Mechanical stress induces release of ATP from Ehrlich ascites tumor cells

Abstract

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700× g for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca 2+, [Ca 2+] i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2.6±0.2 μM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca 2+] i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca 2+-activated K + channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca 2+-activated K + current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, α,β-MeATP, bzATP) in eliciting an increase in [Ca 2+] 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 Ca 2+ influx as well as release and activation of an outwardly rectifying whole-cell current.