Calcium Pathways in Human Neutrophils-The Extended Effects of Thapsigargin and ML-9

In neutrophils, intracellular Ca²⁺ levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca²⁺-ATPase], SOCE (store-operated calcium entry), and ROCE (receptor-operated calcium entry). However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca²⁺ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca²⁺ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca²⁺ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca²⁺ levels due to its influx by SOCE. Moreover, it was obvious that ML-9 enhanced the thapsigargin-induced emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca²⁺ pathways, that, in the future, should be further explored.