Abstract
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.
Highlights
Calcium is a well-known intracellular second messenger with proven involvement in a wide variety of biological processes, and vital for the correct function of cells, tissues and organisms [1,2].The Ca2+ role in immune cells, especially neutrophils, is of great scientific and therapeutic interest, as it has already been proven that this cation is involved in processes like neutrophils activation, oxidative stress, cell death/clearance and inflammation [3,4,5]
This process is generally known as SOCE [4,6,7]
To better understand thapsigargin effects in the human neutrophils Ca2+ flux, five different experimental conditions were tested: (a) thapsigargin alone was incubated with these cells, for 1 h; (b) thapsigargin and ML-9 were simultaneously added and cells were incubated for 1 h; (c) thapsigargin was incubated with the cells for 30 min, after which ML-9 was added for more
Summary
Calcium is a well-known intracellular second messenger with proven involvement in a wide variety of biological processes, and vital for the correct function of cells, tissues and organisms [1,2].The Ca2+ role in immune cells, especially neutrophils, is of great scientific and therapeutic interest, as it has already been proven that this cation is involved in processes like neutrophils activation, oxidative stress, cell death/clearance and inflammation [3,4,5]. Calcium is a well-known intracellular second messenger with proven involvement in a wide variety of biological processes, and vital for the correct function of cells, tissues and organisms [1,2]. The Ca2+ movements into the cell occur through two closely related events: first, a rapid emptying of the Ca2+ stores occur and some intermediate mechanism translates this information to plasma membrane channels that enable Ca2+ entry to refill the depleted stores. This process is generally known as SOCE (store-operated calcium entry) [4,6,7]. In SOCE, the Ca2+ influx occurs by the formation of a ternary complex between STIM
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