Effects of EGTA on calcium signaling in airway epithelial cells

We have studied the effect of the extracellular calcium buffers ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) as well as varying extracellular calcium concentration on the intracellular free calcium response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine stimulates a rapid transient increase in cell calcium followed by a slower second peak lasting several minutes. Bathing cells in nominally calcium-free medium (no added calcium) did not abolish the second peak in the biphasic response to histamine. We examined the effect of including either 1 mM EGTA or BAPTA in the perfusate to investigate whether influx might have been supported by trace amounts of calcium in the nominally calcium-free medium. The second histamine-stimulated peak had a shorter duration but similar amplitude in the presence of BAPTA. In contrast, the second peak was completely abolished by EGTA. To examine if the different histamine responses were due to EGTA's lower dissociation constant or some pharmacological effect of unbound EGTA, extracellular calcium was removed by pretreating the saline with BAPTA covalently bound to polystyrene beads, and effluent from the imaging chamber was collected and analyzed for calcium contamination. Histamine stimulation still produced a biphasic calcium response when extracellular free calcium was approximately 7 nM, a concentration that should reverse the electrochemical gradient for calcium at the plasma membrane. These data suggest that the unbound form of EGTA can interfere with calcium signaling in cells through inhibition of its release from intracellular stores.