antimycin and Leukemia--Basophilic--Acute

The role of ATP in both the activation of store-operated Ca2+ current ICRAC and in Ca2+-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, ICRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4, 5-trisphosphate (InsP3) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of ICRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca2+-dependent vesicular fusion was triggered by dialysing cells with 10 microM Ca2+ and guanosine-5'-O-(3-thiotriphosphate (GTP[gamma-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of ICRAC or for the capacitance increases elicited by high concentrations of intracellular Ca2+.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Antimycin A; Calcium; Cell Membrane; Electric Conductivity; Enzyme Inhibitors; Leukemia, Basophilic, Acute; Membrane Fusion; Oligomycins; Patch-Clamp Techniques; Proton-Translocating ATPases; Rats; Tumor Cells, Cultured