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

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. 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. 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. 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.

Zhou Y ，Wang Y ，Wang X ，Tian X ，Zhang S ，Yang F ，Guo H ，Fan R ，Feng N ，Jia M ，Gu X ，Wang Y ，Li J ，Pei J ... -  《-》

Role of κ-opioid receptor in hypoxic pulmonary artery hypertension and its underlying mechanism.

Zhang L ，Li J ，Shi Q ，Fan R ，Kaye AJ ，Wang Y ，Sun X ，Rivera FB ，Kaye AD ，Pei J ... -  《-》

The effect of activated κ-opioid receptor (κ-OR) on the role of calcium sensing receptor (CaSR) in preventing hypoxic pulmonary hypertension development.

κ-opioid receptor (κ-OR) plays a key role in preventing hypoxic pulmonary hypertension (HPH) development after activated by exogenous agonist U50,488H. Calcium sensing receptor (CaSR) activation induces HPH by promoting vasoconstriction and vascular remodeling. The activated κ-OR is reported to inhibit the expression of CaSR in pulmonary artery smooth muscle cells (PASMCs). Thus, in this study, we aimed to explore the effect of activated κ-OR on the role of CaSR in preventing HPH development. An HPH rat model was constructed using Sprague-Dawley rats. Changes in mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) mediated by κ-OR agonist U50,488H and CaSR inhibitor NPS2143 were observed. The effects of CaSR agonist spermine and inhibitor NPS2143 on pulmonary artery tension were tested. The expression and localization of κ-OR and CaSR were measured in isolated PASMCs. A cell-counting kit-8 assay was performed to evaluate the effect of spermine in PASMC proliferation. Expression of proliferating cell nuclear antigen (PCNA), Erk, and p-Erk was evaluated by western blot analysis. Results showed that κ-OR and CaSR were co-expressed and colocalized in PASMCs under normoxic and hypoxic conditions. Interactions between κ-OR and CaSR were also observed. Spermine improved vasoconstriction in the pulmonary artery in HPH rats, which was abolished by U50,488H. RVP and mPAP were significantly increased in HPH rats under CaSR stimulation, but were significantly reduced when the rats were pretreated with U50,488H and NPS2143 (P < 0.01). Spermine treatment significantly promoted PASMC proliferation, which was significantly inhibited by U50,488H, p38 inhibitor SB203580, JNK inhibitor SP600125, Erk inhibitor SCH772984, and MEK inhibitor U0126, especially Erk inhibitor (P < 0.01). Spermine significantly increased PCNA and P-Erk expression in hypoxic conditions, which was inhibited by U50,488H and NPS2143. κ-OR stimulation prevented HPH development via the CaSR/MAPK signaling pathway.

Li J ，Jia M ，Liu M ，Cao Z ，Wang X ，Feng N ，Gu X ，Zhang S ，Fan R ，Guo H ，Wang Y ，Liu M ，Pei J ... -  《-》

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

Li J ，Zhang P ，Zhang QY ，Zhang SM ，Guo HT ，Bi H ，Wang YM ，Sun X ，Liu JC ，Cheng L ，Cui Q ，Yu SQ ，Kaye AD ，Yi DH ，Pei JM ... -  《-》

Quaternary ammonium salt of U50,488H elicits protective effects against hypoxic pulmonary hypertension.

The present study aimed to investigate the role of quaternary ammonium salt of U50,488H (Q-U50,488H) in hypoxic pulmonary hypertension (HPH) and underlying mechanisms involved. A HPH animal model was established in rats under hypoxia and the mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) were measured. Relaxation of the pulmonary artery in response to Q-U50,488H was determined. In addition, expression and activity of endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) with NO content, Akt expression, total antioxidant capacity (T-AOC), and gp91phox were evaluated. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. We demonstrated that both the molecular weight and solubility of Q-U50,488H were higher than that of U50,488H. Q-U50,488H reduced mPAP and RVP and prevented the development of HPH. Moreover, Q-U50,488H relaxed the pulmonary arteries from both normal and HPH rats in a time-dependent manner. Under hypoxic conditions, Q-U50,488H significantly increased Akt phosphorylation, eNOS phosphorylation, NO content in serum, and T-AOC in pulmonary arteries of HPH rats. In addition, the activity of eNOS was elevated, but the activity of iNOS was reduced when Q-U50,488H was given under hypoxia. Q-U50,488H significantly counteracted the increase of gp91phox expression in pulmonary arteries under hypoxia. In addition, in vitro studies suggested that Q-U50,488H inhibited pulmonary artery smooth muscle cells (PASMCs) proliferation under hypoxic conditions and that the effects of Q-U50,488H were blocked by nor-binaltorphimine (nor-BNI). Thus, our results provided evidence that Q-U50,488H plays a protective role against HPH via κ-opioid receptor stimulation.

Zhou Y ，Tian X ，Wang X ，Wang Y ，Fan R ，Wang Y ，Feng N ，Zhang S ，Guo H ，Gu X ，Jia M ，Yin W ，Hou Z ，Li J ，Pei J ... -  《-》