scholarly journals RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia

2018 ◽  
Vol 314 (2) ◽  
pp. C166-C176 ◽  
Author(s):  
Lindsay M. Herbert ◽  
Thomas C. Resta ◽  
Nikki L. Jernigan
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Ion Channel ◽  
Chronic Hypoxia ◽  
Membrane Localization ◽  
Hypoxic Pulmonary Hypertension ◽  
Plasma Membrane Localization ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+ levels and enhanced RhoA/Rho kinase-dependent Ca2+ sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We previously showed that acid-sensing ion channel 1a (ASIC1a)-mediated Ca2+ entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension. However, the enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from pulmonary hypertensive animals is not dependent on an increase in ASIC1a protein expression, suggesting that chronic hypoxia (CH) stimulates ASIC1a function through other regulatory mechanism(s). RhoA is involved in ion channel trafficking, and levels of activated RhoA are increased following CH. Therefore, we hypothesize that activation of RhoA following CH increases ASIC1a-mediated Ca2+ entry by promoting ASIC1a plasma membrane localization. Consistent with our hypothesis, we found greater plasma membrane localization of ASIC1a following CH. Inhibition of RhoA decreased ASIC1a plasma membrane expression and largely diminished ASIC1a-mediated Ca2+ influx, whereas activation of RhoA had the opposite effect. A proximity ligation assay revealed that ASIC1a and RhoA colocalize in PASMC and that the activation state of RhoA modulates this interaction. Together, our findings show a novel interaction between RhoA and ASIC1a, such that activation of RhoA in PASMC, both pharmacologically and via CH, promotes ASIC1a plasma membrane localization and Ca2+ entry. In addition to enhanced RhoA-mediated Ca2+ sensitization following CH, RhoA can also activate a Ca2+ signal by facilitating ASIC1a plasma membrane localization and Ca2+ influx in pulmonary hypertension.

scholarly journals PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells

2016 ◽  
Vol 310 (5) ◽  
pp. C390-C400 ◽  
Author(s):  
Lindsay M. Herbert ◽  
Carlos H. Nitta ◽  
Tracylyn R. Yellowhair ◽  
Carly Browning ◽  
Laura V. Gonzalez Bosc ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Membrane Localization ◽  
Arterial Smooth Muscle ◽  
Plasma Membrane Localization ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
Pulmonary Arterial Smooth Muscle

Acid-sensing ion channel 1 (ASIC1) contributes to Ca2+ influx and contraction in pulmonary arterial smooth muscle cells (PASMC). ASIC1 binds the PDZ (PSD-95/Dlg/ZO-1) domain of the protein interacting with C kinase 1 (PICK1), and this interaction is important for the subcellular localization and/or activity of ASIC1. Therefore, we first hypothesized that PICK1 facilitates ASIC1-dependent Ca2+ influx in PASMC by promoting plasma membrane localization. Using Duolink to determine protein-protein interactions and a biotinylation assay to assess membrane localization, we demonstrated that the PICK1 PDZ domain inhibitor FSC231 diminished the colocalization of PICK1 and ASIC1 but did not limit ASIC1 plasma membrane localization. Although stimulation of store-operated Ca2+ entry (SOCE) greatly enhanced colocalization between ASIC1 and PICK1, both FSC231 and shRNA knockdown of PICK1 largely augmented SOCE. These data suggest PICK1 imparts a basal inhibitory effect on ASIC1 Ca2+ entry in PASMC and led to an alternative hypothesis that PICK1 facilitates the interaction between ASIC1 and negative intracellular modulators, namely PKC and/or the calcium-calmodulin-activated phosphatase calcineurin. FSC231 limited PKC-mediated inhibition of SOCE, supporting a potential role for PICK1 in this response. Additionally, we found PICK1 inhibits ASIC1-mediated SOCE through an effect of calcineurin to dephosphorylate the channel. Furthermore, it appears PICK1/calcineurin-mediated regulation of SOCE opposes PKA phosphorylation and activation of ASIC1. Together our data suggest PKA and PICK1/calcineurin differentially regulate ASIC1-mediated SOCE and these modulatory complexes are important in determining downstream Ca2+ signaling.

scholarly journals Chronic hypoxia limits H2O2-induced inhibition of ASIC1-dependent store-operated calcium entry in pulmonary arterial smooth muscle

2014 ◽  
Vol 307 (5) ◽  
pp. L419-L430 ◽  
Author(s):  
Danielle R. Plomaritas ◽  
Lindsay M. Herbert ◽  
Tracylyn R. Yellowhair ◽  
Thomas C. Resta ◽  
Laura V. Gonzalez Bosc ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Chronic Hypoxia ◽  
Inhibitory Effect ◽  
Alternative Mechanism ◽  
Arterial Smooth Muscle ◽  
Hypoxic Pulmonary Hypertension ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
Expression And Activity

Our laboratory shows that acid-sensing ion channel 1 (ASIC1) contributes to the development of hypoxic pulmonary hypertension by augmenting store-operated Ca2+ entry (SOCE) that is associated with enhanced agonist-induced vasoconstriction and arterial remodeling. However, this enhanced Ca2+ influx following chronic hypoxia (CH) is not dependent on an increased ASIC1 protein expression in pulmonary arterial smooth muscle cells (PASMC). It is well documented that hypoxic pulmonary hypertension is associated with changes in redox potential and reactive oxygen species homeostasis. ASIC1 is a redox-sensitive channel showing increased activity in response to reducing agents, representing an alternative mechanism of regulation. We hypothesize that the enhanced SOCE following CH results from removal of an inhibitory effect of hydrogen peroxide (H2O2) on ASIC1. We found that CH increased PASMC superoxide (O2·−) and decreased rat pulmonary arterial H2O2 levels. This decrease in H2O2 is a result of decreased Cu/Zn superoxide dismutase expression and activity, as well as increased glutathione peroxidase (GPx) expression and activity following CH. Whereas H2O2 inhibited ASIC1-dependent SOCE in PASMC from control and CH animals, addition of catalase augmented ASIC1-mediated SOCE in PASMC from control rats but had no further effect in PASMC from CH rats. These data suggest that, under control conditions, H2O2 inhibits ASIC1-dependent SOCE. Furthermore, H2O2 levels are decreased following CH as a result of diminished dismutation of O2·− and increased H2O2 catalysis through GPx-1, leading to augmented ASIC1-dependent SOCE.

Chronic hypoxia elevates intracellular pH and activates Na+/H+ exchange in pulmonary arterial smooth muscle cells

2005 ◽  
Vol 289 (5) ◽  
pp. L867-L874 ◽  
Author(s):  
Eon J. Rios ◽  
Michele Fallon ◽  
Jian Wang ◽  
Larissa A. Shimoda
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Intracellular Ph ◽  
Chronic Hypoxia ◽  
Rt Pcr ◽  
Arterial Smooth Muscle ◽  
Gene And Protein Expression ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
Pulmonary Arterial Smooth Muscle

Chronic hypoxia (CH), caused by many lung diseases, results in pulmonary hypertension due, in part, to increased muscularity of small pulmonary vessels. Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to growth factors requires increased intracellular pH (pHi) mediated by activation of Na+/H+ exchange (NHE); however, the effect of CH on PASMC pHi homeostasis is unknown. Thus we measured basal pHi and NHE activity and expression in PASMCs isolated from mice exposed to normoxia or CH (3 wk/10% O2). pHi was measured using the pH-sensitive fluorescent dye BCECF-AM. NHE activity was determined from Na+-dependent recovery from NH4-induced acidosis, and NHE expression was determined by RT-PCR and immunoblot. PASMCs from chronically hypoxic mice exhibited elevated basal pHi and increased NHE activity. NHE1 was the predominate isoform present in mouse PASMCs, and both gene and protein expression of NHE1 was increased following exposure to CH. Our findings indicate that exposure to CH caused increased pHi, NHE activity, and NHE1 expression, changes that may contribute to the development of pulmonary hypertension, in part, via pH-dependent induction of PASMC proliferation.

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