Role of cytoskeletal elements in cytochalasin E-induced superoxide production by human polymorphonuclear leukocytes.

The release of superoxide anions from human polymorphonuclear leukocytes induced by cytochalasin E was greatly enhanced by the pretreatment of the cells either with deuterium oxide or with concanavalin A. Colchicine, vinblastine and cyclic AMP inhibited the release. Cytochalasins A and B also suppressed the superoxide release. These observations suggest the involvement of microfilament-microtubule system in the production and release of superoxide anions induced by cytochalasin E.

Effects of fatty acids on superoxide radical generation in leukocytes.

Acetylated ferricytochrome c was employed for the detection of superoxide radicals (O-2) generated both in intact cells and in subcellular fractions of leukocytes. Certain saturated fatty acids, myristate in particular, induced the production of O-2 in leukocytes, suggesting a correlation between the formation of O-2 and the hydrophobic interaction of fatty acids with the leukocyte plasma membrane. As compared with O-2 radical generation from phagocytizing leukocytes a greater stimulation of O-2 formation was observed in cells in which myristate was added. The enhanced activity which generated O-2 in the cell-free system was located in a particulate fraction but not in the cytosol. The rate of O-2 generation in the particulate fraction was higher in the presence of NADPH than in the presence of NADH. The effects of reagents such as KCN, 2,4-dichlorophenol and aminotriazole on the O-2 generation in this fraction are examined and the nature of the O-2 generating system is discussed.

Effect of hydrogen ion concentration on energy metabolism in pig platelets.

Respiration and glycolysis of pig platelets suspended in a dialyzed plasma were studied at various hydrogen ion concentrations. Respiration of platelets was high at acidic pH and decreased at physiological pH. This pH profile may not be attributed to properties of mitochondria, since the respiratory rate of mitochondria prepared from platelets was maximal at physiological pH. A low respiratory rate at physiological pH seemed to be attributable to depression of respiration by glycolysis, since the addition of glucose further depressed the rate. The Crabtree effect was more prominent at alkaline ph. glycolysis increased with an increase in the pH of the plasma, contrary to oxygen comsumption. The Pasteur effect was less prominent at alkaline pH. The effect of pH on lactate production by the cytosol fraction of platelets was similar to that of whole platelets. The glycolytic intermediate pattern showed that phosphofructolinase was the committed step. Both ATP concentration and ATP formation calculated from respiratory and glycolytic rates were constant at various pH values. These observations may indicate that the pH primarily affects platelet glycolysis at the phosphofructokinase step and the respiration is secondarily controlled by glycolysis.

ATP-induced calcium mobilization in human neutrophils.

Extracellular ATP at 10 microM increased the concentration of cytoplasmic free Ca2+ ( [Ca2+]i) 3-fold in human neutrophils. The [Ca2+]i was measured by fura-2 fluorescence. The effect was rapid but transient: [Ca2+]i reached a maximum within 10 s and then returned to its resting value after 2-3 min. The rise in [Ca2+]i elicited by ATP was unaffected by the removal of extracellular Ca2+, indicating that the primary source of Ca2+ is from intracellular stores. In contrast to ATP, neither ADP nor AMP, at concentrations as high as 100 microM, caused any detectable changes in [Ca2+]i. Among other nucleotide triphosphates tested, UTP was as effective as ATP in causing a transient rise in [Ca2+]i, and prevented a subsequent response to ATP. Similarly, ATP prevented a subsequent response to UTP but the second response could be obtained when the initially added ATP was removed by the addition of hexokinase. Phorbol myristate acetate, the activator of Ca2+, phospholipid-dependent protein kinase, completely inhibited the ATP-induced increases in [Ca2+]i without affecting the basal [Ca2+]i level. The results suggest that extracellular ATP stimulates human neutrophils by causing the release of calcium from intracellular storage pools by mechanisms which can be inhibited by phorbol myristate acetate.

Protein phosphorylation in intact pig leukocytes.

The phosphorylation of proteins in intact pig polymorphonuclear leukocytes loaded with H3(32)PO4 was investigated by two-dimensional gel electrophoresis and subsequent autoradiography. The incorporation of 32P into at least 17 proteins began to increase and into one to decrease, relative to resting cells, upon exposure of the cells to phorbol 12-myristate 13-acetate. These changes in the autoradiographic patterns were accompanied by changes in the protein patterns obtained by staining with Coomassie brilliant blue, including the appearance, the acidic shift and the increase or decrease of the intensity of the spots. Among these proteins, Mr = 64 000, 31 000, 22 000, 21 000, 18 000 and 13 000 proteins were correlated well with the superoxide anion production of the cells in respect to the time-courses and the dose-responses. By taking the effects of EGTA into consideration, the phosphorylation of Mr 64 000 and 21 000 proteins, of which the latter was identified as the light chain of myosin, seemed to be involved in the signal-transmission mechanism of the induction of the NADPH oxidase responsible for the 'respiratory burst'. These two proteins were also phosphorylated in the cells stimulated by NaF or oil droplets opsonized with IgG.

NADPH-dependent reduction of ubiquinone-1 associated with the superoxide-forming oxidase of pig polymorphonuclear leucocytes.

NADPH-dependent ubiquinone-1 reductase activity was present in the phagocytic vesicles of pig polymorphonuclear leucocytes. The apparent Km-value of the reductase for NADPH was 29 microM which is similar to that of the NADPH-dependent superoxide formation. Increase of the quinone-reductase activity by increasing the concentrations of ubiquinone-1 was associated with the decrease of the superoxide forming activity, the rate of the NADPH oxidation being constant independent of the quinone concentration. p-Chloromercuribenzoate inhibited both superoxide formation and reduction of the quinone, whereas low concentrations of cetyltrimethylammonium bromide which inhibit the superoxide formation did not inhibit the reduction of the quinone. The reduction of 2,6-dichlorophenolindophenol which has been shown not to be inhibited by both inhibitors. The quinone-reductase activity could be extracted with a mixture of deoxycholate and Tween 20 which extracts the superoxide forming activity. The observations indicate that a region of the superoxide-forming NADPH oxidase between a mercurial-sensitive site and a site sensitive to the cationic detergent is responsible for the reduction of ubiquinone.

Formation of 20-oxoleukotriene B4 by an alcohol dehydrogenase isolated from human neutrophils.

When 20-hydroxyleukotriene B4 (20-OH-LTB4) is incubated at pH 10.5 in the presence of NAD+ with an alcohol dehydrogenase isolated from human neutrophils, a polar product is formed as detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product is identified as 20-oxo-LTB4 (20-CHO-LTB4) on the basis of its co-elution with the authentic compound on HPLC, ultraviolet spectrometry and gas chromatography-mass spectrometry. The 20-CHO-LTB4-forming activity requires NAD+, but NADP+ scarcely replaces NAD+. The apparent Km for 20-OH-LTB4 is 83 microM and the Vmax is 2.04 mumol/min per mg of protein. The activity is inhibited by omega-hydroxy fatty acids such as 12-hydroxylauric acid, 16-hydroxypalmitic acid and 12(S), 20-dihydroxyeicosatetraenoic acid, but not by 4-methylpyrazole. At pH 7.0 with NADH, the purified dehydrogenase catalyzes the reverse reaction, the reduction of 20-CHO-LTB4 to 20-OH-LTB4.

Characterization of the NADPH-dependent superoxide production activated by sodium dodecyl sulfate in a cell-free system of pig neutrophils.

The NADPH-dependent superoxide production induced by sodium dodecyl sulfate (SDS) in the sonicates of unstimulated pig neutrophils required both membrane fraction and two components of cytosol fraction. The potency of the cytosol fraction in the activation of the superoxide production could be reconstituted dose dependently by mixing two protein components with relative molecular masses of 300 kDa and 50 kDa. Another low-molecular-mass component (1.3 kDa) could substitute the 50-kDa component. In the cell-free system consisting of the 300- and 50-kDa components and the membrane fraction, the superoxide production was markedly enhanced by FAD with a required concentration for half-maximal effect of 0.16 microM and inhibited by divalent cations such as Ca2+, Ba2+, Co2+, Zn2+ and Mn2+ and not Mg2+. ATP was not necessary for the activation, indicating that protein kinases such as protein kinase C are not involved in the SDS-dependent activation of NADPH oxidase. The NADPH oxidase activated by SDS in the cell-free system was recovered in the membrane fraction, and the superoxide formation by the SDS-activated membrane exhibited a Km value for NADPH of 46 microM and optimum pH at 7.0. The formation did not require the addition of SDS and FAD to the reaction mixture and was scarcely inhibited by the divalent cations.

Decrease in the phosphotyrosine phosphatase activity in the plasma membrane of human neutrophils on stimulation by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) induced a decrease in the phosphotyrosine phosphatase (PTPase) activity in human neurophils. The decrease in the activity induced by PMA was blocked by the treatment of the cells with staurosporine, indicating that protein kinase C is involved in the decrease. The PTPase activity was localized in the plasma membrane. The activity in the membrane with the optimum pH at 5.5 had a Km value for phosphotyrosine of 2.2 mM and Vmax of 2.0 mumol/min per mg of protein. No activity was observed against phosphoserine and phosphothreonine. Vanadate, molybdate, zinc and a sulfhydryl reagent, p-chloromercuribenzenesulphonic acid, inhibited the PTPase. The PMA-induced decrease in activity was almost completely recovered by treatment of the plasma membrane with Triton X-100 at low concentrations which did not solubilize the activity from the membrane. When the plasma membrane was treated with trypsin, the PTPase of the membrane from PMA-treated cells was mostly protected from the proteinase attack while that from the resting cells was not protected. Pretreatment of the plasma membrane with Triton X-100 enabled trypsin to gain access to all the PTPase in the membrane from both PMA-treated and resting cells. The PMA treatment affected neither subcellular localization of the PTPase nor the orientation of the plasma membrane vesicles. These findings suggest that conformational changes of the enzyme induced by PMA result in the decrease in PTPase activity.

Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils.

We studied the action of iodinated thyronines on the superoxide (O2-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced O2- production with IC50 values of about 10(-6) M and 7.10(-6) M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the O2- production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O2- production stimulated with either the fifth component of the complement (C5a) or phorbol 12-myristate 13-acetate. L-Thyroxine and L-triiodothyronine were found to block [3H]FMLP binding to its own receptor with IC50 values similar to those for the inhibition of the O2- production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O2- production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.

Phosphorylation of myosin light chain in intact pig leukocytes stimulated by phorbol 12-myristate 13-acetate.

Phosphorylation of myosin light chain was investigated in intact pig polymorphonuclear leukocytes. The labeling of the myosin light chain (Mr = 21000) was increased by exposure of the cells to phorbol 12-myristate 13-acetate. Two-dimensional gel electrophoresis revealed three forms of Mr = 21000 light chain, of which two were phosphorylated. The phosphorylation of the myosin light chain was inhibited by neither N-(6-aminohexyl)-5-chloro-1-naphthalene sufonamide nor trifluoperazine, suggesting that calmodulin is not involved in the phosphorylation.

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4.

Leukotriene B4 stimulated a transient production of superoxide anions (O2-) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B4 and calcium for the half-maximal production were about 1 microM and 200 microM, respectively. 8-(N,N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O2- production stimulated by leukotriene B4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B4 was added to the cells treated with cytochalasin B, the production of O2- was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca2+ concentrations but it could be induced even without the addition of Ca2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca2+-free medium showed a negligible production of superoxide on addition of leukotriene B4, but the production appeared upon addition of CaCl2. These findings suggest that the superoxide production stimulated by leukotriene B4 is associated with the influx of Ca2+.

Cyclic AMP inhibits the respiratory burst of electropermeabilized human neutrophils at a downstream site of protein kinase C.

We studied a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.

NAD+-dependent oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by rat liver cytosol.

20-Hydroxyleukotriene B4 was converted by rat liver homogenates in the presence of NAD+ to a more polar product on reverse-phase high-performance liquid chromatography. The product was identified as 20-carboxyleukotriene B4 by straight-phase high performance liquid chromatography, ultraviolet spectrophotometry and gas chromatography-mass spectrometry. The oxidative activity of the homogenates was located in the cytosol with an optimal pH of 8.0. The activity was dependent on NAD+, and NADP+ could not substitute for NAD+. 1 mol of 20-carboxyleukotriene B4 was formed with the reduction of 2 mol of NAD+. The reaction was inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by various alcohols, such as ethanol, 12-hydroxylaurate, and 20-hydroxyprostaglandin E1. Disulfiram, an inhibitor of aldehyde dehydrogenase, also inhibited the activity. These results suggest that two discrete steps catalyzed by different enzymes, alcohol dehydrogenase and aldehyde dehydrogenase, are involved in the oxidation of 20-hydroxyleukotriene B4 in rat liver cytosol. The enzyme system seems to be different from that of human neutrophils.

Omega-hydroxylation of lipoxin B4 by human neutrophil microsomes: identification of omega-hydroxy metabolite of lipoxin B4 and catalysis by leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega).

Lipoxin B4 (LXB4) is metabolized either by human neutrophils or by the neutrophil microsomes to a polar compound on a reverse-phase high-performance liquid chromatography. The metabolite is identified as 20-hydroxy-lipoxin B4 (20-OH-LXB4), a novel member in the arachidonic acid cascade, on the basis of ultraviolet spectrometry and gas chromatography-mass spectrometry. The neutrophil microsomes convert LXB4 to its 20-hydroxy derivative under aerobic condition in the presence of NADPH. The reaction is inhibited by carbon monoxide, an inhibitor of cytochrome P-450 (P-450), and by antibodies raised against NADPH-P-450 reductase. A P-450 is thus involved in the omega-hydroxylation of LXB4. The P-450 appears to be the one responsible for leukotriene B4 (LTB4) omega-hydroxylation, P-450LTB omega, based on the following observations. The formation of 20-OH-LXB4 is inhibited solely by substrates of P-450LTB omega such as LTB4 and leukotriene B5 among various fatty acids including prostaglandins. The order of the inhibitory potencies of these substances on the LXB4 omega-hydroxylation is the same as that of their affinities for LTB4 omega-hydroxylase. LTB4 inhibits the reaction in a competitive manner with the Ki value of 0.2 microM, which agrees with the Km value for the LTB4 omega-hydroxylation (0.3 microM).

Characterization of the GTP-dependent activation of the superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We characterized the cell-free activating system of the superoxide (O2-)-producing NADPH oxidase of pig neutrophils. Activation of the oxidase required both the membrane and cytosolic fractions in the presence of sodium dodecyl sulfate. Chromatography on 2',5'-ADP-Sepharose resulted in separation of the cytosolic fraction into two fractions, the flow-through and bound fractions, which synergistically supported the O2- production with the membrane fraction in the absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas only the bound fraction besides the membrane fraction was required for the activation in the presence of GTP gamma S. The effective factors in the bound fraction were further purified by gel filtration on Superdex G-200 and anion exchange chromatography on Mono Q and found to be p47-phox and p63-phox. The purified recombinant p47-phox and p65-phox replaced corresponding native factors for the activation. These results suggest that the membrane fraction from pig neutrophils contains the GTP-binding protein responsible for the activation. Furthermore, the presence of the GTP-binding protein for the activation in the flow-through fraction from 2',5'-ADP-Sepharose was also shown on the basis of the findings that extensive dialysis of the flow-through fraction resulted in complete loss of the ability to activate the oxidase with the recombinant factors and the washed membrane of human neutrophils which contained no GTP-binding protein for the activation and the lost ability was recovered by the addition of GTP gamma S. Thus, activation of the oxidase in the cell-free system of pig neutrophils absolutely requires the GTP-binding protein which localizes in the membrane fraction or in the cytosolic fraction.

Involvement of calcium and protein kinase C in the activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP.

We have studied the activation of the Na+/H+ exchanger which leads to the intracellular alkalinization in cultured bovine aortic endothelial cells stimulated by extracellular ATP. The alkalinization induced by ATP was largely dependent on extracellular Ca2+ and the rate of alkalinization was decreased by about 60% in the absence of extracellular Ca2+. ATP caused a rapid and transient increase and a subsequent sustained increase of the intracellular Ca2+ concentration ([Ca2+]i) in the Ca2+ buffer, while only the rapid and transient increase of [Ca2+]i was observed in the absence of extracellular Ca2+. The Ca2+-depleted cells prepared by incubation in Ca2+-free buffer containing 0.1 mM EGTA showed only a slight increase of [Ca2+]i with no alkalinization on stimulation by ATP. The alkalinization was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by another isoquinoline analogue (HA 1004), which has a less inhibitory effect on the kinase. Phorbol 12-myristate 13-acetate also induced the alkalinization by the activation of the Na+/H+ exchanger. Neither dibutyryl cyclic AMP nor dibutyryl cyclic GMP affected the alkalinization induced by ATP. Treatment of the cells by pertussis and cholera toxins had no effect on the alkalinization. The results suggest that the increase in [Ca2+]i is essential for the ATP-induced activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells and a protein kinase C-dependent pathway is involved in the activation.

Cytoplasmic pH change induced by leukotriene B4 in human neutrophils.

Leukotriene B4 induced a biphasic change in the cytoplasmic pH of human neutrophils: an initial rapid acidification followed by an alkalinization. The acidification was slightly reduced by the removal of extracellular Ca2+, but the subsequent alkalinization was not. The leukotriene B4-induced alkalinization was dependent on extracellular Na+ and pH, and was inhibited by amiloride and its more potent analogue, 5-(N,N-hexamethylene)amiloride. These characteristics indicate that the cytoplasmic alkalinization is mediated by the Na+-H+ exchange. Oxidation products of leukotriene B4, 20-hydroxyleukotriene B4, 20-carboxyleukotriene B4, and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) also stimulated the Na+-H+ exchange, but higher concentrations were required. Treatment of the cells with pertussis toxin inhibited both phases of the leukotriene B4-induced pHi change, while cholera toxin did not affect the pHi change. The alkalinization induced by leukotriene B4 was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, but was not inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide which has a less inhibitory effect on protein kinase C. Acidification was not affected by the drugs. These findings suggest that a GTP-binding protein sensitive to pertussis toxin and protein kinase C are involved in the activation of the Na+-H+ exchange stimulated by leukotriene B4.

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system.

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.

Superoxide release and intracellular free calcium of calcium-depleted human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine.

The superoxide release and the change in the intracellular free calcium ions on stimulation with N-formyl-methionyl-leucyl-phenylalanine were studied in human neutrophils deprived of divalent cations by treatment of the cells with ionophore A23187 in the presence of EGTA. The depleted cells showed no release of superoxide on stimulation with the chemotactic peptide when calcium ions were absent in the medium, but the activity was completely recovered when the cells were preincubated with calcium for at least 3 min before the stimulation. The cells pretreated with Cd2+ showed slight activity of the release, but no recovery was observed with other divalent cations such as Mg2+, Sr2+, Co2+, Ba2+ and Zn2+. The recovery with calcium ions was dependent on the time of the addition relative to the time of the stimulation with the chemotactic peptide: a simultaneous addition of both calcium and the peptide elicited about half of the full activity, while no release was observed when calcium was added later than 2 min after the stimulation with the peptide, though a marked elevation of the intracellular free calcium monitored by quin-2 fluorescence was found. Comparison of the time-courses of the superoxide release and the change in the fluorescence suggest that, besides the elevation of intracellular free calcium, a transient reaction which is also dependent on calcium is required for the full induction of the superoxide-producing activity.