The underlying mechanism of hypoxic pulmonary hypertension (HPH) is still not fully understood and an ideal prevention and treatment strategy is still lacking. It is believed that pulmonary artery contraction and smooth muscle proliferation are 2 important processes of HPH pathogenesis. Therefore, molecules that have pulmonary artery vasodilatory property may have certain effects on the prevention and treatment of HPH.
Endogenous opioid peptide plays an important role in the modulation of cardiovascular activities. Radioimmunology competitive binding assay and functional studies also show that κ-opioid receptor is the predominant type of opioid receptors in the heart and peripheral vessels.1 Our previous study2 demonstrated that a selective κ-opioid receptor agonist U50,488H relaxes the aortic artery and lowers the blood pressure of systemic circuit in rats, but the effect of U50,488H on pulmonary circulation has not been previously studied. The purposes of the present study were to determine the effect of U50,488H on pulmonary circulation and investigate its preventive and therapeutic efficacy in HPH. Thus, we observed the effect of U50,488H on the pulmonary artery and also determined the effect of U50,488H on pulmonary artery pressure and right ventricular hypertrophy in both normal and HPH rats.
MATERIALS AND METHODS
Healthy Male Sprague-Dawley rats (250±20 g, from the animal center of the Fourth Military Medical University, Xi'an, China) were used for all experiments. U50,488H (trans-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamide) and nor-binaltorphimine (nor-BNI) were both purchased from Sigma (St Louis, MO).
In Vitro Blood Vessel Perfusion
After decapitation of the rat, the pulmonary aorta was separated and immediately placed in 95% O2/5% CO2 saturated Krebs solution (mmol/L: NaCl 118.4, KCl 4.7, MgCl2·6H2O 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25.0, Glucose 11.1 pH 7.4). Connective tissue was discarded and the vessel ring segment was prepared with a length of 3 mm. The vessel ring was suspended in Krebs solution (8 mL, 37°C) bath and continuously bubbled with a gas mixture of 95% O2 and 5% CO2. Each ring was connected to a FORT-10 force transducer (WPI, Sarasota, FL) and the tension of the vessel ring was recorded via a MacLab data acquisition system. The preload was adjusted to 0.75 g and the fluid in the bath was replaced every 15 min. After equilibration for 1 h, norepinephrine (NE) at l μmol/L was added to the bath to examine the constriction activity of the segment, and those segments with <300 mg developed tension were not used for further study. NE was then washed out from the bath and segment tension was allowed to return to baseline. After another 40 min of equilibration, NE was added again. When the contractive response of the vessel ring reached a steady state (taken as 100% contraction), U50,488H (10–140 μmol/L) was added in an accumulative fashion and concentration-relaxation (reduction in tension after each dose/the maximal NE-induced tension×100%) curve to U50,488H was constructed. The maximal relaxation effect (Emax) and the concentration that produces half-maximal effect (EC50) was determined.
In Vivo Experiment
Rat HPH Model
Hypoxia was performed for 8 h every day by exposing rats (hypoxia group) to low pressure and low oxygen in a homemade automodulating cabin (air pressure 50 kPa, oxygen concentration 10%). The animals in the control group were kept in room air.
Animal Groups and Treatment
Animals were divided into normal control group, hypoxia for 1w group, hypoxia for 2w group, hypoxia for 2w+saline group (0.5 mL saline was intraperitoneally injected every other day) and hypoxia for 2w+U50,488H group (1.25 mg/kg/day U50,488H was intraperitoneally injected every other day).
To investigate the treatment effect, animals were divided into normal control group and hypoxia for 4w group. Then, all of the animals were injected U50,488H (1.25 mg/kg) through vena caudalis.
Pulmonary Hemodynamics and Right Ventricular Hypertrophy Examination
Sodium pentobarbital (40 mg/kg) was injected into the abdomen to anesthetize rat and the mean carotid artery pressure (mCAP) was measured by left carotid artery cannulation. A polyethylene microcatheter was inserted from right external carotid artery to right ventricle or further advanced to pulmonary artery to measure right ventricular pressure (RVP) or mean pulmonary artery pressure (mPAP) as described previously.3 The mCAP, RVP, and mPAP were sampled and digitally processed via a hemodynamic analyzing system (Chengdu Instrument Co, China). After completion of the experiment, hearts were harvested and atria tissue was discarded. Right ventricle (RV), left ventricle (LV), and septum (S) were isolated, and RV and LV+S were weighed after absorbing water using filter paper. The ratios of RV/(LV+S) and RV/BW (body weight) were used as indexes for RV hypertrophy.
Values were expressed as mean±SD. One-way ANOVA was employed to determine the difference among groups. P<0.05 was regarded as the significant difference, whereas P<0.01 was extremely significant difference.
Effects of U50,488H on Pulmonary Artery Isolated From Normal Rats
U50,488H, a selective κ-opioid receptor agonist, caused significant relaxation effect starting at a concentration of 30 μmol/L. U50,488H exhibited a dose-dependent relaxation effect within the concentration range of 30 to 140 μmol/L when added accumulatively. At the maximal concentration tested, pulmonary rings were fully relaxed (near 100%, n=10, Figs. 1A, B).
U50,488H at a single concentration of 70 μmol/L also induced a significant relaxation effect in rat pulmonary artery rings. The relaxation started 1 min after the administration of U50,488H; rapid relaxation occurred within 10 min, which peaked at ≈15 min after administration. The maximal relaxation effect was 61.4±4.2% (n=10, Fig. 2A). This vasorelaxing effect was completely abolished by nor-BNI, a selective κ-opioid receptor antagonist (5 μmol/L; Fig. 2B).
Effect of U50,488H on mPAP in Normal Rats
To verify that the vasorelaxing effect of U50,488H may lead to a decrease in pulmonary blood pressure, we observed the effect of U50,488H on mPAP in normal rats. U50,488H at 1.25 mg/kg was given to normal rats by vena caudalis injection. Within 1 min after injection, mPAP, mCAP, and heart rate (HR) all significantly decreased, and a recovery did not occur until 30 min later. These parameters returned to the normal level approximately 60 min after injection (Table 1). This effect was abolished by intravenous injection of nor-BNI (2 mg/kg, 10 min before U50,488H administration), which itself had no effects on mPAP, mCAP, and HR (data not shown).
Changes of Parameters for Pulmonary Hemodynamics and RV Hypertrophy Induced by Chronic Hypoxia
After the rats were subjected to chronic hypoxia for 1w, 2w, or 4w, mCAP and HR in the hypoxic group showed no great variation compared with control group. However, the mPAP, RVP and ratio of RV/BW were significantly higher in the hypoxic group than those in the control group, and they increased progressively as the time for hypoxia extended (Table 2).
Preventive Effects of U50,488H in HPH Rats
U50,488H was administered intraperitoneally during hypoxia as described in Materials and Methods. Compared with the hypoxia 2w group and hypoxia 2w+NS group, the hypoxia 2w+U50,488H group showed a significant decrease in mPAP, RVP, RV/(LV+S), and RV/BW ratios. However, mCAP and HR showed no significant difference among groups (Table 3).
Therapeutic Effects of U50,488H in HPH Rats
After 4w of chronic hypoxia, rats developed severe pulmonary hypertension. Intravenous administration of a single dose of U50,488H at 1.25 mg/kg markedly reduced mPAP, mCAP, and HR. The time course of response to U50,488H in HPH rats was similar to that observed in normal rats (Table 4). However, the decrease in mPAP (31%) was significantly stronger than that in normal rats (24%).
κ-opioid receptor widely participates in the modulation of various functions of systemic circulation. It has been reported that selective κ-opioid receptor agonist U50,488H significantly relaxes rat abdominal aortic ring in vitro4,5 and reduces blood pressure in vivo. However, little has been unveiled about the relationship between κ-opioid receptor and pulmonary circulation. The present study and our previous observation discovered that U50,488H significantly relaxes rat pulmonary vessel ring in a dose-dependent manner in vitro.6 In the present study, we discovered that U50,488H significantly lowers mPAP in normal rats, and that this effect is totally abolished by nor-BNI. Thus, we conclude that U50,488H modulates pulmonary artery pressure by activating κ-opioid receptors.
Hypoxic pulmonary vasoconstriction is one of the important mechanisms for HPH and the clinical use of drugs that relax the pulmonary artery shows certain preventive and therapeutic effect in HPH. To make it clear whether U50,488H has a preventive and therapeutic effect in HPH, we successfully established a rat HPH model by a hypoxia and hypopressure method. It was found that in chronic hypoxic rats, the level of mPAP began to rise and RV hypertrophy developed in 2w, and HPH became even more typical in 4w. Every other day, we intraperitoneally injected U50,488H to hypoxic rats to investigate its preventive effect on HPH. It was found that compared with control, hypoxia 2w+U50,488H group had a lower level of mPAP and RV hypertrophy. This result demonstrated that U50,488H effectively attenuated the process of HPH and RV hypertrophy, thus exerting preventive effect against HPH in rats.
In severe HPH induced by exposing rats to low oxygen for 4w, intravenous administration of U50,488H lowered mPAP rapidly, and this effect lasted for a relatively long period of time. All of these results suggested that U50,488H has some therapeutic effect on pulmonary hypertension.
Noticeably, U50,488H administrated intravenously lowered blood pressure and HR in both normal and hypoxic rats. This is in agreement with previous results on systemic circulation.2 Preventive application of U50,488H for a long time did not bring down systemic blood pressure and HR while it lowered pulmonary pressure and alleviated heart hypertrophy. Although the underlying mechanism is still unclear, this demonstrates a bright future for the application of U50,488H in pulmonary circulation because it suggests that this compound may have certain selectivity in pulmonary circulation.
Hypoxic pulmonary vasoconstriction and smooth muscle proliferation are 2 important processes in HPH pathogenesis. Our previous study demonstrated that U50,488H depresses growth and proliferation of cardiac cells,7 and the present study proved that U50,488H relaxes pulmonary vessels and lowers HPH. Furthermore, it was also found that U50,488H slows down the process of RV hypertrophy in HPH rats. However, whether U50,488H attenuates pulmonary vessel remodeling in HPH rats through preventing hyperproliferation of pulmonary artery smooth muscle warrants further study.
Pulmonary hypertension, which is an important pathophysiological stage in the genesis and development of many cardiopulmonary diseases, still has no ideal therapeutic method. Given that the KV channel is the key factor in the genesis of pulmonary hypertension, especially HPH, much research has focused on the use of the KV channel agonist to treat HPH. Our recent study8 shows that κ-opioid receptor agonist U50,488H may be effective in the opening of KV channels, thus having great potential as a therapy against pulmonary hypertension, especially HPH. In recent years, the clinical use of opioid peptide as a treatment for cardiovascular disease has increased, so the study of the relationship between κ-opioid receptor and pulmonary circulation and HPH may provide new insight for the treatment of HPH.
In this study, we found that κ-opioid receptor agonist U50,488H relaxes pulmonary artery and decreases the pulmonary artery pressure by activating κ-opioid receptors. We also provide the evidence for the first time that U50,488H has certain therapeutic and preventive effects on hypoxia-induced pulmonary hypertension and RV hypertrophy.
The authors thank Mr M. Z. Li for technical assistance.