Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase

2004 ◽  
Vol 287 (2) ◽  
pp. L352-L359 ◽  
Author(s):  
Anastasia Sobolewski ◽  
Karen B. Jourdan ◽  
Paul D. Upton ◽  
Lu Long ◽  
Nicholas W. Morrell
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Adenylyl Cyclase ◽  
Muscle Cells ◽  
Protein Levels ◽  
Prostacyclin Receptor ◽  
Pulmonary Arterial ◽  
Pulmonary Artery Smooth Muscle

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 μg/ml of cycloheximide. Desensitization was heterologous since desensitization to other Gsα-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 μM), adrenomedullin (100 nM), or bradykinin (1 μM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 μM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 μM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the Gi inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous Gsα desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.

scholarly journals Untargeted Metabolic Profiling Cell-Based Approach of Pulmonary Artery Smooth Muscle Cells in Response to High Glucose and the Effect of the Antioxidant Vitamins D and E

Metabolites ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 87 ◽  
Author(s):  
Abdulwahab Alamri ◽  
Abdulhadi Burzangi ◽  
Paul Coats ◽  
David Watson
Keyword(s):  
Reactive Oxygen Species ◽  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
High Glucose ◽  
Muscle Cells ◽  
Oxygen Species ◽  
Pulmonary Arterial ◽  
Pulmonary Artery Smooth Muscle

Pulmonary arterial hypertension (PAH) is a multi-factorial disease characterized by the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Excessive reactive oxygen species (ROS) formation resulted in alterations of the structure and function of pulmonary arterial walls, leading to right ventricular failure and death. Diabetes mellitus has not yet been implicated in pulmonary hypertension. However, recently, variable studies have shown that diabetes is correlated with pulmonary hypertension pathobiology, which could participate in the modification of pulmonary artery muscles. The metabolomic changes in PASMCs were studied in response to 25 mM of D-glucose (high glucose, or HG) in order to establish a diabetic-like condition in an in vitro setting, and compared to five mM of D-glucose (normal glucose, or LG). The effect of co-culturing these cells with an ideal blood serum concentration of cholecalciferol-D3 and tocopherol was also examined. The current study aimed to examine the role of hyperglycemia in pulmonary arterial hypertension by the quantification and detection of the metabolomic alteration of smooth muscle cells in high-glucose conditions. Untargeted metabolomics was carried out using hydrophilic interaction liquid chromatography and high-resolution mass spectrometry. Cell proliferation was assessed by cell viability and the [3H] thymidine incorporation assay, and the redox state within the cells was examined by measuring reactive oxygen species (ROS) generation. The results demonstrated that PASMCs in high glucose (HG) grew, proliferated faster, and generated higher levels of superoxide anion (O2·−) and hydrogen peroxide (H2O2). The metabolomics of cells cultured in HG showed that the carbohydrate pathway, especially that of the upper glycolytic pathway metabolites, was influenced by the activation of the oxidation pathway: the pentose phosphate pathway (PPP). The amount of amino acids such as aspartate and glutathione reduced via HG, while glutathione disulfide, N6-Acetyl-L-lysine, glutamate, and 5-aminopentanoate increased. Lipids either as fatty acids or glycerophospholipids were downregulated in most of the metabolites, with the exception of docosatetraenoic acid and PG (16:0/16:1(9Z)). Purine and pyrimidine were influenced by hyperglycaemia following PPP oxidation. The results in addition showed that cells exposed to 25 mM of glucose were oxidatively stressed comparing to those cultured in five mM of glucose. Cholecalciferol (D3, or vitamin D) and tocopherol (vitamin E) were shown to restore the redox status of many metabolic pathways.

scholarly journals Upregulation of miR-361-3p suppresses serotonin-induced proliferation in human pulmonary artery smooth muscle cells by targeting SERT

2020 ◽  
Vol 25 (1) ◽  
Author(s):  
Ying Zhang ◽  
Yongbin Chen ◽  
Guo Chen ◽  
Yingling Zhou ◽  
Hua Yao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Pulmonary Vascular Resistance ◽  
Vascular Resistance ◽  
Muscle Cells ◽  
Luciferase Reporter ◽  
Pulmonary Arterial ◽  
Pulmonary Artery Smooth Muscle

Abstract Background Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is a key mechanism in pulmonary arterial hypertension (PAH). Serotonin (5-hydroxytryptamine, 5-HT) can induce abnormal proliferation of PASMCs. The role of miR-361-3p in serotonin-induced abnormal PASMCs proliferation remains unclear. Methods The miR-361-3p level was analyzed in plasma from PAH patients and normal controls and in human PASMCs (hPASMCs) using RT-PCR. The hPASMCs were transfected with an miR-361-3p mimic and then treated with serotonin. Untransfected hPASMCs were used as the control. Cell proliferation was evaluated using an MTS assay and 5-ethynyl-2′-deoxyuridine (EdU) staining. The cell cycle stages were evaluated using flow cytometry. The association between miR-361-3p and serotonin transporter (SERT) was determined using a luciferase reporter assay and anti-AGO2 RNA immunoprecipitation assay. The protein expression was evaluated via western blotting. Results The miR-361-3p level was lower in plasma from PAH patients than in plasma from the any of the normal control subjects. The mean pulmonary arterial pressure, pulmonary vascular resistance and pulmonary vascular resistance index were higher in PAH patients whose miR-361-3p level was lower than the median value for patients than in those whose miR-361-3p level was higher than the median. Serotonin treatment reduced miR-361-3p expression in the hPASMCs. MiR-361-3p overexpression suppressed cell proliferation, promoted apoptosis, induced G1 arrest, and decreased the phosphorylation level of ERK1/2 in serotonin-treated hPASMCs. SERT was identified as an miR-361-3p target. Its overexpression alleviated the effect of miR-361-3p overexpression on serotonin-induced hPASMC proliferation and upregulation of phosphorylated ERK1/2. Conclusions The miR-361-3p level is lower in the plasma of PAH patients. Upregulation of miR-361-3p suppresses serotonin-induced proliferation of hPASMCs by targeting SERT. Our results suggest that miR-361-3p is a potential therapeutic target in PAH.

Abstract 379: Enhancement of TRPC6-mediated Ca 2+ Entry in Pulmonary Artery Smooth Muscle Cells of Idiopathic Pulmonary Arterial Hypertension Patients

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ying Yu ◽  
Olga Safrina ◽  
Oleksandr Platoshyn ◽  
Michael D Cahalan ◽  
Lewis J Rubin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Arterial Hypertension ◽  
Pulmonary Artery ◽  
Arterial Hypertension ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Idiopathic Pulmonary Arterial Hypertension ◽  
Pulmonary Arterial ◽  
Pulmonary Artery Smooth Muscle ◽  
Excessive Proliferation

Background & Hypothesis: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and sustained pulmonary vasoconstriction are thought to play critical roles in the development of idiopathic pulmonary arterial hypertension (IPAH). Recently, we demonstrated that upregulation of the canonical transient receptor potential 6 (TRPC6) channel contributes to excessive proliferation of PASMCs isolated from IPAH patients. This study aimed at characterizing whether up-regulated TRPC6 expression affects resting cytosolic [Ca 2+ ] ([Ca 2+ ] cyt ) level and Ca 2+ entry in PASMCs of IPAH patients. Methods & Results: [Ca 2+ ] cyt was measured by fluorescence ratio video imaging with the Ca 2+ indicator fura-2. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable diacylglycerol analog that activates TRPC6 channels, was used to stimulate channel activities in the presence of extracellular Ca 2+ . The resting [Ca 2+ ] cyt andOAG-mediated increase in [Ca 2+ ]cyt were significantly higher in PASMCs from IPAH patients compared to PASMCs isolated from secondary pulmonary artery hypertension and normotensive patients. In PASMCs from IPAH patients, inhibition of TRPC6 expression by adenovirus-mediated siRNA specifically targeted the human TRPC6 gene led to an approximately 90% reduction of TRPC6 mRNA and protein levels and significantly attenuated OAG-mediated increase in [Ca 2+ ] cyt . Treatment of the IPAH-PASMCs with siRNA also decreased the resting [Ca 2+ ] cyt and significantly inhibited cell proliferation in comparison to cells treated with scrambled control siRNA. Furthermore, exogenous overexpression of human TRPC6 increased the resting [Ca 2+ ] cyt and enhanced OAG-mediated Ca 2+ entry in normal PASMCs. C onclusions: These results suggest that upregulation of TRPC6 channels in PASMC from IPAH patients serves as an important pathway for agonist-mediated Ca 2+ entry, mitogen-mediated PASMC proliferation and, ultimately, pulmonary vascular remodeling. Targeting TRPC6 expression and function in PASMCs would help develop novel therapeutic approaches for IPAH patients.

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