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This Article Full Text Full Text (PDF) All Versions of this Article: 297/1/L17    most recent 00063.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Lu, W. Articles by Sylvester, J. T. PubMed PubMed Citation Articles by Lu, W. Articles by Sylvester, J. T.

Knockdown of stromal interaction molecule 1 attenuates store-operated Ca²⁺ entry and Ca²⁺ responses to acute hypoxia in pulmonary arterial smooth muscle

Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland

Submitted 26 February 2009 ; accepted in final form 19 April 2009

Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca²⁺ in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca²⁺-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca²⁺ depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca²⁺ signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca²⁺-sensitive dye, fura 2, to measure basal intracellular Ca²⁺ concentration ([Ca²⁺]_i), store-operated Ca²⁺ entry (SOCE), and increases in [Ca²⁺]_i caused by acute hypoxia (4% O₂) 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 [Ca²⁺]_i response to hypoxia but did not alter the initial transient [Ca²⁺]_i response to hypoxia, the [Ca²⁺]_i response to depolarization, or basal [Ca²⁺]_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 [Ca²⁺]_i induced by acute hypoxia in these cells required SR Ca²⁺ release and STIM1-dependent activation of SOC.

STIM2; TRPC1; hypoxic pulmonary vasoconstriction; calcium signaling; vascular smooth muscle; KCl