Propionic acid-induced calcium mobilization in human neutrophils

The ability of propionic acid to elicit an increase in the level of cytoplasmic free calcium in human neutrophils was examined in detail. Propionic acid induced a rapid and dose-dependent mobilization of calcium that relied on both internal and external sources of calcium. The effects of propionic acid on the mobilization of calcium were inhibited by pertussis toxin, but not cholera toxin, implicating a guanine nucleotide binding protein. Furthermore, preincubation of the neutrophils with phorbol 12-myristate 13-acetate resulted in a decreased mobilization of calcium. This inhibitory activity of phorbol myristate acetate was antagonized by the protein kinase C inhibitor H-7. Preincubation of the cells with the synthetic chemotactic factor fMet-Leu-Phe caused a reduction in the magnitude of the calcium transient elicited by propionic acid. However, the calcium response to propionic acid was not affected by antagonists of fMet-Leu-Phe and platelet-activating factor binding or by an inhibitor of leukotriene synthesis. Propionic acid did not elicit a mobilization of calcium in monocytes, platelets, lymphocytes, or undifferentiated HL-60 cells. However, the treatment of the HL-60 cells with dimethylsulfoxide resulted in the appearance of a calcium response to propionic acid. The potential physiological significance of these findings are discussed.     

Desensitization of calcium mobilization and cell function in human neutrophils.

Neutrophils pretreated with the chemoattractant formylmethionyl-leucyl-phenylalanine become unresponsive when re-exposed to the same ligand, a process termed desensitization. We have examined whether desensitization of transduction (Ca2+ mobilization) or of other cell functions (superoxide generation, enzyme release, or aggregation) occurs synchronously. Simultaneous studies of Ca2+ mobilization and aggregation by using Fura-2-loaded cells indicate that, under conditions where the aggregation response is abolished, most of the Ca2+ mobilization is unaltered. Further studies were then carried out to ascertain whether desensitization of Ca2+ mobilization could in fact be induced. Desensitization was observed, and was dependent on the number of exposures of the cells to the ligand, the concentration of the ligand, and whether the ligand was left in the medium or was removed. The pattern of resensitization was dependent on the experimental design. Under conditions where ligand was continuously present, no recovery of the Ca2+-mobilization response was seen with subsequent challenges. In contrast, on removal of ligand, this response showed partial recovery. Whereas complete desensitization of aggregation was noted, enzyme release showed a markedly lesser degree of desensitization and required more frequent exposures to the ligand before it was observed. Little or no desensitization of superoxide generation was observed regardless of the conditions utilized. Studies using phorbol myristate acetate as the ligand showed that Ca2+ mobilization and aggregation could be simultaneously inhibited. Our results suggest that discrete mechanisms of desensitization are possible in human neutrophils, and that desensitization of one particular function (aggregation) does not imply concomitant desensitization of other functions.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Actin polymerization in human eosinophils,unlike human neutrophils,depends on intracellular calcium mobilization

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca²⁺]_i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca²⁺-depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes. © 1996 Wiley-Liss, Inc.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Comparative effect of leukotriene B4 and leukotriene B5 on calcium mobilization in human neutrophils

Leukotriene B4 (LTB4) is reported to exert its biological activity in neutrophils through the increase in cytosolic free calcium that follows binding to its specific receptor. Leukotriene B5 has been shown to be far less active than LTB4. Therefore we compared the capacity of LTB4 and LTB5 to stimulate the rise in cytosolic free calcium using fura-2-loaded human neutrophils, to assess the relationship between the calcium mobilizing activity and biological potency of LTB4 and LTB5. At any concentration tested, LTB5 was less active than LTB4 in increasing cytosolic free calcium. ED50 for LTB4 and LTB5 were 5 X 10(-10) M and 5 X 10(-9) M, respectively. The difference in the binding affinities of LTB4 and LTB5 to the LTB4 receptor has been reported to explain the difference in their biological activities. In the present study we further demonstrated that the calcium mobilizing activity of LTB4 and LTB5 also correlates the different biological activity of the two compounds.     

Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

Calcium fluxes and calcium buffering in human neutrophils

Neutrophils loaded with the calcium indicator quin-2 and challenged with the ionophore ionomycin or the chemotactic peptide fMet-Leu-Phe were examined in the light of a theory that relates time-dependent changes in the fluorescence of the indicator to cytosolic calcium fluxes and levels. The cytosolic binding capacity was estimated from the theory to be 1.5 +/- 0.6 X 10(8) sites/cell (0.76 mM based on a cell volume of 330 micron 3, irrespective of water content and the distribution of sites), each site having an apparent average single class dissociation constant of 0.55 +/- 0.2 microM. Some 20% of the total available cytosolic calcium sites of the normal resting cell appear to be occupied when no quin-2 is present. In a calcium-free medium, the amount of calcium released by fMet-Leu-Phe from storage pool locations that are distinct from the cytosolic sites is sufficient to further raise the cytosolic site occupancy level to 50%, at which point the calcium buffering capacity of the cytosol is maximal. In a calcium-containing medium, however, simultaneous influx from the outside appears to supply enough additional calcium to saturate most of the remaining sites. The combined initial rate of storage pool calcium release plus influx through the plasma membrane was roughly twice the initial rate at which calcium was released from storage locations alone, suggesting that stimulus-induced influx from the outside may be comparable in importance to storage pool mobilization in determining physiological calcium levels in stimulated cells.     

Hydrogen peroxide activates calcium influx in human neutrophils

The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H₂O₂ produced a rapid influx of Mn²⁺ and an increase of intracellular calcium. The H₂O₂ was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca²⁺ to H₂O₂-treated cells suspended in a free Ca²⁺/EGTA buffer resulted in a great increase in [Ca²⁺]_i reflecting influx of Ca²⁺ across the cell membrane. The increase of intracellular calcium was inhibited by Ni²⁺, La³⁺, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H₂O₂ by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.     

Auranofin dissociates chemotactic peptide-induced generation of inositol 1,4,5-trisphosphate from the subsequent mobilization of intracellular calcium in intact human neutrophils

Auranofin, an antiarthritic gold compound, modulates a number of chemotactic factor-induced inflammatory responses in human neutrophils. In order to unravel the mechanism involved, the present study investigated the effects of auranofin on early signal transduction events in these cells. Auranofin did not affect the chemotactic peptide (fMetLeuPhe)-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), neither in the presence nor in the absence of extracellular calcium ions. In contrast, there was a progressive inhibition by auranofin on the fMet-Leu-Phe-induced mobilization of intracellular calcium. This demonstrates that auranofin can dissociate the generation of Ins(1,4,5)P3 from the subsequent release of intracellular calcium, perhaps by interfering with the intracellular binding of Ins(1,4,5)P3 to its receptor. In experiments performed in electro-permeabilized cells, however, a relatively high concentration of the drug failed to abolish the specific binding of Ins(1,4,5)P3. In addition, in the same system, auranofin also failed to abolish the Ins(1,4,5)P3-induced release of Ca2+. Consequently, auranofin-mediated dissociation of fMLP-induced Ins(1,4,5)P3 formation and intracellular calcium release can not be explained merely by an antagonistic effect of auranofin on the Ins(1,4,5)P3 receptor. Instead the interaction between auranofin and the plasma membrane seems to be an initial and important part of the mechanism by which this drug interferes with the transduction signalling system.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10^?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up ⁴⁵Ca²⁺ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O^?₂ (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O^?₂ generation and aggregation (30–35 s).     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils. Relationship to calcium mobilization, aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to 'stage' events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10(-7) M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 microgram/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up 45Ca2+ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O2-. (10-15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O2-. generation and aggregation (30-35 s).     

Mechanisms of ATP-induced calcium signaling and growth arrest in human prostate cancer cells

This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.     

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: Evidence for an involvement of phospholipase D-derived phosphatidic acid

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.     

Arachidonic acid-induced release of calcium in permeabilized human neutrophils

The addition of arachidonic acid to a suspension of digitonin-permeabilized human neutrophils was found to induce, in a dose-dependent manner (ED50 about 15 microM), the release of calcium from internal stores. Arachidic acid was without effect, while linoleic acid and linolenic acid were (on a concentration basis) at least 5-times less active than arachidonic acid. The activity of arachidonic acid appears to be due to the fatty acid itself and not to one of its metabolites. The pool of calcium mobilized by arachidonic acid includes that sensitive to inositol 1,4,5-triphosphate. These results demonstrate a significant intracellular role for arachidonic acid at the level of the internal mobilization of calcium in human neutrophils.     

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.     

State-dependent calcium mobilization by urotensin-II in cultured human endothelial cells

Human endothelial cells express urotensin-II (U-II) as well as its receptor GPR14. Using microfluorimetric techniques, the effect of human U-II on cytosolic Ca2+ concentrations [Ca2+]i in cultured human aortic endothelial cells (HAECs) loaded with Fura-2 was evaluated in static or flow conditions. Under the static state, U-II (100 nM) abolished spontaneous Ca2+ oscillations, which occurred in a population of cultured HAEC. Similarly, U-II reduced thrombin-, but not ATP-induced calcium responses, suggesting that the peptide does not alter the Gq/11/IP3 pathway; rather, it modifies the coupling between protease-activated receptors and Gq/11/IP3. Under the flow condition, U-II (1, 10 and 100 nM) produced a dose-dependent increase in [Ca2+]i, which was subjected to desensitization. The result demonstrates a state-dependent effect of U-II in cultured HAEC, which may explain the variable responses to U-II under different experimental conditions.     

Deactivation of the effects of F-Met-Leu-Phe and leukotriene B4 on calcium mobilization in rabbit neutrophils

Preincubation of rabbit neutrophils with the synthetic chemotactic factor f-Met-Leu-Phe has been found to diminish the ability of these cells to mobilize calcium upon subsequent stimulation by f-Met-Leu-Phe or by leukotriene B₄. The preexposure of the neutrophils to leukotriene B₄ on the other hand results in a diminished subsequent response to itself but an unaltered response to f-Met-Leu-Phe. These results demonstrate that deactivation can be observed at the level of calcium mobilization, strengthen the postulated second messenger role of calcium in neutrophils and imply that neutrophil activation by chemotactic factors can bypass the arachidonic acid metabolic pathway.     

Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl-leucyl-phenylalanine in human neutrophils

It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration.