sphingosine-1-phosphate and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

We report the activation of outwardly rectifying anion currents by sphingosine-1-phosphate (S1P) in the murine macrophage cell line RAW 264.7. The S1P-induced current is mainly carried by anions, because the reversal potential of the current was shifted by replacement of extracellular Cl(-) by glutamate(-) but not when extracellular Na(+) was substituted by Tris(+). The inhibition of the current by hypertonic extracellular or hypotonic intracellular solution as well as the inhibitory effects of NPPB, tamoxifen, and glibenclamide indicates that the anion current is mediated by volume-regulated anion channels (VRAC). The S1P effect was blocked by intracellular GDPβS and W123, which points to signaling via the S1P receptor 1 (S1PR1) and G proteins. As cytochalasin D diminished the action of S1P, we conclude that the actin cytoskeleton is involved in the stimulation of VRAC. S1P and hypotonic extracellular solution induced secretion of ATP from the macrophages, which in both cases was blocked in a similar way by typical VRAC blockers. We suppose that the S1P-induced ATP secretion in macrophages via activation of VRAC constitutes a functional link between sphingolipid and purinergic signaling in essential processes such as inflammation and migration of leukocytes as well as phagocytosis and the killing of intracellular bacteria.

Topics: Adenosine Triphosphate; Animals; Cell Line; Cell Size; Chlorides; Cytochalasin D; Glyburide; GTP-Binding Proteins; Ion Channels; Lysophospholipids; Macrophages; Mice; Nitrobenzoates; Receptors, Lysosphingolipid; Sodium; Sphingosine; Tamoxifen

It is well established that transforming growth factor (TGF)-beta stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (I(Cl-LPA)) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of I(Cl-LPA) on fibroblast-to-myofibroblast differentiation. We recorded I(Cl-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-alpha-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-beta (myofibroblasts) had significantly elevated alpha-SMA levels and I(Cl-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 microM). DIDS and NPPB, in a dose-dependent manner, significantly reduced alpha-SMA levels in HLF stimulated with TGF-beta. 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 I(Cl-LPA) activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Actins; Adenosine Triphosphate; Cell Differentiation; Cells, Cultured; Chloride Channels; Electrophysiology; Fetus; Fibroblasts; Humans; Lung; Lysophospholipids; Muscle, Smooth; Nitrobenzoates; Receptors, Lysophosphatidic Acid; Sphingosine; Transforming Growth Factor beta

This study was designed to examine the effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on Cl(-) currents (ICl(LPA)) in cultured corneal keratocytes isolated from the corneas of New Zealand White rabbits.. ICl(LPA) and resting voltages were recorded with the amphotericin perforated-patch technique. Phenotype was determined with antibodies to alpha-smooth muscle actin.. Keratocytes cultured in serum have a phenotype (myofibroblast) and ionic currents similar to those of keratocytes isolated directly from corneas during wound healing. LPA and S1P both activated ICl(LPA) in a dose-dependent manner, and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (DGPP) blocked the LPA response, but not the S1P response. In addition, a relatively inactive form of LPA (LPA 8:0) was relatively ineffective in activating ICl(LPA). Activation of ICl(LPA) significantly depolarized the cells, and this depolarization was reversed by blocking ICl(LPA) with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).. These results demonstrate that activation of ICl(LPA) by LPA in cultured corneal keratocytes is receptor mediated and that ICl(LPA) can also be activated by S1P. From a functional standpoint, this work confirms that the current, which is typically thought of as purely volume-activated, can be activated through a receptor. In addition, activation of ICl(LPA) results in depolarization of the keratocyte. Finally, this work demonstrates that cultured corneal keratocytes can act as a model for the study of ion channel function in keratocytes during corneal wound healing.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cells, Cultured; Chloride Channels; Cornea; Electrophysiology; Immunohistochemistry; Ion Channel Gating; Lysophospholipids; Nitrobenzoates; Rabbits; Receptors, Cell Surface; Sphingosine