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 ICl-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. ICl-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 ICl-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.