The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway

Hypoxic pulmonary hypertension (HPH) is a common clinical pathophysiological process as well as an important pathological event involved in the development of various heart and lung diseases, such as chronic obstructive pulmonary disease, chronic pulmonary heart disease, and high altitude pulmonary hypertension. HPH is characterized by pulmonary artery vasoconstriction and hyperproliferative remodeling, which leads to right heart failure and early death [1-3]. It has been documented that proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) is a critical process underlying pulmonary vascular remodeling in HPH [4-6]. Therefore, attenuation of PASMC proliferation and pathogenic vascular remodeling is critical for both the prevention and treatment of HPH [7].

Autophagy is a conserved lysosome-associated degradation process responsible for the turnover of organelles and proteins. It is closely linked to the occurrence and development of several lung diseases, including chronic obstructive pulmonary disease, pulmonary hypertension, acute lung injury and lung cancer [8-11]. Inhibiting autophagy decreases cell proliferation under hypoxic conditions and has a protective effect during the pathogenesis of HPH [12, 13]. Furthermore, autophagy suppression appears to inhibit the proliferation and to increase the apoptosis of PASMCs in vitro, and also attenuates the development and progression of monocrotaline-induced pulmonary hypertension in vivo [14]. Jin et al. reported that autophagy can regulate the balanced generation of vascular smooth muscle and endothelial cells in pulmonary hypertension [15]. These results indicate that autophagy plays an important pathogenic role in the development of pulmonary hypertension.

κ-opioid receptors are a class of opioid receptors first reported by Martin which have been detected in the vascular system [16, 17]. κ-opioid receptors have been identified in pulmonary arteries and their expression levels increase during hypoxia [18]. Our previous studies showed that U50, 488H (trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacet-amidel), a selective κ-opioid receptor agonist, relaxed the pulmonary artery in a dose-dependent manner and inhibited PASMC proliferation [19, 20]. In addition, we found that double membrane vacuolar structures, which display the morphological features of autophagosomes, appeared in U50, 488H-treated pulmonary artery endothelial cells under hypoxic conditions (for all online suppl. material, see www.karger.com/doi/10.1159/000485886, Fig. S1). However, whether U50, 488H induces autophagy in PASMC under hypoxic conditions is still unclear, and the molecular mechanisms underlying U50, 488H-mediated HPH protective effects need to be analyzed.

Adenosine monophosphate-activated protein kinase (AMPK) is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits [21-23]. Hypoxia leads to the activation of AMPK, and AMPK can also be activated by several chemicals independently of energy imbalance conditions [24-26]. The mammalian target of rapamycin (mTOR) negatively regulates autophagy during nutrient-rich conditions. AMPK negatively regulates mTOR and therefore positively regulates autophagy in response to hypoxia [27-29]. However, whether the AMPK-mTOR pathway is involved in the modulation of autophagy by U50, 488H in PASMC under hypoxic conditions remains unknown.

The present study aimed to investigate the modulation of autophagy by U50, 488H under hypoxia and the underlying mechanisms, focusing especially on the AMPK–mTOR pathway.

U50, 488H(trans-3, 4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cy-clohexyl]benzeacetamide), a selective κ-opioid receptor agonist, and nor-binaltorphimine (nor-BNI), a selective κ-opioid receptor antagonist, were purchased from Tocris Cookson. All compounds were dissolved in normal saline (0.85% NaCl solution) before use. Antibodies against p-mTOR, mTOR, caspase 3, p62, LC3B and β-actin were purchased from Cell Signaling Technology (CST). Antibodies against p-AMPK and AMPK were purchased from Abcam. The secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG conjugated with HRP) were purchased from Boster Company. AICAR and rapamycin were obtained from Selleck Chemicals. The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). The Protein Quantitation Kit and chemiluminescence kits were purchased from the Pierce Company.

Sprague-Dawley rats (200±20 g) were purchased from the Animal Center at the Fourth Military Medical University. Animals were allowed ad libitum access to food and water for the duration of the experiments. This study followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, NIH Publication NO.85-23, revised 1996. Ethical approval was also granted by the Ethics Committee of the Fourth Military Medical University.

Animals were randomly divided into the following groups: (1) control (normoxic) group; (2) hypoxia group (rats exposed to hypobaric and hypoxic condition for 3 weeks and intraperitoneally injected every other day with normal saline, 2.0 ml/kg, 10 min prior to hypoxia); (3) hypoxia + chloroquine group (same conditions as the hypoxia group but intraperitoneally injected every other day with 50 mg/kg of chloroquine, a specific inhibitor of autophagy, 10 min prior to hypoxia) [14]; (4) hypoxia + U50, 488H group (same conditions as the hypoxia group but intraperitoneally injected every other day with 1.25 mg/kg of U50, 488H, a selective κ-opioid receptor agonist, 10 min prior to hypoxia) [30]; (5) hypoxia + U50, 488H + nor-BNI group (same conditions as the hypoxia group but injected with both nor-BNI and U50, 488H, as follows: 2.0 mg/kg of nor-BNI was intraperitoneally injected first, followed by 1.25 mg/kg of U50, 488H intraperitoneally 15 minutes later). These injections were performed 10 minutes before exposure to hypoxia every other day.

An automatic hypoxia equipment was used to generate hypobaric and hypoxic pulmonary hypertension in these animals [18, 20, 31, 32]. Rats were exposed to both low pressure and low oxygen (air pressure 50 kpa, oxygen concentration 10%) for 8 hours every day. Rats in the control group were maintained in room air.

After hypoxia, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg). Supplemental doses of sodium pentobarbital were given when needed to maintain a uniform level of anesthesia. The mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured by inserting a polyethylene microcatheter into the pulmonary artery and the right ventricle via the right external jugular vein [33].

Lung tissue was fixed in 10% formalin (pH 7.4) for 1 week, paraffin-embedded and sliced serially. Tissues were stained with HE (hematoxylin and eosin). Morphological changes in the peripheral pulmonary artery were assessed using a light microscope and a computerized morphometric system. The external diameter, medial wall thickness, medial cross-sectional area, vessel lumen cross-sectional area and total arterial cross-sectional area of the peripheral pulmonary artery were measured. The ratio of vascular medial wall thickness to external diameter, and the ratio of vascular medial cross-sectional area to total arterial cross-sectional area, were calculated to assess the degree of pulmonary artery remodeling.

Rats (180∼200 g) were anesthetized by intraperitoneal injection of pentobarbital sodium (100 mg/ kg), and the skin was sterilized with 75% alcohol. The organs were rinsed several times with D-Hanks solution (Ca²⁺ and Mg²⁺-free, pH: 7.4) at 4°C. The pulmonary arteries were dissected under sterile conditions. Collected PASMCs were cultured following previously reported methods [34]. Briefly, the outer spheres were peeled and the microtubules were sniped visually, then the endothelium was shaved slightly for 2∼3 times to remove endothelial cells. The tunica media was cut into small fragments (1 mm³) in Dulbecco’s Modified Eagle Medium (DMEM). PASMCs were cultured at 37°C and 5% CO₂ in DMEM containing 20% fetal bovine serum. The purity and identity of PASMCs were determined based on their typical morphological pattern and by immunofluorescence staining using a specific antibody against α-actin. All experiments were performed using cells with passage number between 3 and 6. Cells were placed in a hypoxic incubator (95% N₂, 5% CO₂, 37°C) for 24 hours. Then, total proteins were extracted.

Cell proliferation was quantified by counting the cells directly and by means of the CCK-8 assay. PASMCs were seeded onto 96-well plates in a total volume of 100 µl of medium per well before exposure to hypoxia. After exposure to hypoxia for 24 hours, CCK-8 was added to the wells (10 µl/well) and the absorbance at 450 nm was measured to determine the number of viable cells.

The Annexin V-PI Apoptosis Detection Kit (MaiBio, Shanghai, China) was used to evaluate the effects of chloroquine or U50, 488H on PASMC apoptosis. The fluorescence signals from Annexin V and PI were measured using a flow cytometer (Beckman Coulter; Miami, USA), and the data were analyzed using the Expo32 software (Beckman Coulter).

Proteins were extracted from the pulmonary arteries and PASMCs and concentrations determined by means of the BCA assay. For Western blot analysis, equal amounts of protein (30 g of protein/lane) were electrophoresed on 10% and 12% SDS-polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA). Non-specific binding sites were blocked with 10% non-fat dry milk in buffer (10 mM Tris-HCl [pH 7.6], 100 mM NaCl and 0.1% Tween 20) for 1 hr at room temperature and then incubated with the desired primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibodies for 1 hr at room temperature. The immunoblotting was detected using an enhanced chemiluminescence detection kit (Millipore) and the ChemiDocXRS imaging system (Bio-Rad Laboratory, Hercules, CA). The blot densities were analyzed with Quantity One Software.

PASMCs were transduced for 48 h with an adenovirus expressing mRFP-GFP-LC3 (tfLC3) (Hanbin, Shanghai, China) using a MOI of 100 and then treated with other agents or left untreated. The cells were viewed under a fluorescence microscope (Nikon Eclipse E800). The number of autolysosome dots were determined by counting 50 cells in a minimum of 5 experiments.

Data analysis was carried out using Graph Pad Prism software 5.0. All quantitative data are presented as the mean ± SEM. Statistical significance was determined using one-way analysis of variance (ANOVA). A probability value of P<0.05 was considered to be statistically significant.

After being exposed to hypobaric hypoxia for three weeks, rats showed increased mPAP and RVP values. Chloroquine administered at 50 mg/kg every other day for 3 weeks prevented the elevation of mPAP and RVP in rats exposed to hypoxia (Fig. 1A, B). Representative RVP traces are shown in Fig. 1C. In addition, the thickness of the pulmonary artery wall was significantly reduced in rats treated with chloroquine for 3 weeks, as evidenced by light microscopy and transmission electron microscopy analysis (see online suppl. material, Fig. S2).

Previous studies have demonstrated that chloroquine can prevent acidification of lysosomes, thereby interfering with the processing of autophagosomes and inhibiting autophagy in PASMCs [14]. To investigate further the process of autophagy in HPH, we used immunoblotting to analyze the expression of two autophagy markers, LC3B, and p62, in pulmonary arteries obtained from control rats or from rats of the hypoxia/saline and hypoxia/chloroquine experimental groups. In pulmonary arteries obtained from control rats, the LC3B- II/-I ratio was low. This ratio increased in the pulmonary arteries of hypoxic rats, and chloroquine significantly enhanced the LC3B-Π/-I ratio (Fig. 1D, E). Moreover, the autophagy-associated degradation of p62 was increased in the pulmonary arteries of hypoxic rats compared with control rats, and chloroquine prevented this reduction in p62 expression, an outcome that was consistent with inhibition of autophagy (Fig. 1D, F). These data suggest that HPH is associated with increased autophagy, and that inhibiting autophagy with chloroquine produces a beneficial effect in HPH.

Three weeks after being exposed to chronic hypoxia, rats developed severe pulmonary hypertension. Treatment with U50, 488H significantly reduced the mPAP and RVP of rats exposed to chronic hypoxia, and these effects were inhibited by nor-BNI, a selective κ-opioid receptor antagonist (Fig. 2A, B). Representative RVP traces are shown in Fig. 2C. Analysis by light microscopy and transmission electron microscopy showed that the thickness of the pulmonary artery wall was significantly reduced in rats treated with U50, 488H (see online suppl. material, Fig. S3). Next, we evaluated the effects of U50, 488H on autophagy. As a membrane receptor, κ-opioid receptor stimulation would inhibit autophagy by signaling upstream of autophagy. Compared with the hypoxia group, the LC3B-II/-I was markedly decreased and p62 expression was increased in pulmonary artery samples obtained from U50, 488H-treated rats and these changes were significantly abolished by nor-BNI (Fig. 2D-F). These data suggest that U50, 488H inhibits autophagy, exerting a protective effect against HPH, and that this effect is mediated by the κ-opioid receptor.

To investigate whether U50, 488H inhibits autophagy in PASMCs under hypoxic conditions, PASMCs were treated with U50, 488H for 24 hours under hypoxia or normoxia. The expression of the autophagy markers LC3B and p62 was analyzed by immunoblotting. As shown in Fig. 3A-C, the LC3B-II/-I ratio increased after hypoxia, and U50, 488H inhibited the increase in the LC3B-II/-I ratio in a dose-dependent manner. In contrast, p62 expression levels were decreased in the hypoxia group, and U50, 488H dose-dependently counteracted this reduction. Consistently, mRFP-GFP-LC3 puncta formation assays showed that U50, 488H dose-dependently attenuated autolysosome puncta formation in treated PASMCs (Fig. 3D-E). Our results indicate that U50, 488H inhibits autophagy in a dose-dependent manner in PASMCs exposed to hypoxic conditions.

When quiescent cells grown in DMEM with 5% serum were exposed to hypoxic conditions for 24 hours, the absorbance in the CCK-8 assay increased by 49% compared with cells from the normoxic group (P <0.01) (Fig. 4A). We investigated the effects of U50, 488H on the proliferation of PASMCs. When quiescent cells grown in DMEM with 5% serum were treated with U50, 488H and exposed to hypoxic conditions for 24 hours, the CCK-8 assay showed that U50, 488H significantly inhibited PASMC proliferation in a dose-dependent manner (10, 40, 70 µM) (Fig. 4A). Flow cytometry experiments also confirmed that U50, 488H treatment produced a dose-dependent increase in PASMC apoptosis when compared with the untreated hypoxia group (Fig. 4B, C). To further investigate the effects of U50, 488H on apoptosis, we analyzed caspase 3 protein expression by immunoblot. Consistently, U50, 488H dose-dependently increased caspase 3 expression levels in PASMCs exposed to hypoxia (Fig. 4D, E). Our results indicate that U50, 488H significantly induces caspase-dependent apoptosis of PASMCs exposed to hypoxic conditions.

To confirm the role of autophagy in PASMCs exposed to hypoxia, we pretreated PASMCs with chloroquine, a specific autophagy inhibitor, and then exposed them to normoxic or hypoxic conditions. We examined the effects of chloroquine on the proliferation and apoptosis of PASMCs. As shown in Fig. 5A-C, treatment with chloroquine profoundly inhibited the proliferation of PASMCs in hypoxic conditions and induced apoptosis of PASMCs under both normoxic and hypoxic conditions.

Our experiments also showed that rapamycin, a selective mTOR inhibitor (also called the autophagy agonist), counteracted the suppression of PASMC proliferation induced by U50, 488H (Fig. 5D). Consistently, U50, 488H-induced apoptosis decreased with rapamycin, as evidenced by flow cytometry (Fig. 5E, F). These data demonstrate that U50, 488H inhibits autophagy and in turn suppresses proliferation and induces apoptosis of PASMCs.

We investigated the role of the κ-opioid receptor in the autophagy and proliferation effects of U50, 488H on PASMCs grown under hypoxic conditions. PASMCs were treated under hypoxic conditions with U50, 488H in the absence or presence of nor-BNI. The protein expression analysis showed that nor-BNI significantly increased the LC3B-Π/-I ratio and decreased p62 expression when compared with the U50, 488H-only treatment group (Fig. 6A-C). The mRFP-GFP-LC3 puncta formation assays showed that more autolysosome dots were present in the nor-BNI treated cells after 24 hours of hypoxia than in the U50, 488H treated cells, suggesting that nor-BNI counteracted the inhibitory effect of U50, 488H on PASMC autophagy under hypoxic conditions (Fig. 6D, E). These results demonstrate that U50, 488H significantly decreases PASMCs proliferation during hypoxia and that this effect can be attenuated by nor-BNI, a selective κ-opioid receptor antagonist (Fig. 6F).

Next, we investigated whether the reduced autophagy induced by U50, 488H was dependent on the AMPK-mTOR signaling pathway. Hypoxia increased AMPK phosphorylation and reduced mTOR phosphorylation. In contrast, AMPK phosphorylation was reduced after U50, 488H treatment and mTOR phosphorylation was prominently elevated. These effects of U50, 488H were abolished by nor-BNI (Fig. 7).

To confirm that the AMPK-mTOR pathway was involved in the U50, 488H-induced inhibition of autophagy, AICAR (a selective AMPK agonist) and rapamycin (a selective mTOR inhibitor) were administered together with U50, 488H to PASMCs under hypoxic conditions. As shown in Fig. 8A-C, rapamycin (100 nM) markedly increased the LC3B-II/-I ratio and decreased p62 expression when compared with cells treated with U50, 488H only. Furthermore, activation of AMPK with AICAR (1mM) inhibited mTOR activity, increased the LC3B-II/-I ratio and decreased p62 expression in PASMCs treated with U50, 488H under hypoxic conditions (Fig. 8D-G). These results indicate that the AMPK-mTOR pathway is possibly implicated in the inhibition of autophagy induced by U50, 488H under hypoxic conditions.

In this study we found that inhibiting autophagy with chloroquine could suppress pulmonary artery remodeling, thereby preventing the development of HPH. This effect correlated with decreased proliferation and increased apoptosis of PASMCs exposed to hypoxic conditions. Moreover, our study also demonstrated that U50, 488H, a specific κ-opioid receptor agonist, reduced both the mPAP and the RVP in HPH rats. U50, 488H inhibited pulmonary artery remodeling, suppressed PASMC proliferation, and induced PASMC apoptosis in response to hypoxia. It also decreased the level of autophagy under hypoxia. The inhibition of autophagy and PASMC proliferation seen with U50, 488H were mediated by the κ-opioid receptor, since these effects could be blocked by nor-BNI, a selective κ-opioid receptor antagonist. Our results also indicated that the AMPK-mTOR pathway was involved in mediating these effects of U50, 488H on autophagy.

Autophagy is a membrane-dependent mechanism for the sequestration, transport, and lysosomal turnover of subcellular components, including proteins and organelles [35, 36]. Increased expression of LC3B-II has been reported previously in the lungs of mice exposed to chronic hypoxia and in the lungs of patients with pulmonary hypertension of various etiologies [13]. However, the role of autophagy in hypoxia-induced pulmonary hypertension is still controversial. In our study we demonstrated that activation of autophagy is involved in the development of HPH. Treatment with chloroquine, a specific inhibitor of autophagy, increased the LC3B-II/-I ratio and decreased p62 expression in the pulmonary arteries of rats subjected to hypoxia, and resulted in the inhibition of pulmonary artery remodeling as well as reduced mPAP and RVP in this animal model of HPH. We also showed that inhibition of autophagy in vitro suppressed PASMC proliferation and increased PASMC apoptosis. These results are consistent with the notion that the role of autophagy may be to enhance arterial remodeling and accelerate disease progression in HPH.

κ-opioid receptor expression is localized in the pulmonary artery and its levels increase during chronic hypoxia [18]. Previously, we demonstrated ex vivo that U50, 488H relaxed rat pulmonary vessel rings in a dose-dependent manner [19]. Our laboratory has also demonstrated in vivo the preventive and therapeutic effects of U50, 488H on HPH [20, 31, 32]. Although autophagy seems to be an adaptive process necessary in HPH, it is still unclear whether autophagy is involved in U50, 488H-mediated protection. Interestingly, our data showed that U50, 488H led to an inhibition of autophagy. We demonstrated that U50, 488H decreased the LC3-II/LC3B-I ratio and increased p62 levels with respect to the untreated hypoxia group. Importantly, the mRFP-GFP-LC3 puncta formation assay results were consistent with the protein expression results, suggesting that the autophagic flux was blocked. Thus, our data indicate that the beneficial effects of U50, 488H may depend on the inhibition of autophagy. Furthermore, the κ-opioid receptor antagonist nor-BNI counteracted the inhibitory effects of U50, 488H on autophagy, indicating that these effects were mediated by the κ-opioid receptor. In addition, and in agreement with previous studies, our study showed that U50, 488H significantly inhibited pulmonary artery remodeling and attenuated the elevation of mPAP and RVP in HPH rats. We also showed that these effects of U50, 488H were inhibited by nor-BNI, indicating that they were mediated by the κ-opioid receptor.

Hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling are two key processes in HPH pathogenesis. Long-term hypoxia can cause vasoconstriction and trigger marked proliferation of smooth muscle cells in the pulmonary vessels [37]. Our previous study demonstrated that U50, 488H suppressed the growth of cardiac cells in a concentration-dependent manner [38]. In the present study, we found that U50, 488H can significantly inhibit the proliferation and increase the apoptosis of PASMCs after 24 hours of hypoxia exposure in a dose-dependent manner (10∼70 µM). It also decreased the level of autophagy under hypoxia, suggesting that the effects of U50, 488H on PASMCs may be related to the regulation of autophagy. This effect was also suppressed by nor-BNI, highlighting the specificity of the anti-pulmonary artery remodeling effect of U50, 488H.

AMPK is a metabolic master switch that maintains cellular energy homeostasis and is known to activate autophagy. On the other hand, the mTOR complex controls essential pathways that lead to autophagy and receives input from many cellular processes [12, 39, 40]. The AMPK-mTOR pathway is required for autophagy activation: activation of AMPK can inhibit mTOR (the negative regulator of autophagy) and thus stimulate autophagy [29]. Both AICAR and rapamycin counteracted the decrease in autophagy induced by U50, 488H in PASMCs under hypoxia, supporting the idea that the inhibition of AMPK and the activation of mTOR were essential to produce the effects of U50, 488H on autophagy under hypoxia. Furthermore, nor-BNI counteracted the inhibition of AMPK phosphorylation and the increased mTOR phosphorylation induced by U50, 488H, reversing its effects on the proliferation and autophagy of PASMCs. These results indicate that the regulatory effects of U50, 488H on autophagy mediated by the AMPK-mTOR pathway depend on activation of the κ-opioid receptor.

Autophagy may be a crucial component of U50, 488H-induced protection against HPH. U50, 488H inhibited autophagy through the inhibition of AMPK and the activation of mTOR in a κ-opioid receptor-dependent manner. Our findings provide evidence that κ-opioid receptor modulation may be a potential new strategy to regulate autophagy, a cellular process that plays a central role in the development of pulmonary hypertension.

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81470248, 81270102, 81770243) and by a grant from Shaanxi Province (No. 2016KTCL03-11).

The authors declare that they have no conflicts of interest to disclose.

Y. Zhou, Y. Wang and X. Wang contributed equally to this work.