Distribution of ╬║-Opioid Receptor in the Pulmonary Artery and its Changes During Hypoxia.код для вставкиСкачать
THE ANATOMICAL RECORD 292:1062–1067 (2009) Distribution of j-Opioid Receptor in the Pulmonary Artery and its Changes During Hypoxia PAI PENG,1,2 LU-YU HUANG,3 JUAN LI,2 RONG FAN,2 SHU-MIAO ZHANG,2 YUE-MIN WANG,2 YU-ZHEN HU,2 XIN SUN,2 ALAN DAVID KAYE,4,5 AND JIAN-MING PEI2* 1 Department of Plastic Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China 2 Department of Physiology, National Key Discipline of Cell Biology, Fourth Military Medical University, Xi’an, China 3 Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, China 4 Department of Anesthesiology, LSU School of Medicine, New Orleans, USA 5 Department of Pharmacology, LSU School of Medicine, New Orleans, USA ABSTRACT The present study evaluated the distribution of j-opioid receptors (j-ORs) in pulmonary arteries (PAs) in rats and investigated whether j-ORs are altered in PAs during hypoxia. An animal model of hypobaric/ hypoxic pulmonary hypertension and a pulmonary artery smooth muscle cell (PASMC) model of hypoxia were utilized. Distribution of j-ORs was determined by ﬂuorescence immunohistochemistry and changes in j-ORs expression in PAs and PASMCs were determined by ﬂuorescence immunohistochemistry or Western blot techniques. The j-ORs were primarily distributed in the smooth muscle layer of the PAs and in the nucleus of PASMCs. The expression of the j-ORs were increased in PAs of rats subjected to hypoxia for 1–4 week (P < 0.01). Accordingly, the expression of j-ORs in PASMCs were also increased when subjected to hypoxia for 12– 36 hr (P < 0.05). The present study has provided evidence for the ﬁrst time of the precise location of j-ORs in PAs and PASMCs of rats and that hypoxia upregulates expression of j-ORs. Anat Rec, 292:1062–1067, C 2009 Wiley-Liss, Inc. 2009. V Key words: j-opioid receptor; pulmonary artery; hypoxia The j-opioid receptor (j-OR) is a class of opioid receptor ﬁrst described by Martin (1983). j-ORs have been identiﬁed in the brain, the spinal cord, and the periphery (Gray et al., 2005), including the vascular system. Radioimmunology competitive binding assays and functional studies have demonstrated that the j-ORs are the predominant type of opioid receptors, which exist in peripheral vessels (Tai et al., 1991; Wittert et al., 1996). Seelhorst and Starke (1986) showed that it was likely that receptors in the pulmonary artery of the rabbits are at least, predominantly kappa-type. Our previous study demonstrated for the ﬁrst time that a selective j-opioid receptor agonist, U50,488H, relaxed pulmonary arteries C 2009 WILEY-LISS, INC. V Grant sponsor: Department of Health, General Department of Logistic, PLA; Grant number: 01MB129, 06MA203; Grant sponsor: National Natural Science Foundation, China; Grant number: 30770802. *Correspondence to: Jian-Ming Pei, Department of Physiology, Fourth Military Medical University, Changle west Road 17#, Xi’an, Shaan Xi, China. Fax: 86-029-84774519. E-mail: firstname.lastname@example.org The ﬁrst three authors contributed equally to this work. Received 18 November 2008; Accepted 27 February 2009 DOI 10.1002/ar.20911 Published online 21 May 2009 in Wiley InterScience (www. interscience.wiley.com). j-OR IN PULMONARY ARTERY in vitro, and decreased pulmonary pressure in vivo in rats (Pei et al., 2006; Sun et al., 2006), indicating that jORs may exist in pulmonary arteries (PAs) in rats. However, the exact distribution of j-ORs in PAs and how they change during hypoxia still remains largely uncertain. In the present study, our ﬁrst aim was to investigate the distribution of j-ORs in the pulmonary artery and its changes under hypoxic conditions in rats. This objective was approached by western-blot and ﬂuorescence immunohistochemistry. Then, we tested these results in primary cultured pulmonary artery smooth muscle cells (PASMCs). The present study revealed a deﬁnite location of j-ORs in the PASMCs, which is different in cardiomyocytes. In addition, the expression of the j-ORs was altered during hypoxic conditions. MATERIALS AND METHODS Animal Groups and Rat HPH Model Thirty-two male Sprague-Dawley rats (250 20 g) from the animal center of the Fourth Military Medical University (This study conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health) were divided into 4 groups: (1) normoxic group, normal control group; (2) hypoxia for 1-week group, the rats were exposed to hypobaric and hypoxic conditions for 1 week; (3) hypoxia for 2-week group, the rats were exposed to hypobaric and hypoxic conditions for 2 weeks; (4) hypoxia for 4-week group, the rats were exposed to hypobaric and hypoxic conditions for 4 weeks. The animal model of hypobaric and hypoxic pulmonary hypertension was performed with automatic hypoxia equipment (Pei et al., 2006). Hypoxia was performed for 8 hr every day by exposing rats to low pressure and low oxygen (air pressure 50 kpa, oxygen concentration 10%). The normoxic group of rats was kept in room air. Tissue Treatment After hypoxia, rats were anesthetized with peritoneal injection with 30 g L1 pentobarbital sodium (1.5 mL kg1, ip). The skin was sterilized with 75% alcohol, then the chest was opened, and the heart and lungs were removed. The organs were placed into PBS (pH 7.4, 4 C); The studied tissues were extracted from the same position of the inferior lobe of the right lung, and ﬁxed in 10% formalin (pH 7.4) for 1 week. PA were gently isolated and not stretched during the preparation. A three grade branch of PA were picked, ﬁxed in 4% paraform solution for 48 hr, and then the artery were removed and un-hydrated in sucrose solution step by step (10%, 20%, 30%), then 6-lm frozen sections were prepared, and tissue slices were stored at 80 C. The rest of the tissues were held in liquid nitrogen for 10 min and then were stored at 80 C. 1063 right ventricular pressure (RVP) were measured. Right ventricle (RV), left ventricle (LV), and septum (S) were isolated, and after the absorbing of water using ﬁlter paper, RV and LVþS was weighed. The RV/(LVþS) and RV/ BW (body weight) ratios were calculated and used as indexes for right ventricular hypertrophy. Fluorescence Immunohistochemistry The frozen sections were taken out from the refrigerator (80 C), placed in room temperature for 30 min, and then they were soaked in 1% PBS for 5 min and endogenous peroxidase were inactivated with 3% H2O2 (diluted with methanol) for 30 min. After washed with 1% PBS, the sections were blocked with 10% goat blood serumþ5% bovine albumin for 20 min, then the sections were incubated overnight with rabbit polyclonal IgG [jOR-1 (H-70), Santa Cruz Biotechnology] at 4 C, after surplus IgG[j-OR-1 (H-70)] was washed off, the samples were incubated with a second rabbit IgG(HþL)/TRITC for 45 min at 37 C, surplus anti-rabbit IgG (HþL)/ TRITC were washed off, the sections were blocked with 50% glycerol, and PAs were imaged with an Olympus ﬂuorescent microscope (400). Ten ﬁelds of view were selected in every sample randomly (400); positive cells were counted with image pro-plus software. Determination the Expression of j-ORs by Western-Blot The PAs were minced and homogenized in lysis buffer (Tris 20 mM, NaCl 50 mM, NaF 50 mM, Na4P2O710H2O 5 mM, C12H22O11 25 mM, DTT 1 mM, Na3VO4 2 mM and 1% protease inhibitor cocktail, pH 7.4) by a Heidolph DIA900 tissue homogenizer (Heidolph Instruments GmbH, KG, Schwabach, Germany) (Li et al., 2007). The homogenate was centrifuged (12,000g at 4 C for 10 min), the supernatant was decanted, and total protein level was determined with a BSA assay kit. After the aforementioned protocol, equal amounts of protein was electrophoresed in SDS-polyacrylamide gel, and electrophoretically transferred to a poly (vinylidene diﬂuoride) membrane (Millipore, Billerica, MA). After blocking with 5% skim milk in TBS at room temperature for 1 hr, the membrane was incubated with a rabbit polyclonal IgG[jOR-1 (H-70)] overnight at 4 C. The membrane was then washed with PBS and incubated with a HRP-conjugated IgG antibody for 1 hr at 37 C. b-actin was selected as a housekeeping protein and was analyzed following the same procedure, using a speciﬁc antib-actin mouse monoclonal antibody. The straps were developed with an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL). Immunoblotting was visualized with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA), and the relative value of the protein densities were analyzed with Lab Image software. Hemodynamics in Hypoxia After hypoxia for 30 min, rats were anesthetized with peritoneal injection with 30 g L1 pentobarbital sodium (1.5 mL kg1, ip). According to Michelakis et al. (2002), a microcatheter was inserted into right ventricle and pulmonary artery through right external jugular vein, and the mean pulmonary arterial pressure (mPAP) and Primary PASMCs Culture and Immunohistochemistry Staining of Actin Sprague–Dawley male rats (180–200 g) were anesthetized with a peritoneal injection of 30 g L1 pentobarbital sodium (1.5 mL kg1, ip) and skin was sterilized with 75% alcohol. Chest was opened, and heart and lung 1064 PENG ET AL. TABLE 1. Changes in hemodynamics in rats exposed to chronic hypoxia Group Control 1-Week hypoxia 2-Week hypoxia 4-Week hypoxia mPAP (mmHg) 15.2 25.5 27.9 32 2.3 1.8a 2.2a 3.3a RVP (mmHg) 25.6 34.8 35.6 36.8 1.7 2.1a 1.7a 2.0a RV/(LVþS) 0.26 0.30 0.38 0.41 0.04 0.04 0.06a,b 0.07a,b,c RV/BW (mg/g) 0.58 0.65 0.90 0.99 0.06 0.07 0.06a,b 0.08a,b,c N ¼ 8; 1 mmHg ¼ 0.133 kPa. mPAP, mean pulmonary artery pressure; RVP, right ventricular pressure; RV, right ventricle; LV, left ventricle; S, septum; BW, body weight. a P < 0.01 versus control. b P < 0.01 versus 1-week hypoxia group. c P< 0.01 versus 2-week hypoxia group. were removed. The organs were placed into D-hanks solution (Ca2þ and My2þ-free, pH 7.4, 4 C) and rinsed for several times. The PAs were segregated in a sterile manner, and then the obtained PASMCs were cultured as previously described (Campbell et al., 1979). Brieﬂy, the outer spheres were peeled and the microtubules were sniped visually, endotheliums were shaved slightly for 2–3 times to remove endothelial cells. The tunica medias were placed in a DMEM culture solution, then prepared into scraps (1 mm3) where these primary PASMCs were cultured (37 C, 5%CO2; the culture solution was DMEM containing 20% fetal bovine serum). PASMCs were found to be surrounding the scraps of the PA about 4–7 days later. The third to sixth generations of cells were used for the experiments. The PASMCs were seeded onto glass coverslips, then they were ﬁxed in pure liquor pyroaceticus for 20 min, washed with distilled water, and the endogenous peroxydase was inactivated by 0.5%H2O2 (diluted with methanol) for 30 min. After washed with distilled water, the PASMCs were blocked with 10% goat blood serum for 20 min at room temperature, and then incubated overnight with rabbit polyclonal a-actin (Sigma Company) at 4 C. After washed off with 1%PBS, the samples were incubated with 1% anti-rabbit IgG for 20 min at 37 C, then the surplus anti-rabbit IgG were washed off with 1% PBS, and then the samples were incubated with 1% SABC for 20 min at 37 C. After been washed, the samples were stained with DAB and hematoxylin, cells were imaged with a light microscope. Light microscopic observation of cultured rat PASMCs, PASMCs crept from tissue pieces like Fusiform shape, After 4–7 days, they demonstrate typical morphological patterns (multilayer sheets and ‘‘hills and valleys’’). Immunohistochemistry staining of actin in rat PASMC showed that a-actin distributed in cytoplasm in yellow. Cells were divided into 5 groups: (1) normal oxygen state, (2) hypoxia for 12 hr, (3) hypoxia for 24 hr, (4) hypoxia for 36 hr, (5) hypoxia for 48 hr. Hypoxic groups were cultured in an incubator (37 C, 2%O2, 5% CO2, 93%N2) (Kendro, Germany). The hypoxia exposures were initiated at different times according to the durance of the hypoxia exposure so the hypoxia exposures were ended at the same time. After hypoxia, experiments for all the ﬁve groups were performed at the same time. Cell Fluorescence Immunohistochemistry The PASMCs were seeded onto glass coverslips, ﬁxed in 4% paraform solution for 20 min, then washed in 1% PBS for 5 min 3 times, and inactivated by endogenous peroxidase with 3%H2O2 (diluted with methanol) for 30 min. After washed in 1% PBS for 5 min 3 times, the samples were blocked with 10% goat blood serum and 5% bovine albumin for 20 min, then incubated overnight with rabbit polyclonal IgG [jOR-1 (H-70)] at 4 C. After surplus rabbit IgG [jOR-1 (H-70)] were washed off, the samples were incubated with anti-rabbit IgG (HþL)/ TRITC for 45 min at 37 C, then the surplus anti-rabbit IgG (HþL)/TRITC was washed off, and ﬁnally the samples were incubated with Hoechst 33258 for 5 min, and rinsed for 5 min 3 times. Cover slips were blocked with 50% glycerol and cells were imaged with the Olympus ﬂuorescent microscope (400). The value of the protein densities were analyzed with image pro-plus software (Media Cybernetics). Mean ﬂuorescence intensity of TRITC was measured by a gray scale value (Changez et al., 2006). Ten ﬁelds of view in each sample were selected at random (400) to measure cell ﬂuorescence intensity with image pro-plus software. Antibody and Kit Rabbit polyclonal IgG [jOR-1 (H-70)] (sc-9112) (j-OR-1 is a rabbit polyclonal antibody raised against amino acids 1–70 of j-OR-1 of human origin, and its molecular weight is 58 kDa), and Actin primary antibody (sc1616r) were purchased from Santa Cruz Biotechnology. The secondary antibody (goat anti-rabbit antibody) conjugated with HRP was purchased from Boshide company. Rabbit IgG (HþL)/TRITC made by Dingguo Company in Beijing. Protein Quantitation Kit and chemiluminescence kits were purchased from Pierce Company. Statistical Analysis Values are presented as mean SD. One-way ANOVA was employed to determine the difference among groups. Signiﬁcance level was set at P < 0.05. RESULTS Hemodynamics in Hypoxia Pulmonary hypertension developed after 1 week of hypoxia in the rats. Compared with the normal oxygen state control group, the mPAP (Gong et al., 2004), the RVP, the RV/ (LVþS) ratio and the RV/BW ratio of the hypoxia state group all increased signiﬁcantly in all three hypoxic groups. Aforementioned changes were typical at 2 weeks and more signiﬁcant at 4 weeks (Table 1). j-OR IN PULMONARY ARTERY 1065 Fig. 2. Changes of expression of j-ORs protein in hypoxia (N ¼ 6). P < 0.01 vs. control group; bP < 0.01 versus hypoxia for 2-week group. (Molecular weight of j-OR-1: 58 kDa). a Fig. 1. Expression of j-opioid receptors in PAs (400, N ¼ 6). Expression of j-opioid receptors in inner membrane (*), in outer membrane (#), and mainly in the layer of SMC (arrows) in PAs. Panel a: A, Negative; B, 10% goat blood serum replaced IgG [jOR-1 (H-70)]; C, normaxic group; D, Hypoxia for 1 week group; E, Hypoxia for 2-week group; F, Hypoxia for 4-week group. Panel b: Group results, aP < 0.01 versus control group, bP < 0.01 versus hypoxia for 2 week group. Expression of j-ORs in PAs During Hypoxia The j-OR was distributed in all three layers of the PAs. Results of ﬂuorescence immunohistochemistry (Fig. 1) demonstrated that j-ORs were mainly distributed in the layer of the smooth muscle. Results of ﬂuorescence immunohistochemistry (Fig. 1b) and Western-Blot (Fig. 2) demonstrated that expression of the j-OR increased signiﬁcantly in the PA during hypoxia (P < 0.01). Expression of the j-ORs reached maximum after 2 week of hypoxia (P < 0.01). The j-ORs expression returned to the 1 week level after prolonged hypoxia for 4 week. Expression of j-ORs in PASMCs During Hypoxia The j-ORs were located in the nucleus of normal PASMCs, appearing as a relatively large particle, while the expression of the j-ORs increased during hypoxia (P < 0.05, N ¼ 10). Expression of the j-ORs reached maximum after 24 hr of hypoxia (P < 0.01). Additionally, the morphology of the j-ORs appeared as a relatively small particle under hypoxic conditions (Fig. 3a,b). We used the same method in primary cultured cardiomyocytes and found that j-ORs are located in plasma membrane and cytoplasm in cardiomyocytes (Fig. 3aI). DISCUSSION The major ﬁndings in the present study are: (1) j-ORs were mainly distributed in the smooth muscle layer of the PAs; (2) j-ORs were mainly located in the nucleus in the normal PASMCs, appearing as a relatively large particle, and the morphology of the j-ORs was altered into a relatively small particle under hypoxia conditions; (3) expression of the j-ORs in the PAs and the PASMCs were signiﬁcantly increased during hypoxia. Our previous studies (Pei et al., 2006; Sun et al., 2006) showed that j-ORs might be located in the PAs of the rat. The present study has demonstrated that j-ORs are located in the PAs of the rat. Further, the j-ORs were mainly distributed in the smooth muscle layer of the PAs, and correspondingly, the j-ORs were mainly located in the nucleus of the normal PASMCs. It is well known that opioid receptors belong to a super family of G-protein coupled receptors, which are characterized by seven transmembrane hydrophobic regions and it has been reported that the j-ORs exist in cardiac tissue (Ventura et al., 1989; Tai et al., 1991; Jin et al., 1995; Peart et al., 2008). Consistent with these studies, we utilized the same method in the primary cultured myocardial cell and found that j-ORs were located in the plasma membrane and the cytoplasm in myocardial cells. Svingos and Colago et al. (2002) have reported that j-ORs are localized in intracellular compartments and plasma membranes of individual dendrites. OP4 (ORL1, NOP1) receptors particularly concentrate in the perinuclear area with a granular pattern in endothelial cells from various vascular regions (Granata et al., 2003). The construction of OP4 receptors is similar to that of j-ORs. Therefore, we hypothesize that the distribution of the j-OR may be dissimilar in different types of cells. Our previous studies (Liu et al., 2008) have showed that the expression of j-ORs upregulated in the heart of I/R (ischemia and reperfusion) rats. Bhargava et al. (1997) showed that j-opioid receptors density was 1066 PENG ET AL. Fig. 3. Expression of j-ORs in PASMCs and cardiomyocytes with ﬂuorescence immunohistochemistry (400, N ¼ 10). Panel a: A, Negative; B, Control; C, Hypoxia for 24 h; D, Hypoxia for 12 h; E, Hypoxia for 12 hr (Hoechst 33258); F, Hypoxia for 12 hr (merge D and E); G, Hypoxia for 48 hr; H, nucleus (incubated with Hoechst 33258) of PASMCs with the same view of E; I, cardiomyocytes. Arrows showing the j-ORs (red) in PASMCs and cardiomyocytes, nucleus (blue) incubated with Hoechst 33258. To testify that our method for determination of j-ORs distribution in PASMCs is correct, we used the same method in primary cultured cardiomyocytes and found that j-ORs locate in plasma membrane and cytoplasm in cardiomyocytes (I). Panel b: Group results of IOD (OD/lm2),cP < 0.05, aP < 0.01 versus control group, bP < 0.05 versus hypoxia for 12 hr, dP < 0.01 versus hypoxia for 24 hr. augmented in sensitized lung parenchyma, while l- and d-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. In the present study, we found that expression of j-ORs in the SMC layer of PAs were increased during hypoxia. In order to further determine changes of j-ORs expression in hypoxia, we cultured primary PASMC and found that the expression of j-ORs was increased in hypoxia. The results are consistent with the above mentioned results in vivo. Qing, et al. (2001) reported an upregulation of CGRP and ADM receptors in the lung of rats exposed to 1 and 2 week of chronic hypobaric hypoxia. Other investigators have shown that ETA receptors are increased in the lungs of chronically hypoxic rats (Li et al., 1994; Soma et al., 1999). These studies indicate that receptors of vasomotor factors may signiﬁcantly increase in hypoxia. But the underlying mechanisms are still unknown. Numerous laboratories have shown that the plasma level of Dynorphin A, an endogenous j-OR agonist, has been shown to increase signiﬁcantly after hypoxia (Lu et al., 1997; Shen et al., 1998). And our previous research revealed that Dynorphin A can relax the PA (Sun et al., 2006) and also showed that U50,488H, a selective j-OR agonist, decreased mean mPAP in vivo in rats. Thus, it is possible that an increased level of Dynorphin stimulates the expression of j-ORs during hypoxia. Further, the lasting release of endogenous Dynorphin down regulates the expression of j-ORs in the PAs after hypoxia for 2 week. It should be noted that the mPAP, the RVP, the RV/ (LVþS) ratio and the RV/BW ratio of hypoxia groups were all increased signiﬁcantly compared with the normal oxygen control group. These changes were typical at 2 weeks and more signiﬁcant at 4 weeks. Lachmanová et al. (2005) showed that free radical injury can happen in the pulmonary vascular wall in early under hypoxic exposure. We hypothesize that the injury induced by hypoxia may also impact the expression of j-ORs after hypoxia for 2 week. But the underlying mechanism of the increasing expression of the j-ORs during hypoxia needs further investigation. In the future, we hope to elucidate whether the release of endogenous Dynorphin changes during hypoxia, and/or whether nor-binaltorphimine, a selective j-opioid receptor antagonist, inhibits the expression of j-ORs in the PAs of the rat exposed to hypoxia for 2 week compared with normal oxygen control group. In conclusion, the present study has provided evidence for the ﬁrst time of the precise location of the j-ORs in the PAs and PASMCs in rats, and that hypoxia induces an increased expression of j-ORs in the PAs. 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