Volume-sensitive release of organic osmolytes in the human lung epithelial cell line A549: role of the 5-lipoxygenase

Abstract

Holm JB, Grygorczyk R, Lambert IH. Volume-sensitive release of organic osmolytes in the human lung epithelial cell line A549: role of the 5-lipoxygenase. Am J Physiol Cell Physiol 305: C48-C60, 2013. First published March 13, 2013; doi:10.1152/ajpcell.00412.2012.-Pathophysiological conditions challenge cell volume homeostasis and perturb cell volume regulatory mechanisms leading to alterations of cell metabolism, active transepithelial transport, cell migration, and death. We report that inhibition of the 5-lipoxygenase (5-LO) with AA861 or ETH 615-139, the cysteinyl leukotriene 1 receptor (CysLT1) with the antiasthmatic drug Zafirlukast, or the volume-sensitive organic anion channel (VSOAC) with DIDS blocks the release of organic osmolytes (taurine, meAIB) and the concomitant cell volume restoration following hypoosmotic swelling of human type II-like lung epithelial cells (A549). Reactive oxygen species (ROS) are produced in A549 cells upon hypotonic cell swelling by a diphenylene iodonium-sensitive NADPH oxidase. The swelling-induced taurine release is suppressed by ROS scavenging (butylated hydroxytoluene, N-acetyl cysteine) and potentiated by H 2O2. Ca 2ϩ mobilization with ionomycin or ATP stimulates the swelling-induced taurine release whereas calmodulin inhibition (W7) inhibits the release. Chelation of the extracellular Ca 2ϩ (EGTA) had no effect on swelling-induced taurine release but prevented ATP-induced stimulation. H2O2, ATP, and ionomycin were unable to stimulate the taurine release in the presence of AA861 or Zafirlukast, placing 5-LO and CysLT1 as essential elements in the swelling-induced activation of VSOAC with ROS and Ca 2ϩ as potent modulators. Inhibition of tyrosine kinases (genistein, cucurbitacin) reduces volume-sensitive taurine release, adding tyrosine kinases (Janus kinase) as regulators of VSOAC activity. Caspase-3 activity during hypoxia is unaffected by inhibition of 5-LO/CysLT1 but reduced when swelling-induced taurine loss via VSOAC is prevented by DIDS excess extracellular taurine, indicating a beneficial role of taurine under hypoxia. 5-lipoxygenase; lung adenocarcinoma; cysteinyl leukotriene 1 receptor; hypoxia; ischemia; cell volume regulation; reactive oxygen species; taurine homeostasis WITHIN RECENT YEARS it has become clear that cell volume regulation under isotonic conditions and cell volume restoration following osmotic perturbations are important for regulation of the intracellular milieu and cellular functions, such as cell metabolism, cell migration, and control of cell death (see refs. 16, 39). Cells maintain a steady cell volume by regulating the intracellular concentrations of ions (mainly Na ϩ , K ϩ , and Cl Ϫ ) and organic osmolytes (16,39). A consequence of hypotonic cell swelling is net loss of KCl and amino acids including the ␤-amino ethane sulfonic acid, taurine.Taurine: a multifunctional amino acid. Taurine is the most abundant free amino acid of the human body and has been estimated to account for up to 38% of the total free amino acid pool in rat heart ce...

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“…[ 3 H]taurine (PerkinElmer, Waltham, MA) was used to estimate the uptake of taurine via TauT as well as the efflux under isotonic and hypotonic conditions as previously described (19,61). All the experiments were performed at room temperature.…”

“…In contrast to the active taurine uptake system involving TauT, taurine release involves 1) volume-sensitive leak pathways, activated by osmotic cell swelling [termed the volumesensitive organic anion channel (VSOAC)]; and 2) volumeinsensitive leak pathways, activated during anoxia, apoptosis, and cholesterol depletion (27,31,45,61). With the use of EATC and the human lung epithelial cell line A549, it has been demonstrated that swelling-induced activation of VSOAC involves phospholipase A 2 (PLA 2 ) and 5-lipoxygenase (5-LO) activity as well as binding of eicosanoids to the cysteinyl leukotriene 1 receptor CysLT1 (19,27). Once activated VSOAC activity is modulated by phosphatases, i.e., inhibition of vanadate-sensitive protein tyrosine phosphatases by ROS, which are generated in a variety of cells in response to osmotic perturbation, or inhibition of the phosphatidylinositol (3,4,5)-trisphosphate phosphatase PTEN (phosphatase and tensin homologue), leads to significant potentiation of taurine loss following exposure to hypotonic conditions (12,26,28,31).…”

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