In the current study, activation of neutrophils with LTB4 (20 nM), FMLP (10 nM) and PAF (100 nM) resulted in the characteristic immediate increase in [Ca2+]i. The peak concentration of [Ca2+]i in LTB4 stimulated neutrophils was very short lived (60 s, Fig. 1A) in comparison to FMLP treated cells, which took longer to reach basal levels (Fig. 2A). The prolongation of the peak [Ca2+]i to reach basal levels in FMLP stimulated
MLN8237 probably reflects a delayed extrusion of [Ca2+]i from intracellular stores in comparison to [Ca2+]i responses with LTB4. This prolongation of [Ca2+]i to return to basal values was the most notable with neutrophils stimulated with PAF in which a much broader response was observed that took much longer to reach basal levels (Fig. 2C). This biphasic component in response to PAF is in agreement with a previous study [18] and has been attributed to the secondary production of LTB4 after PAF stimulation.In rabbit neutrophils it has been established that PMA activates the NADPH oxidase and causes membrane depolarization with no apparent rise in [Ca2+]i[19]. In addition, in a more recent study it was shown that phorbol esters potently inhibit divalent cation influx (PMA IC50 ～50 pM) at concentrations lower than required for stimulation of delayed superoxide formation [20]. The results in the present study are in agreement with these previous studies. In addition, the binding affinity values of PMA were very similar for all the agonists. Our findings show that PMA inhibits [Ca2+]i release in neutrophils stimulated with FMLP. Previously, it was shown that PMA only partially inhibited [Ca2+]i release in response to 10 nM FMLP [21]. In the present study we demonstrate that PMA at concentrations greater than 1 nM completely inhibited [Ca2+]i release in response to FMLP at concentrations of 10-fold less than that was observed in the previous study [21].Our data show that the NADPH oxidase inhibitors DPI and apocynin can suppress [Ca2+]i release in human neutrophils stimulated with FMLP. An interesting variation of our current results is the ability of NADPH oxidase inhibitors to suppress [Ca2+]i release. Neutrophils treated with these inhibitors mimic the CGD phenotype and CGD cells are proposed to have higher levels of [Ca2+]i influx [11] and [22]. In one study it was shown that CGD cells stimulated with FMLP had higher levels of cytosolic calcium in comparison to healthy cells and therefore led to the proposal that CGD neutrophils are prone to calcium overload [11]. In contrast, our findings demonstrate that DPI and apocynin treated neutrophils had significantly lower [Ca2+]i release levels than untreated cells. The proposed theory of the NADPH oxidase in preventing Ca2+ overload [11] and [22] needs to be treated with caution. A previous report using Quin-2 to measure changes in [Ca2+]i release from patients with CGD [23] demonstrated that [Ca2+]i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal
cells suggesting that a functional oxidase was not responsible for preventing [Ca2+]i overload. This finding along with the data presented in the current study therefore casts some uncertainty on the theory proposed by other studies [11] and [22]. Taken together these findings suggest that the role for a functional NADPH oxidase in preventing Ca2+ overload in FMLP stimulated human neutrophils is not as clear as was previously thought. For the purposes of the present study we have predominantly focussed on the direct affect of specific NADPH oxidase inhibitors on FMLP induced changes in [Ca2+]i release in human neutrophils. FMLP is considered to be the gold standard for investigating different responses in human neutrophils and in addition our study shows that LTB4 and PAF can increase [Ca2+]i. Investigating the direct interaction of NADPH oxidase inhibitors on LTB4 and PAF induced changes in [Ca2+]i release is not the purpose of the present study but no doubt would be an important avenue to explore in future studies.What do our currents finding suggest for the neutrophil NADPH oxidase and changes in [Ca2+]i release? A possible interpretation of the current findings could be linked to the role of membrane potential in regulating G-protein coupled receptors. It has been documented that FMLP receptors are dependent on Gi, whereas both LTB4 receptor and PAF receptors utilise both Gi and pertussis toxin insensitive G-proteins [24]. In addition, membrane potential is thought to have an important role in regulating G-protein coupled receptors in several cell types [25]. Although this has not yet been documented in neutrophils, findings from another non-excitable cell; the megakaryocyte have demonstrated that membrane depolarisation can modulate Ca2+ mobilisation evoked by G-protein coupled receptors [26]. It is possible in neutrophils that after activation of the NADPH oxidase, concomitant membrane depolarisation may differentially regulate the activities of G-proteins coupled to FMLP receptors and therefore modulate the [Ca2+]i release. In the presence of NADPH oxidase inhibitors, membrane depolarisation would be abrogated [7] and therefore, would effect how different G-protein coupled receptors modulate the way [Ca2+]i is released from stores in response to different agonists.In conclusion, our study demonstrates that inhibition of the NADPH oxidase can suppress [Ca2+]i release in response to FMLP in human neutrophils and suggests a complex interaction occurs between FMLP receptors and the NADPH oxidase in activated human neutrophils.AcknowledgmentsWe thank Nicholas Seeley and Kevin Clough for their technical assistance.
