The main aim of this review is to understand the mechanisms of Ca2+ influx which occur in neutrophils upon stimulation by pro-inflammatory mediators. It is well explained the process of Ca2+ influx by store operated calcium entry, which is activated by any agent that depletes intracellular Ca2+ stores. However, the authors also show another mechanism of Ca2+ entry that is probably dependent on receptors present on the plasma membrane. In light of these considerations, the review outlines the possible pharmacological compounds that are useful to distinguish whether they are involved in store or receptor operated calcium entry. Therefore, these findings are extremely important in order to allow a new target for the development of novel anti-inflammatory drugs.
In this journal several pharmacological approaches have been used to demonstrate the importance of Receptor operated calcium entry in human neutrophils. A way to study Ca2+ dynamics in neutrophils could have been using ER-aequorin, which is a photoprotein that target specifically the ER and is useful to measure [Ca2+] inside the lumen of the endoplasmic reticulum. (Button et al.1996)
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However, reconstitution of aequorin with its essential part coelenterazine requires previous CA2+ store depletion, thereby making this technique useless to study mechanism of SOCE (Alvarez et al. 2002). Previous researches have shown there is indeed a non-capacitative pathway of calcium influx upon stimulation with inflammatory mediators such as FMLP, LTB4 and PAF. Examples of such cells are smooth muscle cells where different substances have shown to activate receptor operated cation influx. (McFadzean et al. 2002)
In this review are displayed different pharmacological approaches to distinguish between SOCE and ROCE.
The first compound used is ML-9, which has been previously shown to inhibit SOCE in monocytes and macrophages. Different experiments in human neutrophils have shown that ML-9 enhances both the release of Ca2+ from stores and the subsequent Ca2+ influx in the presence of agonists (FMLP, LTB4, and PAF). A possible explanation is that ML-9 can also interact with Ca2+ release from IP3-sensitive stores. However, it is a question that still needs to be answered.
In contrast, whilst ML-9 also enhances thapsigargin-stimulated Ca2+ stores emptying, it also inhibits the subsequent thapsigargin stimulated calcium, thereby showing that ML-9 is implicated in SOCE. Therefore, a consistently hypothesis is that ML-9 might interact with STIM1 on the plasma membrane.
Another pharmacological compound used is gadolinium (Gd3+). In previous studies it has been shown that at very low concentrations (1uM), Gd3+ is able to inhibit totally store operated calcium entry. In this paper different experiments have been performed in human neutrophils in order to compare the different mechanisms involved in calcium entry. It is shown that 1 uM Gd3+ does not affect Ca2+ release by any of the three agonists, FMLP, PAF or LTB4 but partially inhibits the subsequent influx following the addition of CaCl2.
In comparison, whilst Gd3+ does not affect either thapsigargin- stimulated Ca2+ release, it completely abolishes the subsequent Ca2+ influx.
This study enables us to understand that receptor-operated entry has a substantial role in modulating Ca2+ fluxes in human neutrophils.
Further experiments have been conducted using Strontium (Sr2+). In fact, it well known that Sr2+ can substitute for Ca2+ in several cellular functions. It is also used to study Ca2+ kinetics in a variety of cell types.
Data collected from human neutrophils after stimulation with agonists and then thapsigargin have shown that the SOCE mechanism is unable to support Sr2+ entry thereby suggesting that Sr2+entry is characteristic of ROCE. This interpretation comes from the result that whilst after agonist stimulation there is a similar divalent cation influx as with Ca2+ influx, addition of SrCl2 following thapsigargin failed to produce any significant divalent cation influx.
Another compound used to study the mechanisms of SOCE and ROCE is 2-APB. Established that 2-APB works as an inhibitor of InsP3 mediated Ca2+ release, in this paper it is also analyzed its role in distinguishing between store operated calcium entry and receptor operated. Itagaki et al. (2002) have shown this, inhibiting InsP3 with 2-APB, there was a complete inhibition of Sr2+ influx whilst Ca2+ remained the same. This suggested that 2-APB could have been used as a tool to study SOCE mechanisms and that Sr2+ influx did not depend on SOCE. What it is interesting is that 2-APB has shown a biphasic effect in the current implicated in SOCE. Among the several CRAC channel inhibitors characterized so far, 2-APB has been most commonly used and is a well-established CRAC inhibitor that, at lower concentrations, also acts as an activator of this current  J.P. Kukkonen, P.E. Lund and K.E. Akerman, 2-Aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca(2+) discharge and store-operated Ca(2+) influx, Cell Calcium 30 (2001), pp. 117ââ‚¬"129. Abstract | PDF (349 K) | View Record in Scopus | Cited By in Scopus (56). This prominent bimodal effect has been utilized to pharmacologically identify CRAC currents. (Goto et al.2010)
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2-APB activates ICRAC, at low micro-molar concentrations whilst inhibiting it at doses higher than 30ÎÂ¼M. In particular, after agonist stimulation, there is not visible effect on Ca2+ influx, while there is an increase after thapsigargin stimulated Ca2+ influx. This means that 2-APB represents a stimulation of the SOCE mechanism.
However, recent evidence has proposed that 2-APB might also be implicated in ROCE. 2-APB can at least partially inhibit TRPC3 channels thus suggesting that an intimate relationship could exist between 2-APB and components of the ROCE pathway. Both this utility in SOCE and ROCE makes 2-APB not the best compound used to study those mechanisms.
Finally, experiments using Pyr3, which is a specific TRPC channel inhibitor, have been performed. In neutrophils TRPC3 is of particular interest because it is one of the TRPC proteins reported to be activated by diacylglycerol and thus potentially the basis for a G-protein-phospholipase C linked ROCE mechanism in neutrophils.
Pyr3 partially reduced Ca2+ influx with agonists. This was more apparent in FMLP stimulated neutrophils in comparison to those stimulated with PAF and LTB4.
In thapsigargin stimulated neutrophils, the reduction in Ca2+ influx was far greater and nearly abolished with Pyr3.
If indeed Pyr3 acts to specifically inhibit TRPC3 channels then these findings strongly suggest that this protein is associated with SOCE in human neutrophils.
TRPC3 may therefore be linked to the store-operated Ca2+ entry pathway and can be used as a potential target to discriminate between receptor- and store-operated influxes in activated human neutrophils. Experiments from Schleifer et al. (2009) have shown connection of TRPC3 and SOCE also in rat basophil leukemia cells.
TRPC channels are considered the strongest candidates to further elucidate the mechanisms of store operated and receptor operated calcium entry.
However, it is not perfectly clear whether TRPC channels are involved only in SOCE or can take also part in ROCE.
Recently, TRPC1 has been reported to interact with the ER Ca2+ sensor STIM1 and the PM CRAC protein Orai1, suggesting the participation of TRPC1 in SOCE. (Salido et al. 2009)