Cell swelling-induced ATP release and gadolinium-sensitive channels

Abstract

ATP release induced by hypotonic swelling is an ubiquitous phenomenon in eukaryotic cells, but its underlying mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has been implicated because gadolinium (Gd³⁺), an inhibitor of stretch-activated channels, suppressed ATP efflux monitored by luciferase bioluminescence. We examined the effect of Gd³⁺on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells. We found that luciferase was inhibited by ≤10 μM Gd³⁺, and this may have contributed to the previously reported inhibition of ATP release. In ATP efflux experiments, luciferase inhibition could be prevented by chelating Gd³⁺with EGTA before luminometric ATP determinations. Using this approach, we found that 10–100 μM Gd³⁺, i.e., concentrations typically used to block MS channels, actually stimulated hypotonically induced ATP release from fibroblasts. Inhibition of ATP release required at least 500, 200, or 100 μM Gd³⁺ for fibroblasts, A549 cells, and 16HBE14o⁻ cells, respectively. Such biphasic and cell-specific effects of Gd³⁺ are most consistent with its action on membrane lipids and membrane-dependent processes such as exocytosis.