Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis.

Inflammation and oxidative stress are important factors in the pathogenesis of diabetes and contribute to the pathogenesis of diabetic complications. Periodontitis is an inflammatory disease that is characterized by increased oxidative stress, and the risk for periodontitis is increased significantly in diabetic subjects. In this study, we examined the superoxide (O(2)(-))-generating reduced nicotinamide adenine dinucleotide phosphate-oxidase complex and protein kinase C (PKC) activity in neutrophils. Fifty diabetic patients were grouped according to glycemic control and the severity of periodontitis. Neutrophils from diabetic patients with moderate [amount of glycated hemoglobin (HbA(1c)) between 7.0% and 8.0%] or poor (HbA(1c) >8.0%) glycemic control released significantly more O(2)(-) than neutrophils from diabetic patients with good glycemic control (HbA(1c) <7.0%) and neutrophils from nondiabetic, healthy individuals upon stimulation with 4beta-phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. Depending on glycemic status, neutrophils from these patients also exhibited increased activity of the soluble- and membrane-bound forms of PKC, elevated amounts of diglyceride, and enhanced phosphorylation of p47-phox during cell stimulation. In addition, we report a significant correlation between glycemic control (HbA(1c) levels) and the severity of periodontitis in diabetic patients, suggesting that enhanced oxidative stress and increased inflammation exacerbate both diseases. Thus, hyperglycemia can lead to a novel form of neutrophil priming, where elevated PKC activity results in increased phosphorylation of p47-phox and O(2)(-) release.

p90-RSK and Akt may promote rapid phosphorylation/inactivation of glycogen synthase kinase 3 in chemoattractant-stimulated neutrophils.

Stimulation of neutrophils with the chemoattractant fMet-Leu-Phe (fMLP) triggers phosphorylation/inactivation of the a- and beta-isoforms of glycogen synthase kinase 3 (GSK-3) with phosphorylation of the alpha-isoform predominating. These reactions were monitored with a phosphospecific antibody that only recognized the alpha- or beta-isoforms of GSK-3 when these proteins were phosphorylated on serine residues 21 and 9, respectively. Inhibitor studies indicated that phosphorylation of GSK-3alpha may be catalyzed by the combined action of p90-RSK and Akt and may represent a new strategy by which G protein-coupled receptors inactivate GSK-3. Inactivation of GSK-3 may be one of the mechanisms that delay apoptosis in fMLP-stimulated neutrophils.

Phosphorylation of the activation loop of gamma p21-activated kinase (gamma-Pak) and related kinases (MSTs) in normal and stressed neutrophils.

Neutrophils stimulated with a variety of chemoattractants exhibit a rapid activation of two p21-activated kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). A number of in vitro studies suggest that modification of Thr(402) in the activation loop (AL) of gamma-Pak can play a critical role in the regulation of this kinase under certain circumstances. A phosphospecific Ab was generated to this region of Pak (pPak(AL)Ab). This Ab reacted with activated gamma- and alpha-Pak from fMLP-stimulated neutrophils that contain the sequence KRXT(P)XXGTP in their ALS: The rapid but transient activation of Paks in normal stimulated neutrophils coincided with phosphorylation and dephosphorylation at the ALs of these enzymes. In contrast, stressed cells exhibited a prolonged phosphorylation at Thr(402) in both intact gamma-Pak and a proteolytic fragment of this kinase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like kinases (MSTs) (members of the Pak family) in osmotically stressed neutrophils and neutrophils treated with certain apoptotic agents (i.e., tumor promoters that inhibit type 1 and 2A protein phosphatases) but not in normal fMLP-stimulated cells. Thus, our results indicate that the AL of gamma-Pak undergoes transient phosphorylation during normal neutrophil stimulation and chronic phosphorylation in stressed cells. In addition, we demonstrate that a number of MSTs are present in neutrophils and also undergo phosphorylation during stressful circumstances.

Antagonists of calcium fluxes and calmodulin block activation of the p21-activated protein kinases in neutrophils.

Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), the intracellular Ca(2+) channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N:-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC(50) values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca(2+)/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.

Ca2+-calmodulin-dependent protein kinase II-dependent activation of contractility in ferret aorta.

1. The present study was undertaken to determine whether Ca2+-calmodulin-dependent protein kinase II (CaMKII) participates in the regulation of vascular smooth muscle contraction, and if so, to investigate the nature of the downstream effectors. 2. The contractility of isolated ferret aorta was measured while inhibiting CaMKII either with antisense oligodeoxynucleotides against CaMKII or with the CaMKII inhibitor KN93. 3. Treatment with antisense oligodeoxynucleotides against CaMKII resulted in, on average, a decrease in protein levels of CaMKII to 56 % of control levels and significantly decreased the magnitude of the contraction in response to 51 mM potassium physiological saline solution (KCl). Contraction in response to the phorbol ester DPBA was not significantly affected. 4. The CaMKII blocker KN93 also resulted in a significant decrease in the force induced by 51 mM KCl but caused no significant change in the contraction in response to DPBA or the alpha-adrenoceptor agonist phenylephrine. 5. During contraction with 51 mM KCl, both CaMKII and mitogen-activated protein kinase (MAPK) activity increased, as determined by phospho-specific antibodies. The MAPK phosphorylation level was inhibited by KN93, PD098059 (a MAPK kinase (MEK) inhibitor) and calcium depletion. 6. Myosin light chain (LC20) phosphorylation also increased during contraction with KCl and the increase was significantly blocked by PD098059 as well as by both KN93 and antisense oligodeoxynucleotides to CaMKII. 7. The data indicate that CaMKII plays a significant role in the regulation of smooth muscle contraction and suggest that CaMKII activates a pathway by which MAPK activation leads to phosphorylation of LC20 via activation of myosin light chain kinase.

A protein kinase from neutrophils that specifically recognizes Ser-3 in cofilin.

Cofilin promotes the depolymerization of actin filaments, which is required for a variety of cellular responses such as the formation of lamellipodia and chemotaxis. Phosphorylation of cofilin on serine residue 3 is known to block these activities. We now report that neutrophils contain a protein kinase that selectively catalyzes the phosphorylation of cofilin on serine 3 (>/=70%) and a nonspecific kinase that recognizes multiple sites in this protein. The selective serine 3 cofilin kinase binds to a deoxyribonuclease I affinity column, whereas the nonspecific cofilin kinase does not. Deoxyribonuclease I forms a very tight complex with actin, and deoxyribonuclease affinity columns have been utilized to identify a variety of proteins that interact with the cytoskeleton. The serine 3 cofilin kinase did not react with antibodies to LIM kinase 1 or 2, which can catalyze the phosphorylation of cofilin in other cell types. The activity of the serine 3 cofilin kinase was insensitive to a variety of selective antagonists of protein kinases but was blocked by staurosporine. This pattern of inhibition is similar to that observed for the kinase that is active with cofilin in intact neutrophils. Thus, neutrophils contain a protein kinase distinct from LIM kinase-1/2 that selectively recognizes serine 3 in cofilin.

Activation of p90RSK and cAMP response element binding protein in stimulated neutrophils: novel effects of the pyridinyl imidazole SB 203580 on activation of the extracellular signal-regulated kinase cascade.

Neutrophils stimulated with the chemoattractant FMLP or the phorbol ester PMA are known to exhibit activation of a 90-kDa renaturable protein kinase. Activation of this kinase was maximal at approximately 1-3 min after cell stimulation and the time course for activation was similar to that of the extracellular-regulated kinases (ERKs) and p38-mitogen activated protein kinase (p38MAPK). Compounds that block activation of ERK-1/2 (PD 98059) or that inhibit the activity of p38MAPK (SB 203580) blocked activation of this 90-kDa kinase. SB 203580 is a highly selective inhibitor of p38MAPK in vitro and is under intense study as a lead compound for developing novel anti-inflammatory agents. However, we demonstrate that SB 203580 at concentrations >/=10 microM can also inhibit activation of ERK-1/2 in neutrophils. An Ab to the protein kinase p90RSK2 (also referred to as MAPKAP-K1b, or p90rsk) immunoprecipitated the active 90-kDa kinase from lysates of stimulated neutrophils. No activity was observed for this enzyme in immunoprecipitates obtained from unstimulated cells, and the amounts of activity were markedly reduced if the cells were treated with PD 98059 or SB 203580 before stimulation. Neutrophils stimulated with FMLP exhibited phosphorylation of the cAMP response element binding protein (CREB), and this reaction was inhibited by SB 203580 and PD 98059. These data establish that the renaturable 90-kDa protein kinase is p90RSK2 and that CREB may be a substrate for this enzyme in these cells. Novel effects of compound SB 203580 on stimulated neutrophils are also described.

Neutrophils stimulated with a variety of chemoattractants exhibit rapid activation of p21-activated kinases (Paks): separate signals are required for activation and inactivation of paks.

Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the alpha and betagamma subunits of complex G proteins.

Products of sphingolipid catabolism block activation of the p21-activated protein kinases in neutrophils.

Neutrophils stimulated with the chemoattractant FMLP are known to exhibit a rapid and transient activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa. Paks can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 to 331 of the 47-kDa subunit of the nicotinamide-adenine dinucleotide phosphate-oxidase complex (p47-phox) fixed within a gel. In this study, we demonstrate that N-acetylsphingosine (C2-ceramide) and a variety of sphingoid bases (e.g., D-erythrosphingosine) block activation of the 63- and 69-kDa Paks in neutrophils. The concentrations of these lipids that were effective in blocking Pak activation were similar to those that inhibit a variety of neutrophil responses. Activation of the 63- and 69-kDa Paks was also markedly reduced in neutrophils treated with sphingomyelinase before stimulation. Moreover, we report that addition of C2-ceramide or D-erythrosphingosine to neutrophils after stimulation with FMLP markedly enhances the rate of Pak inactivation. These effects were not mimicked by arachidonate, which is a potent disorganizing agent of neutrophil membranes. These data support and extend the proposal that sphingoid bases may establish a set point in neutrophils for positive stimuli.

Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation.

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2-) release. Immunofluorescent studies revealed that cofilin was present throughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

Activation of the p21-activated protein kinases from neutrophils with an antibody that reacts with the N-terminal region of Pak 1.

Neutrophils contain two renaturable p21-activated protein kinases (Paks) with molecular masses of ca. 69 and 63 kDa that undergo rapid activation upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. We now report that these kinases undergo a massive, ATP-dependent activation in lysates of unstimulated neutrophils during immunoprecipitation with an antibody generated to residues 2-21 of the N-terminal region of Pak1. This activation was specific as it was completely blocked by a peptide that corresponds to residues 2-21 of Pak1 and was not observed with an antibody generated to the C-terminal region of Pak 1. The properties of the Paks activated with the antibody were virtually identical to those observed for these kinases from stimulated neutrophils, or activated in vitro with Rac-GTPgammaS plus ATP. These data indicate that perturbation of the N-terminal region of Pak can trigger activation of this enzyme, and that both the 69 and 63 kDa kinases may represent forms of Pak 1 that differ in their content of phosphate.

The renaturable 69- and 63-kDa protein kinases that undergo rapid activation in chemoattractant-stimulated guinea pig neutrophils are p21-activated kinases.

Neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) are known to exhibit rapid activation of four protein kinases with molecular masses of approximately 69, approximately 63, approximately 49, and approximately 40-kDa. Activation of these kinases is blocked by antagonists of phosphatidylinositol 3-kinase and type 1 and/or type 2A protein phosphatases. These enzymes can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297-331 of the 47-kDa subunit of the NADPH-oxidase complex fixed within a gel. In this report, we demonstrate that an antibody generated to a fusion protein containing amino acid residues 175-306 of p21-activated protein kinase 1 (Pak1) reacts with three proteins in guinea pig neutrophils with molecular masses in the 60-70-kDa range during Western blotting. This antibody immunoprecipitates both the 69- and 63-kDa renaturable kinases from lysates of stimulated cells along with a minor 60-kDa kinase. No activities were observed for any of these enzymes in immunoprecipitates from unstimulated neutrophils. However, addition of ATP and activated Rac 1 or Cdc42 to immunoprecipitates from unstimulated cells resulted in the stimulation of two renaturable kinases with molecular masses in the 69- and 63-kDa range. These immunoprecipitates also contained two novel protein kinases with masses of approximately49 and 40 kDa that were selectively activated by Cdc42. In contrast, the 69- and 63-kDa kinases were not immunoprecipitated from lysates of stimulated neutrophils with an antibody to Pak2 or with nonimmune serum. These data indicate that the renaturable 69- and 63-kDa kinases are Paks and reveal some of the upstream events that are necessary for the rapid activation of this family of protein kinases in neutrophils.

In vitro activation of a 60-70 kDa histone H4 protein kinase from neutrophils by limited proteolysis.

Neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe (fMLP) are known to exhibit a rapid and transient activation of a histone H4 kinase that may function in a stimulatory pathway downstream of phosphatidylinositol 3-kinase. The activity of this histone kinase in unstimulated neutrophils and cells treated with 1.0 microM fMLP for 10 sec was 8.8 +/- 5 and 43 +/- 2 pmol P/min per 10(7) cells, respectively. In this paper, we report that unstimulated neutrophils contain a latent H4 kinase in the 100,000 x g soluble fraction that can be markedly activated by treatment with trypsin. The values for the untreated and trypsin treated enzyme were 5.5 +/- 1.0 and 63.6 +/- 18 pmol P/min per 10(7) cell-equivalents, respectively. This kinase was insensitive to a selective antagonist of protein kinase C (i.e., 50 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) but completely blocked by 100 nM staurosporine. Only a single peak of activity was observed for this enzyme when the 100,000 x g supernatant fraction was fractionated on either an exclusion (KW-803) or an anion exchange column (DEAE), or during isoelectric focusing. The molecular weight of the latent kinase was 64 +/- 6 kDa and the isoelectric point was 7.6 +/- 0.1. During all fractionation procedures, the H4 kinase co-chromatographed with a trypsin-activated kinase that catalyzed the phosphorylation of a peptide which corresponds to residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex (p47-phox). The properties of the trypsin-activated H4 kinase from unstimulated neutrophils are very similar to those reported for this enzyme from fMLP-stimulated cells.

Protein phosphorylation in neutrophils from patients with p67-phox-deficient chronic granulomatous disease.

Neutrophils are known to contain a major 67-kD protein that undergoes enhanced phosphorylation and translocation to the membrane during cell stimulation. Recent studies have assumed that this 67-kD phosphoprotein is the 67-kD subunit of the phagocyte oxidase (p67-phox). We compare here the protein phosphorylation patterns in lysates of normal neutrophils and neutrophils from patients with chronic granulomatous disease (CGD) that are completely deficient in p67-phox. The phosphoproteins were labeled by incubation of the cells with radioactive inorganic phosphate (32Pi) or by the addition of [gamma-32P]ATP to electropermeabilized neutrophils. With either method, stimulation of the normal or CGD cells always resulted in an enhanced incorporation of 32p into two proteins in the 67-kD area. The extent of phosphorylation of these two proteins was very similar in the normal and CGD cells when permeabilized neutrophils loaded with [gamma -32P]ATP were compared. Moreover, no overall differences in the protein phosphorylation patterns were observed between the normal and CGD cells. Our data indicate that the major 67-kD phosphoproteins observed in stimulated neutrophils are clearly different from p67-phox.

Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases.

Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils.

Several novel protein kinases are known to be rapidly activated in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP). These kinases include a histone H4 protein kinase and several renaturable kinases with molecular masses of about 69, 63, 49, and 40 kDa. The renaturable kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH-oxidase system (p47-phox). Previous studies have indicated that the activation of all of these protein kinases involves an uncharacterized stimulatory pathway and/or novel second messenger. The studies reported herein were undertaken to determine if phosphatidylinositol 3-kinase (PI3-K) is a component of this pathway. We report that certain chromosome derivatives (e.g. 2-(4-morpholinyl)-8-phenylchromone (LY294002)) and wortmannin, which inhibit PI3-K by distinct mechanisms, blocked activation of all of these novel kinases. These antagonists also inhibited the phosphorylation of p47-phox (about 50%) and O2.- release (about 80%) in cells stimulated with fMLP, but not with 4 beta-phorbol 12-myristate 13-acetate. A strong correlation exists between the amounts of these antagonists required to produce 50% inhibition of PI3-K in vitro and O2.- release in vivo. In contrast, a single atom substitution of LY294002 produced a compound (LY303511) that did not inhibit PI3-K. Compound LY303511 did not appreciably inhibit the activation of the novel protein kinases or O2.- generation. These data strongly suggest that PI3-K is involved in the activation of several novel protein kinases in neutrophils, one or more of which may be involved in O2.- release.

Farnesyl thiotriazole, a potent neutrophil agonist and structurally novel activator of protein kinase C.

Farnesylcysteine derivatives can initiate or inhibit superoxide (O2-) release in neutrophils. The mechanism by which one of these derivatives, farnesyl thiotriazole (FTT), initiates O2- release in neutrophils is the subject of this paper. Treatment of guinea pig neutrophils with FTT results in the rapid release of O2- by a route shown to be independent of the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) receptor. The signal transduction pathway utilized by the chemoattractant fMLP is generally accepted as the paradigm for receptor-mediated stimulation of O2- production. Antagonists of fMLP had no effect on FTT-induced O2- release, and pretreatment of neutrophils with fMLP had no effect on the ability of FTT to trigger further O2- generation. In fact, FTT behaves like a typical protein kinase C (PKC) activator. It promotes phosphorylation of the 47-kDa subunit of the NADH oxidase complex (p47-phox) in neutrophils, and this phosphorylation is specifically blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an antagonist of PKC. FTT is also shown to activate PKC in vitro in a specific and saturable fashion. FTT is approximately equipotent with (S)-diolein, a physiologically relevant activator of this kinase. FTT represents a new, and quite novel, structure for a PKC activator. PKC activators include diglycerides and the structurally diverse tumor promoters.

The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view.

The NADPH oxidase complex catalyzes the formation of superoxide (O2.-) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed.

Wortmannin and 1-butanol block activation of a novel family of protein kinases in neutrophils.

Neutrophils contain four uncharacterized protein kinases with molecular masses of ca. 69, 63, 49 and 40 kDa that are rapidly activated upon stimulation of these cells with the chemoattractant fMet-Leu-Phe [Ding, J. and Badwey, J.A. (1993) J. Biol. Chem. 268, 17326-17333]. We now report that wortmannin and 1-butanol block activation of all four of these kinases. These reagents are known to inhibit superoxide generation in neutrophils stimulated with this agonist. Wortmannin inhibits phosphatidylinositol 3-kinase and blocks activation of phospholipase D, whereas 1-butanol can reduce the generation of phosphatidate in cells by serving as a substrate for phospholipase D. These data suggest that phosphatidylinositol 3-kinase and phospholipase D may be involved in the activation of several novel protein kinases in neutrophils and that one or more of these kinases is/are involved in superoxide release.