Induction of endogenous arachidonic acid metabolism in human neutrophils with snake venom phospholipase A2, immune complexes, and A23187

The stimuli responsible for eicosanoid secretion of phagocytes in chronic inflammatory disorders like rheumatoid arthritis and chronic inflammatory bowel disease are unknown. Phospholipase A2 (PLA2), found in Russelli vipera snake venom, has been proposed to be more than 100 times more potent on a molar basis than A23187 in releasing leukotriene B4 (LTB4) from porcine neutrophils. Therefore, this enzyme was investigated as a challenger of human neutrophils (PMNs) and compared with immune complexes and A23187. 1-14C-Arachidonic acid (AA) was incorporated into purified human PMNs until steady state conditions were obtained. AA release and metabolism were stimulated with either PLA2 isoenzyme of Russelli vipera, immune complexes, or A23187. The radioactive eicosanoids released were extracted and separated by thin layer chromatography, followed by autoradiography and quantitative laser densitometry. Stimulation with PLA2, immune complexes, or A23187 resulted in LTB4 formation of 0%, 1.8%, and 5.3%, respectively, of total released radioactivity. In conclusion, Russelli vipera PLA2 does not stimulate AA-release and metabolism in human PMNs, and immune complexes are weak as compared to the unphysiologic challenger A23187 in this respect.     

Effects of 60 Hz magnetic field exposure on polymorphonuclear leukocyte activation

We have investigated the effects of a sinusoidal 60 Hz magnetic field on free radical (superoxide anion) production, degranulation (beta-glucuronidase and lysozyme release) and viability in human neutrophils (PMNs). Experiments were performed blindly in very controlled conditions to examine the effects of a magnetic field in resting PMNs and in PMNs stimulated with a tumor promoter: phorbol 12-myristate 13-acetate (PMA). Exposure of unstimulated human PMNs to a 60 Hz magnetic field did not affect the functions examined. In contrast, exposure of PMNs to a 22 milliTesla (mT), 60 Hz magnetic field induced significant increases in superoxide anion (O2-) production (26.5%) and in beta-glucuronidase release (53%) when the cells were incubated with a suboptimal stimulating dose of PMA. Release of lysozyme and lactate dehydrogenase was unchanged by the magnetic field, whether the cells were stimulated or not. A 60 Hz magnetic field did not have any effect on O2- generation by a cell-free system xanthine/xanthine oxidase, suggesting that a magnetic field could upregulate common cellular events (signal transduction) leading to O2- generation and beta-glucuronidase release. In conclusion, exposure of PMNs to a 22 mT, 60 Hz magnetic field potentiates the effect of PMA on O2- generation and beta-glucuronidase release. This effect could be the result of an alteration in the intracellular signaling.     

Metabolic activation of chlorpromazine by stimulated human polymorphonuclear leukocytes. Induction of covalent binding of chlorpromazine to nucleic acids and proteins.

Human polymorphonuclear leukocytes (PMNs) have been stimulated with either phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 or a combination of both to induce the respiratory burst and myeloperoxidase (MPO) release. Chlorpromazine (CPZ) but not chlorpromazine sulfoxide (CPZSO) inhibited the respiratory burst as measured with lucigenin chemiluminescence. The inhibition was due to interference with processes in the cell leading to the respiratory burst and not to scavenging of produced oxygen radicals that provoke the luminescence. CPZ was metabolized by stimulated PMNs. HPLC analysis revealed formation of CPZSO and an unidentified product. Both products result from decay of chlorpromazine radical cation (CPZ+.), indicating formation of this radical intermediate in CPZSO oxidation by stimulated PMNs. CPZ conversion correlated with H2O2 production and MPO release. The largest CPZ conversion was observed with phorbol ester plus A23187 stimulation. The conversion was reduced by catalase and sodium azide, an inhibitor of MPO, with 70% and 40%, respectively. This indicates only partial involvement of extracellularly released MPO in CPZ metabolism by PMNs. Considerable covalent binding of [3H]CPZ to nucleic acids and proteins of intact stimulated PMNs was observed. This binding was larger upon co-stimulation with phorbol ester and A23187. Azide did not reduce covalent binding. This indicates that covalent binding is not mediated by extracellularly released MPO and that CPZ is probably activated intracellularly. Activation of PMNs and production of H2O2 is a prerequisite for both CPZ conversion and covalent binding. This study demonstrates that phagocytic cells might contribute to drug metabolism and drug-induced toxicity.     

Effect of isorhapontigenin on respiratory burst of rat neutrophils

The effect of Isorhapontigenin (Iso) isolated from Belamcanda chinensis on respiratory burst of rat neutrophils was investigated. Iso (1, 10, 100 mmol/l) showed an inhibitory effect on superoxide anion and hydrogen peroxide production in phorbol myristate acetate (PMA) activated rat neutrophils in a concentration-dependent manner. Scanning electron microscopy detected that Iso (100 mmol/l) protected against surface changes in rat neutrophils stimulated with PMA. Also, 100 mmol/l Iso inhibited the release of beta-glucuronidase from the activated neutrophils. Electron-spin resonance (ESR) detected that Iso scavenged oxygen free radicals generated in the PMA activated Neutrophils. These results suggest that Iso inhibits respiratory burst of PMA-activated rat neutrophils by scavenging oxygen free radicals.     

Inhibition of protein kinase C by H-7 potentiates the release of oleic, linoleic and arachidonic acids in A23187-stimulated human neutrophils

This present report describes the effect of H-7, a protein kinase C inhibitor, on the release of oleic, linoleic and arachidonic acids in A23187-stimulated neutrophils. Surprisingly, the inhibitor potentiated the release of all three unsaturated fatty acids in neutrophils stimulated with A23187 alone. In contrast, released oleic acid, linoleic acid and arachidonic acid in phorbol 12-myristate 13-acetate-primed neutrophils were attenuated by 35, 47 and 33%, respectively, in the presence of H-7 (300 microM). Phorbol 12-myristate 13-acetate (PMA) had no effect on A23187-stimulated release of saturated fatty acids. Both PMA and H-7 when used alone had no effect on the release of saturated or unsaturated fatty acids. We, therefore, conclude that H-7 may have effects other than inhibiting PMA-primed responses including superoxide generation, degranulation and arachidonic acid release in human neutrophils.     

Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1-oleoyl-2-acetylglycerol from human neutrophils

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet-Leu-Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction--calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1-oleoyl-2-acetylglycerol). Prostaglandin E1 (3 X 10(-5) M) decreased fMet-Leu-Phe-stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1-oleoyl-2-acetylglycerol. Similar enhancement was obtained with prostaglandin E2.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

Phospholipase A2-mediated superoxide production of murine peritoneal macrophages induced by chrysotile stimulation

In the present study, we investigated how chrysotile-stimulated macrophages generate superoxide using murine peritoneal macrophages, with special attention to the modulatory role of phospholipase A(2) (PLA(2)). We examined the effects of the following inhibitors and antagonists for signaling molecules on the superoxide anion (O(-)(2)) production of chrysotile-stimulated macrophages: p-bromophenacyl bromide (pBPB) and mepacrine for PLA(2); islet-activating protein (IAP) for G-protein; H-7 for protein kinase C (PKC); AA861 for 5-lipoxygenase (5-LO); indomethacin for cyclo-oxygenase (COX); ETYA for both 5-LO and COX; hexanolamine PAF for platelet-activating factor (PAF). The PLA(2) and PKC inhibitors effectively inhibited the chrysotile-induced superoxide anion production of macrophages, but not the G-protein inhibitor, the 5-LO and COX inhibitors, and the PAF antagonist. We also examined the effects of the PLA(2) inhibitors on macrophages stimulated by phorbol 12-myristate 13-acetate (PMA) which directly activates PKC. The two structurally different PLA(2) inhibitors showed differential effects on the PMA-induced superoxide generation: pBPB inhibited it but mepacrine did not. These results suggested that (1) PLA(2) and PKC modulate the chrysotile-induced O(2) production, and (2) two different kinds of PLA(2) work upstream and downstream of PKC, but (3) G-protein, 5-LO and COX metabolites, and PAF have no modulatory role in the reaction.     

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung

Alveolar macrophages (AM) differ from other macrophage (m phi) populations in their profile of eicosanoids synthesized from arachidonic acid (AA)3. Little information is available regarding possible differences in the regulation of AA metabolism among various m phi populations. In our study, we compared the ability of cultured resident rat AM and peritoneal m phi (PM) to release and metabolize AA in response to exogenous activators of protein kinase C (PKC). When stimulated with PMA, prelabeled PM released free [3H]AA in a dose-dependent manner over the concentration range 1 to 100 nM. As assessed by HPLC, PMA-stimulated PM metabolized AA to a variety of predominantly cyclooxygenase products. The dose-dependent synthesis of PGE2 by unlabeled PM stimulated with PMA was confirmed using RIA. The ability of PMA to trigger AA release and metabolism in PM was a function of its capacity to activate PKC, as indicated by the following: 1) an additional activator of PKC, oleoyl acetylglycerol, also triggered PM AA metabolism, whereas phorbol didecanoate, which lacks the ability to activate PKC, did not; 2) two structurally unrelated inhibitors of PKC activation (staurosporine and sphinganine) both abrogated PMA induced AA release in PM; and 3) pretreatment for 18 h with high dose PMA (used to deplete cellular PKC), but not phorbol didecanoate, rendered PM refractory to subsequent PMA stimulation of AA release. In contrast to PM, AM cultured in identical fashion failed to release or metabolize AA in response to either PMA or oleoyl acetylglycerol. PM and AM were also compared for their ability to release extracellular superoxide anion in response to PMA; once again, PM exhibited significantly greater release than did AM. Inasmuch as this unresponsiveness to activation of PKC distinguishes AM from other m phi populations, we conclude that it is a unique consequence of m phi differentiation in the lung. Moreover, because both AA metabolism and the respiratory burst are affected, this refractoriness appears to reflect a defect at some proximal level in PKC-mediated signaling.     

Secretory and cytosolic phospholipase A(2)regulate the long-term cytokine-induced eicosanoid production in human keratinocytes

The involvement of cytosolic phospholipase A(2)(cPLA(2)) and secretory non-pancreatic PLA(2)(npPLA(2)) in release of arachidonic acid (AA) preceding eicosanoid formation in the human keratinocyte cell line HaCaT was examined. Interleukin 1beta (IL-1beta) and tumour necrosis factor-alpha (TNF), phorbol myristate acetate (PMA) and calcium ionophore A(23187)increased the extracellular AA release, and stimulated eicosanoid synthesis as determined by HPLC analysis. The main metabolites after stimulation with IL-1beta, PMA or A(23187)were PGE(2), an unidentified PG and LTB(4), while TNF stimulated HETE-production. Both cPLA(2)and npPLA(2)message and enzyme activity were detected in unstimulated HaCaT cells. IL-1beta, PMA and TNF increased both cPLA(2)enzyme activity and expression, but did not lead to any increase in npPLA(2)expression or activity. The selective npPLA(2)inhibitors LY311727 and 12-epi-scalaradial, or the cPLA(2)inhibitor arachidonyl trifluoro methyl ketone (AACOCF(3)) reduced IL-1beta-induced eicosanoid production in a concentration dependent manner. The results presented strongly suggest that both cPLA(2)and npPLA(2)contribute to the long-term generation of AA preceding eicosanoid production in differentiated, human keratinocytes. Inhibitors against npPLA2 or cPLA2 enzymes should be useful in treating inflammatory skin diseases, such as psoriasis.     

Modulation by phorbol myristate acetate of arachidonic acid release and leukotriene synthesis by human polymorphonuclear leukocytes stimulated with A23187

Phorbol myristate acetate augmented the release of 3H-AA and the synthesis of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human polymorphonuclear leukocytes stimulated by A23187. PMA alone had no effect. Enhancement of the response to A23187 was not seen when the inactive phorbol ester 4-alpha phorbol didecanoate was added with A23187. These data are consistent with the hypothesis that activation of protein kinase C enhances AA release and metabolism in stimulated polymorphonuclear leukocytes.     

Studies of signal transduction in the respiratory burst-associated stimulation of fMet-Leu-Phe-induced tubulin tyrosinolation and phorbol 12-myristate 13-acetate-induced posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils and HL-60 cells

A specific stimulation of tubulin tyrosinolation in human neutrophils (PMNs) is induced by the synthetic peptide chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and this stimulation is closely associated with activation of the NADPH oxidase-mediated respiratory burst (Nath, J., and Gallin, J. I. (1983) J. Clin. Invest. 71, 1273-1281). In contrast, along with tubulin tyrosinolation, a distinctly different respiratory burst-associated random posttranslational incorporation of tyrosine into multiple PMN proteins is observed in PMNs stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol (DAG). In studies exploring the mechanism(s) of signal transduction for these distinct neutrophil responses, we found that the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation in PMNs and in differentiated HL-60 cells is completely blocked by pertussis toxin, while the PMA-induced random incorporation of tyrosine is not inhibited. We also found that expression of the fMet-Leu-Phe-mediated stimulation of tubulin tyrosinolation in HL-60 cells is correlated with increases in the specific activity of protein kinase C and with the acquisition of respiratory burst activity which occur during induced myeloid maturation of these cells. Furthermore, both the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation and the PMA or DAG-induced random posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils, were found to be reversibly inhibited (greater than 70%) by the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)piperazine (C-I) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), in parallel with inhibition of superoxide (O2-) generation. In related studies, we also found that fMet-Leu-Phe-stimulated O2- production is comparably inhibited by C-I and H-7, but in a highly temperature-dependent manner. Inhibition was observed only when C-I or H-7 is added to PMNs at physiologic temperature, i.e. 37 degrees C. Interestingly, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. Considered together, these findings argue against the suggestion that there is a protein kinase C-independent pathway for activation of the respiratory burst in neutrophils stimulated with N-formyl peptides.     

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.     

Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release in luminol-dependent photoemission from stimulated neutrophils.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore

In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2-)release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated "protein kinase C." PMA enhanced O2- release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2- release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2-.