Activation of ATP secretion via volume-regulated anion channels by sphingosine-1-phosphate in RAW macrophages

Abstract

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.

ATP release during cell swelling activates a Ca2+-dependent Cl− current by autocrine mechanism in mouse hippocampal microglia

“…The release of ATP through volume regulated channels has been proposed long time ago in hepatoma 39 and endothelial cells 52 and more recently in astrocytes and Raw macrophages 44, 53, 54 . Our conclusion that ATP is released by microglia under hypotonic stimulation is based on severeal lines of evidence.…”

The identification of a volume‐regulated anion channel: an amazing Odyssey

“…, Burow et al . ). This unique permeation profile of VRAC is important for its role in non‐synaptic cellular communication which modulates neuronal excitability and synaptic activity in the brain.…”

“…Volume‐independent activation of VRAC is also induced by sphingosine‐1‐phosphate, which is generated by S‐kinase activated by bacterial lipopolysaccharide (LPS), PDGF, TNF α , thrombin, IgE‐bound antigen and importantly ATP (Burow et al . ).…”

Hypotonic stress response of human keratinocytes involves LRRC8A as component of volume‐regulated anion channels

“…Electrophysiological studies already showed that hypotonicity‐induced Cl − currents of HaCaT cells are sensitive to various Cl − channel inhibitors . Different efficiencies and incomplete blocking have been described for several VRAC inhibitors in many cell types including HaCaT cells and NHEKs . In our hsYFP‐based measurements in HaCaT cells, CBX almost completely blocked hypotonicity‐induced I − influx (90%).…”

“…[11] Different efficiencies and incomplete blocking have been described for several VRAC inhibitors in many cell types including HaCaT cells and NHEKs. [11,33,41,42] In our hsYFP-based measurements in HaCaT cells, CBX almost completely blocked hypotonicity-induced I − influx (90%). A comparable degree of inhibition (80%) of VRAC-mediated Cl − currents was reported for CBX in cultured rat cortical astrocytes.…”