Chloride channel activity in human lung fibroblasts and myofibroblasts

Zhaohong Yin, Mitchell Aaron Watsky

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30 Citations (Scopus)

Abstract

It is well established that transforming growth factor (TGF)-β stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (ICl-LPA) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of ICl-LPA on fibroblast-to-myofibroblast differentiation. We recorded ICl-LPA using the amphotericin perforated-patch technique. We activated I Cl-LPA using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-α-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-β (myofibroblasts) had significantly elevated α-SMA levels and ICl-LPA current density compared with control fibroblasts. I Cl-LPA activation was blocked by DIDS, 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 μM). DIDS and NPPB, in a dose-dependent manner, significantly reduced α-SMA levels in HLF stimulated with TGF-β. These results demonstrate the receptor-mediated activation of I Cl-LPA by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal ICl-LPA activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume288
Issue number6 32-6
DOIs
StatePublished - Jun 1 2005
Externally publishedYes

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Chloride Channels
Myofibroblasts
Human Activities
Fibroblasts
Lung
Transforming Growth Factors
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Smooth Muscle
Actins
lysophosphatidic acid
Lysophosphatidic Acid Receptors
Growth Factor Receptors
Amphotericin B
Glycerol
Phospholipids
Western Blotting
Immunohistochemistry

Keywords

  • α-smooth muscle actin
  • Lysophosphatidic acid
  • Sphingosine-1-phosphate
  • Transforming growth factor-β

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Cell Biology
  • Physiology

Cite this

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title = "Chloride channel activity in human lung fibroblasts and myofibroblasts",
abstract = "It is well established that transforming growth factor (TGF)-β stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (ICl-LPA) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of ICl-LPA on fibroblast-to-myofibroblast differentiation. We recorded ICl-LPA using the amphotericin perforated-patch technique. We activated I Cl-LPA using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-α-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-β (myofibroblasts) had significantly elevated α-SMA levels and ICl-LPA current density compared with control fibroblasts. I Cl-LPA activation was blocked by DIDS, 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 μM). DIDS and NPPB, in a dose-dependent manner, significantly reduced α-SMA levels in HLF stimulated with TGF-β. These results demonstrate the receptor-mediated activation of I Cl-LPA by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal ICl-LPA activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.",
keywords = "α-smooth muscle actin, Lysophosphatidic acid, Sphingosine-1-phosphate, Transforming growth factor-β",
author = "Zhaohong Yin and Watsky, {Mitchell Aaron}",
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AU - Yin, Zhaohong

AU - Watsky, Mitchell Aaron

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N2 - It is well established that transforming growth factor (TGF)-β stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (ICl-LPA) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of ICl-LPA on fibroblast-to-myofibroblast differentiation. We recorded ICl-LPA using the amphotericin perforated-patch technique. We activated I Cl-LPA using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-α-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-β (myofibroblasts) had significantly elevated α-SMA levels and ICl-LPA current density compared with control fibroblasts. I Cl-LPA activation was blocked by DIDS, 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 μM). DIDS and NPPB, in a dose-dependent manner, significantly reduced α-SMA levels in HLF stimulated with TGF-β. These results demonstrate the receptor-mediated activation of I Cl-LPA by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal ICl-LPA activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.

AB - It is well established that transforming growth factor (TGF)-β stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (ICl-LPA) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of ICl-LPA on fibroblast-to-myofibroblast differentiation. We recorded ICl-LPA using the amphotericin perforated-patch technique. We activated I Cl-LPA using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-α-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-β (myofibroblasts) had significantly elevated α-SMA levels and ICl-LPA current density compared with control fibroblasts. I Cl-LPA activation was blocked by DIDS, 5-nitro-2-(3- phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 μM). DIDS and NPPB, in a dose-dependent manner, significantly reduced α-SMA levels in HLF stimulated with TGF-β. These results demonstrate the receptor-mediated activation of I Cl-LPA by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal ICl-LPA activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.

KW - α-smooth muscle actin

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