Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle.
Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca(2+) in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca(2+)-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca(2+) depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca(2+) signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca(2+)-sensitive dye, fura 2, to measure basal intracellular Ca(2+) concentration ([Ca(2+)](i)), store-operated Ca(2+) entry (SOCE), and increases in [Ca(2+)](i) caused by acute hypoxia (4% O(2)) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and abolished the sustained [Ca(2+)](i) response to hypoxia but did not alter the initial transient [Ca(2+)](i) response to hypoxia, the [Ca(2+)](i) response to depolarization, or basal [Ca(2+)](i). The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca(2+)](i) induced by acute hypoxia in these cells required SR Ca(2+) release and STIM1-dependent activation of SOC.
Lu
Wenju
W
Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland 21224, USA.
Wang
Jian
J
Peng
Gongyong
G
Shimoda
Larissa A
LA
Sylvester
J T
JT
D000208:Acute Disease; D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects; D002712:Chlorides / Q000494:pharmacology; D005453:Fluorescence; D016257:Fura-2 / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology; D007211:Indoles / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D011189:Potassium Chloride / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D002712:Chlorides; D007211:Indoles; D017895:Manganese Compounds; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; C518857:Stim1 protein, rat; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D011189:Potassium Chloride; C025340:manganese chloride; D016257:Fura-2; C000543:cyclopiazonic acid