Mechanical stress induces release of ATP from Ehrlich ascites tumor cells

The supernatant from a suspension of Ehrlich cells exposed to centrifugatio n at 700xg for 45 s induced a transient increase in the intracellular conce ntration of free, cytosolic Ca2+, [Ca2+](i), as well as activation of an ou twardly rectifying whole-cell current when added to a suspension of non-sti mulated cells. These effects were inhibited by suramin, a non-specific P2 r eceptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revea led that the supernatant from Ehrlich cells exposed to centrifugation conta ined 2.6 +/- 0.2 mu M ATP, and that the mechanical stress-induced release o f ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprot ein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a sur amin-sensitive, transient, concentration-dependent increase in [Ca2+](i), a ctivation of an outwardly rectifying whole-cell current and a hyperpolariza tion of the plasma membrane. The ATP-induced hyperpolarization of the plasm a membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimula tion of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. T he relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alph a,beta-MeATP, bzATP) in eliciting an increase in [Ca2+](i), as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y(1) and P2Y(2) receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current. (C) 1999 Elsevier Science B.V. All rights re served.