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

In neutrophils, intracellular Ca levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca-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 Ca pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca 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 Ca fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca 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 Ca pathways, that, in the future, should be further explored.