EFFECTS OF EGTA ON CALCIUM SIGNALING IN AIRWAY EPITHELIAL-CELLS

We have studied the effect of the extracellular calcium buffers ethyle ne glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) a s well as varying extracellular calcium concentration on the intracell ular free calcium response to histamine in a cystic fibrosis airway ep ithelial cell line (CF/T43). Histamine stimulates a rapid transient in crease in cell calcium followed by a slower second peak lasting severa l minutes. Bathing cells in nominally calcium-free medium (no added ca lcium) did not abolish the second peak in the biphasic response to his tamine. mie examined the effect of including either 1 mM EGTA or BAPTA in the perfusate to investigate whether influx might have been suppor ted by trace amounts of calcium in the nominally calcium-free medium. The second histamine-stimulated peak had a shorter duration but simila r amplitude in the presence of BAPTA. In contrast, the second peak was completely abolished by EGTA. To examine if the different histamine r esponses were due to EGTA's lower dissociation constant or some pharma cological effect of unbound EGTA, extracellular calcium was removed by pretreating the saline with BAPTA covalently bound to polystyrene bea ds, and effluent from the imaging chamber was collected and analyzed f or calcium contamination. Histamine stimulation still produced a bipha sic calcium response when extracellular free calcium was similar to 7 nM, a concentration that should reverse the electrochemical gradient f or calcium at the plasma membrane. These data suggest that the unbound form of EGTA can interfere with calcium signaling in cells through in hibition of its release from intracellular stores.