Delayed activation of plasma membrane Ca pump in human neutrophils

The interplay between Ca²⁺ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium (Ca²⁺]_i) was studied in suspensions of human neutrophils loaded with the Ca²⁺]_i indicator, Fura-2. To reveal Ca²⁺ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of Ca²⁺]_i were caused exclusively by PM Ca²⁺ transporters. Following preincubation, the addition of CaCl₂ induced, mediated by ionomycin, a transient rise of Ca²⁺]_i, a spike, eventually decreasing to an intermediary Ca²⁺]_i level. The ATP-dependent decrease of Ca²⁺]_i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na⁺-free medium, suggesting that neither activity of protein kinase C nor exchange was necessary for generation of the Ca²⁺ spike. In conclusion, the PM Ca²⁺ pump was responsible for the Ca²⁺ spike by responding to the rapid rise of Ca²⁺]_i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular Ca²⁺]_i spikes in general.