, and partially inhibited that of EKA/B. However, there was no significant difference between naloxone + EKA/B + MCRT and EKA/B + MCRT (Fig. 4). L-NAME significantly increased MAP by 52.17 ± 1.97 mmHg. It was found to partially abolish the depressor effects of EKA/B, MCRT, and EKA/B + MCRT (Fig. 4).

4. Discussion

The co-existence of SP and endomorphins as well as the co-localization of mu opioid receptor and NK1 receptor indicated that tachykinins might interact with opioid peptides closely (Aicher et al., 2000; Greenwell et al., 2007; Luo et al., 2014; Wu et al., 2015). However, the details of the interactions between tachykinins and opioid peptides remain unclear. EKA/B (a NK1 agonist) and MCRT (a mu/delta opioid and NPFF receptors agonist) were reported to decrease the blood pressure at peripheral level (Abdelrahman et al., 2005; Jin et al., 2011; Li et al., 2013). And also, the close and complicated coactions of EKA/B and MCRT on pain regulation have been reported newly (He et al., 2016). Thus, we co-injected EKA/B and MCRT to study their interactions and regulatory mechanisms in cardiovascular system.

In the present study, MCRT dose-dependently decreased MAP as we reported before (Li et al., 2013). Besides opioid receptors and NO pathway, SR140333B, GR159897 and SR142801 were found to partially antagonize the depressor effect of MCRT, indicating that the depressor effect was partially mediated through NK1, NK2 and NK3 receptors.

Consistent with our previous work, the dose-response curve of EKA/B was a reverse parabola that was mediated by tachykinin receptors, opioid receptors, and NO pathway (Jin et al., 2011). The reverse parabola was also observed in the depressor effects of human hemokinin-1 (hHK-1) but not hHK-1(4-11) (Kong et al., 2008). EKA/B and hHK-1(4-11) are the N-terminally truncated peptides of hHK-1,

indicating that the depressor effect of EKA/B diminished at high dose is mainly due to the N-terminal region of EKA/B (Kong et al., 2008).

When co-injected EKA/B and MCRT, our results showed that the dose dependent effects of MCRT were abolished by EKA/B (10 nmol/kg), suggesting that EKA/B obscures the depressor effects of MCRT. On the other hand, different doses of MCRT range from 50 to 400 nmol/kg attenuated the depressor effects of EKA/B (10 nmol/kg) and the degree of inhibition was similar, suggesting that MCRT may play a regulatory role related to EKA/B on blood pressure and the effective concentration is no more than 50 nmol/kg. Subsequently, we found that naloxone did not block the depressor effects of EKA/B + MCRT, while it completely blocked the hypotensive effect of MCRT. It means that EKA/B obscuring the depressor effects of MCRT is due to the nonparticipation of opioid receptors. L-NAME slightly inhibited the depressor effect of EKA/B + MCRT, meaning that the NO pathway participates in the effect. These results are coincident with the cardiovascular study of the co-injection of EKA/B and EM-1 that was mediated partially by NO pathway, but not by opioid receptors (Jin et al., 2011). Furthermore, blocking the tachykinin receptors could inhibit the depressor effects of EKA/B + EM-1 effectively and the restraining levels were NK1 receptor > NK2 receptor > NK3 receptor (Jin et al., 2011). Differently, SR140333B fully abolished the effect of EKA/B + MCRT, while GR159897 had little influence on that. SR142801 completely blocked the effect of EKA/B + MCRT indicating that SR142801 potentiated the restraining level of MCRT on EKA/B (10 nmol/kg). The phenomenon may be due to the difference of MCRT and EM-1.

Moreover, we found that MCRT (50 nmol/kg) had little influence on the depressor effect of EKA/B (1 nmol/kg), but strongly potentiated that of EKA/B (100 nmol/kg). The results were similar to the changes in MAP induced by co-injection of EKA/B + EM-1 (Jin et al., 2011). However, EM-1 (30 nmol/kg) attenuated the half recovery time of EKA/B (100 nmol/kg), while MCRT (50 nmol/kg) potentiated that (Jin et al., 2011). The conflict could also be ascribed to the difference of MCRT and EM-1. MCRT is a chimeric peptide composed of morphiceptin and PFRTic-NH2 through sharing of one proline (Li et al., 2013; Li et al., 2012). Morphiceptin is an EM-2 analogue substituted by Pro4 (Chang et al., 1981). Although morphiceptin, EM-1 and EM-2 showed high affinity with mu opioid receptors, they functioned as selective endogenous mu opioid receptor ligands (Gao et al., 2006; Goldberg et al., 1998; Zadina et al., 1997; Stone et al., 1997). In addition, EM-1 showed weak but significant affinity with NK1 and NK2 receptors, while EM-2 presented affinity only for NK2 receptor (Kosson et al., 2005). Thus, we presumed that the exact cause behind the difference might be the different affinities of MCRT and EM-1 for tachykinin receptors. However, in the present study, SR140333B, GR159897, and SR142801 inhibited the depressor effect of MCRT similar to the former study, in which blocking of the three tachykinin receptors effectively inhibited the depressor effect of EM-1 (Jin et al., 2011). On the other hand, as PFRTic-NH2 derived from NPFF dose-dependently decreased the MAP, MCRT may interact with NPFF receptors (Huang et al., 2000; Li et al., 2013; Tan et al., 1999). In addition, NPFF significantly reduced the analgesic effect of EM-1 via NPFF2 receptor, but enhanced

that of EM-2 via both NPFF1 and NPFF2 receptors (Wang et al., 2014). Based on these results, we presumed that the difference of MCRT and EM-1 may be resulted from the presence or absence of NPFF receptors’ agonists. Further studies should be carried out in the future.

In summary, the interactions between EKA/B and MCRT on the depressor effects at peripheral level were investigated in this study. MCRT presented a unique regulatory effect on the depressor effects induced by EKA/B. It had little influence on the depressor effect of EKA/B (1 nmol/kg), slightly inhibited that of EKA/B (10 nmol/kg), and strongly potentiated that of EKA/B (100 nmol/kg). The depressor effect induced by EKA/B + MCRT was mainly regulated by NK1 and NK3 receptors, partially by the NO pathway, and possibly by NPFF receptors. A graphical representation of the receptors/peptides interaction was presented in Fig. 2S. Co-injection is widely studied in practice. These findings may pave the way for a new strategy about investigating the interaction between various vasodilators.

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities (lzujbky-2013-154) and partially supported by the grant from the foundation of Key Laboratory for Gastrointestinal Diseases of Gansu Province (gswcky-2012-006). The authors thank Sanofi-Aventis for the kind gift of SR140333B and SR142801.

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Fig. 1. (A) Decreases in MAP induced by i.v. injection of MCRT (50, 100, 200, 400 nmol/kg), EKA/B (10 nmol/kg) and co-injection of MCRT (50, 100, 200, 400 nmol/kg) and EKA/B (10 nmol/kg) in rats; (B) the half of the recovery time. N = 6 – 9; ### *

P < 0.001, statistically significant difference between MCRT + EKA/B and MCRT;

P < 0.05, statistically significant difference between MCRT + EKA/B and EKA/B;

ΔΔ

P < 0.01, statistically significant difference between drugs and the previous

concentration of drugs. Fig. 2. (A) Decreases in MAP induced by i.v. injection of MCRT (50 nmol/kg), EKA/B (1, 10, 100 nmol/kg) and co-injection of MCRT (50 nmol/kg) and EKA/B (1, 10, 100 nmol/kg) in rats; (B) the half of the recovery time. N = 6 – 9; ### P < 0.001, statistically significant difference between MCRT + EKA/B and MCRT; * P < 0.05, statistically significant difference between MCRT + EKA/B and EKA/B; Δ P < 0.05, ΔΔ

P < 0.01, statistically significant difference between drugs and the previous

concentration of drugs. Fig. 3. Effects of SR140333B, GR159897, SR142801 on the regulation of MAP induced by i.v. injection of MCRT (400 nmol/kg), EKA/B (10 nmol/kg) and

co-injection of EKA/B + MCRT (10 + 50 nmol/kg) in anesthetized rats. N = 6 – 9; ## P < 0.01, ### P < 0.001, statistically significant difference between drugs and MCRT (400 nmol/kg); *** P < 0.001, statistically significant difference between drugs and EKA/B + MCRT (10 + 50 nmol/kg); Δ P < 0.05, ΔΔΔ P < 0.01, ΔΔΔ P < 0.001, statistically significant difference between drugs and EKA/B (10 nmol/kg);

＆＆＆

P<

0.001, statistically significant difference between drugs and EKA/B with the pretreatment of antagonists. Fig. 4. Effects of L-NAME on the regulation of MAP induced by i.v. injection of MCRT (400 nmol/kg), EKA/B (10 nmol/kg) and co-injection of EKA/B + MCRT (10 + 50 nmol/kg) in anesthetized rats. N = 6 – 9; ## P < 0.01, ### P < 0.001, statistically significant difference between drugs and MCRT (400 nmol/kg); *** P < 0.001, statistically significant difference between drugs and EKA/B + MCRT (10 + 50 nmol/kg); Δ P < 0.05, ΔΔΔ P < 0.01, ΔΔΔ P < 0.001, statistically significant difference between drugs and EKA/B (10 nmol/kg).