Studies on molecular regulation of phagocytosis and activation of the NADPH oxidase in neutrophils. IgG- and C3b-mediated ingestion and associated respiratory burst independent of phospholipid turnover and Ca2+ transients

The role of messengers derived from hydrolysis of phosphoinositides and other phospholipids, of the basal level of [Ca2+]i and of the increase in [Ca2+]i in phagocytosis and respiratory burst was investigated, using normal neutrophils and neutrophils Ca2(+)-depleted by pretreatment with Quin2/AM and EGTA. 1) Phagocytosis and respiratory burst in control neutrophils challenged with yeast opsonized with IgG or C3b/bi were associated with a stimulation of the production of inositol phosphates, diacylglycerol, phosphatidic acid, arachidonic acid, and rise in [Ca2+]i. 2) In Ca2(+)-depleted neutrophils (basal [Ca2+]i 10 to 20 nM) the phagocytosis of yeast-IgG was similar to that in control neutrophils, the respiratory burst was slightly depressed (-30%), while the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, and phosphatidic and arachidonic acid did not occur. 3) In Ca2(+)-depleted neutrophils the phagocytosis of yeast-C3b/bi was slightly lower than that in control neutrophils, and the respiratory burst, related to the same number of particles ingested, was depressed by about 60%, whereas the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, phosphatidic acid, and arachidonic acid release did not occur. These findings demonstrate that transmembrane signaling pathways involving the hydrolysis of phosphoinositides by phospholipase C and D and of other phospholipids by phospholipase C and Az, and the rise in [Ca2+]i are not essential processes for triggering the ingestion of yeast particles opsonized with IgG and C3b/bi and the activation of the NADPH oxidase.     

Double stimulation with FMLP and Con A restores the activation of the respiratory burst but not of the phosphoinositide turnover in Ca2+-depleted human neutrophils. A further example of dissociation between stimulation of the NADPH oxidase and phosphoinositide turnover

The results reported here show that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A) may occur with a stimulus response coupling sequence that bypasses the activation of phosphoinositide hydrolysis, monitored as accumulation of inositol phosphates and glycerophosphoinositol, and the increase in [Ca2+]i. In fact: in Ca2+-depleted neutrophils FMLP and Con A do not induce the respiratory burst and the activation of phosphoinositide hydrolysis. The addition of Ca2+ restores both the respiratory and the phosphoinositide responses; the double treatment of Ca2+-depleted neutrophils with FMLP and Con A in sequence, before FMLP and then Con A and vice versa, or simultaneously, restores the capacity to respond to the second stimulus with the respiratory burst but not with the activation of phosphoinositide hydrolysis. These findings suggest that, for the activation of the NADPH oxidase by FMLP and by Con A: the transduction pathway including the stimulation of phosphoinositide turnover, the Ca2+ changes and the activity of the protein kinase C is not required, or is not the unique, and one stimulus may trigger more than one transduction pathway. Possible transduction pathways are discussed.     

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.     

Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate

Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca2+ changes. In fact: i) Ca2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca2+. ii) In Ca2+-depleted neutrophils phorbol-myristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatment of Ca2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed.     

Fluoride can activate the respiratory burst independently of Ca2+, stimulation of phosphoinositide turnover and protein kinase C translocation in primed human neutrophils

Evidences have been provided in our laboratory that in neutrophils different signal transduction sequences for the activation of O2(-)-forming NADPH oxidase can be triggered by the same stimulus (Biochem. Biophys. Res. Commun. 1986, 135, 556-565; 1986, 135, 785-794; 1986, 140, 1-11). The results presented here show that the transduction sequence triggered by fluoride via dissociation of G-proteins and involving messengers produced by stimulation of phosphoinositide turnover, Ca2+ changes and translocation of protein kinase C from the cytosol to the plasmamembrane, can be bypassed when a primed state of neutrophils is previously induced. In fact: i) fluoride causes a pertussis toxin insensitive and H-7 sensitive respiratory burst in human neutrophils, which is linked to the activation of hydrolysis of PIP2, rise in [Ca2+]1 and translocation of PKC. In Ca2+-depleted neutrophils these responses to fluoride do not occur and are restored by addition of CaCl2. ii) The pretreatment of Ca2+-depleted unresponsive neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase by fluoride but not the turnover of phosphoinositides and PKC translocation. The nature of the alternative transduction sequence, the reactions different from phospholipase C activated by G-protein for the alternative sequence and the role of these discrete pathways for NADPH oxidase activation are discussed.     

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phosphatidylinositol turnover measured as modification of incorporation of [32P]Pi and [3H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca2+, without a decrease in the total quantity of Ca2+ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca2+ concentration [Ca2+]i, without a decrease in the magnitude of the final increase of [Ca2+]i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca2+-phospholipid-dependent protein kinase C.     

Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.     

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.     

fMet-Leu-Phe-induced activation of phospholipase D in human neutrophils. Dependence on changes in cytosolic free Ca2+ concentration and relation with respiratory burst activation.

In this study, we have investigated the Ca2+ requirements for the activation of phospholipase D by the tripeptide fMet-Leu-Phe (fMLP) in human neutrophils. EGTA inhibited the activation of phospholipase D (PLD) by 55% (n = 4). When the initial transient rise in [Ca2+]i was prevented by loading the cells with limited amounts of the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), PLD activation was inhibited by 92% (n = 4). In the presence of both chelators, PLD activation was only 4% of control. In electropermeabilized neutrophils, too, the activation of PLD after the addition of fMLP strongly depends on the Ca2+ concentration, being almost absent with 100 nM free Ca2+ present and reaching maximum activation with a free [Ca2+] of 500 nM. We subsequently investigated the relationship between PLD activation and the activation of the respiratory burst. In neutrophils loaded with BAPTA/AM (10 microM), in which PLD activation was almost absent, a respiratory burst could be induced by fMLP, albeit with a much longer lag time. A respiratory burst could also be elicited by fMLP in electropermeabilized neutrophils incubated with 100 nM free Ca2+. This response, however, was strongly enhanced in the presence of 1 microM Ca2+. Our results indicate that changes in [Ca2+]i are essential for the activation of PLD by fMLP, but probably do not constitute the sole activation signal. In addition, our data provide evidence that PLD activation is important, but not necessary, for activation of the neutrophil respiratory burst.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Role of Ca2+ in phosphatidylinositol response and arachidonic acid release in formylated tripeptide- or Ca2+ ionophore A23187-stimulated guinea pig neutrophils

The role of Ca2+ in phospholipid metabolism and arachidonic acid release was studied in guinea pig neutrophils. The chemotactic peptide formylmethionyl-leucyl-phenyl-alanine (fMLP) activated [32P]Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) without any effects on the labeling of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). This activation was observed in Ca2+-free medium. Even in the neutrophils severely deprived of Ca2+ with EGTA and Ca2+ ionophore A23187, the stimulated labeling was not inhibited. When [3H]arachidonic acid-labeled neutrophils were stimulated by fMLP, a loss of [3H]arachidonic acid moiety in PI and the resultant increase in [3H]arachidonyl-diacylglycerol (DG), -PA, and free [3H]arachidonic acid was marked within 3 min. With further incubation, a loss of [3H]arachidonic acid in PC and PE became significant. These results suggest the activation of phospholipase C preceded the activation of phospholipase A2. In Ca2+-free medium, the decrease in [3H]arachidonyl-PI and the increase in [3H]arachidonyl-PA were only partially inhibited, although the release of [3H]arachidonic acid and a loss of [3H]arachidonyl-PC and -PE was completely blocked. These results show that PI-specific phospholipase C was not as sensitive to Ca2+ deprivation as arachidonic acid cleaving enzymes, phospholipase A2, and diacylglycerol lipase. Ca2+ ionophore A23187, which is known as an inducer of secretion, also stimulated [32P]Pi incorporation into PI and PA, although the incorporation into other phospholipids, such as PC and PE, was inhibited. This stimulated incorporation seemed to be caused by the activation of de novo synthesis of these lipids, because the incorporation of [3H]glycerol into PA and PI was also markedly stimulated by Ca2+ ionophore. But the chemotactic peptide did not increase the incorporation of [3H]glycerol into any glycerolipids including PI and PA. Thus, it is clear that fMLP mainly activates the pathway, PI leads to DG leads to PA, whereas Ca2+ ionophore activates the de novo synthesis of acidic phospholipids. When [3H]arachidonic acid-labeled neutrophils were treated with Ca2+ ionophore, the enhanced release of arachidonic acid and the accumulation of [3H]arachidonyl-DG, -PA with a concomitant decrease in [3H]arachidonyl-PC, -PE, and -PI were observed. Furthermore, the Ca2+ ionophore stimulated the formation of lysophospholipids, such as LPC, LPE, LPI, and LPA nonspecifically. These data suggest that Ca2+ ionophore releases arachidonic acid, unlike fMLP, directly from PC, PE, and PI, mainly by phospholipase A2. When neutrophils were stimulated by fMLP, the formation of LPC and LPE was observed by incubation for more than 3 min. Because a loss of arachidonic acid from PI occurred rapidly in response to fMLP, it seems likely the activation of PI-specific phospholipase C occurred first and was followed by the activation of phospholipase A2 when neutrophils are activated by fMLP...     

Possible Existence of Two Subsets of Platelet-Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells

Platelet-activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca(2+)-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of 3H-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8) M, respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of 3H-inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two transduction sequences are necessary for neutrophil activation by receptor agonists

The effects of 17-hydroxywortmannin (HWT), a powerful inhibitor of the respiratory burst associated with phagocytosis (Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P. H., and Payne, T. G. (1987) Exp. Cell Res. 169, 408-418), were studied in human neutrophils stimulated with chemotactic agonists or phorbol myristate acetate. At nanomolar concentrations HWT inhibited superoxide production and the release of granule contents induced by N-formyl-Met-Leu-Phe, C5a, platelet-activating factor, and leukotriene B4, but not by phorbol myristate acetate, indicating that it interferes with receptor-mediated activation of the neutrophils, without directly affecting protein kinase C (Ca2+/phospholipid-dependent enzyme), the NADPH-oxidase, or the process of granule exocytosis. Moreover, HWT did not influence agonist-induced [Ca2+]i changes, indicating that it does not interfere with the function of agonist receptors, G-proteins or the phosphatidylinositol-specific phospholipase C. By studying the effect of HWT on the respiratory burst elicited in normal and Ca2+-depleted cells by combined stimulation with N-formyl-Met-Leu-Phe and phorbol myristate acetate, evidence was obtained that two transduction sequences, both of which are G-protein-dependent, are necessary for the induction of the response by receptor agonists. One sequence is Ca2+-dependent, HWT-insensitive, and leads to activation of protein kinase C, the other is Ca2+-independent and HWT-sensitive. Ca2+ depletion, which blocks the first, and HWT, which blocks the second, can be used to show that both processes must be functional for the transduction of agonist signals into a respiratory burst response.     

Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.     

De novo synthesis of diacylglycerol from glucose. A new pathway of signal transduction in human neutrophils stimulated during phagocytosis of beta-glucan particles.

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.     

Correlation of intracellular and extracellular calcium ion concentrations with synergy between 1,2-dioctanoyl-sn-glycerol and ionomycin in platelet arachidonic acid mobilization

The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites. This hypothesis was confirmed by simultaneous measurements of [Ca2+]i increases, and the production of the arachidonic acid metabolites. With the aequorin method, the optimal concentrations of [Ca2+]i fell to between 10 microM and 20 microM, and with the fura-2 method, it fell to between 800 nM and 1800 nM. Direct measurements of [14C]arachidonic acid release suggested that the DiC8-potentiated production of arachidonic acid metabolites induced by ionomycin was attributable to increased arachidonic acid release. Since ionomycin and DiC8 induced relatively low levels of phosphatidic acid production, an indicator of phospholipase C activation, it was suggested that the increased arachidonic acid release was largely dependent upon phospholipase A2. Synergy between DiC8 and ionomycin was also observed with aggregation and serotonin release. Aggregation was induced by lower concentrations of ionomycin, and appeared to be more dependent upon extracellular Ca2+, while serotonin release required higher concentrations of ionomycin, and variations in extracellular Ca2+ affected the response minimally. These findings suggest that the mechanisms underlying the synergy between protein kinase C activation and Ca2+ mobilization differ among the three functions evaluated in this study.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Calcium channel blockers nifedipine and diltiazem inhibit Ca2+ release from intracellular stores in neutrophils.

We have undertaken a detailed study of the mechanisms of maintenance of intracellular Ca2+ homeostasis in human polymorphonuclear neutrophils (PMN) and its implications for phagocytosis and IgG Fc receptor (FcR) signaling. When PMN were incubated in Ca(2+)-free medium, cytoplasmic calcium concentration ([Ca2+]i) was markedly depressed and intracellular stores were depleted of calcium. [Ca2+]i in these depleted cells increased within 1 min when PMN were placed in medium containing Ca2+ and then decreased to a level close to the normal basal [Ca2+]i, replenishing the intracellular Ca2+ pools. LaCl3 prevented entry of Ca2+ into Ca(2+)-depleted PMN, but the calcium channel blockers nifedipine, diltiazem, and verapamil did not. Nifedipine and diltiazem but not verapamil inhibited the movement of Ca2+ from cytosol to intracellular stores. Nifedipine and diltiazem inhibited the normal increase in [Ca2+]i from aggregated IgG binding to FcR and also prevented formyl-methionyl-leucyl-phenyl-alanine (fMLP)-induced [Ca2+]i rise. Verapamil had no effect on either an fMLP- or IgG-mediated increase in [Ca2+]i. Consistent with this, nifedipine and diltiazem inhibited fMLP-stimulated phagocytosis (which is dependent on an increase in [Ca2+]i) when PMN had repleted intracellular stores. In contrast, LaCl3 inhibited fMLP-stimulated ingestion only in PMN which had intracellular store depleted. None of these compounds had any effect on phorbol dibutyrate-stimulated ingestion (which is independent of a [Ca2+]i rise). In summary, these data show that Ca2+ is in rapid equilibrium between intracellular and extracellular compartments in PMN. Exchange of cytoplasmic Ca2+ with the extracellular space is inhibited by LaCl3, while exchange of Ca2+ between the cytosol and intracellular stores is inhibited by the dihydropyridine nifedipine and the benzothiazepine diltiazem. These data suggest that these drugs, which are known to regulate some plasma membrane Ca2+ channels in excitable cells, can also regulate Ca2+ release from intracellular stores in PMN and that this regulation may have significant effects on PMN function.     

Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.