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

Wenju Lu, Jian Wang, Gongyong Peng, Larissa Shimoda, J. T. Sylvester

Research output: Contribution to journalArticle

Abstract

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 Ca 2+-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 [Ca 2+]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 [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 Ca2+ release and STIM1-dependent activation of SOC.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume297
Issue number1
DOIs
StatePublished - Jul 2009

Fingerprint

Smooth Muscle
Small Interfering RNA
Lung
Smooth Muscle Myocytes
Cell Hypoxia
Reticulum
Messenger RNA
Fura-2
Real-Time Polymerase Chain Reaction
Microscopy
Membrane Proteins
Proteins
Western Blotting
Hypoxia
Stromal Interaction Molecule 1
Polymerase Chain Reaction

Keywords

  • Calcium signaling
  • Hypoxic pulmonary vasoconstriction
  • KCl
  • STIM2
  • TRPC1
  • Vascular smooth muscle

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology
  • Physiology

Cite this

@article{fe7b431937684bb7b9609f2773dbf8b9,
title = "Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle",
abstract = "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 Ca 2+-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 [Ca 2+]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 [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 Ca2+ release and STIM1-dependent activation of SOC.",
keywords = "Calcium signaling, Hypoxic pulmonary vasoconstriction, KCl, STIM2, TRPC1, Vascular smooth muscle",
author = "Wenju Lu and Jian Wang and Gongyong Peng and Larissa Shimoda and Sylvester, {J. T.}",
year = "2009",
month = "7",
doi = "10.1152/ajplung.00063.2009",
language = "English (US)",
volume = "297",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "1",

}

TY - JOUR

T1 - Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle

AU - Lu, Wenju

AU - Wang, Jian

AU - Peng, Gongyong

AU - Shimoda, Larissa

AU - Sylvester, J. T.

PY - 2009/7

Y1 - 2009/7

N2 - 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 Ca 2+-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 [Ca 2+]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 [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 Ca2+ release and STIM1-dependent activation of SOC.

AB - 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 Ca 2+-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 [Ca 2+]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 [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 Ca2+ release and STIM1-dependent activation of SOC.

KW - Calcium signaling

KW - Hypoxic pulmonary vasoconstriction

KW - KCl

KW - STIM2

KW - TRPC1

KW - Vascular smooth muscle

UR - http://www.scopus.com/inward/record.url?scp=67650079717&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67650079717&partnerID=8YFLogxK

U2 - 10.1152/ajplung.00063.2009

DO - 10.1152/ajplung.00063.2009

M3 - Article

VL - 297

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 1

ER -