Diacylglycerol Accumulation and Superoxide Anion Production in Stimulated Human Neutrophils*

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY Q 1987 by The American Society of Biological Chemists, nc. Vol. 262, No. 12, ssue of April 25, pp ,1987 Printed in U.S.A. Diacylglycerol Accumulation and Superoxide Anion Production in Stimulated Human Neutrophils* (Received for publication, September 30, 1986) Lisa G. Rider$$ and James E. NiedelSl From the $Department of Medicine, Division of Clinical Pharmacology and the lldepartments of Microbiology, mmunology, and Pharmacology, Duke University Medical Center, Durham, North Carolina Exogenous diacylglycerols stimulate neutrophil su- stimulated translocation of protein kinase C from the cytosol peroxide anion production, suggesting that endogenous to the particulate fraction of human neutrophils correlates diacylglycerols may function as second messengers for with the activation of NADPH oxidase in a time- and dosethis biological response. We have measured the diacylglycerol mass in human neutrophils stimulated by met-leu-phe, ionomycin, and concanavalin A and have correlated the kinetics and magnitude of the diacylglycerol response with those for superoxide anion production. For each stimulus, no increase in diacyl- glycerol mass was detected prior to the onset of superoxide anion generation. However, large sustained increases in diacylglycerol concentration ( % of basal levels) occurred in parallel with the rise in superoxide anion. The cessation or continuation of diacylglycerol accumulation and superoxide anion production also correlated. The diacylglycerol response was proportionaltothestimulusconcentrationand correlated with the concentration dependence for superoxide anion. Pretreatment of neutrophils with cytochalasin B enhanced both superoxide anion and diacylglycerol responses with all three stimuli. These data support the hypothesis that diacylglycerol func- tions as a modulator of superoxide anion generation causing a sustained or augmented respiratory burst. Human neutrophils stimulated by the synthetic peptide Wet-Leu-Phe, the calcium ionophore ionomycin, and the lectin concanavalin A (ConA) reduce molecular oxygen to reactive metabolites, such as superoxide anion (0;)(1-5). This respiratory burst, essential to the cell s bactericidal activity and significant in the pathogenesis of some inflammatory diseases, is catalyzed by NADPH oxidase and results from oxidation of NADPH generated via the hexosemonophosphate shunt. The biochemical mechanisms underlying the activation of the NADPH oxidase are not well understood, but recent evidence has implicated the calcium and phospholipid-dependent protein kinase, protein kinase C, as a central component of this pathway. Phorbol myristate acetate (PMA), a potent stimulator of the respiratory burst (6), directly binds to and activates protein kinase C (7-9). Moreover, the PMA- * This work was supported by United States Public Health Service Grants CA35680 and A The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by a Medical Student Research Fellowship from the Pharmaceutical Manufacturers Association Foundation. The abbreviations used are: Met-Leu-Phe, N-formylmethionylleucylphenylalanine; ConA, concanavalin A; OF, superoxide anion; PMA, phorbol myristate acetate; DG, sn-1,2-diacylglycerol; Hepes, 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid dependent manner (10). PMA also induces the phosphorylation of a 31.5-kDa protein, which may be cytochrome b245 of the NADPH oxidase (11). Recently, Cox et al. (12) have been able to measure 0; production in purified neutrophil membranes upon addition of protein kinase C, phosphatidylserine, PMA, or l-oleoyl-2-acetylglycerol, ATP, Ca2+, and M$+, thus demonstrating that activation of protein kinase C is sufficient to stimulate the NADPH oxidase. Protein kinase C is markedly stimulated by sn-1,2-diacylglycerols (DG), which increase the affinity of the enzyme for Ca2+ and phosphatidylserine, resulting in enzyme activation at intracellular Ca2+ levels (13-15). Evidence supporting a second messenger function for DG in activating neutrophils has emerged from studies employing cell-permeable synthetic DGs. sn-1,2-dioctanoylglycerol and other synthetic DGs containing short chain fatty acids, as well as 1-oleoyl-2-acetylglycerol, its palmitoyl and myristoyl analogs, and oleate stimulate 0; production or oxygen consumption in a manner similar to PMA (16-20). 1-Oleoyl-2-acetylglycerol and the calcium ionophore A23187 are synergistic in generating O;, suggesting roles for both DG and calcium in activation of the NADPH oxidase (21). DG is produced from hydrolysis of phosphoinositides, and perhaps other phospholipids, in cells stimulated by Met-Leu- Phe and the calcium ionophores ionomycin and A23187 (22-31). With fmet-leu-phe stimulation, inositol 1,4,5-trisphosphate, which releases Ca2+ from intracellular stores, is also transiently generated (32-34). While extensive data support the role of inositol lipid metabolism in signal transduction in neutrophils, there has been little direct evidence correlating endogenous DG production with 0; generation. n this communication, we have measured the mass of DG in lipid extracts of human neutrophils stimulated with Met- Leu-Phe, ionomycin, and ConA and have attempted to correlate the kinetics and magnitude of the DG response with those for 0; production. For each stimulus, no increment in DG was detected prior to the onset of 0; generation. However, the later kinetics of DG closely parallel those for 0;. The DG response was dependent on the stimulus concentration and correlated with the concentration dependence for 0;. Pretreatment of neutrophils with cytochalasin B enhanced both the DG and 0; responses with all three stimuli. These data provide evidence in support of a role for DG in sustaining or augmenting 0; production. EXPERMENTAL PROCEDURES Materials-Met-Leu-Phe, cytochalasin B, concanavalin A, horse heart ferricytochrome c (type ), and superoxide dismutase were obtained from Sigma; R59022 from Janssen (Beerse, Belgium); DL-

2 5604 Kinetics of Diacylglycerol Superoxide and Production dithiothreitol from Bachem (Torrence, CA); ionomycin from Behring Diagnostics; Merck Silica Gel 60 F254 thin layer chromatography plates from VWR Scientific (Atlanta, GA); and 13 X 100-mm borosilicate glass culture tubes from Scientific Products (McGraw Park, L). lonomycin was dissolved in anhydrous ethanol and stored at 4 "C, desiccated, and protected from light. Fresh ionomycin was prepared monthly. Met-Leu-Phe and cytochalasin B were stored as 2 mm stock solutions in dimethyl sulfoxide and diluted into buffer before use. Solutions of ConA were prepared immediately before use. The purity of each batch of [y3']atp (4500 Ci/mmol; CN Radiochemicals, rvine, CA) was determined by thin layer chromatography on polyethyleneimine cellulose with 0.75 M potassium phosphate, ph 3.4. Radioactive impurities were corrected for when calculating mass of DG; batches less than 80% pure were discarded. sn-1,2-dioleoyl- glycerol (diolein) prepared by phospholipase C digestion of ~-adioleoyl-sn-glycerol 3-phosphocholine (Avanti) and Escherichia coli strain N4830/pJW10 membranes prepared as previously described (35) were the generous gifts of Dr. Robert Bell and Dr. Carson Loomis, Duke University, Durham, NC. Deionized, ultrafiltered, endotoxinfree water from an Ultra 35 pure water system (Feed Water Systems, Richmond, VA) was used to prepare all solutions. All other materials are as previously described (30). Neutrophil Preparation-Neutrophil suspensions containing 96% neutrophils were prepared from heparinized venous blood (10 units of heparin/ml of blood) obtained from healthy adult donors. A modification of the method of Boyum (36) was employed, using lymphocyte separation medium gradients followed by dextran sedimentation and hypotonic lysis. Prior to hypotonic lysis, the cells were maintained in a buffered salt solution consisting of 138 mm Na+, 4 mm K+, 10 mm PO?, and 140 mm C1-, ph 7.4. Following hypotonic lysis, the cells were suspended in a Hepes buffer, ph 7.4, having the composition of 150 mm Na+, 5 mm K+, 1.3 mmca", 1.2 mm Me, 155 mmc1-, and 10 mm Hepes. Cell viability, determined by trypan blue exclusion, was consistently greater than 98% at the outset and greater than 95% at the conclusion of an experiment. Diacylglycerol Assay-sn-1,2-Diacylglycerol levels in crude lipid extracts of neutrophils were measured by the method of Preiss et al. as previously described (30, 37). Briefly, 6.25 X 10' cells/data point were preincubated at 37 "C in the presence or absence of 5 pg/ml cytochalasin B for 5 min prior to stimulation. At the desired time after stimulation, cells were extracted by a modification of the method of Bligh and Dyer (38). For incubations less than 4 s, a metronome was used as a timer. Mixed micelles were prepared by solubilizing an aliquot of the dried crude lipid extract in 20 pl of 7.5% OCtyl-8-Dglucoside and 5 mm cardiolipin by water bath sonication. The mixed micelles were then reacted with E. coli membranes containing sn-1,2- diacylglycerol kinase and with 5 mm [Y-~~PATP described as (30, 37). After neutral lipid extraction, an aliquot of the lipid phase was subjected to thin layer chromatography on a Silica Gel 60 F254 plate developed with chloroform:methanol:acetic acid (65:15:5, v/v/v), fol- lowed by autoradiography and liquid scintillation counting. The amount of sn-1,2-diacylglycerol present in the original sample was calculated from the amount of [32P]phosphatidic acid produced, the sample volumes, and the specific activity and purity of [Y-~~P]ATP. Samples of sn-1,2-diolein similarly labeled and treated were spotted onto each plate as controls. This demonstrated that diacylglycerol recovery and conversion were consistently greater than 90%. n separate controls, the addition of cytochalasin B, Met-Leu-Phe, ionomycin, ConA, or R59022 did not inhibit the recovery or conversion of a known quantity of diolein. The diacylglycerol assay was linear in the range from 10' to lo7 cells/data point. All data points were measured in duplicate. Superoxide Anion Generation-The generation of 0: by stimulated neutrophils was measured as superoxide dismutase-inhibitable cytochrome c reduction, modified from the batch (39) and kinetic methods (40,41) previously described. Duplicate reaction mixtures containing X lo6 neutrophils and 75 p~ ferricytochrome c (horse heart type 111) were incubated for 5 min at 37 "C in the presence or absence of 5 pg/ml cytochalasin B. Reference cuvettes contained, in addition, 10 pg/ml superoxide dismutase. After incubation with appropriate stimuli, the reactions were terminated by centrifuging at 4 "C for 5 min at 1200 X g. For kinetic studies, stimuli were added at time 0, and the reduction of cytochrome c was monitored continuously at 550 nm in a Varian DMS 100 dual-beam spectrophotometer. 02 generation, expressed as nmol of cytochrome c reduced per lo6 cells, was calculated by using an absorbance coefficient of 21.1 mm-l cm-' at 550 nm (reduced-oxidized). RESULTS Effect of fmet-leu-phe on DG Accumulation and 0; Production-The time courses of the DG and 0; responses in neutrophils treated with three different stimuli were determined to establish whether DG may function as a second messenger for 0; production based on the temporal relationship of the responses. An early time course of DG levels in neutrophils preincubated with cytochalasin B and stimulated with lo-' M Met-Leu-Phe is shown in Fig. 1. No detectable increase in DG (n = 25) occurred prior to the onset of 0; at 8 f 1 s (n = 8). A small increase in DG was occasionally observed within the first 2-5 s, but this occurred in both the met-leu-phe-stimulated and control cells and appeared to result from agitation of the cells. The inability to detect the early (40 s) Net-Leu-Phe-stimulated increase in DG expected from studies of phosphatidylinositol bisphosphate hydrolysis and inositol 1,4,5-trisphosphate release may have been caused by rapid conversion of DG to phosphatidic acid catalyzed by endogenous neutrophil DG kinase. To test this hypothesis, neutrophils were preincubated for 15 s with R59022, an inhibitor of eukaryotic DG kinase (42), and then stimulated with Met-Leu-Phe. At a concentration of R59022 (5 WM) that enhanced met-leu-phe-mediated 0; production, we were still unable to detect an early increase in DG. However, R59022 did augment the DG response to met-leu-phe at later times (n = 5, data not shown). Examination of a full-time course of DG and 0; production (Fig. 2) shows that Met-Leu-Phe stimulated a rapid linear rise in DG, beginning at 17 f 1.4 s (n = 6), which coincided with the activation time of s for 0; (n = 11). Activation time, as defined by Cohen and Chovaniec (40), is the time to the onset of a linear rate of cytochrome c reduction, while latency, as defined by Sklar et al. (411, is the time to onset of 0; production. We have adopted these kinetic terms to DG accumulation. 0; generation ceased between 5 and 8 min, as previously reported (43,44). DG accumulation ceased slightly earlier, at 4-5 min, with maximal levels % (n = 6) above basal. DG returned toward, but not to, basal levels between 10 and 15 min. The concentration dependence of met-leu-phe-stimulated DG accumulation and 0; production at 5 min is shown in Fig. 3. The DG and 0; responses demonstrated identical w TME (seconds) FG. 1. Early time course of DG accumulation by neutrophils in response to met-leu-phe stimulation. Human neutrophils were preincubated with 5 pg/ml cytochalasin B for 5 min at 37 "C and stimulated with 10- M fmet-leu-phe (time 0) , cytochalasin B alone;."-., Met-Leu-Phe plus cytochalasin B- stimulated cells. Data points are the average of20-25 individual determinations from seven separate experiments. Error bars depict S.E.

3 m o p,,,,,,,,,,,,,,,,, 0 05 O " TME manuter) FG. 2. Time course of 0; production and DG accumulation by neutrophils in response to met-leu-phe stimulation. Human neutrophils were preincubated with 5 pg/ml cytochalasin B for 5 min at 37 "C and stimulated with lo6 M met-leu-phe (time 0). The arrow indicates the onset of 0; production. Representative data from one of three experiments is shown. CONCENTRATON FMLP (M FG. 3. Met-Leu-Phe concentration dependence of DG accumulation and 0; production by neutrophils. Human neutrophils were preincubated with 5 pg/ml cytochalasin B for 5 min at 37 "C and stimulated for 5 min with varying concentrations of met- Leu-Phe. -, DG; A-A, 07. Data are the average f S.E. of two separate experiments performed in duplicate. thresholds of lo-' M and ED,, values of 2.3 X M with Met-Leu-Phe stimulation. 0; generation was maximal at 10"j M Wet-Leu-Phe, whereas DG levels were maximal in response to 5 X M. Both responses exhibited slight inhibition at higher stimulus concentrations. There was no direct correlation between the magnitude of the 0; and DG responses. While two different donors generated identical peak DG levels of 225 pmol/107 cells/5 min, their maximal 0; responses were widely discrepant: 7.0 versus 25.4 nmol/ O6 cells/5 min, although both gave identical 0; responses to PMA stimulation. Correlation of onomycin-stimulated DG Accumulation and 0; Production-onomycin-stimulated cells, in the presence of 5 pg/ml cytochalasin B, showed a latency for 0; generation of 41.5 f 3.3 s (n = 6). n Fig. 4A, the average of six separate experiments, a DG increase to 66 f 17% above basal levels occurred at 40.8 f 2.5 s (n = 12), essentially coincident with the onset of 0; production (p > 0.05). Moreover, in several individual experiments, 0; production preceded the increase in DG. With ionomycin stimulation in the absence of cytochalasin B, the latency for 0; production remained unchanged, while the latency for DG accumulation was increased to 50 f 5.2 s (n = 6), but the difference in latencies was not statistically significant ( p > 0.05) (Fig. 4B). The average activation time for DG accumulation with ionomycin stimulation was 51 f 2.2 s (n = 9) in the presence of cytochalasin B preceding the 0; activation time of 62.2 f 5.5 s (n = 5) (p < 0.05) (Fig. 4A). n the absence of cytochalasin B, however, the onset of a linear rate of DG at 62.5 f 5.7 s (n = 6) did not differ significantly from the 0; activation Kinetics of Diacylglycerol and Superoxide Production 5605 time of 67 f 3.6 s (n = 3) ( p > 0.05) (Fig. 4B). n the presence of cytochalasin B, ionomycin stimulated a large (20-fold above basal) continuous increase in DG measured out to 15 min. 0; production also continuously increased out to 7 min but could not be followed beyond this point due to depletion of cytochrome c. n the absence of cytochalasin B, the large (10-fold above basal) increase in ionomycinstimulated DG also continued out to 15 min. However, 0; production ceased between 6 and 9 min. A dose response of ionomycin-stimulated DG accumulation and 0; production is shown in Fig. 5. Both in the presence and absence of cytochalasin B, the threshold for DG and 0; responses occurred at 100 nm ionomycin. While the peak responses and general shape of the DG and 0; curves correlated, the EDbo values were different. n the presence of cytochalasin B, the EDb0 for 0; generation was 82.5 f 4 nm (n = 4) ionomycin, while the EDSo for DG accumulation was 270 f 31 nm (n = 4). n the absence of cytochalasin B, 195 f 26 nm (n = 4) ionomycin produced a half-maximal 0; response, but 555 f 66 nm (n = 4) was required for half-maximal accumulation of DG. Cytochalasin B enhances calcium ionophore A23187-stimulated neutrophil 0; generation (45, 46). Cytochalasin B also enhances met-leu-phe-stimulated DG accumulation (30). With ionomycin stimulation, cytochalasin B shifted the doseresponse curves to the left. n the presence of cytochalasin B, DG peaked at 500 nm ionomycin, while in its absence, the maximal DG response occurred at 1 p ~ Similarly,. 0; production was maximal at 750 nm ionomycin in the presence of cytochalasin B and at 2 PM in its absence. Cytochalasin B also augmented the magnitude of the DG and 0; responses, although the extent of the enhancement varied. At 500 nm ionomycin, for example, cytochalasin B increased ionomycinstimulated DG 1.6-fold and augmented 0; generation 2.7- fold. At high concentrations of ionomycin, greater than 2 PM, 0; production was inhibited, yet DG was elevated above basal levels. This was especially striking at 5 and 10 p ~ The. cells remained viable, as judged by their ability to exclude trypan blue. To test whether the cells were still capable of generating 02, a saturating dose of PMA (160 nm) was added after the cells were stimulated with 5 pm ionomycin for 5 min. No 0; was generated in response to PMA (data not shown). The high intracellular Ca2+ generated by the ionophore had apparently caused the cells to become refractory to endogenous DG or exogenous PMA as stimuli for 0; production. DG Accumulation and 0; Production in ConA-stimulated Neutrophils-The average time course of DG accumulation and 0; production in neutrophils pretreated with cytochalasin B and stimulated with 200 pg/ml ConA is shown in Fig. 6. The latency for 0; production was 31.5 f 0.7 s (n = 4), while the onset of a DG increase above basal levels occurred at 34.2 f 7.5 s (n = 8). As evidenced by the large standard error, in four separate experiments the latency for DG accumulation was highly variable, ranging from 5 to 60 s, while 0; generation consistently began at s. n two experiments, there was no consistent increase in DG before the onset of 02, while in two other experiments, increases of 15-60% above basal occurred prior to the onset of 0;. Our conclusion from these data is that no consistent increase in DG preceded the onset of 0; production. The average activation time for DG accumulation with ConA stimulation was s (n = 6), preceding the onset of a linear rate of 0; production at 59.4 f 4.1 s (n = 5) (p < 0.05) (Fig. 6). ConA consistently produced a large (500%

4 5606 Kinetics of Diacylglycerol Superoxide and Production A / FG. 4. A, time course of 0; production and DG accumulation by neutrophils in response to ionomycin and cytochalasin B. Human neutrophils were preincubated with 5 pg/ml cytochalasin B for 5 min at 37 "C and stimulated with ionomycin (time 0). Data are the average f S.E. of six separate experiments performed in duplicate. B, time course of 0; production and DG accumulation by neutrophils in response to ionomycin alone. Human neutrophils were thermally equilibrated to 37 "C for 5 min and then stimulated with ionomycin (time 0). The initiation of 0; generation is indicated by an arrow. Data are the average f S.E. of three separate experiments performed in duplicate. f 700[ 600 CONCENTRATON ONOMYCN(p.NJ FG. 5. onomycin concentration dependence of DG accumulation and 0; production by neutrophils. Human neutrophils were preincubated in the presence or absence of 5 pg/ml cytochalasin B for 5 min at 37 "C and stimulated for 5 min with varying concentrations of ionomycin. M, DG in cells preincubated with cytochalasin B; , DG in cells in the absence of cytochalasin B; A-A, 0; in cells preincubated with cytochalasin B; A- - -A, 0; in cells in the absence of cytochalasin B. Representative data from one of two identical experiments is shown. above basal) prolonged increase in DG measured out to 15 min. 0; also increased out to 5 min beyond which the cytochrome c was depleted. Cytochalasin B greatly augments 0; production with ConA stimulation (47). n the absence of cytochalasin B, 200 pg/ml ConA produced no DG or 0;. The concentration dependence of ConA-stimulated DG accumulation and 0; generation at 5 min in the presence of cytochalasin B is shown in Fig. 7. The threshold for 0, generation was 30 pg/ml ConA, which was lower than the DG response threshold of 100 pg/ml. DSCUSSON Several observations suggested that DG may function as a second messenger for 0; production in neutrophils. Exogenous DG stimulated 0; production in intact neutrophils without generating a Ca2+ signal (16, 20). Furthermore, purified neutrophil membranes, reconstituted with protein kinase C and stimulated with DG, were able to generate 0; (12). Finally, rapid (5-10 s) degradation of PP2 with generation of P3 has been detected in cells stimulated with Met-Leu-Phe TME (mmuter) FG. 6. Time course of DG accumulation and 0; production by neutrophils in response to ConA and cytochalasin B. Human neutrophils were preincubated with 5 pg/ml cytochalasin B for 5 min at 37 'C and stimulated with 200 pg/ml ConA (time 0). 02 latency is indicated with an arrow. Data are the average & S.E. of four separate experiments performed in duplicate. CONCENTRATON CON A (po/ml) FG. 7. ConA concentration dependence of DG accumulation and 0; production by neutrophils. Human neutrophils were preincubated with 5 pg/ml cytochalasin B for 5 min at 37 "C and stimulated for 5 min with varying concentrations of ConA. M, DG; A-A, 0;. Data are the average k S.E. of three separate experiments performed in duplicate.

5 (32-34, a), suggesting the occurrence of an early increment in DG, equimolar with released inositol phosphates. Based on these observations, we tested the hypothesis that cell surface stimuli that cause 0; production utilize DG as a second messenger in triggering the neutrophil respiratory burst. n an attempt to establish a role for DG as a second messenger in the activation sequence for 0; generation, we have correlated the kinetics of DG accumulation and 0, production. When large numbers of experiments were averaged, the start of a rise in DG mass coincided with or followed, but did not precede, the initiation of 0; production in met- Leu-Phe, ionomycin, and ConA-stimulated cells. n several individual experiments, furthermore, the onset of 0; generation clearly preceded a change in DG mass for each stimulus. The DG response in neutrophils thus differs considerably from that of platelets, in which there is a %fold rise in DG mass by 5 s after stimulation, with a peak response at s (37,49). However, DG should be produced within the first few seconds after Met-Leu-Phe stimulation, with kinetics identical to those for inositol 1,4,5-trisphosphate formation (32-34, 48). Our inability to detect this early increment in DG may have been caused by rapid conversion of the DG to phosphatidic acid catalyzed by neutrophil DG kinase. But pretreatment of met-leu-phe-stimulated neutrophils with R59022, an inhibitor of eukaryotic DG kinase (42), did not unmask an early DG signal. Also, in unpublished studies, we have found that when 3 p~ dioctanoylglycerol was added to 5 X O6 neutrophils, it was not metabolized at 5 min? again suggesting that rapid metabolism does not mask an early DG signal. Alternatively, the early increase in DG may be of such small magnitude as to be missed by our assay above the basal level ofdg. Cockcroft and Allan (26) were also unable to detect an early increase in DG mass using a different method of analysis (26). Using ['4C]glycerol and [3H]arachidonic acid labeling, however, other workers have reported a rise in DG of 30-60% occurring 2-5 s after met-leu-phe stimulation and an increase in DG of 22% within 5 s after ConA stimulation (29, 50). The phosphatidylinositol bisphosphate hydrolysis and inositol 1,4,5-trisphosphate generation studies in neutrophils have also relied on radiolabeling techniques, which may improve the sensitivity in detecting a relatively small signal by preferentially measuring those lipid pools involved in signal transduction. When our studies of DG mass are combined with the results of numerous radiolabeling studies, we must conclude that an early DG increment precedes 0; generation but that it is of very small magnitude compared to the large subsequent increase in DG mass. Whether this small early increment in DG is sufficient to initiate 0; production cannot be answered at present. Although our data do not support a role for DG in initiating 0; production, the data suggest that DG serves as a modulator of the total 0; response. Kojima et al. (51), working in the adrenal glomerulosa cell, first reported a role for DG in the sustained phase of aldosterone secretion. That DG may be important in sustaining neutrophil 0; generation is suggested by the close correlation of the later kinetics of DG and 0;. With met-leu-phe, ionomycin, or ConA treatment in the presence of cytochalasin B, the activation time of DG preceded or coincided with that for O;, and DG increased in parallel with, but prior to, the rise in 0; at later times. Further evidence suggesting that DG is involved in sustaining 0; comes from the concentration dependence curves at 5 min, a Rider, L. G., and Niedel, J. E. (1987) J. Zmmuml., in press. Kinetics of Diucylglycerol and Superoxide Production 5607 late time point at which there was close correlation of the thresholds and maximal DG and 02 responses with met- Leu-Phe and ionomycin stimulation. The cessation or continuation of DG and 0; also paralleled each other. n ConA- and ionomycin-treated cells, in the presence of cytochalasin B, DG continued to accumulate at late time points, and this led to further 0; production. With met-leu-phe stimulation, 0; generation ceased between 5 and 8 min, correlating with the cessation of DG accumulation. A role for DG in sustaining or augmenting 0; production is also suggested by work from our laboratory using cytochalasin B. Pretreatment of neutrophils with cytochalasin B augmented and sustained the ionomycin-stimulated DG accumulation and correspondingly enhanced and prolonged the 0; response. We have reported similar results in met-leu- Phe-treated neutrophils (30). With ConA stimulation, furthermore, neither DG nor 0; were produced in the absence of cytochalasin B, while large prolonged responses were obtained in cytochalasin B-pretreated cells. The data suggest that cytochalasin B may enhance neutrophil 0; production through an augmentation of the DG response. This correlation of cytochalasin B enhancement of both responses further suggests that DG itself may be involved in augmenting 0; generation. The possibility that other mediators, in addition to DG, may be involved in the generation of 0; is suggested by the ability of ConA to produce 0; in the absence of a DG response. At low doses of ConA ( Fg/ml), 0; was generated without any increment in DG. The recent work of Gerard et al. (52) has also demonstrated that inhibition of protein kinase C does not alter met-leu-phe or C5a-stimulated 0; production, suggesting that alternate pathways of activation may exist in parallel to DG and protein kinase C. Nevertheless, the data from our laboratory, when correlated with published data on the translocation of protein kinase C, thus far demonstrate a relationship between the ability of stimuli to generate 02, produce large accumulations of DG, and translocate the enzyme from cytosol to membranes (53, 54). 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