ASIC1 contributes to pulmonary vascular smooth muscle store-operated Ca²⁺entry

Abstract

Acid-sensing ion channels (ASIC) are voltage-insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). It is possible that ASIC contribute to vascular reactivity via Na(+) and Ca(2+) conductance; however, their function in VSM is largely unknown. In pulmonary VSM, store-operated Ca(2+) entry (SOCE) plays a significant role in vasoregulatory mechanisms such as hypoxic pulmonary vasoconstriction and receptor-mediated arterial constriction. Therefore, we hypothesized that ASIC contribute to SOCE in pulmonary VSM. We examined SOCE resulting from depletion of intracellular Ca(2+) stores with cyclopiazonic acid in isolated small pulmonary arteries and primary cultured pulmonary arterial smooth muscle cells by measuring 1) changes in VSM [Ca(2+)](i) using fura-2 indicator dye, 2) Mn(2+) quenching of fura-2 fluorescence, and 3) store-operated Ca(2+) and Na(+) currents using conventional whole cell patch-clamp configuration in voltage-clamp mode. The role of ASIC was assessed by the use of the ASIC inhibitors, amiloride, benzamil, and psalmotoxin 1, or siRNA directed towards ASIC1, ASIC2, or ASIC3 isoforms. We found that store-operated VSM [Ca(2+)](i) responses, Mn(2+) influx, and inward cationic currents were attenuated by either pharmacological ASIC inhibition or treatment with ASIC1 siRNA. These data establish a unique role for ASIC1 in mediating SOCE in pulmonary VSM and provide new insight into mechanisms of VSM Ca(2+) entry and pulmonary vasoregulation.

The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway

“…However, ASIC1b (a splice variant of ASIC1) and ASIC3 are not permeable to Ca 2ϩ (7,17). As our results indicate the functional significance of ASIC1 and ASIC3 as downstream effectors of phenamil, we speculate that activation of the calcineurin-NFAT pathway by phenamil might be a result of sodium imbalance.…”

“…ASICs are voltage-insensitive, pH-sensitive cationic channels permeable to Ca 2ϩ , Na ϩ , and K ϩ . Phenamil is known to inhibit ASICs more potently than other amiloride analogs (17). Four different ASIC genes have been identified, and ASIC proteins form homomeric or hetereomeric cation channels that are proton gated.…”

“…Ca 2؉ depletion/repletion assay. The calcium depletion/repletion assay was performed as described previously (17). Briefly, PAC1 cells were plated onto a 10-cm plate and stimulated with different concentrations of phenamil for 24 h. The cells were trypsinized and resolubilized in Ca -sensitive fluorescent indicator fura-2 AM (2 M) (Molecular Probes), excess dye was washed off, and the cells were resolubilized in PSS containing diltiazem and CPA at 1 ϫ 10 6 cells/ml.…”

“…ASICs are essential for vSMC migration both at basal and PDGF-stimulated conditions (14). ASIC1 protein level is also elevated in pulmonary arteries under chronic-hypoxia treatment (17).…”

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

“…ture, our laboratory has recently shown that ASIC1 is an important facilitator of G protein-coupled receptor signaling via store-operated Ca 2ϩ entry (SOCE) in pulmonary arterial smooth muscle cells (PASMC) (21,22). Furthermore, ASIC1-mediated Ca 2ϩ entry in PASMC appears to be an important constituent of both the active vasoconstrictor and vascular remodeling components of chronic hypoxic-induced pulmonary hypertension (21,31).…”