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The cardiovascular effects of a chimeric opioid peptide based on morphiceptin and PFRTic-NH2
Peptides 39 (2013) 89–94
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The cardiovascular effects of a chimeric opioid peptide based on morphiceptin and PFRTic-NH2 Meixing Li a,1 , Lanxia Zhou b,1 , Guoning Ma a , Shuo Cao a , Shouliang Dong a,c,∗ a b c
Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China The Core Laboratory of the First Affiliated Hospital, Lanzhou University, 1 Donggang West Road, Lanzhou 730000, China Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China
a r t i c l e
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Article history: Received 22 September 2012 Received in revised form 30 October 2012 Accepted 30 October 2012 Available online 14 November 2012 Keywords: Mean arterial pressure Heart rate Opioid receptors Nitric oxide pathway
a b s t r a c t MCRT (YPFPFRTic-NH2 ) is a chimeric opioid peptide based on morphiceptin and PFRTic-NH2 . In order to assess the cardiovascular effect of MCRT, it was administered by intravenous (i.v.) injection targeting at the peripheral nervous system and by intracerebroventricular (i.c.v.) injection targeting at the central nervous system. Naloxone and l-NAME were injected before MCRT to investigate possible interactions with MCRT. Results show that administration of MCRT by i.v. or i.c.v. injection could induce bradycardia and decrease in mean arterial pressure (MAP) at a greater degree than that with morphiceptin and PFRTic-NH2 . When MCRT and NPFF were coinjected, we observed a dose-dependent weakening of these cardiovascular effects by MCRT. Because naloxone completely abolished the cardiovascular effects of MCRT, we conclude that opioid receptors are involved in regulating the MAP of MCRT regardless of modes of injection. The effect of MCRT on heart rate is completely dependent on opioid receptors when MCRT was administered by i.c.v. instead of i.v. The central nitric oxide (NO) pathway is involved in regulating blood pressure by MCRT under both modes of injection, but the peripheral NO pathway had no effect on lowering blood pressure mediated by MCRT when it was administered by i.c.v. Based on the results from different modes of injection, the regulation of heart rate by MCRT mainly involves in the central NO pathway. Lastly, we observed that the cardiovascular effects of MCRT such as bradycardia and decrease of blood pressure, were stronger than that of its parent peptides. Opioid receptors and the NO pathway are involved in the cardiovascular regulation by MCRT, and their degree of involvement differs between intravenous and intracerebroventricular injection. © 2012 Elsevier Inc. All rights reserved.
1. Introduction MCRT (YPFPFRTic-NH2 ) is a chimeric peptide composed of a Pro4 substituted morphiceptin (YPFP-NH2 ), an endomorphin II (END II) analogue, and a neuropeptide FF (NPFF) derivative (PFRTicNH2 ) through sharing of one proline. Similar to other mixed /␦ agonists peptides [3], MCRT, with its mixed mu opioid receptor (MOR) and delta opioid receptor (DOR) agonist activity, produces a very impressive analgesic effect with a low effective dose (ED50 ) of 1.47 nmol/kg. MCRT’s analgesia property is significantly more than that of morphiceptin and PFRTic-NH2 . Therefore, its analgesic mechanism is still via the classical opioid system involving both MOR and DOR [14].
∗ Corresponding author at: Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China. Tel.: +86 931 8912428; fax: +86 931 8912428. E-mail address: [email protected] (S. Dong). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.10.014
Considerable evidence suggests that opioids and neuropeptides play an important role in regulating cardiovascular function [2,9,12,16,20–22]. First, opioid receptors distributed in the central and peripheral nervous system are involved in the regulation of cardiovascular activity. In addition, opioid receptor agonists regulate mean arterial pressure (MAP) and heart rate. Moreover, NPFF receptors distributed in the central nervous system and heart and its analogues have potent cardiovascular effects. Based on autoradiography studies, NPFF receptors are distinct from all types of opioid receptors and located in the rat cardiac tissue [1,2]. Although the role of NPFF in cardiovascular regulation is still unclear, assessment of cardiovascular parameters could be useful for the functional characterization of NPFF analogues. Indeed, both parent peptides (morphiceptin and PFRTic-NH2 ) induce hypotension and bradycardia in anesthetized rats [11,15,25]. Morphiceptin (1–50 nmol/kg, intravenous (i.v.) or intracerebroventricular (i.c.v.)) produces a dose-related hypotensive response and bradycardia in normotensive male Wistar Albino Glaxo rats [24], but morphiceptin (100–320 nmol/kg) microinjected into the region of the nucleus of tractus solitarius (NTS) in anesthetized cats has no effect on MAP
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[10]. PFRTic-NH2 was initially considered to be an antagonist of NPFF because of its depressor response, but recently, some studies show that it is a super agonist of NPFF receptor 1 and NPFF receptor 2 [8]. However, its ability to lower MAP is dose-dependent and significant at various doses (0.2–0.9 mg) of PFRTic-NH2 [11]. In the present study, we examined the effects of MCRT injected by i.v. and i.c.v. on the MAP and heart rate (HR) of anesthetized rats. We also examined the effects of morphiceptin and PFRTicNH2 injected by i.v. on the MAP and HR and then compared the measurements with that of MCRT. Multiple dosages of NPFF and MCRT were administered jointly to investigate whether there is an interaction between MCRT and NPFF receptors on the observed cardiovascular effects. Naloxone and l-NAME were injected prior to MCRT to determine the mechanism underlying the cardiovascular effects of MCRT and its related peptides. 2. Materials and methods 2.1. Chemicals MCRT, morphiceptin, PFRTic-NH2 and NPFF 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 RPHPLC and characterized by ESI-MS. l-NAME (N -nitro-l-arginine methylester hydrochloride, a nitric oxide synthase inhibitor) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Naloxone hydrochloride dihydrate (a non-specific antagonist of opioid receptors, Fluka, Beijing, China), naltrindole hydrochloride (a specific antagonist of DOR, Tocris Bioscience, Bristol, UK) and cyprodime hydrochloride (a specific antagonist of MOR, Tocris Bioscience, Bristol, UK) were dissolved in normal saline (NS). All compounds were dissolved in sterile saline and 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. 2.2. Animals Male Wistar rats weighed 200–300 g (Animal Center of Lanzhou University, Lanzhou, China) were used. Animals were housed in a temperature-controlled environment (18–22 ◦ C) under standard 12 h light/dark cycle and received food and water ad libitum. All experiments were carried out in accordance with the Principles of Laboratory Animal Care and Guidelines of the Ethics Committee of Lanzhou University. 2.3. Surgical procedure Rats were anesthetized by intraperitoneal administration of ethyl carbamate (1.5 g/kg). Supplemental doses of ethyl carbamate were given as needed to maintain a uniform level of anesthesia. To insert an indwelling cannula into the lateral cerebral ventricle, a small hole was drilled through the skull at 1.0 mm caudal and 1.5 mm lateral from bregma for inserting a guide cannula. The guide cannula was set to a depth of 4.0 mm from the top of the skull, and accurate placement into the ventricle was verified by observing the outflow of the cerebrospinal fluid induced by withdrawing the cannula on a microsyringe. A second small hole was drilled 4–5 mm caudal from the guide cannula for inserting an anchor screw. A small amount of dental cement was applied to the screw and the guide cannula to hold it in the certain place and make sure it gets hardened before closure of the wound and recovery from anesthesia. A polyethylene catheter was inserted into the external jugular vein for i.v. administration of drugs. The trachea was incised to facilitate spontaneous respiration as well as to get rid of mucus. The BP and HR was 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 BL310 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. Each rat was used only once in one experiment. After surgery, animals were allowed to remain stable for at least 30 min, and then the experiment was started. 2.4. Drug administration MCRT, morphiceptin, NPFF and PFRTic-NH2 of various concentrations were dissolved in isotonic saline. 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 of saline, then they were injected by i.c.v. in a constant volume of 0.01 ml within 1 min. A time interval of 30–35 min was allowed between each injection for recovery of blood pressure to basal level. Separate experiments were carried out for HR. Only a single dose of the peptides was used to examine its effect on HR. Those doses have been shown previously to be sufficient to produce significant effects on BP. Naloxone (2 mg/kg), naltrindole (3 mg/kg), cyprodime (3 mg/kg) and l-NAME (30 mg/kg) were injected through the jugular vein 10 min before the second drug administration. 2.5. Physiological measurements With the polyethylene cannula inserted into the rat carotid artery, MAP and HR were measured with a pressure transducer (PT-100) connected to BL-310 and directly recorded the parameters throughout the experiments. Once the blood pressure (BP, 85–115 mmHg) and HR (280–400 beats/min) get stable, the injection of the test peptide was performed. Change of pressure (mmHg; maximum BP minus basal BP) was used to express the effect of the injection. In HR experiments, the HR was counted from the injection of drug until the BP returned to the basal rate (the duration of drug effect). The same period before the drug administration was sampled to establish the basal value of HR. The change of HR was then obtained and converted to the percentage of the basal. 2.6. Statistics Student’s t-tests were used to evaluate the significance of the change of BP and HR. The drug-induced increase or decrease of BP (mmHg) and HR (%) was compared with the basal values by using an unpaired Student’s t-test (one side) and one-way ANOVA. In all cases, a P value <0.05 was considered statistically significant. Area under the curve (AUC) data was calculated for the interval of time in which drug administered by i.v. or i.c.v. induced MAP change. The results shown are means ± S.E.M. 3. Results The decrease in MAP in response to MCRT (50, 100, 200, 400 nmol/kg, i.v.) in anesthetized rats are shown in Fig. 1A. MCRT administered by i.c.v. or i.v. dose-dependently lowered the MAP significantly when the MCRT’s doses ranged from 0.25 to 250 nmol/kg (P < 0.01, vs saline, Fig. 1B). Based on these arterial blood pressure data, we selected 400 nmol/kg MCRT (i.v.) and 250 nmol/kg MCRT (i.c.v.) for further comparison, as shown in Fig. 1C and D. There was no significant difference (P > 0.05) between the peak of the decrease in MAP induced by 400 nmol/kg (i.v.) and 250 nmol/kg MCRT (i.c.v.); the peak at the dose of 400 nmol/kg was −18.96 ± 1.90 mmHg and appeared at 0.5 min while the peak at of 250 nmol/kg was −23.80 ± 2.48 mmHg and appeared at 4 min. Intravenous infusion of 400 nmol/kg MCRT resulted in a decrease in MAP that occurred
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Fig. 1. (A and B) Dose-dependent decrease in the MAP effects induced by MCRT (50–400 nmol/kg, i.v. or 0.25–250 nmol/kg, i.c.v.) in rats. Control group, n = 8; other groups, n = 12. ***P < 0.001, statistically significant difference between MCRT and saline. (C) Decrease in the MAP effects induced by MCRT (250 nmol/kg, i.c.v., 400 nmol/kg, i.v.) at each time point in rats. (D) Change in MAP is represented as AUC, bars indicate the means ± S.E.M. ***P < 0.001, statistically significant difference between MCRT (250 nmol/kg, i.c.v.) and MCRT (400 nmol/kg, i.v.).
within 4 min while i.c.v. infusion of 250 nmol/kg MCRT resulted in a decrease in MAP that was sustained for approximately 23 min before returning to normal levels. Injections of MCRT, morphiceptin and PFRTic-NH2 all produced a dose-related decrease in MAP (Fig. 2). The decrease in MAP
Fig. 2. Dose-dependent decrease in the MAP effects induced by MCRT, PFRTicamide, morphiceptin and morphiceptin + PFRTic-amide (50, 100, 200, 400 nmol/kg, i.v.) at the nanomolar level in rats. 8 rats were used in each group; *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference between morphiceptin, PFRTic-amide and MCRT, ## P < 0.01, ### P < 0.001, statistically significant difference between MCRT and saline.
induced by MCRT was significantly more potent than that induced by morphiceptin and PFRTic-NH2 (P < 0.01), with that by PFRTicNH2 less potent than that by morphiceptin. As illustrated in Fig. 3, blockade of MOR, opioid receptor (KOR) and DOR with an intravenous dose of 2 mg/kg of naloxone, 3 mg/kg of naltrindole, 3 mg/kg of cyprodime and 30 mg/kg l-NAME were injected prior to MCRT to determine the mechanism of the MAPlowering effects of MCRT. The use of 50 mg/kg l-NAME resulted in the same outcome as that of 30 mg/kg. Fig. 3A shows that pretreatment of rats with naloxone almost abolished the hypotensive effects of MCRT (i.v.) at all doses. Naloxone by itself caused little or no change in the BP of anesthetized rats. Consistent with a previous finding [19], i.v. administration of l-NAME at a dose of 30 mg/kg evoked an increase in MAP (Fig. 3D), and consequently, the decrease in MAP in response to MCRT was partially but significantly reduced. Fig. 3B shows that pretreatment of rats with naloxone completely abolished the hypotensive effects of MCRT (i.c.v.) at all doses, and after administration of 30 mg/kg l-NAME (i.v.), the decrease in arterial blood pressure in response to MCRT by i.c.v. injection was significantly abolished. However, 30 mg/kg lNAME administered by i.c.v. injection did not significantly affect the hypotensive effect of MCRT (i.c.v.). Naltrindole and cyprodime were administered with MCRT (i.v.) to confirm whether ␦ and receptors are involved (Fig. 3C). Naltrindole could significantly antagonize the decrease in MAP induced by i.v. MCRT, but cyprodime had no or little effect. Naltrindole or cyprodime administered alone could evoke decrease in MAP (Fig. 3E). Administration of NPFF into the lateral ventricles resulted in a dose-dependent elevation of mean arterial blood pressure (MAP). Intracerebroventricular injection of 25 nmol/kg NPFF did
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Fig. 3. (A and B) Effects of l-NAME (30 mg/kg) and naloxone (2 mg/kg) on the regulation of MAP induced by i.v. or i.c.v. administration of MCRT in anesthetized rats. (C) Effects of naltrindole (3 mg/kg) and cyprodime (3 mg/kg) on the regulation of MAP induced by i.v. administration of MCRT in anesthetized rats. (D) Effects of l-NAME (30 nmol/kg, i.v. or i.c.v.) on MAP in anesthetized rats. (E) Effects of cyprodime (3 mg/kg, i.v.) and naltrindole (3 mg/kg, i.v.) on MAP in anesthetized rats. 8 rats were used in each group; *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference between MCRT + l-NAME, MCRT + naloxone and MCRT, # P < 0.05, ## P < 0.01, ### P < 0.001, statistically significant difference between MCRT and saline.
not significantly change MAP compared to control animals receiving saline (P > 0.6, data not shown). In rats receiving 50, 100, 200, 250 or 400 nmol/kg NPFF, there was a significant increase in MAP compared to control (P < 0.05). There was no significant increase of MAP with administration of 250 nmol/kg or 400 nmol/kg NPFF (P > 0.2). NPFF (50, 100, 200, 250, 320 nmol/kg) could dose-dependently inhibit the decrease in MAP induced by 250 nmol/kg MCRT (Fig. 4) and could completely block this decrease by NPFF at the doses of 250 nmol/kg and 320 nmol/kg. Administration of 50 nmol/kg NPFF did not alter the effect of 250 nmol/kg MCRT on MAP (P > 0.05). After co-injection of MCRT and different concentrations of NPFF, the decrease in MAP due to MCRT was blocked and could not be reversed even with NPFF at 400 nmol/kg.
MCRT, MCRT + naloxone, MCRT + l-NAME, or morphiceptin and PFRTic-NH2 were i.v. injected to explore whether there are differences in the effects of MCRT and parent peptides on HR (Fig. 5). The most effective doses were chosen based on the BP data. Our data show that MCRT could induce bradycardia both by i.v. or i.c.v. injection, but the effect of MCRT at 250 nmol/kg (i.c.v.) on HR was more effective than MCRT at 400 nmol/kg (i.v.). One of the parent peptides PFRTic-NH2 could lower HR to the same degree as MCRT, whereas morphiceptin did not significantly change HR. In the following experiments, naloxone and l-NAME were injected prior to MCRT for further investigation of the possible mechanisms involved. l-NAME (20 mg/kg) did not change the effects of MCRT (administered by i.v. or i.c.v. injection) on HR. It is surprising that naloxone (2 mg/kg, i.v.) significantly antagonized the bradycardia
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Fig. 4. Effects of i.c.v. administered MCRT + NPFF (50, 100, 200, 250, 400 nmol/kg) on the regulation of MAP in anesthetized rats. 250 nmol/kg MCRT was used. 8 rats were used in each group; *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference between drugs group and saline.
caused by i.c.v. injected MCRT (P < 0.001), but had no influence on bradycardia induced by i.v. injected MCRT (P > 0.05). 4. Discussion MCRT was initially synthesized to obtain high analgesic activity through interactions between the opioid system and neuropeptide system, and both opioids and neuropeptides could cause (arouse) some changes in the cardiovascular system. As expected, MCRT, with its enhanced analgesic activity [14], could result in a dose-response MAP decrease when administered at various concentrations by i.v. and i.c.v. injection. The same doses of morphiceptin and PFRTic-NH2 could also lower MAP by i.v. injection but at a lesser degree. Thus, MCRT administered by i.v. induces a more effective cardiovascular response than its parent peptides at the same dose. This may be because the cardiovascular system mainly has DOR and KOR, and MCRT’s affinity to MOR is similar to that of morphiceptin, but its affinity to DOR is more higher [14]. Thus, activating DOR in the periphery could decrease myocardial contractility. In addition, as PFR(Tic)amide decreases the BP in a dose-dependent manner with an estimated ID50 value
Fig. 5. The effects of MCRT, morphiceptin, PFRTic-NH2 on the heart rate and effects of naloxone, naltrindole, cyprodime and l-NAME on the regulation of HR induced by i.v. administration of MCRT (400 nmol/kg) and i.c.v. administration of MCRT (250 nmol/kg) in rats. Naloxone (i.v.) and l-NAME (i.v. or i.c.v.) were injected 10 min before administration of MCRT. 8 rats were used in each group; **P < 0.01, statistically significant difference between drugs group and saline. ## P < 0.01, ### P < 0.001, statistically significant difference between (antagonist + MCRT) and MCRT.
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of 909.8 nmol (0.525 mg) [11], MCRT may interact with NPFF receptors. Another noticeable property of MCRT was its prolonged effect when administered by i.c.v. injection compared to that by i.v. injection. This could be partially explained by its improved metabolic stability observed in our previous study. Whether administered by i.v. or i.c.v. injection, MCRT caused a dose-dependent fall in blood pressure. However, the peak and duration varied widely after i.v. and i.c.v. injection, with a sustained effect observed only with i.c.v. injection. Our results indicate that the downstream mechanisms and pathways differ between the two injection modes. We used naloxone (i.v.), naltrindole (i.v.), cyprodime (i.v.) and lNAME (i.v. or i.c.v.) to observe the interactions between MCRT and an antagonist of opiate receptor, and between MCRT and a nitric oxide synthase inhibitor. NO in the central nervous system has been recognized as a novel regulatory messenger with important physiological functions in many organs. It is released in varying amounts in several parts of the brain where its many regulatory functions are under active investigation [5]. l-NAME (30 mg and 300 mg i.c.v.) induces a dose-dependent increase in mean arterial pressure and heart rate in anesthetized normotensive rats, and i.c.v. injection of l-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo [4,17]. Pretreatment with naloxone nearly abolished the cardiovascular effects of MCRT, indicating that opioid receptors are involved. Naltrindole could significantly antagonize the change in MAP induced by MCRT (i.v.) whereas cyprodime had no or little effect. This indicates that DOR is mainly involved in the MAP regulation of the peripheral system. However, l-NAME could only antagonize the cardiovascular effect of MCRT (i.v.) to a certain degree, and had no function on the effect of MCRT (i.c.v). We all know that l-NAME, which acts as a competitive inhibitor of NOS due to its structural similarity to arginine, the substrate of NOS, may act as such an agent [6]. Thus l-NAME could competitively inhibit endothelium-dependent vasodilation, as a result of the inhibition of NO releasing from vascular endothelial cells, that a single injection of l-NAME could cause an increase in blood pressure. This indicates that the endothelial NO pathway is involved in the response after i.v. injection of MCRT, while i.c.v. injection of MCRT might be unrelated to NO release in the vascular endothelium. The results from co-injection of l-NAME (i.c.v.) with MCRT (i.c.v.) illustrate that lNAME (i.c.v.) could completely abolish the cardiovascular effect of MCRT, that is MCRT (i.c.v.) plays a cardiovascular effect mainly via the central NO pathway. The cardiovascular response to NPFF (i.c.v.) observed here is consistent with previously published data [13]. We observed a dose-dependent increase in BP. Quantitative differences in magnitude and duration might be related to the different strains of rats used in these experiments. In a previous experiment of analgesia, antagonistic interactions between MCRT and NPFF might be based on their different effects on both receptors. To know whether an interaction between NPFF and MCRT plays a role in the cardiovascular effect of MCRT, we co-administered MCRT (250 nmol/kg) and different concentrations of NPFF in the ventricle. NPFF could dose-dependently antagonize the blood pressure effect of MCRT, but when the concentration of NPFF is greater than or equal to 250 nmol/kg, NPFF could completely antagonize but could not reverse the MCRT’s antihypertensive effect. The mutual antagonistic effect of NPFF and MCRT on blood pressure may be due to the NPFF receptor-mediated regulation of MCRT. MCRT contains PFRTic-amide, which is an NPFF receptor antagonist in the brain. Interactions exist between NPFF and opioids receptors, and the regulation of opioid analgesia by activation of receptors could increase NPFF receptors. These results indicate that besides the activation of opioid receptors, other mechanisms might be involved. It is also possible that differences between central and peripheral opioid receptors contribute to the divergence. When NPFF concentration
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was more than that of MCRT, the combined effect still does not raise blood pressure, indicating that their receptor binding capacity is not the same, and their interaction does not simply cancel each other out, and thus, a complex mechanism exists. Based on previous studies, the effects of drugs, especially opioids, to blood pressure and heart rate is almost synergistic [7,18,23], and that changes in the direction and magnitude of blood pressure and heart rate are relatively consistent. MCRT, administered either by i.v. or i.c.v. injection, slows the heart rate. However, the two concentrations of MCRT resulted in the same decrease in MAP, but reduced the heart rate to a higher degree when injected by i.c.v. than by i.v. Intriguingly, pre-injection of naloxone antagonized the effect of MCRT injected through i.c.v. on heart rate. Different concentrations of MCRT had no impact on heart rate. Injection of l-NAME (i.v.) had no influence in the decrease of heart rate induced by MCRT. Besides, the lowering of BP induced by MCRT was significantly reduced by l-NAME, indicating that NO pathway participates in BP regulation. We also found that the same concentration of MCRT and PFRTic-NH2 administered by i.v. injection regulated heart rate similarly, while the same concentration of morphiceptin by i.v. injection had little effect on heart rate. Naloxone did not inhibit the effect of MCRT on heart rate after i.v. injection, that the heart rate effect of MCRT administered by i.v. injection was similar to that with PFRTic-NH2 , as reported in the previous literature [11]. We demonstrate that MCRT could induce bradycardia and decrease in the MAP to a higher degree than that with morphiceptin and PFRTic-amide. NPFF could dose-dependently weaken the cardiovascular effects of MCRT. Opioid receptors seem to be involved in the MAP and heart rate regulation by i.v. or i.c.v. injected MCRT. However, the effect of MCRT by peripheral injection on the heart rate had no relationship with opioid receptors. The NO pathway is involved in the MCRT-mediated regulation of blood pressure under both modes of injection, but the peripheral NO pathway had no effect on the blood pressure changes induced by MCRT administered by i.c.v. injection. The peripheral NO pathway is not involved in the heart rate effect induced by MCRT, but the central NO pathway is involved in the regulation of the heart rate by MCRT administered by central injection. Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities and partially supported by the grant from the National Natural Science Foundation of China (No. 30870526). References [1] Allard M, Geoffre S, Legendre P, Vincent JD, Simonnet G. Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Res 1989;500:169–76. [2] Allard M, Labrouche S, Nosjean A, Laguzzi R. Mechanisms underlying the cardiovascular responses to peripheral administration of NPFF in the rat. J Pharmacol Exp Ther 1995;274:577–83.
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Peptides journal homepage: www.elsevier.com/locate/peptides
The cardiovascular effects of a chimeric opioid peptide based on morphiceptin and PFRTic-NH2 Meixing Li a,1 , Lanxia Zhou b,1 , Guoning Ma a , Shuo Cao a , Shouliang Dong a,c,∗ a b c
Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China The Core Laboratory of the First Affiliated Hospital, Lanzhou University, 1 Donggang West Road, Lanzhou 730000, China Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 22 September 2012 Received in revised form 30 October 2012 Accepted 30 October 2012 Available online 14 November 2012 Keywords: Mean arterial pressure Heart rate Opioid receptors Nitric oxide pathway
a b s t r a c t MCRT (YPFPFRTic-NH2 ) is a chimeric opioid peptide based on morphiceptin and PFRTic-NH2 . In order to assess the cardiovascular effect of MCRT, it was administered by intravenous (i.v.) injection targeting at the peripheral nervous system and by intracerebroventricular (i.c.v.) injection targeting at the central nervous system. Naloxone and l-NAME were injected before MCRT to investigate possible interactions with MCRT. Results show that administration of MCRT by i.v. or i.c.v. injection could induce bradycardia and decrease in mean arterial pressure (MAP) at a greater degree than that with morphiceptin and PFRTic-NH2 . When MCRT and NPFF were coinjected, we observed a dose-dependent weakening of these cardiovascular effects by MCRT. Because naloxone completely abolished the cardiovascular effects of MCRT, we conclude that opioid receptors are involved in regulating the MAP of MCRT regardless of modes of injection. The effect of MCRT on heart rate is completely dependent on opioid receptors when MCRT was administered by i.c.v. instead of i.v. The central nitric oxide (NO) pathway is involved in regulating blood pressure by MCRT under both modes of injection, but the peripheral NO pathway had no effect on lowering blood pressure mediated by MCRT when it was administered by i.c.v. Based on the results from different modes of injection, the regulation of heart rate by MCRT mainly involves in the central NO pathway. Lastly, we observed that the cardiovascular effects of MCRT such as bradycardia and decrease of blood pressure, were stronger than that of its parent peptides. Opioid receptors and the NO pathway are involved in the cardiovascular regulation by MCRT, and their degree of involvement differs between intravenous and intracerebroventricular injection. © 2012 Elsevier Inc. All rights reserved.
1. Introduction MCRT (YPFPFRTic-NH2 ) is a chimeric peptide composed of a Pro4 substituted morphiceptin (YPFP-NH2 ), an endomorphin II (END II) analogue, and a neuropeptide FF (NPFF) derivative (PFRTicNH2 ) through sharing of one proline. Similar to other mixed /␦ agonists peptides [3], MCRT, with its mixed mu opioid receptor (MOR) and delta opioid receptor (DOR) agonist activity, produces a very impressive analgesic effect with a low effective dose (ED50 ) of 1.47 nmol/kg. MCRT’s analgesia property is significantly more than that of morphiceptin and PFRTic-NH2 . Therefore, its analgesic mechanism is still via the classical opioid system involving both MOR and DOR [14].
∗ Corresponding author at: Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China. Tel.: +86 931 8912428; fax: +86 931 8912428. E-mail address: [email protected] (S. Dong). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.10.014
Considerable evidence suggests that opioids and neuropeptides play an important role in regulating cardiovascular function [2,9,12,16,20–22]. First, opioid receptors distributed in the central and peripheral nervous system are involved in the regulation of cardiovascular activity. In addition, opioid receptor agonists regulate mean arterial pressure (MAP) and heart rate. Moreover, NPFF receptors distributed in the central nervous system and heart and its analogues have potent cardiovascular effects. Based on autoradiography studies, NPFF receptors are distinct from all types of opioid receptors and located in the rat cardiac tissue [1,2]. Although the role of NPFF in cardiovascular regulation is still unclear, assessment of cardiovascular parameters could be useful for the functional characterization of NPFF analogues. Indeed, both parent peptides (morphiceptin and PFRTic-NH2 ) induce hypotension and bradycardia in anesthetized rats [11,15,25]. Morphiceptin (1–50 nmol/kg, intravenous (i.v.) or intracerebroventricular (i.c.v.)) produces a dose-related hypotensive response and bradycardia in normotensive male Wistar Albino Glaxo rats [24], but morphiceptin (100–320 nmol/kg) microinjected into the region of the nucleus of tractus solitarius (NTS) in anesthetized cats has no effect on MAP
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[10]. PFRTic-NH2 was initially considered to be an antagonist of NPFF because of its depressor response, but recently, some studies show that it is a super agonist of NPFF receptor 1 and NPFF receptor 2 [8]. However, its ability to lower MAP is dose-dependent and significant at various doses (0.2–0.9 mg) of PFRTic-NH2 [11]. In the present study, we examined the effects of MCRT injected by i.v. and i.c.v. on the MAP and heart rate (HR) of anesthetized rats. We also examined the effects of morphiceptin and PFRTicNH2 injected by i.v. on the MAP and HR and then compared the measurements with that of MCRT. Multiple dosages of NPFF and MCRT were administered jointly to investigate whether there is an interaction between MCRT and NPFF receptors on the observed cardiovascular effects. Naloxone and l-NAME were injected prior to MCRT to determine the mechanism underlying the cardiovascular effects of MCRT and its related peptides. 2. Materials and methods 2.1. Chemicals MCRT, morphiceptin, PFRTic-NH2 and NPFF 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 RPHPLC and characterized by ESI-MS. l-NAME (N -nitro-l-arginine methylester hydrochloride, a nitric oxide synthase inhibitor) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Naloxone hydrochloride dihydrate (a non-specific antagonist of opioid receptors, Fluka, Beijing, China), naltrindole hydrochloride (a specific antagonist of DOR, Tocris Bioscience, Bristol, UK) and cyprodime hydrochloride (a specific antagonist of MOR, Tocris Bioscience, Bristol, UK) were dissolved in normal saline (NS). All compounds were dissolved in sterile saline and 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. 2.2. Animals Male Wistar rats weighed 200–300 g (Animal Center of Lanzhou University, Lanzhou, China) were used. Animals were housed in a temperature-controlled environment (18–22 ◦ C) under standard 12 h light/dark cycle and received food and water ad libitum. All experiments were carried out in accordance with the Principles of Laboratory Animal Care and Guidelines of the Ethics Committee of Lanzhou University. 2.3. Surgical procedure Rats were anesthetized by intraperitoneal administration of ethyl carbamate (1.5 g/kg). Supplemental doses of ethyl carbamate were given as needed to maintain a uniform level of anesthesia. To insert an indwelling cannula into the lateral cerebral ventricle, a small hole was drilled through the skull at 1.0 mm caudal and 1.5 mm lateral from bregma for inserting a guide cannula. The guide cannula was set to a depth of 4.0 mm from the top of the skull, and accurate placement into the ventricle was verified by observing the outflow of the cerebrospinal fluid induced by withdrawing the cannula on a microsyringe. A second small hole was drilled 4–5 mm caudal from the guide cannula for inserting an anchor screw. A small amount of dental cement was applied to the screw and the guide cannula to hold it in the certain place and make sure it gets hardened before closure of the wound and recovery from anesthesia. A polyethylene catheter was inserted into the external jugular vein for i.v. administration of drugs. The trachea was incised to facilitate spontaneous respiration as well as to get rid of mucus. The BP and HR was 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 BL310 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. Each rat was used only once in one experiment. After surgery, animals were allowed to remain stable for at least 30 min, and then the experiment was started. 2.4. Drug administration MCRT, morphiceptin, NPFF and PFRTic-NH2 of various concentrations were dissolved in isotonic saline. 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 of saline, then they were injected by i.c.v. in a constant volume of 0.01 ml within 1 min. A time interval of 30–35 min was allowed between each injection for recovery of blood pressure to basal level. Separate experiments were carried out for HR. Only a single dose of the peptides was used to examine its effect on HR. Those doses have been shown previously to be sufficient to produce significant effects on BP. Naloxone (2 mg/kg), naltrindole (3 mg/kg), cyprodime (3 mg/kg) and l-NAME (30 mg/kg) were injected through the jugular vein 10 min before the second drug administration. 2.5. Physiological measurements With the polyethylene cannula inserted into the rat carotid artery, MAP and HR were measured with a pressure transducer (PT-100) connected to BL-310 and directly recorded the parameters throughout the experiments. Once the blood pressure (BP, 85–115 mmHg) and HR (280–400 beats/min) get stable, the injection of the test peptide was performed. Change of pressure (mmHg; maximum BP minus basal BP) was used to express the effect of the injection. In HR experiments, the HR was counted from the injection of drug until the BP returned to the basal rate (the duration of drug effect). The same period before the drug administration was sampled to establish the basal value of HR. The change of HR was then obtained and converted to the percentage of the basal. 2.6. Statistics Student’s t-tests were used to evaluate the significance of the change of BP and HR. The drug-induced increase or decrease of BP (mmHg) and HR (%) was compared with the basal values by using an unpaired Student’s t-test (one side) and one-way ANOVA. In all cases, a P value <0.05 was considered statistically significant. Area under the curve (AUC) data was calculated for the interval of time in which drug administered by i.v. or i.c.v. induced MAP change. The results shown are means ± S.E.M. 3. Results The decrease in MAP in response to MCRT (50, 100, 200, 400 nmol/kg, i.v.) in anesthetized rats are shown in Fig. 1A. MCRT administered by i.c.v. or i.v. dose-dependently lowered the MAP significantly when the MCRT’s doses ranged from 0.25 to 250 nmol/kg (P < 0.01, vs saline, Fig. 1B). Based on these arterial blood pressure data, we selected 400 nmol/kg MCRT (i.v.) and 250 nmol/kg MCRT (i.c.v.) for further comparison, as shown in Fig. 1C and D. There was no significant difference (P > 0.05) between the peak of the decrease in MAP induced by 400 nmol/kg (i.v.) and 250 nmol/kg MCRT (i.c.v.); the peak at the dose of 400 nmol/kg was −18.96 ± 1.90 mmHg and appeared at 0.5 min while the peak at of 250 nmol/kg was −23.80 ± 2.48 mmHg and appeared at 4 min. Intravenous infusion of 400 nmol/kg MCRT resulted in a decrease in MAP that occurred
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Fig. 1. (A and B) Dose-dependent decrease in the MAP effects induced by MCRT (50–400 nmol/kg, i.v. or 0.25–250 nmol/kg, i.c.v.) in rats. Control group, n = 8; other groups, n = 12. ***P < 0.001, statistically significant difference between MCRT and saline. (C) Decrease in the MAP effects induced by MCRT (250 nmol/kg, i.c.v., 400 nmol/kg, i.v.) at each time point in rats. (D) Change in MAP is represented as AUC, bars indicate the means ± S.E.M. ***P < 0.001, statistically significant difference between MCRT (250 nmol/kg, i.c.v.) and MCRT (400 nmol/kg, i.v.).
within 4 min while i.c.v. infusion of 250 nmol/kg MCRT resulted in a decrease in MAP that was sustained for approximately 23 min before returning to normal levels. Injections of MCRT, morphiceptin and PFRTic-NH2 all produced a dose-related decrease in MAP (Fig. 2). The decrease in MAP
Fig. 2. Dose-dependent decrease in the MAP effects induced by MCRT, PFRTicamide, morphiceptin and morphiceptin + PFRTic-amide (50, 100, 200, 400 nmol/kg, i.v.) at the nanomolar level in rats. 8 rats were used in each group; *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference between morphiceptin, PFRTic-amide and MCRT, ## P < 0.01, ### P < 0.001, statistically significant difference between MCRT and saline.
induced by MCRT was significantly more potent than that induced by morphiceptin and PFRTic-NH2 (P < 0.01), with that by PFRTicNH2 less potent than that by morphiceptin. As illustrated in Fig. 3, blockade of MOR, opioid receptor (KOR) and DOR with an intravenous dose of 2 mg/kg of naloxone, 3 mg/kg of naltrindole, 3 mg/kg of cyprodime and 30 mg/kg l-NAME were injected prior to MCRT to determine the mechanism of the MAPlowering effects of MCRT. The use of 50 mg/kg l-NAME resulted in the same outcome as that of 30 mg/kg. Fig. 3A shows that pretreatment of rats with naloxone almost abolished the hypotensive effects of MCRT (i.v.) at all doses. Naloxone by itself caused little or no change in the BP of anesthetized rats. Consistent with a previous finding [19], i.v. administration of l-NAME at a dose of 30 mg/kg evoked an increase in MAP (Fig. 3D), and consequently, the decrease in MAP in response to MCRT was partially but significantly reduced. Fig. 3B shows that pretreatment of rats with naloxone completely abolished the hypotensive effects of MCRT (i.c.v.) at all doses, and after administration of 30 mg/kg l-NAME (i.v.), the decrease in arterial blood pressure in response to MCRT by i.c.v. injection was significantly abolished. However, 30 mg/kg lNAME administered by i.c.v. injection did not significantly affect the hypotensive effect of MCRT (i.c.v.). Naltrindole and cyprodime were administered with MCRT (i.v.) to confirm whether ␦ and receptors are involved (Fig. 3C). Naltrindole could significantly antagonize the decrease in MAP induced by i.v. MCRT, but cyprodime had no or little effect. Naltrindole or cyprodime administered alone could evoke decrease in MAP (Fig. 3E). Administration of NPFF into the lateral ventricles resulted in a dose-dependent elevation of mean arterial blood pressure (MAP). Intracerebroventricular injection of 25 nmol/kg NPFF did
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Fig. 3. (A and B) Effects of l-NAME (30 mg/kg) and naloxone (2 mg/kg) on the regulation of MAP induced by i.v. or i.c.v. administration of MCRT in anesthetized rats. (C) Effects of naltrindole (3 mg/kg) and cyprodime (3 mg/kg) on the regulation of MAP induced by i.v. administration of MCRT in anesthetized rats. (D) Effects of l-NAME (30 nmol/kg, i.v. or i.c.v.) on MAP in anesthetized rats. (E) Effects of cyprodime (3 mg/kg, i.v.) and naltrindole (3 mg/kg, i.v.) on MAP in anesthetized rats. 8 rats were used in each group; *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference between MCRT + l-NAME, MCRT + naloxone and MCRT, # P < 0.05, ## P < 0.01, ### P < 0.001, statistically significant difference between MCRT and saline.
not significantly change MAP compared to control animals receiving saline (P > 0.6, data not shown). In rats receiving 50, 100, 200, 250 or 400 nmol/kg NPFF, there was a significant increase in MAP compared to control (P < 0.05). There was no significant increase of MAP with administration of 250 nmol/kg or 400 nmol/kg NPFF (P > 0.2). NPFF (50, 100, 200, 250, 320 nmol/kg) could dose-dependently inhibit the decrease in MAP induced by 250 nmol/kg MCRT (Fig. 4) and could completely block this decrease by NPFF at the doses of 250 nmol/kg and 320 nmol/kg. Administration of 50 nmol/kg NPFF did not alter the effect of 250 nmol/kg MCRT on MAP (P > 0.05). After co-injection of MCRT and different concentrations of NPFF, the decrease in MAP due to MCRT was blocked and could not be reversed even with NPFF at 400 nmol/kg.
MCRT, MCRT + naloxone, MCRT + l-NAME, or morphiceptin and PFRTic-NH2 were i.v. injected to explore whether there are differences in the effects of MCRT and parent peptides on HR (Fig. 5). The most effective doses were chosen based on the BP data. Our data show that MCRT could induce bradycardia both by i.v. or i.c.v. injection, but the effect of MCRT at 250 nmol/kg (i.c.v.) on HR was more effective than MCRT at 400 nmol/kg (i.v.). One of the parent peptides PFRTic-NH2 could lower HR to the same degree as MCRT, whereas morphiceptin did not significantly change HR. In the following experiments, naloxone and l-NAME were injected prior to MCRT for further investigation of the possible mechanisms involved. l-NAME (20 mg/kg) did not change the effects of MCRT (administered by i.v. or i.c.v. injection) on HR. It is surprising that naloxone (2 mg/kg, i.v.) significantly antagonized the bradycardia
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Fig. 4. Effects of i.c.v. administered MCRT + NPFF (50, 100, 200, 250, 400 nmol/kg) on the regulation of MAP in anesthetized rats. 250 nmol/kg MCRT was used. 8 rats were used in each group; *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference between drugs group and saline.
caused by i.c.v. injected MCRT (P < 0.001), but had no influence on bradycardia induced by i.v. injected MCRT (P > 0.05). 4. Discussion MCRT was initially synthesized to obtain high analgesic activity through interactions between the opioid system and neuropeptide system, and both opioids and neuropeptides could cause (arouse) some changes in the cardiovascular system. As expected, MCRT, with its enhanced analgesic activity [14], could result in a dose-response MAP decrease when administered at various concentrations by i.v. and i.c.v. injection. The same doses of morphiceptin and PFRTic-NH2 could also lower MAP by i.v. injection but at a lesser degree. Thus, MCRT administered by i.v. induces a more effective cardiovascular response than its parent peptides at the same dose. This may be because the cardiovascular system mainly has DOR and KOR, and MCRT’s affinity to MOR is similar to that of morphiceptin, but its affinity to DOR is more higher [14]. Thus, activating DOR in the periphery could decrease myocardial contractility. In addition, as PFR(Tic)amide decreases the BP in a dose-dependent manner with an estimated ID50 value
Fig. 5. The effects of MCRT, morphiceptin, PFRTic-NH2 on the heart rate and effects of naloxone, naltrindole, cyprodime and l-NAME on the regulation of HR induced by i.v. administration of MCRT (400 nmol/kg) and i.c.v. administration of MCRT (250 nmol/kg) in rats. Naloxone (i.v.) and l-NAME (i.v. or i.c.v.) were injected 10 min before administration of MCRT. 8 rats were used in each group; **P < 0.01, statistically significant difference between drugs group and saline. ## P < 0.01, ### P < 0.001, statistically significant difference between (antagonist + MCRT) and MCRT.
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of 909.8 nmol (0.525 mg) [11], MCRT may interact with NPFF receptors. Another noticeable property of MCRT was its prolonged effect when administered by i.c.v. injection compared to that by i.v. injection. This could be partially explained by its improved metabolic stability observed in our previous study. Whether administered by i.v. or i.c.v. injection, MCRT caused a dose-dependent fall in blood pressure. However, the peak and duration varied widely after i.v. and i.c.v. injection, with a sustained effect observed only with i.c.v. injection. Our results indicate that the downstream mechanisms and pathways differ between the two injection modes. We used naloxone (i.v.), naltrindole (i.v.), cyprodime (i.v.) and lNAME (i.v. or i.c.v.) to observe the interactions between MCRT and an antagonist of opiate receptor, and between MCRT and a nitric oxide synthase inhibitor. NO in the central nervous system has been recognized as a novel regulatory messenger with important physiological functions in many organs. It is released in varying amounts in several parts of the brain where its many regulatory functions are under active investigation [5]. l-NAME (30 mg and 300 mg i.c.v.) induces a dose-dependent increase in mean arterial pressure and heart rate in anesthetized normotensive rats, and i.c.v. injection of l-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo [4,17]. Pretreatment with naloxone nearly abolished the cardiovascular effects of MCRT, indicating that opioid receptors are involved. Naltrindole could significantly antagonize the change in MAP induced by MCRT (i.v.) whereas cyprodime had no or little effect. This indicates that DOR is mainly involved in the MAP regulation of the peripheral system. However, l-NAME could only antagonize the cardiovascular effect of MCRT (i.v.) to a certain degree, and had no function on the effect of MCRT (i.c.v). We all know that l-NAME, which acts as a competitive inhibitor of NOS due to its structural similarity to arginine, the substrate of NOS, may act as such an agent [6]. Thus l-NAME could competitively inhibit endothelium-dependent vasodilation, as a result of the inhibition of NO releasing from vascular endothelial cells, that a single injection of l-NAME could cause an increase in blood pressure. This indicates that the endothelial NO pathway is involved in the response after i.v. injection of MCRT, while i.c.v. injection of MCRT might be unrelated to NO release in the vascular endothelium. The results from co-injection of l-NAME (i.c.v.) with MCRT (i.c.v.) illustrate that lNAME (i.c.v.) could completely abolish the cardiovascular effect of MCRT, that is MCRT (i.c.v.) plays a cardiovascular effect mainly via the central NO pathway. The cardiovascular response to NPFF (i.c.v.) observed here is consistent with previously published data [13]. We observed a dose-dependent increase in BP. Quantitative differences in magnitude and duration might be related to the different strains of rats used in these experiments. In a previous experiment of analgesia, antagonistic interactions between MCRT and NPFF might be based on their different effects on both receptors. To know whether an interaction between NPFF and MCRT plays a role in the cardiovascular effect of MCRT, we co-administered MCRT (250 nmol/kg) and different concentrations of NPFF in the ventricle. NPFF could dose-dependently antagonize the blood pressure effect of MCRT, but when the concentration of NPFF is greater than or equal to 250 nmol/kg, NPFF could completely antagonize but could not reverse the MCRT’s antihypertensive effect. The mutual antagonistic effect of NPFF and MCRT on blood pressure may be due to the NPFF receptor-mediated regulation of MCRT. MCRT contains PFRTic-amide, which is an NPFF receptor antagonist in the brain. Interactions exist between NPFF and opioids receptors, and the regulation of opioid analgesia by activation of receptors could increase NPFF receptors. These results indicate that besides the activation of opioid receptors, other mechanisms might be involved. It is also possible that differences between central and peripheral opioid receptors contribute to the divergence. When NPFF concentration
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was more than that of MCRT, the combined effect still does not raise blood pressure, indicating that their receptor binding capacity is not the same, and their interaction does not simply cancel each other out, and thus, a complex mechanism exists. Based on previous studies, the effects of drugs, especially opioids, to blood pressure and heart rate is almost synergistic [7,18,23], and that changes in the direction and magnitude of blood pressure and heart rate are relatively consistent. MCRT, administered either by i.v. or i.c.v. injection, slows the heart rate. However, the two concentrations of MCRT resulted in the same decrease in MAP, but reduced the heart rate to a higher degree when injected by i.c.v. than by i.v. Intriguingly, pre-injection of naloxone antagonized the effect of MCRT injected through i.c.v. on heart rate. Different concentrations of MCRT had no impact on heart rate. Injection of l-NAME (i.v.) had no influence in the decrease of heart rate induced by MCRT. Besides, the lowering of BP induced by MCRT was significantly reduced by l-NAME, indicating that NO pathway participates in BP regulation. We also found that the same concentration of MCRT and PFRTic-NH2 administered by i.v. injection regulated heart rate similarly, while the same concentration of morphiceptin by i.v. injection had little effect on heart rate. Naloxone did not inhibit the effect of MCRT on heart rate after i.v. injection, that the heart rate effect of MCRT administered by i.v. injection was similar to that with PFRTic-NH2 , as reported in the previous literature [11]. We demonstrate that MCRT could induce bradycardia and decrease in the MAP to a higher degree than that with morphiceptin and PFRTic-amide. NPFF could dose-dependently weaken the cardiovascular effects of MCRT. Opioid receptors seem to be involved in the MAP and heart rate regulation by i.v. or i.c.v. injected MCRT. However, the effect of MCRT by peripheral injection on the heart rate had no relationship with opioid receptors. The NO pathway is involved in the MCRT-mediated regulation of blood pressure under both modes of injection, but the peripheral NO pathway had no effect on the blood pressure changes induced by MCRT administered by i.c.v. injection. The peripheral NO pathway is not involved in the heart rate effect induced by MCRT, but the central NO pathway is involved in the regulation of the heart rate by MCRT administered by central injection. Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities and partially supported by the grant from the National Natural Science Foundation of China (No. 30870526). References [1] Allard M, Geoffre S, Legendre P, Vincent JD, Simonnet G. Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Res 1989;500:169–76. [2] Allard M, Labrouche S, Nosjean A, Laguzzi R. Mechanisms underlying the cardiovascular responses to peripheral administration of NPFF in the rat. J Pharmacol Exp Ther 1995;274:577–83.
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