Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils

In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.     

Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated. 2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.33 +/- 0.05 microgram ml-1 and 0.49 +/- 0.04 microgram ml-1, respectively. 3. Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.- in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. 4. Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8 +/- 0.2 micrograms ml-1) and the generation of inositol trisphosphate (IP3) (IC50 10.6 +/- 2.0 micrograms ml-1) in response to fMLP. 5. Abruquinone A did not affect the enzyme activaties of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6. Abruquinone A had no effect on intracellular guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/ CB was inhibited by abruquinone A with IC50 values of 2.2 +/- 0.6 micrograms ml-1 and 2.5 +/- 0.3 micrograms ml-1, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73-78 kDa in activated neutrophils. 8. Abruquinone A inhibited both the O2.- generation in PMA-activated neutrophil particulate NADPH oxidase (IC50 0.6 +/- 0.1 microgram ml-1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC50 1.5 +/- 0.2 micrograms ml-1) 9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.     

Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated. 2. Kazinol B concentration-dependently stimulated the superoxide anion (O2*-) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2+/-1.4 nmol O2*- 10 min(-1) per 10(6) cells) was observed at 10 microM kazinol B. 3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O2*- generation following the subsequent stimulation of cells with kazinol B. 4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O2*- generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response. 5. Kazinol B significantly stimulated [Ca2+]i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP2 and IP3) formation with a 1 min lag time. 6. The membrane-associated PKC-alpha and PKC-theta but not PKC-iota were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-theta than PKC-alpha by kazinol B. Kazinol B partially inhibited the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to the neutrophil cytosolic PKC. 7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O2*- generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein. 8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Regulation of Src family tyrosine kinase activities in adherent human neutrophils. Evidence that reactive oxygen intermediates produced by adherent neutrophils increase the activity of the p58c-fgr and p53/56lyn tyrosine kinases

Src family tyrosine kinases have been implicated in the adhesion-dependent activation of neutrophil functions (Yan, S. R., Fumagalli, L., and Berton, G. (1995) J. Inflamm. 45, 297-312; Lowell, C. A., Fumagalli, L., and Berton, G. (1996) J. Cell Biol. 133, 895-910). Because the activity of tyrosine kinases can be affected by oxidants, we investigated whether reactive oxygen intermediates (ROI) produced by adherent neutrophils regulate Src family kinase activities. Inhibition of ROI production by diphenylene iodonium, an inhibitor of NADPH oxidase, or degradation of H2O2 by exogenously added catalase inhibited the adhesion-stimulated activities of p58(c-fgr) and p53/56(lyn). In addition, adhesion-stimulated p58(c-fgr) and p53/56(lyn) activities were greatly reduced in neutrophils from patients with chronic granulomatous disease (CGD) that are deficient in the production of ROI. Exogenously added H2O2 increased p58(c-fgr) and p53/56(lyn) activities in nonadherent neutrophils. Although ROI regulated the activities of p58(c-fgr) and p53/56(lyn), they did not affect the redistribution of the two kinases to a Triton X-100-insoluble, cytoskeletal fraction that occurs in adherent neutrophils. Tyrosine phosphorylation of proteins in adherent, CGD neutrophils was only partially inhibited, suggesting that the full activation of p58(c-fgr) and p53/56(lyn), which depends on endogenously produced ROI, does not represent an absolute requirement for protein tyrosine phosphorylation. The adhesion-stimulated activity of the tyrosine kinase p72(syk) was not affected by catalase in normal neutrophils, and it was comparable in normal and CGD neutrophils. These findings suggest that ROI endogenously produced by adherent neutrophils regulate Src family kinases activity selectively and establish the existence of a cross-talk between reorganization of the cytoskeleton, production of ROI, and Src family tyrosine kinase activities in signaling by adhesion.     

Possible target components for the inhibitory effect of N-ethylmaleimide on the activation of neutrophil NADPH oxidase

Potential target components for the inhibitory effect of covalent sulfhydryl-modifying reagent N-ethylmaleimide (NEM) on the activation of NADPH oxidase in human neutrophils was studied in a cell-free system. The capacity of both cytosol and membrane fractions to induce the translocation of cytosolic components and O2-generation in the cell-free activation system was affected by NEM. The phosphorylation of p47phox, which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM-treated p47phox was as effective as untreated p47phox both in the kinase-dependent and in the amphiphile-dependent cell-free activation systems. In addition, phosphatidic acid-dependent phosphorylation of cytosol including p47phox was not affected by NEM. The inhibition of cytosol's capacity to activate NADPH oxidase was partially reversed by an addition of the fraction containing G-protein rac. Taken together, the data suggest that membrane component cytochrome b558 and cytosolic component rac may be the potential targets for the NEM effect on the activation of NADPH oxidase.     

Suppression of tumor promoter-induced oxidative stress and inflammatory responses in mouse skin by a superoxide generation inhibitor 1'-acetoxychavicol acetate

Double applications of phorbol esters trigger excessive reactive oxygen species (ROS) production in mouse skin. Previously reported data suggest that the two applications induce distinguishable biochemical events, namely, priming and activation. The former is characterized as a recruitment of inflammatory cells, such as neutrophils, by chemotactic factors to inflammatory regions and edema formation. The latter is responsible for ROS generation. Thus, inhibitory effects of 1'-acetoxychavicol acetate (ACA), previously reported to be a superoxide generation inhibitor in vitro, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammatory responses in mouse skin model were examined using a double application of ACA. We demonstrated that two pretreatments and pretreatment with ACA (810 nmol) in the activation phase suppressed double TPA application-induced H2O2 formation in mouse skin. ACA exhibited no inhibitory effects on edema formation and the enhancement of myeloperoxidase activity during the first TPA treatment, whereas the anti-inflammatory agent genistein administered at the same dose inhibited both biomarkers. No inhibitory potential of ACA for TPA-induced H2O2 formation in the priming phase was confirmed. On the other hand, in the in vitro study, ACA inhibited ROS generation in differentiated HL-60 cells more strongly than did 1'-hydroxychavicol, which showed no inhibition by pretreatment in the activation phase. In addition, allopurinol did not inhibit double TPA application-induced H2O2 formation in mouse skin. These findings suggest that the NADPH oxidase system of neutrophils rather than the epithelial xanthine oxidase system is dominant for the O2--generating potential in double TPA-treated mouse skin. ACA significantly inhibited mouse epidermis thiobarbituric acid-reacting substance formation, known as an overall oxidative damage biomarker. Moreover, histological studies demonstrated that ACA inhibited double TPA treatment-induced morphological changes reflecting inflammatory response, such as edema formation, leukocyte infiltration, hyperplasia, and cell proliferation. Furthermore, pretreatment with ACA but not 1'-hydroxychavicol in the activation phase inhibits double TPA application-induced increases in both number of leukocytes and proliferating cell nuclear antigen index. These results suggested that ROS from leukocytes including O2- plays an important role for continuous and excessive production of chemotactic factors, leading to chronic inflammation and hyperplasia, which are inhibitable by ACA. Thus, we concluded that O2- generation inhibitors are agents that effectively inhibit oxidative stress and inflammatory responses in mouse skin.     

Superoxide generation links protein kinase C activation to impaired ATP-sensitive K+ channel function after brain injury

BACKGROUND AND PURPOSE--Endothelin-1, in concentrations similar to that present in cerebrospinal fluid after fluid percussion brain injury (FPI), increases superoxide anion (O2-) production. Endothelin-1 also contributes to altered cerebral hemodynamics after FPI through impairment of ATP-sensitive K+ (KATP) channel function through protein kinase C (PKC) activation. Generation of O2- additionally occurs after FPI. Nitric oxide and cGMP elicit pial artery dilation through KATP channel activation. The present study was designed to determine whether PKC activation generates O2-, which, in turn, could link such activation to impaired KATP channel function after FPI. METHODS--Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O2- generation. RESULTS--Phorbol 12, 13-dibutyrate (10(-6) mol/L), a PKC activator, increased superoxide dismutase-inhibitable NBT reduction from 1+/-1 to 37+/-5 pmol/mm2. Staurosporine (10(-7) mol/L), a PKC antagonist, blocked the NBT reduction after phorbol 12,13-dibutyrate and blunted the NBT reduction observed after FPI (1+/-1 to 15+/-2 versus 1+/-1 to 5+/-1 pmol/mm2 after FPI in the absence versus presence of staurosporine). Exposure of the cerebral cortex to a xanthine oxidase O2--generating system increased NBT reduction in a manner similar to FPI and blunted pial artery dilation to the KATP channel agonists cromakalim and calcitonin gene-related peptide, the nitric oxide releasers sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and the cGMP analogue 8-bromo-cGMP (10+/-1% and 21+/-1% versus 4+/-1% and 9+/-1% for 10(-8) and 10(-6) mol/L cromakalim before and after activated oxygen-generating system exposure). CONCLUSIONS--These data show that PKC activation increases O2- production and contributes to such production observed after FPI. These data also show that an activated system that generates an amount of O2- similar to that observed with FPI blunted pial artery dilation to KATP channel agonists and nitric oxide/cGMP. These data suggest, therefore, that O2- generation links PKC activation to impaired KATP channel function after FPI.     

Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.     

Characterization of the mechanism of action of tachykinins in rat striatal cholinergic interneurons

Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.     

Staurosporine and its derivatives enhance f-Met-Leu-Phe-induced superoxide production via phospholipase D activation in human polymorphonuclear leukocytes

Staurosporine (STAR) is one of the most potent inhibitors of protein kinase C (PKC). It is known that in human polymorphonuclear leukocytes (PMNs), the phorbol ester-induced generation of superoxide anion (respiratory burst) is effectively inhibited by STAR in a dose-dependent manner, whereas superoxide generation induced by chemoattractants, e.g. n-formyl-methionyl-leucyl-phenylalanine (FMLP) or PAF, is regulated biphasically by STAR. We compared the effects of STAR and K252a on FMLP-induced superoxide production from PMNs and examined the effects of propranolol, a inhibitor of phosphatidic acid (PA) phosphohydrolase, on the potentiation of the production by STAR. We also examined the effects of some derivatives of STAR and K252a on the production and the alteration of the effects induced by propranolol pretreatment. When PMNs were stimulated with FMLP, STAR potentiated superoxide production by 240.5 +/- 30.9% at a low concentration (100 nmol/l). Propranolol pretreatment specifically inhibited the potentiation. When phorbol-12-myristate-13-acetate (PMA) was used as a stimulant, STAR inhibited superoxide production dose-dependently and did not enhance the production. K252a inhibited PMA or FMLP-induced superoxide production dose-dependently and did not enhance FMLP-induced superoxide production. STAR derivatives showed potentiation of FMLP-induced superoxide production similar to that of STAR at concentrations ranging from 10-100 nmol/l, and propranolol (200 mumol/l) effectively inhibited it. K252a derivative NA332 did not show any potentiative effect on the production. PMA-induced superoxide production was inhibited by all compounds dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase

Neutrophils contain a multicomponent NADPH oxidase system that is involved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enzyme to produce superoxide and also has been shown to result in activation of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neutrophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in complete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; FMLP-stimulated oxidant production in adherent cells was also abolished. Treatment of human neutrophils with PMA resulted in production of superoxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY294002 did not inhibit cellfree NADPH oxidase activation, CD11b-dependent adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction pathway of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemotactic peptide. Furthermore, Ptdlns 3-kinase may be located directly upstream of protein kinase C or other protein kinases, which in turn activate the NADPH oxidase system.     

Regulation of phosphatidic acid phosphohydrolase by epidermal growth factor. Reduced association with the EGF receptor followed by increased association with protein kinase Cepsilon

An important component of receptor-mediated intracellular signal transduction is the generation of lipid second messengers. Lipid second messenger production is a complex process involving a variety of regulatory enzymes that control the intracellular response to the extracellular signal. Phosphatidic acid (PA) is generated in response to phospholipase D and can be converted to other lipid second messengers including diacylglycerol (DG) and lysophosphatidic acid. PA is converted to DG by PA phosphohydrolase (PAP). We report here that PAP activity can be detected in epidermal growth factor (EGF) receptor immunoprecipitates. Following treatment with EGF, there is a substantial reduction in the PAP activity that co-precipitates with the EGF receptor. The loss of EGF receptor-associated PAP activity occurs with a concomitant increase in PAP activity associated with the epsilon isoform of protein kinase C (PKC). The PAP activity associated with PKCepsilon was dependent upon the PKC co-factors phosphatidylserine and DG but was independent of the kinase activity of PKCepsilon. These data suggest a novel signaling mechanism for the regulation of lipid second messenger production and implicate PAP as an important regulatory component for lipid second messenger production in receptor-mediated intracellular signaling.     

Effects of a protein kinase C inhibitor, Ro 31-7549, on the activation of human leukocytes by particulate stimuli

1. A new specific protein kinase C (PKC) inhibitor, Ro 31-7549, was used to explore the mechanisms by which particulate stimuli, quartz and chrysotile, stimulate human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen metabolites (ROM). Also soluble stimuli, formyl-Methionyl-Leucyl-Phenylalanine (fMLP) and phorbol myristate acetate (PMA) were used. 2. Ro 31-7549 inhibited chrysotile-induced free intracellular calcium ([Ca2+]i) elevations but did not have an effect on quartz-induced elevations of [Ca2+]i. Both quartz and chrysotile induced production of ROM were partially inhibited by Ro 31-7549. fMLP-induced elevation of [Ca2+]i was inhibited by Ro 31-7549 whereas PMA did not affect [Ca2+]i. Ro 31-7549 strongly inhibited fMLP-induced ROM production, and completely abolished that induced by PMA. 3. These result suggest that PKC may have an important role in the activation of PMNL to produce ROM by particulate and soluble stimuli. However, the inhibition of chrysotile-, but not of quartz-induced [Ca2+]i elevations by Ro 31-7549 provides evidence that both PKC-dependent and -independent mechanisms may play a role in the activation of human leukocytes to produce ROM.     

Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ

Like neutrophils, Epstein-Barr virus (EBV)-immortalized B lymphocytes express all constituents of the NADPH oxidase complex necessary to generate superoxide anion O2-. The NADPH oxidase activity in EBV-B lymphocytes is only 5% of that measured in neutrophils upon PMA stimulation. Cytochrome b558 is the sole redox membrane component of NADPH oxidase; it is the protein core around which cytosolic factors assemble in order to mediate oxidase activity. In the present study, we have compared the structural and functional properties of cytochrome b558 from EBV-B lymphocytes and neutrophils. Cytochrome b558 from EBV-B lymphocyte plasma membrane, like that from neutrophils, is characterized by a heterodimeric structure with a highly glycosylated beta subunit, known as gp91-phox. While the amount of cytochrome b558 recovered after purification from EBV-B lymphocytes (approximately 0.24 nmol from 1010 cells) was low compared to that recovered from neutrophils (approximately 10 nmol), the biochemical properties of purified cytochrome b558 from both EBV-B lymphocytes and neutrophils were quite similar with respect to their differential spectra, redox potential, and FAD binding site. Once cytochrome b558 was extracted from the EBV-B lymphocyte membrane, it was able to mediate, in a reconstituted system of O2- production the same oxidase turnover as that found for cytochrome b558 extracted from neutrophils. A comparison between membrane bound and soluble cytochrome b558 suggested that the weak oxidase activity measured in intact EBV-B cells might be the result not only of the small amount of expressed cytochrome b558, but also of a defect of the activation process in lymphocyte membrane.     

Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils

We examined the ability of opsonized zymosan (OPZ) to stimulate translocation of protein kinase C (PKC) isoforms in human neutrophils. Neutrophils express five PKC isoforms (alpha, betaI, betaII, delta, and zeta), but little is known of their individual roles in neutrophil activation. As determined by immunoblotting, OPZ caused a time-dependent translocation of the predominant PKC isoforms (betaII, delta, and zeta) to neutrophil membranes, with a concomitant loss from the cytosol. Maximal translocation of all three isoforms occurred by 3 min. No PKC immunoreactivity was observed in a crude nuclear fraction, but PKC-delta and -zeta were found in the granule fraction after degranulation (10 min). PKC activity (Ca2+-dependent and -independent) increased 50- and 19-fold, respectively, by 10 min in the granules from OPZ-stimulated cells. Curiously, no immunoreactive cPKC (alpha and beta(I/II)) could be localized in the granule fraction to account for the Ca2+-dependent PKC activity. Localization of PKC isoforms in the neutrophil membranes and granules suggests their involvement in the regulation of functional responses triggered by OPZ. PKC isoform translocation to membranes from OPZ-stimulated cells preceded both p47phox (a cytosolic component of the NADPH oxidase) translocation and NADPH oxidase assembly. The presence of both PKC isoforms and p47phox in the membrane was transient, with the loss of p47phox occurring sooner than either the loss of membrane-associated PKC or that of NADPH oxidase activity. The apparent EC50 values for PKC translocation and NADPH oxidase assembly were similar. These data suggest that PKC isoforms regulate the assembly and activation of NADPH oxidase induced by OPZ.     

Home alone--meeting the needs of mothers after cesarean birth

The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the functional role of the four cysteines in p47PHOX, one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47PHOX-deficient Epstein-Barr virus-transformed B lymphoblasts, and O2- production by these transfected cells was measured. The activity of the mutant C98A was similar to that of wild type, but the maximum rate of O2- production by C196A was significantly larger than seen with wild type. The other two mutants (i.e., C111A and C378A) differed from wild type not only in maximum O2- production, but also in the time required for activation, which was considerably delayed with both of these mutants. The similarity in the time courses of oxidase activation with the C111A and C378A mutants, and the finding that C378A occurs in the sequence CSE, raises the possibility that these cysteines may be involved in redox regulation of oxidase activity.     

Effect of serum amyloid A on selected in vitro functions of isolated human neutrophils

Serum amyloid A (SAA) is an acute phase reactant whose levels in the blood rise as part of the body's response to stress and inflammation. Previous studies have suggested that SAA may carry an anti-inflammatory potential. We evaluated the effects of SAA on human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. At concentrations higher than 10 microg/mL, SAA inhibited neutrophil myeloperoxidase (MPO) release. This effect was located in the N-terminal--that is, amino acid residues 1-14--of the SAA molecule. Directed neutrophil migration was inhibited at the same SAA concentrations. Several amino acid residues (1-14, 15-104, 83-104) contributed to this effect. Neutrophil O2- production was inhibited at low concentrations of SAA (0.1 to 1 microg/ml) and was stimulated at concentrations higher than 50 microg/mL. Neutrophil O2- production induced by phorbol myristate acetate (PMA) and O2- generated by the xanthine-xanthine oxidase reaction were not affected by SAA. These results add to previous data suggesting that SAA, at concentrations recorded in the serum during inflammation, modulates neutrophil function; thus it may play a role in the down-regulation of the inflammatory process.     

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O2.- release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an IC50 value of 60.7+/-8.2 microM. A comparable effect was also demonstrated in the inhibition of O2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O2.- generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca2+]i, but it accelerated the rate of [Ca2+]i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetra hydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinde n-1-one, (KT 5720), a PKA inhibitor, the inhibition of O2.- generation by YT-1, as well as by prostaglandin E1 (PGE1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE1. Moreover, the ability of PGE1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.     

The inhibitory effect of noradrenaline on thyrotrophin-stimulated 3,5,3''-tri-iodothyronine and thyroxine release is mediated through a Ca(2+)-dependent process in the thyroid gland of the mouse

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.     

Angiotensin II induces superoxide anion production by mesangial cells

BACKGROUND: The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O2-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O2- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O2-production and (b) if increased O2- production elicits growth responses in MC. METHODS: MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein. RESULTS: Ang II (10(-5) M to 10(-8) M) dose dependently increased MC O2- production up to 125% above control (ED 50 5 x 10(-7) M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O2- production. Ang II dose dependently increased MC 3H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as 3H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC 3H-leucine incorporation, thereby establishing the specificity of Ang II induced O2- in driving MC hypertrophy. CONCLUSIONS: The current studies demonstrate a previously unrecognized link between Ang II and MC O2- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.