Mechanisms underlying ATP-induced Ca²⁺ mobilization in human neutrophils

The effect of ATP on Ca²⁺ mobilization in human neutrophils was examined by using fura-2 as a Ca²⁺ indicator. ATP (0.1-100 μM) caused a significant [Ca²⁺]_i increase in a concentration-dependent manner. The [Ca²⁺]_i signal comprised an initial rise followed by a plateau. Removal of external Ca²⁺ diminished the peak value of the [Ca²⁺]_i signal. In Ca²⁺-free medium, pretreatment with an endoplasmic reticulum Ca²⁺ pump inhibitor, thapsigargin, prevented ATP from releasing Ca²⁺. In contrast, thapsigargin still increased [Ca²⁺]_i after pretreatment with 10 μM ATP. These results indicate that 10 μM ATP released Ca²⁺ mainly from thapsigargin-sensitive stores. Adding 3 mM Ca²⁺ induced a concentration-dependent increase in [Ca²⁺]_i after pretreatment with ATP or thapsigargin in Ca²⁺-free medium, suggesting ATP induced Ca²⁺ influx via capacitative Ca²⁺ entry. ATP (10 μM)-induced Ca²⁺ release was abolished by inhibiting phospholipase C with 2 μM U73122, indicating that inositol-1,4,5-trisphosphate (IP₃) mediates ATP-induced Ca²⁺ release. Conversely, ATP-induced [Ca²⁺]_i increase was abolished by activating protein kinase C (PKC) with 10 nM phorbol myristate acetate (PMA), but was not altered by inhibiting PKC with 2 μM GF 109203X. This implies ATP-induced [Ca²⁺]_i increase is a PMA-linked event. Together, the results suggest ATP increases [Ca²⁺]_i in human neutrophils by releasing Ca²⁺ from IP₃-coupled, thapsigargin-sensitive Ca²⁺ stores, and inducing Ca²⁺ influx via the process of capacitative Ca²⁺ entry. The ATP-induced Ca²⁺ signal is a PMA-linked event.