Hydrogen peroxide is a critical regulator of the hypoxia-induced alterations of store-operated Ca(2+) entry into rat pulmonary arterial smooth muscle cells.

To investigate the association between store-operated Ca2+ entry (SOCE) and reactive oxygen species (ROS) during hypoxia, this study determined the changes of transient receptor potential canonical 1 (TRPC1) and Orai1, two candidate proteins for store-operated Ca2+ (SOC) channels and their gate regulator, stromal interaction molecule 1 (STIM1), in a hypoxic environment and their relationship with ROS in pulmonary arterial smooth muscle cells (PASMCs). Exposure to hypoxia caused a transient Ca2+ spike and subsequent Ca2+ plateau of SOCE to be intensified in PASMCs when TRPC1, STIM1, and Orai1 were upregulated. SOCE in cells transfected with specific short hairpin RNA (shRNA) constructs was almost completely eliminated by the knockdown of TRPC1, STIM1, or Orai1 alone and was no longer affected by hypoxia exposure. Hypoxia-induced SOCE enhancement was further strengthened by PEG-SOD but was attenuated by PEG-catalase, with correlated changes to intracellular hydrogen peroxide (H2O2) levels and protein levels of TRPC1, STIM1, and Orai1. Exogenous H2O2 could mimic alterations of the interactions of STIM1 with TRPC1 and Orai1 in hypoxic cells. These findings suggest that TRPC1, STIM1, and Orai1 are essential for the initiation of SOCE in PASMCs. Hypoxia-induced ROS promoted the expression and interaction of the SOC channel molecules and their gate regulator via their converted product, H2O2.

Chen TX ，Xu XY ，Zhao Z ，Zhao FY ，Gao YM ，Yan XH ，Wan Y ... -  《-》

Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.

Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.

Lin AH ，Sun H ，Paudel O ，Lin MJ ，Sham JS ... -  《-》

TRPC1 and Orai1 interact with STIM1 and mediate capacitative Ca(2+) entry caused by acute hypoxia in mouse pulmonary arterial smooth muscle cells.

Ng LC ，O'Neill KG ，French D ，Airey JA ，Singer CA ，Tian H ，Shen XM ，Hume JR ... -  《-》

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels.

Cheng KT ，Liu X ，Ong HL ，Ambudkar IS ... -  《-》

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells.

Shi J ，Miralles F ，Kinet JP ，Birnbaumer L ，Large WA ，Albert AP ... -  《-》