Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells

A novel mechanism of hypoxic pulmonary hypertension

Mo Jun Lin, George P H Leung, Wei Min Zhang, Xiao Ru Yang, Kay Pong Yip, Chung Ming Tse, James Sham

Research output: Contribution to journalArticle

Abstract

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca 2+]i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

Original languageEnglish (US)
Pages (from-to)496-505
Number of pages10
JournalCirculation Research
Volume95
Issue number5
DOIs
StatePublished - Sep 3 2004

Fingerprint

Pulmonary Hypertension
Smooth Muscle Myocytes
Thapsigargin
Up-Regulation
Lung
Cations
Glycerol
Pulmonary Artery
Blood Vessels
Fura-2
Nifedipine
Small Interfering RNA
Reverse Transcription
Hypoxia
Homeostasis
Western Blotting
Cell Proliferation
Polymerase Chain Reaction
Genes

Keywords

  • Pulmonary hypertension
  • Receptor-operated Ca channels
  • Store-operated Ca channels
  • Transient receptor potential channels

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells : A novel mechanism of hypoxic pulmonary hypertension. / Lin, Mo Jun; Leung, George P H; Zhang, Wei Min; Yang, Xiao Ru; Yip, Kay Pong; Tse, Chung Ming; Sham, James.

In: Circulation Research, Vol. 95, No. 5, 03.09.2004, p. 496-505.

Research output: Contribution to journalArticle

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abstract = "Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca 2+]i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.",
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AU - Yang, Xiao Ru

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AU - Tse, Chung Ming

AU - Sham, James

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N2 - Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca 2+]i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

AB - Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca 2+]i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

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