Role Of κ-opioid Receptor In Hypoxic Pulmonary Artery Hypertension And Its Underlying Mechanism

Background:Hypoxic pulmonary hypertension (HPH) is a progressive disorder characterized byabnormally high blood pressure in the pulmonary artery caused by hypoxemia after allkinds of respiratory system diseases. Both chronic obstructive pulmonary disease andliving in the plateau for a long time can lead to HPH. Hypoxia-induced pulmonaryvasoconstriction and pulmonary vascular remodeling are two key pathophysiologicprocesses in HPH. First, hypoxia-induced pulmonary vasoconstriction is a compensatorymechanism, redirecting blood flow from alveoli with a lower oxygen content to alveoliwith a higher oxygen content and improving ventilation/perfusion ratio. However,long-term hypoxia can result in irreversible vasoconstriction in the pulmonary artery andfurther lead to HPH. Then, hypoxia upsets the balance between the vasoconstrictor (ET-1, Ang Ⅱ) and vasodilator (NO, PGI), triggering endothelial dysfunction and graduallyleading to vascular remodeling characterized by proliferation of pulmonary artery smoothmuscle cells. In the end, HPH and right ventricular hypertrophy come into being.Therefore, dilating pulmonary artery, preventing endothelial dysfunction and inhibitingsmooth muscle cell proliferation are potential strategies for the prevention and treatmentof HPH.It has been demonstrated that endogenous opioid peptide and its receptor playsignificant role in cardiovascular system, of which the main subtype is κ opioid receptor(κ-OR). Our previous study demonstrates that selective κ-OR agonist U50,488Hadministered after ischemia/reperfusion exhibits an anti-arrhythmic effect, decreasescardiomyocyte necrosis and apoptosis and inhibits inflammation after reperfusion. Theprotective effects of κ-OR are associated with the activation of PI3K-Akt-eNOS pathwaymediated by κ-OR, which increases NO synthesis. The impairment of Akt-eNOS pathwayis one of the important mechanisms of endothelial dysfunction. However, it remainsunclear whether κ-OR ameliorates endothelial function through the activation ofAkt-eNOS pathway. Moreover, hypoxia-induced pulmonary vasoconstriction andpulmonary vascular proliferation are two key processes in HPH. Our previous studydemonstrates that U50,488H has a definite dilating effect on pulmonary artery and adefinite inhibitive effect on pulmonary artery smooth muscle cell proliferation. However,whether those effects exist in HPH and whether those are mediated by κ-OR remainobscure.By utilizing rat HPH model, this study aims to determine whether κ-OR activationantagonizes HPH and further improves endothelial function and pulmonary artery dilation,and inhibits pulmonary artery smooth muscle proliferation and to elucidate the role ofκ-OR in antagonizing HPH and its underlying mechanism. The results may provide newtherapeutic target for the prevention and treatment of HPH and provide experimentalevidence for the clinical application of opioid in the prevention and treatment of HPH,which reveals significant theoretical and clinical implication. Aims:1. To investigate the effect of κ-OR activation on antagonizing HPH.2. To investigate the effect of κ-OR activation on dilating pulmonary artery in HPH rats.3. To clarify the effect of κ-OR activation on the pulmonary artery smooth muscle cellproliferation induced by hypoxia.4. To investigate the effect of κ-OR activation on endothelial dysfunction in HPH rats.Methods:1. Hypoxic condition was created for8hours every day with the exposure of the rats toboth low pressure and low oxygen (air pressure50kpa, oxygen concentration10%), sothe HPH rat model was established. The effects of κ-OR agonist U50,488H and κ-ORantagonist nor-BNI were studied in HPH rats in vivo.2. After hypoxia, rats were anesthetized via peritoneal injection with pentobarbitalsodium (60mg/kg, IP injected). A micro-catheter (diameter=1mm) was inserted intothe pulmonary artery through the right external jugular vein. Then the meanpulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) weremeasured.3. The hearts and blood were then harvested. Each of the following was isolated in orderto calculate the right ventricular hypertrophy index (RVHI): body weight (BW), rightventricle (RV), left ventricle (LV), and septum (S). The RVHI itself was expressed asthe tissue weight ratio of RV/(LV+S) and RV/BW. Expression of κ-OR was determinedby Western-Blot, and concentration of endogenous dynorphin A was determined byELISA.4. The pulmonary artery was carefully isolated and cleaned of fat and connective tissue.In vitro vascular ring perfusion was utilized. U50,488H and dynorphin A were addedrespectively to determine its relaxation effect on the artery rings in HPH rats.5. The effect of U50,488H on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) under hypoxic condition was measured by MTT and [3H]-thymidine(3H-TdR) incorporation assay.6. Isolated perfusion of pulmonary artery ring was used to determine the reaction of theartery ring to ACh, which is an endothelium-dependent vasodilator. Besides, the effectof U50,488H i.p. administration on the reaction of the artery ring to ACh.7. The serum NO was determined by measuring the concentration of nitrite, a stablemetabolite of nitric oxide, through a modified Griess reaction method. The underlyingmechanism was investigated by several inhibitors such as nor-BNI (a selective κ-ORantagonist), wortmannin (a selective PI3K antagonist), AI (a selective PI3Kantagonist), and L-NAME (a non-selective NOS inhibitor).8. The primary culture and subculture of pulmonary microvascular endothelial cells(PMVECs) was performed. The effect of U50,488H on PMVECs apoptosis induced byhypoxia was determined by TUNEL staining. The phosphorylation of Akt and eNOSin the PMVECs was detected by Western-Blot after U50,488H administration inhypoxia.Results:1. The HPH was established after exposing rats to chronic hypoxia for2weeks. ThemPAP and RVP of rats in hypoxia were significantly higher compared with both themPAP and RVP of normoxic rats. Compared with the hypoxia2w group, the hypoxia2w+U50,488H group showed a significant decrease in mPAP, RVP, RV/(LV+S) andRV/BW, and the effect of U50,488H was abolished by nor-BNI.2. Compared with the normoxia group, the expression of κ-OR in pulmonary artery wasincreased in the hypoxia group. U50,488H further up-regulated the expression of κ-ORin the hypoxia groups, which was abolished by nor-BNI.3. The level of dynorphin A was increased at1wk and2wks after hypoxia, whereas itreturned to below baseline level at4wks exposure to hypoxia.4. U50,488H exhibited both time-dependent and dose-dependent relaxation effect on HPH rat pulmonary artery rings. This vasorelaxing effect was abolished by nor-BNIand L-NAME. Dynorphin A also showed time-dependent relaxation effect and theeffect was completely abolished by nor-BNI.5. When PASMCs were exposed to hypoxia, the quantity of3H-TdR incorporation andOD optimum in MTT detection increased significantly compared to the normoxicgroup. U50,488H significantly inhibited the PASMCs proliferation in a dose dependentmanner (10~100μmol/L), which was also abolished by nor-BNI.6. Chronic hypoxia resulted in a significant endothelial dysfunction. This dysfunctionwas demonstrated through decreased vasorelaxation in response to ACh. U50,488Hadministration significantly improved the pulmonary artery relaxation response toACh.7. Hypoxia resulted in a significant decrease in serum NO compared with that in thenormoxic group. U50,488H pretreatment significantly restored serum NO content inHPH rats, which was abolished by nor-BNI, wortmannin (a PI3K inhibitor), AI (aselective Akt inhibitor) and L-NAME (a non-selective NOS inhibitor).8. U50,488H antagonized endothelial cell apoptosis, which was abolished by nor-BNI,wortmannin (a PI3K inhibitor), AI (a selective Akt inhibitor) and L-NAME (anon-selective NOS inhibitor).9. In the cultured PMVECs that were exposed to hypoxia, U50,488H treatment increasedAkt phosphorylation and endothelial nitric oxide synthase (eNOS) phosphorylationsignificantly, which were abolished by both nor-BNI and wortmannin.Conclusions:1. κ-opioid receptor activation exhibits definite effect against HPH. Hypoxia at an earlystage stimulates the release of dynorphin and upregulates κ-OR expression, which maybe a compensatory reaction but needs further investigation.2. The mechanism underlying the effect of antagonizing HPH mediated by κ-ORinvolves dilating pulmonary artery in HPH rats, improving pulmonary artery endothelial dysfunction in HPH rats and inhibiting excessive proliferation of PASMCsinduced by hypoxia, etc.3. U50,488H administration further activates κ-OR. For one thing, U50,488H dilatespulmonary artery through NOS pathway. For another, U50,488H improves endothelialdysfunction through PI3K-Akt-eNOS pathway.