Knockdown of Stromal Interaction Molecule 1 (STIM‐1) Decreases Store‐operated Calcium Entry (SOCE) and Attenuates Hypoxic Calcium Response in Pulmonary Artery Smooth Muscle Cells (PASMC)

Abstract

The increased intracellular Ca2+ concentration ([Ca2+]i) caused by acute hypoxia in PASMC requires Ca2+ release from sarcoplasmic reticulum (SR) and influx through store‐operated Ca2+ channels (SOCC). Recent evidence in a variety of cell types, including smooth muscle, suggests that STIM‐1 senses Ca2+ concentration within the SR lumen and transduces activation of SOCC upon SR Ca2+ release and depletion. To determine if STIM‐1 plays a role in the [Ca2+]i response to hypoxia in PASMC, we measured [Ca2+]i with the fluorescent Ca2+‐sensitive dye, Fura‐2, in primary cultures of rat distal PASMC treated with nontargeted small interfering RNA (siRNA) or siRNA targeted to STIM‐1. Compared to nontargeted siRNA, STIM‐1 siRNA caused: 1) a 92% decrease in STIM‐1 mRNA, as measured by real‐time quantitative PCR; 2) an 81% decrease in STIM‐1 protein, as measured by Western blotting; 3) no change in mRNA for STIM‐2, a related protein that may regulate STIM‐1; 4) 74% inhibition of the increase in [Ca2+]i caused by restoration of extracellular Ca2+ in cells perfused with Ca2+‐free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete SR Ca2+ stores and nifedipine to block Ca2+ entry through L‐type voltage‐operated Ca2+ channels; 5) 77% inhibition of increase in [Ca2+]i caused by hypoxia (4% O2) in PASMC perfused with normal KRBS; and 6) no inhibition of the increase in [Ca2+]i caused by equimolar (60 mM) substitution of KCl for NaCl in the normal KRBS perfusate. These results indicate that STIM‐1 is expressed in rat distal PASMC and plays an important role in SOCE and the [Ca2+]i response to acute hypoxia in these cells.