Shu-Miao Zhang

Myocardial Apoptosis and Infarction after Ischemia/Reperfusion Are Attenuated by κ-Opioid Receptor Agonist

BACKGROUND AND AIMS It remains unclear whether U50488H (a selective kappa-opioid receptor agonist) produces anti-apoptotic effect during ischemia and reperfusion (I/R). Therefore, the effect of U50488H on myocardial apoptosis was investigated in the present study. METHODS Rats were subjected to 45min coronary artery occlusion and 180min of reperfusion. U50488H (1.5mg/kg IV) was given prior to occlusion. Nor-Binaltorphimine (nor-BNI) (2mg/kg IV), a selective kappa-opioid receptor antagonist, was given 10min prior to U50488H. Cardiac apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay and in situ identification of nuclear DNA fragmentation. RESULTS The ultrastructure injury of myocardium, myocardial infarct size, and plasma CK and LDH were reduced significantly with administration of U50488H before I/R, whereas the effects of U50488H were abolished by nor-BNI. DNA fragments were visualized by agarose electrophoresis, and clear DNA ladder formation was observed in myocardial tissue from hearts subjected to I/R. Administration of U50488H before ischemia exerted a significant anti-apoptotic effect as evidenced by markedly weaker DNA ladder formation. TUNEL staining showed U50488H treatment before I/R significantly reduced the percentage of apoptotic cells, which was blocked by 5-HD, a mitochondrial k(ATP) channel blocker. In accordance, U50488H treatment significantly inhibited I/R-induced elevated activities of caspase-3 and caspase-9. U50488H also produced an increase in Bcl-2 and a decrease in Bax protein expression in the I/R heart, and the anti-apoptotic effects of U50488H were all blocked by nor-BNI. CONCLUSIONS U50488H reduces myocardial necrosis and apoptosis after I/R and activation of kappa-opioid receptor may mediate a role in U50488H-induced myocardial protection.

Effects of U50,488H on hypoxia pulmonary hypertension and its underlying mechanism

The aim of the present study was to determine whether U50,488H, a selective kappa-opioid receptor agonist, inhibits the remodeling of the pulmonary artery (PA). In addition, changes in the concentrations of nitric oxide (NO), endothelin (ET) and angiotensin II (AngII) in hypoxic pulmonary hypertensive (HPH) rats were investigated to explore the mechanisms underlying the effects of U50, 488H on HPH. We found that intraperitoneal administration of U50,488H (every other day) during hypoxia depressed mean pulmonary arterial pressure (mPAP) and attenuated right ventricular pressure (RVP) and right ventricular hypertrophy, at the same time it inhibited remodeling of the PA compared with hypoxia for 2 wk. Moreover, U50,488H also inhibited proliferation of the pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia for 48 h in a dose-dependent manner. Compared with the 2 wk hypoxia group, U50,488H increased the concentration of NO and decreased the production of ET and AngII (P<0.01). In addition, acute intravenous administration of U50,488H after hypoxia for 4 wk decreased mPAP. Our results suggest that effects of anti-remodeling of the PA and anti-proliferation of the PASMC, and regulation of the vasomotor factors in both blood and pulmonary tissues of HPH rats may be critical mechanisms underlying the preventive and therapeutic effects of U50,488H in HPH rats.

κ-Opioid receptor stimulation modulates TLR4/NF-κB signaling in the rat heart subjected to ischemia-reperfusion.

It is well documented that the Toll-like receptor 4 (TLR4)/NF-κB signaling mediates early inflammation during myocardial ischemia and reperfusion. Our previous study has demonstrated that κ-opioid receptor stimulation with U50,488H produces cardioprotective and anti-inflammatory effects. The aim of the present study was to investigate whether κ-opioid receptor stimulation could modulate the TLR4/NF-κB signaling and reduce neutrophil accumulation and TNF-α induction in an ischemia-reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia and reperfusion (MI/R), and MI/R+U50,488H in the absence or presence of Nor-BNI, a selective κ-opioid receptor antagonist. The results demonstrated that after MI/R, the expressions of myocardial TLR4 and NF-κB increased significantly both in ischemia area and risking area. Compared with MI/R, κ-opioid receptor stimulation with U50,488H significantly attenuated the expressions of TLR4 and NF-κB. At the mean time, it also reduced myeloperoxidase (MPO) levels, both serum and myocardial TNF-α production, myocardial infarct sizes (INF/AAR%) and myocardial apoptosis induced by MI/R, all the effects of U50,488H were abolished by Nor-BNI. These data provide evidence for the first time that κ-opioid receptor stimulation inhibits TLR4/NF-κB signaling in the rat heart subjected to MI/R.

Antiarrhythmic effect mediated by κ-opioid receptor is associated with Cx43 stabilization.

Objective:Acute myocardial ischemia induces electrical and chemical uncoupling of gap junctions, which contributes to conduction abnormalities and re-entrant arrhythmias. We tested the hypothesis that structure and function of Connexin43 may vibrate during acute myocardial ischemia and reperfusion and &kgr;-opioid receptor stimulation may stabilize the alteration of Connexin43. Design:An animal intervention study was conducted with comparison to a control group. Setting:University preclinical research laboratory. Subjects:Age-, weight-, and sex-matched Sprague-Dawley rats. Interventions:Adult rat hearts were subjected to ischemia or ischemia/reperfusion, which was induced by temporary occlusion of the left main coronary artery. U50488H was given 10 mins before tissue specimens were taken or before ischemia (1.5 mg/kg, intravenous) and nor-BNI was given 15 mins before tissue specimens were taken or before ischemia (2 mg/kg, intravenous). Tissue samples came from left ventricular myocardium of the rat hearts. Measurements and Main Results:Electrocardiogram, immunohistochemistry, immunoblotting, and reverse transcription–polymerase chain reaction were used to measure changes of arrhythmias, protein, and gene expression of Connexin43, respectively. &kgr;-opioid receptor activation with U50 decreased arrhythmia in a model of myocardial ischemia and reperfusion. In normal hearts, immunohistochemical data showed reduced amount and lateralization of Connexin43 induced by &kgr;-opioid receptor activation, whereas immunoblotting data demonstrated no significant changes between control and U50 group. During ischemia, however, Connexin43 protein underwent dephosphorylation and degradation, and Connexin43 mRNA was upregulated. These alterations were significantly attenuated on &kgr;-opioid receptor stimulation. During ischemia and reperfusion, Connexin43 protein underwent dephosphorylation and degradation and recovered slowly during reperfusion. Activation of &kgr;-opioid receptor accelerated recovery of phosphorylated and total Connexin43. Conclusions:In normal rat hearts, Connexin43 translocates from intercellular junctions to intracellular locations on &kgr;-opioid receptor activation. In rat hearts experiencing acute myocardial ischemia and reperfusion, protein and gene expression of Connexin43 undergo vibration. This phenomenon is stabilized when &kgr;-opioid receptor is activated and by the fact that &kgr;-opioid receptor produces antiarrhythmic effects.

Distribution of κ-Opioid Receptor in the Pulmonary Artery and its Changes During Hypoxia

The present study evaluated the distribution of κ‐opioid receptors (κ‐ORs) in pulmonary arteries (PAs) in rats and investigated whether κ‐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 κ‐ORs was determined by fluorescence immunohistochemistry and changes in κ‐ORs expression in PAs and PASMCs were determined by fluorescence immunohistochemistry or Western blot techniques. The κ‐ORs were primarily distributed in the smooth muscle layer of the PAs and in the nucleus of PASMCs. The expression of the κ‐ORs were increased in PAs of rats subjected to hypoxia for 1–4 week (P < 0.01). Accordingly, the expression of κ‐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 κ‐ORs in PAs and PASMCs of rats and that hypoxia upregulates expression of κ‐ORs. Anat Rec, 2009.

U50,488H inhibits neutrophil accumulation and TNF-α induction induced by ischemia–reperfusion in rat heart

The role of the κ-opioid receptor in inflammation is not well understood. The aim of this study was to investigate whether the κ-opioid receptor agonist U50,488H modulates neutrophil accumulation and TNF-α induction in an ischemia-reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia-reperfusion (MI/R) alone, MI/R+U50,488H, MI/R+U50,488H+Wortmannin, and MI/R+U50,488H+L-NAME. The results demonstrated that compared to MI/R, U50,488H reduced myocardial infarction area, myocardial myeloperoxidase (MPO) levels, serum creatinine kinase (CK) levels, and both serum and myocardial TNF-α production. Increases were seen in NOx levels in the myocardium subjected to MI/R injury. All demonstrated effects of U50,488H were abolished by Nor-BNI, a selective κ-opioid receptor antagonist; Wortmannin, a specific PI3K inhibitor; or L-NAME, a nitric oxide synthase (NOS) inhibitor. In summary, κ-opioid receptor stimulation with U50,488H produces both cardioprotective and anti-inflammatory effects. These effects may be associated with an increase in NO production and the inhibition of neutrophil accumulation and TNF-α induction via a PI3K sensitive pathway in myocardium subjected to MI/R.

Modulation of intracellular calcium transient in response to β-adrenoceptor stimulation in the hearts of 4-wk-old rats during simulated weightlessness

Modulation of intracellular calcium ([Ca(2+)](i)) transient in response to beta-adrenoceptor stimulation in the hearts of hindlimb unweighted (HLU) rats during simulated weightlessness has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness. Effects of simulated microgravity on beta-adrenoceptor responsiveness were then studied. Mean arterial blood pressure, left ventricular pressure (LVP), systolic function [maximum positive change in pressure over time (+dP/dt(max))], and diastolic function [maximum negative change in pressure over time (-dP/dt(max))] were monitored during the in vivo experiment. beta-Adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. +/-dP/dt(max), myocyte contraction, intracellular [Ca(2+)](i) transient, and L-type calcium current in response to beta-adrenoceptor stimulation with isoproterenol were measured. Compared with the control group, no significant changes were found in heart weight, body weight, and mean arterial blood pressure, whereas LVP and +/-dP/dt(max) were significantly reduced. LVP and +/-dP/dt(max) were significantly attenuated in the HLU group in response to isoproterenol administration. In the in vitro study, the beta-adrenoceptor density was unchanged. Effects of isoproterenol on electrically induced single-cell contraction and [Ca(2+)](i) transient in myocytes of ventricles in HLU rats were significantly attenuated. The enhanced L-type Ca(2+) current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The above results indicate that impaired function of L-type Ca(2+) current and decreased [Ca(2+)](i) transient cause the depressed responsiveness of the beta-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.

Modulation of β-adrenoceptor signaling in the hearts of 4-wk simulated weightlessness rats

The modulation of beta-adrenoceptor signaling in the hearts of hindlimb unweighting (HU) simulated weightlessness rats has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness; then the effects of simulated microgravity on beta-adrenoceptor signaling were studied. Mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (-dP/dtmax) were monitored in the course of the in vivo experiment. Single rat ventricular myocyte was obtained by the enzymatic dissociation method. Hemodynamics, myocyte contraction, and cAMP production in response to beta-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured, and Gs protein was also determined. Compared with the control group, no significant changes were found in heart weight, body weight and ABP, while LVP and +/-dP/dtmax were significantly reduced. The ABP decrease, LVP increase, and +/-dP/dtmax in response to isoproterenol administration were significantly attenuated in the HU group. The effects of isoproterenol on electrically induced single-cell contraction and cAMP production in myocytes of ventricles in the HU rats were significantly attenuated. The biologically active isoform, Gsalpha (45 kDa) in the heart, was unchanged. Both the increased electrically induced contraction and cAMP production in response to forskolin were also significantly attenuated in the simulated weightlessness rats. Above results indicated that impaired function of adenylyl cyclase causes beta-adrenoceptor desensitization, which may be partly responsible for the depression of cardiac function.

κ-opioid receptor activation prevents against arrhythmias by preserving Cx43 protein via alleviation of intracellular calcium.

&kgr;-opioid receptor (&kgr;-OR) activation with U50,488H, a selective &kgr;-OR agonist, has been previously demonstrated to prevent against cardiac arrhythmias via stabilizing the synthesis and degradation of an integral membrane protein, Cx43, in gap junctions. However, the exact prevention mechanism remains unclear. The present study tested the hypothesis that the kappa OR agonist U50,488H mediates the prevention of arrhythmia through the regulation of intracellular calcium leading to the preservation of Cx43 protein. By performing electrocardiogram monitoring and immunoblotting in isolated Langendorff-perfused rat hearts, high concentrations of calcium-perfused rat hearts exhibited increased cardiac arrhythmias. Diminished expression of Cx43 protein was observed. The utilization of a whole-cell patch clamp technique revealed that U50,488H inhibited L-type calcium current in single ventricular myocytes in a dose-dependent manner. These effects were blocked by nor-binaltorphimine, potent and selective &kgr;-OR antagonists. Administration of U50,488H before myocardial ischemia resulted in an attenuated of total arrhythmia scores. The attenuation effect was blocked by nor-binaltorphimine. The attenuation effect was antagonized both by Bay K8644, a L-type calcium channel agonist, and also by the Cx43 uncoupler heptanol. Finally, immunoblotting data demonstrated that the preservation of Cx43 protein conferred by U50,488H was reversed in the presence of Bay K8644. In summary, the present study demonstrates &kgr;-OR activation with U50,488H may confer antiarrhythmic effects via modulation of the calcium-Cx43 pathway.

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

Background/Aims: In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms. Methods: Rats were exposed to 10% oxygen for three weeks to induce HPH. After hypoxia, the mean pulmonary arterial pressure (mPAP) and the right ventricular pressure (RVP) were measured. Cell viability was monitored using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry and Western blot. Autophagy was assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay and by Western blot. Results: Inhibition of autophagy by the administration of chloroquine prevented the development of HPH in the rat model, as evidenced by significantly reduced mPAP and RVP, as well as decreased autophagy. U50,488H mimicked the effects of chloroquine, and the effects of U50,488H were blocked by nor-BNI, a selective κ-opioid receptor antagonist. In vitro experiments showed that the inhibition of autophagy by chloroquine was associated with decreased proliferation and increased apoptosis of PASMCs. Under hypoxia, U50,488H also significantly inhibited autophagy, reduced proliferation and increased apoptosis of PASMCs. These effects of U50,488H were blocked by nor-BNI. Moreover, exposure to hypoxic conditions significantly increased AMPK phosphorylation and reduced mTOR phosphorylation, and these effects were abrogated by U50,488H. The effects of U50,488H on PASMC autophagy were inhibited by AICAR, a selective AMPK agonist, or by rapamycin, a selective mTOR inhibitor. Conclusion: Our data provide evidence for the first time that κ-opioid receptor stimulation protects against HPH by inhibiting PASMCs autophagy via the AMPK-mTOR pathway.