291, No. 1, November
15, pp. 31-37,199l
Stimulation of Superoxide Anion Production in Guinea Pig Polymorphonuclear Leukocytes by Hypotonic Conditions in Combination with Protein Kinase C Activators’ Masanori Masafumi
Hiura, Masaki Ozawa, Toshiaki Yamaguchi, Naoki Okamura,’
18, 1991, and in revised
School of Medicine,
i This work was supported in part by a grant from Education, Science, and Culture of Japan. 2 To whom correspondence should be addressed. $3.00 0 1991 by Academic Press, Inc. of reproduction in any form reserved.
Conditions for superoxide anion (0,) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, 0, production was significantly enhanced by concurrent treatment with low concentrations of loleoyl-2acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced 0; production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic 0, production with hypotonicity. Phosphorylation of 46kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 PM OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in 0, production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase. 0 1001 Academic PEWS. Inc.
ooo3-9&x/91 Copyright All rights
On exposure to bacteria as well as to certain membrane stimulants, polymorphonuclear leukocytes (PMNL)3 manifest a so-called respiratory burst, including the production of potent bactericidal oxygen metabolites, such as the superoxide anion (O;), H202, and the hydroxyl radical (1, 2), through an activation of the membranebound NADPH oxidase system. Although the mechanism for NADPH oxidase activation is still a matter of debate, it has been considered that changes in the structure of the plasma membranes associated with phagocytosis or with perturbation by the stimulants play a key role in the activation. Evidence has been afforded that metabolism of phosphoinositides to 1,2diacylglycerol (DG) and inositol phosphates by phospholipase C is involved in signal transduction for the activation in PMNL (3,4). It is well known that DG is a physiological activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C). It has been reported from several laboratories that the activation of NADPH oxidase is accompanied by the phosphorylation of 44- to 4%kDa proteins (5-7), and activated protein kinase C is responsible for the phosphorylation of such proteins (8). Thus, it is plausible that protein kinase C plays an important role in signal transduction for the activation of NADPH oxidase in PMNL (9-13).
3 Abbreviations used: PMNL, polymorphonuclear leukocytes; OF, superoxide anion; PMA, phorboll2-myristate la-acetate, 4-a-PMA, 4-ophorbol 12-myristate 13-acetate, OAG, 1-oleoyl-2-acetylglycerol; DG, 1,2diacylglycerol; DGi*,,, l,%-dicaprin; DG,,,, 1,2-diolein; Pipes, 1,4piperazinediethanesulfonic acid; DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; fMLP, N-formyl-methionyl-leucyl-phenylalanine; PI, phosphatidylinositol; PIP, phosphatidylinositol4-phosphate; PIPx, phosphatidylinositol4,5bisphosphate; PA, phosphatidate; [Ca*‘li, intracellular calcium concentration; LDH, lactate dehydrogenase; EGTA, ethylene glycol [email protected]
ether) N,N’-tetraacetic acid. 31
In this study, we found that PMNL exposed to the hypotonic medium showed a marked increase in 0, production, especially in the presence of a protein kinase C activator, the concentration of which was too low to alone stimulate production. The hypotonic treatment caused the release of cellular phosphatidate (PA). These results will be discussed with the view that the change in cell membrane structures accompanied by the increase in PA and protein kinase C activation is associatively involved in the stimulation of 0; production in PMNL. MATERIALS
Materiuk Ferricytochrome c (type III), superoxide dismutase, DGia and H-7 were purchased from Sigma Chemical Co.; NADPH was from kohjin Co.; PMA, 4-a-PMA and DGlo:,, were from Funakoshi Pharmaceutical Co.; OAG was from Nakalai Tesque, Inc.; staurosporine was from Boehringer-Mannheim; QuinZ/AM was from Dojin Laboratories; and Silica Gel-60Fxs, plates were from E. Merck. [32P]Orthophosphate was purchased from JRIA and [sH]arachidonate (180-240 Ci/mmol) from New England Nuclear. Other chemicals were all of reagent grade from standard commercial sources. DGs and phorbols were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in the assay system was adjusted to 1% throughout. Guinea pig PMNL. PMNL were obtained from the peritoneal cavities of female guinea pigs of the Hartley strain as reported previously (5). The content of PMNL in the preparation was more than 90%. Suspending medium. Hypotonic media were prepared by gradually decreasing the Naf concentration in Hanks’-Pipes buffer (pH 7.3), originally containing 8 mM Pipes, 137 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO1, 1.28 mM CaCle, 0.43 mM Na,HPO,, 0.44 mM KHsPO,, and 5.5 mM glucose. Either original Hanks’-Pipes buffer or some isotonic media in which Na+ was partially replaced with K+, Li+, or choline ion were employed for comparison with hypotonicity. Measurement of 0; production in PMNL. 0; production was measured on the basis of superoxide dismutase-inhibitable reduction offerricytochrome c by the anion produced (1). A total of 50 ~1 of PMNL (5 X 10’ cells) in isotonic medium was preincubated for 5 min, 450 pl of the above-mentioned prewarmed medium containing 0.1 mM ferricytochrome c was added to the PMNL suspension, and then stimulants were added to the medium successively. In the case of treatment of PMNL with protein kinase C inhibitors, the stimulants were added after the treatment of PMNL with the inhibitors at 37°C for 5 min. For 0; measurement, the mixture was incubated at 37°C for 5 min with ferricytochrome c either in the presence or in the absence of 100 units/ ml superoxide dismutase. Then, the reaction was stopped by chilling in an ice-water bath, and the mixture was centrifuged at 4°C and 120g for 10 min to precipitate PMNL. Reduced cytochrome c in the supernatant was measured on the basis of an increase in absorbance at 550 nm. In time-course experiments, the reaction mixture in a cuvette was thermostatically controlled, and an absorbancy change at 550 nm in reference to 540 nm was followed on a recorder. 0, production was calculated on the basis of an absorption coefficient of 21.0 mM-‘cm-‘. Lactate dehydrogenase (LDH) release from PMNL. Release of LDH from PMNL was examined during the incubation of PMNL in hypotonic medium with or without OAG as described above. LDH activity (14) was measured in the supernatant after centrifugation of the incubation mixture. LDH activity in 0.05% Triton X-100 treated and sonicated PMNL was assayed as the total activity, and the ratio of released to total activity was calculated. Proteinphasphorylation in PMNL. PMNL were labeled with carrierfree 3zPi as described previously (5). The labeled PMNL (10’ cells/ml) were suspended in the isotonic Hanks-Pipes buffer with phosphate-free 16 mM Pipes. The PMNL suspension was preincubated at 37’C for 5 min. After the preincubation, the PMNL suspension was diluted 10 times with either isotonic Hanks’-Pipes buffer or 40 mu NaCl-containing
hypotonic Hanks’-Pipes buffer (to adjust the NaCl concentration to 50 mM), and the PMNL were treated with 10 MM OAG for 2 min at 37°C. Phosphorylated proteins were analyzed by two-dimensional electrophoresis and autoradiography as described previously (5, 15) using an isoelectric focusing gel containing 0.45% Bio-Lyte (pH 3-5) and 1.55% Bio-Lytc (pH 3-10) for the first run and an SDS-polyacrylamide slab gel for the second run. All gels were subjected to autoradiography with the use of an intensifying screen for the same period at -80°C. Measurement of cytosolic Cd’ in PMNL. PMNL were loaded with Quin2 as described previously (16). Briefly, PMNL (5 X 10’) were incubated with 15 pM QuinZ/AM in Hanks’-Pipes buffer at 37°C. After 15 min, the cell suspension was diluted five times with warm isotonic Hanks’-Pipes buffer, and the incubation was continued for 45 min. After the incubation the Quin2-loaded PMNL were washed twice and resuspended in isotonic Hanks’-Pipes buffer to a concentration of 5 X lo7 cells/ml. The PMNL suspension was diluted 10 times with either isotonic or 40 mM NaCl-containing hypotonic Hanks’-Pipes buffer. Changes in intracellular calcium concentration ( [Ca2’li) were calculated according to the previously reported method (17). Lipid metabolism in PMNL. PMNL were labeled with carrier-free 32P. as described previously (18). The labeled PMNL were suspended in phosphate-free Hanks’-Pipes buffer containing 16 mM Pipes, 137 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO,, 1.28 mu CaClx, and 5.5 mM glucose (pH 7.3) to a concentration of lo7 cells/ml. For measurement of [3H]DG formation, PMNL (1.25 X lo7 cells/ml) were suspended in Ca2+- and Mg’+-free Hanks’-Pipes buffer (pH 7.3) containing [3H]arachidonate (5 &i/ml) and incubated at 37°C for 1 h to incorporate [3H]arachidonate into PMNL. At the end of the incubation, PMNL were washed twice and resuspended to a concentration of l-2 X 108 cells/ml in isotonic Hanks’-Pipes buffer (pH 7.3). By these procedures, more than 80% of [3H]arachidonate was incorporated into PMNL. Labeled PMNL were preincubated at 37°C for 5 min and diluted 10 times with either isotonic or 40 mM NaCl-containing Hanks’-Pipes buffer. Then PMNL were treated with stimulant for 30 8. The reaction was terminated by mixing 500 ~1 of the reaction mixture with 4.5 ml of CHC13/CH30H/0.75 N HCl(20/40/8, by volume). Lipids were extracted by a reported procedure (19). An aliquot of the lipids extracts was subjected to thin layer chromatography on Silica Gel 60 Fzl plates. Neutral lipids were separated by a solvent system of n-hexaneldiethyl ether/ acetic acid (50/50/l, by volume). [3H]DG was identified by cochromatography with DGls,. (detected by iodine staining) (20), and the location was confirmed by measuring the specific increase in the radioactivity after the treatment of PMNL prelabeled with [3H]arachidonate with 1 unit/ml phospholipase C (from Cbstridium perfringens) for 2 min at 37°C (data not shown). For phospholipid analysis, the sheets were eluted with CHC13/CH30H/20% methylamine (60/35/10, by volume). 32P-labeled phospbolipids were visualized by autoradiography using Kodak X-Omat films and by iodine staining, and identified by referring with standards run in parallel. The radioactive spots corresponding to each lipid were cut out and the radioactivity was measured with a scintillation spectrophotometer. [sHjAra&idonate release from PMNL. PMNL (5 X lo7 cells/ml) in Ca’+and Mg*+-free Hanks’-Pipes buffer were labeled with [3H]arachidonate (5 pCi/ml) for 50 min as described above and resuspended to l-2 X lo7 cells/ml in isotonic Hanks’-Pipes buffer. The labeled PMNL suspension (50 ~1) in a microcentrifuge tube was preincubated at 37°C for 5 min, and then diluted 10 times with either isotonic or 40 mM NaCl-containing Hanks’-Pipes buffer and incubated with stimulants for 5 min. The reaction was stopped by an addition of 0.6 ml of cold Hanks’-Pipes buffer, and the mixture was immediately centrifuged at 7000g for 10 s. Each aliquot (1 ml) was taken, and radioactivity released during the incubation was measured with a scintillation spectrophotometer.
Effect of the Combination of Hypotonic and OAG Treatments on 0; Production OAG is known as a cell-permeable diacylglycerol that activates protein kinase C (21). Figure 1 shows 0, pro-
Effect of Monovalent
duction in PMNL suspended in the hypotonic (50 mM NaCl) or isotonic (137 mM NaCl) medium. Although the OAG and hypotonic treatment alone stimulated the 0, production little in 30 min, 10 PM OAG significantly increased the 0, production in PMNL, and the lag time was less than 30 s under hypotonic conditions. After OAG addition, enhanced 0; production lasted even if the medium was changed to an isotonic one. However, such a hypotonic effect disappeared within 1 min after the change from the hypotonic medium to the isotonic one prior to OAG addition (data not shown). Next, the NaCl concentration in the medium was gradually changed. A decrease in NaCl promoted a stimulatory effect of OAG on 0; production (Fig. 2). Maximum 0;
137.0 50.0 50.0 50.0 50.0
5.4 5.4 92.4 5.4 5.4
Choline+ 0.0 0.0 0.0 87.0 0.0
0; production (nmol/5 min/lO’ Li+ 0.0 0.0 0.0 0.0 87.0
-0AG 0.18 2.19 2.70 1.34 1.42
-I + + It +
0.42 0.72 0.77 0.49 0.46
3.82 30.46 10.56 12.33 11.96
f 0.99 + 3.22 + 2.02 IIZ 2.04 f 2.97
FIG. 1. Time course of 0; production in PMNL stimulated by the combination of OAG and hypotonic treatments. Prewarmed PMNL in the isotonic medium was diluted 10 times with 40 mM Na+-containing hypotonic or isotonic medium, and OAG was added immediately after the dilution. Then cytochrome c reduction was monitored continuously (for details, see Materials and Methods). Incubation in 137 mM Na+containing isotonic medium (A); incubation in 50 mM Na+-containing hypotonic medium (B). Treatment with 10 @M OAG in the isotonic medium (C); treatment with 10 pM OAG in the hypotonic medium (D).
Cations on 0, Production in PMNL
Cation concentration in the medium ([email protected]
FIG. 2. 0, production and lactate dehydrogenase (LDH) release from PMNL in the hypotonic medium. PMNL were suspended in the medium containing the indicated concentration of Nat in the presence (0) or absence (0) of 10 pM OAG. Solid lines indicate 0, production, and dashed lines indicate LDH activity. 0; production and the release of LDH were assayed as described under Materials and Methods. The values are the mean f SE of at least three experiments. The absence of a vertical bar means that SE is within the range of the symbol.
Note. PMNL were suspended in Hanks’-Pipes buffer with the indicated concentration of monovalent cations in the presence or absence of 10 pM OAG. The values are the mean + SE of three experiments.
production was observed at 30 mM NaCl. But, in the absence of OAG, 0; production increased only slightly even at this NaCl concentration. Since the difference in 0, production between the presence and the absence of OAG was most noticeable at 50 mM NaCl, the medium with 50 mM NaCl was used as the hypotonic medium in the later experiments. The increased 0; production in the hypotonic medium was not due to the disruption of the cells because the release of lactate dehydrogenase, a cytosolic enzyme, was slightly increased in the hypotonic medium and was not affected by OAG (Fig. 2). The fact that little difference was found in the 0; production between 10 and 100 PM OAG in the isotonic medium suggested that the increase in permeation of OAG into the cell was little responsible for the enhancement of 0; production, even if the permeation might be increased under hypotonic conditions (data not shown). Effect of the Replacement of NaCl with Other Monovalent Cations on the Synergism between OAG and Change in the Tonicity of the Medium in Stimulating Oi Production It has been reported that 0, production induced by a chemotactic peptide is progressively increased by substituting Na+ for K+ in a medium (22). 0, production induced by 10 PM OAG was also potentiated by substituting Na+ with K+, Li+, or choline ions in the isotonic medium (Table I). But these increases in production were about a third of that by the hypotonic treatment. These results seem to suggest that one third of the marked increase in 0; production by the combination of OAG and hypotonic treatment is related to Na’ deficiency, while the remainder is due to hypotonicity. Involvement of Protein Kinase C in 0; Production under Hypotonic Conditions OAG, a protein kinase C activator, markedly increased 0; production under hypotonic conditions. This indicates that protein kinase C is responsible for synergic 0, pro-
duction with hypotonicity. To confirm the association of protein kinase C with 0; production, the following experiment was conducted using other protein kinase C activators and inhibitor in combination with hypotonic treatment (Fig. 3). Low concentration of both DGleO, an OAG-like cell-permeable diacylglycerol, and PMA also induced a marked increase in 0, production in guinea pig PMNL suspended in the hypotonic medium, although these agents at the concentrations used were insufficient in stimulating production in the isotonic medium. But neither DGia., which does not permeate into cells, nor 4-CY-PMA, which has no effect on protein kinase C, stimulated 0; production in the hypotonic medium. Pretreatment of the cells with H-7, a protein kinase C inhibitor (23), suppressed most 0; production. Another protein kinase C inhibitor, staurosporine, also suppressed production dose-dependently (Fig. 4). Staurosporine inhibited production more effectively than H-7. These results seem to indicate that the activation of protein kinase C is related to enhancement of 0; production in PMNL in the hypotonic medium. Effect of the Combination Treatments on 46-kDa
of Hypotonic and OAG Protein Phosphorylation
We have reported that both translocation of 46-kDa protein(s) from cytosol to cell membranes accompanied by protein-kinase-C-catalyzed phosphorylation and some change such as in membrane charge are involved in 0, production (24, 25). In the present study, we measured the state of phosphorylation of 46-kDa protein in guinea pig PMNL under hypotonic conditions. OAG stimulated the phosphorylation of 46-kDa protein in both the isotonic and the hypotonic media (Fig. 5), and no significant difference was observed in the phosphorylation between isotonic and hypotonic conditions. The results indicated that hypotonic treatment itself did not activate protein ki-
PMA [email protected]
DG,O:O 4-a-PMA 1OpM
FIG. 3. Effect of protein kinase C activators and inhibitor on 0; production in PMNL in the hypotonic or isotonic medium. PMNL were suspended in 137 mM Na+-containing isotonic medium (open bar), in 50 mM Na+-containing hypotonic medium (hatched bar), or in the same hypotonic medium containing 200 pM H-7 (closed bar) and then were treated with the agents of the indicated concentrations. The values are the mean + SE of three experiments.
c‘120e -E 8 100 % E
inhibitory effects of protein kinase C inhibFIG. 4. Dose-dependent itors, staurosporine and H-7, on 0; production in PMNL stimulated synergically by OAG and hypotonic treatments. PMNL were suspended in 50 mM Na+-containing hypotonic medium together with these inhibitors at the indicated concentration, and then treated with 10 gM OAG. Values are expressed as percentages of the control (no inhibitor) values, and the mean f SE of four experiments.
Effect of Hypotonic
It is known that OAG and calcium-mobilizing agents, such as A23187 and fMLP, show synergism in stimulating 0; production in PMNL (26, 27). So we examined if an increase in [Ca’+]i was involved in the above-mentioned stimulatory effect of hypotonic treatment. fMLP was used as a positive control for this experiment. The addition of 10e7~ fMLP markedly increased [Ca’+]i. However, the hypotonic treatment caused a slight decrease in [Ca’+]i rather than an increase (Table II). OAG (10 PM) itself did not affect intracellular Ca2+ levels (28). On the other hand, the removal of extracellular Ca2+ by the addition of EGTA did not inhibit the 0; production stimulated by the combination of OAG and hypotonic treatment (data not shown). These results indicate that the change in 0;production does not depend on the change in [Ca2+]i. Effect of Hypotonic from PMNL
We have reported that a combination of OAG and a fatty acid, such as arachidonate, oleate, or palmitate, synergically activated 0, production (28). It is plausible that hypotonic treatment activates phospholipase A2, this enzyme releases fatty acids from membrane phospholipids, and OAG causes 0; production synergically with these fatty acids. So we measured the release of arachidonate from PMNL. A23187, a potent arachidonate-releasing agent (25), actually stimulated arachidonate release, but there was no significant difference in the rate of arachidonate release between the hypotonic and the isotonic conditions irrespective of the presence of OAG (Table III), indicating that the effect of hypotonic treatment was not mediated by the increase in the rate of arachidonate release.
Effects of Hypotonicity and OAG on Arachidonate Release in PMNL Arachidonate release (% of total incorporated)
Stimulants 137 mM Na+-containing isotonic 50 mM Na+-containing hypotonic medium 137 mM Na+-containing isotonic + 10 /.tM OAG 50 mM Na+-containing hypotonic medium + 10 pM OAG 137 mM Na+-containing isotonic + 1 PM A23187
2.69 + 0.21 3.41 + 0.46
medium 3.96 + 0.95 3.49 k 0.18 medium 7.25 f 0.62
Note. [3H]Arachidonate-prelabeled PMNL were diluted 10 times with 137 mM Na+-containing isotonic medium or 40 mM Na+-containing hypotonic medium. After the dilution, 10 gM OAG or 1 pM A23187 was added to the PMNL suspension immediately, and then cells were incubated for 5 min. [3H]Arachidonate was measured by the procedure described under Materials and Methods. Arachidonate release is expressed as a percentage of the total radioactivity incorporated into PMNL at the time of the prelabeling. Values are the mean + SE of at least four experiments.
oxidase activation. In the present, study, we investigated membrane phospholipid metabolism in relation to the effect of hypotonic treatment. At first, we measured production of [3H]DG in PMNL prelabeled with [3H]arachidonate (Table IV). Any of the incubations under hypotonic or isotonic conditions for 30 s in the absence or presence of OAG caused an almost similar change in DG formation. fMLP, known to increase DG formation at 1 PM, did not stimulate DG formation
FIG. 5. Autoradiogram of a part of two-dimensional electropherograms of 32P-labeled proteins from PMNL stimulated with OAG, hypotonicity, or both. 32P-preloaded PMNL in 137 or 50 mM Naf-containing medium were incubated at 37’C for 2 min with or without 10 pM OAG. Phosphorylated proteins were separated by two-dimensional SDS-PAGE and detected by autoradiography. Arrows indicate a 46-kDa protein(s). Incubation in the isotonic medium (A); incubation in the hypotonic medium (B). Treatment with 10 jtM OAG in the isotonic medium (C); treatment with 10 WM OAG in the hypotonic medium (D).
Effect of Hypotonic Metabolism
Since it is known that the addition of phospholipase C or several receptor agonists increasing the activity of this enzyme stimulates 0, production in PMNL, an increase in phospholipid turnover may be responsible for NADPH
of Hypotonic Treatment in Quin2-loaded PMNL
Treatments 137 mM Na+-containing 50 mM Na+-containing 137 mM Na+-containing + 100 nM fMLP
[ca’+]i isotonic hypotonic isotonic
medium medium medium
) 3 3
1170 * 158
Note. Quin2-loaded PMNL were diluted 10 times with 137 or 40 mM Na’-containing Hanks’-Pipes buffer. Values are calculated from the peak of fluorescence intensity and shown as the mean + SE of three experiments (for details, see Materials and Methods).
Effects of Hypotonicity DG Accumulation
Note. [3H]Arachidonate-prelabeled 137 mM Na+-containing isotonic potonic medium. Immediately fMLP was added to the PMNL for 30 s. [$H]DG was measured ods. Values, calculated as the was about 8000 cpm/107 cells, periments.
[3H]DG formation (% of control)
Treatments 137 mM Na+-containing isotonic 50 mM Na+-containing hypotonic medium 137 mM Na+-containing isotonic + 10 pM OAG 50 mM Na+-containing hypotonic medium + 10 pM OAG 137 mM Na*-containing isotonic + 30 nM fMLP
and OAG in PMNL
100 91.7 + 16.7
medium 86.7 f 12.4 86.7 + 12.4 medium 112.3 f
PMNL were diluted 10 times with medium or 40 mM Na+-containing hyafter the dilution, 10 pM OAG or 30 nM suspension, and then cells were incubated as described under Materials and Methpercentage of the isotonic group which are the mean + SE of at least three ex-
significantly at 30 nM under the present experimental conditions. However, we previously reported that DG kinase inhibitor R59022 markedly enhanced the level of DG which was slightly increased by fMLP (18). DG formed by fMLP stimulation may be immediately converted to other species such as PA, as will be described below. On the other hand, this inhibitor had no effect on DG formation in the hypotonic medium (data not shown), suggesting that DG is not increased significantly by hypotonic treatment. Next, we measured concentrations of other phospholipid-related compounds, such as phosphatidylinositol (PI), phosphatidylinositol4-phosphate (PIP) plus phosphatidylinositol4,Sbisphosphate (PIP2), and PA, which are known to result from PI turnover. Hypotonic treatment alone slightly decreased the concentrations of PIP and PIP2, but the combination of OAG and hypotonic treatments did not modify the effect of hypotonicity alone. OAG treatment slightly increased the PI level, but little increase in PI was observed after hypotonic treatment (Table V). These findings indicate that PI turnover is not involved in the effect of the combination of hypotonicity and OAG. The results, however, do not indicate the lack of PI turnover in guinea pig PMNL, since fMLP markedly decreased the sum of PIP and PIP2 contents. On the other hand, the level of PA was increased three times by fMLP. Hypotonic treatment also markedly increased PA content by 75 and 90%. This effect was observed irrespective of the presence of OAG. These results indicate that hypotonic treatment causes changes in cell membranes in concurrence with the increase in PA levels through the DG-independent pathway. DISCUSSION
It has been shown that the activation of NADPH oxidase is accompanied by the phosphorylation of 44- to 48kDa proteins (57), and activated protein kinase C is responsible for the phosphorylation of such proteins (8). Thus, protein kinase C may play a central role in the
activation of NADPH oxidase in PMNL (29-31). However, when guinea pig PMNL are stimulated by OAG, a synthetic cell-permeable diacylglycerol, alone, higher concentrations of OAG (more than millimolar) are required to induce significant 0; production. In other words, lower concentrations of OAG, enough to cause the full phosphorylation of 46-kDa protein(s), does not induce a detectable change in 0; production (26). These findings imply that protein kinase C can not trigger the activation of NADPH oxidase by itself. Recently, many reports have been published that the NADPH oxidase of human neutrophil consists of membrane factors, including cytochrome bS5s, flavoproteins and lipids, and cytosolic factors including 46- to 48-kDa and 64- to 67-kDa proteins at least. It has also been reported that a 46- to 48-kDa cytosolic factor is phosphorylated by protein kinase C (32,33). It is known that such a protein is absent in the PMNL of some types of chronic granulomatous disease patients in whom the activation of NADPH oxidase is almost nil. Protein-kinase-C-independent NADPH oxidase activation mechanisms have been pointed out by several investigators (34, 35). We are also analyzing such a mechanism. But in this paper, a protein-kinase-C-dependent mechanism for NADPH oxidase was focused on. On the other hand, it was reported recently that membrane charges were also an important factor for the NADPH oxidase activation in PMNL in regulating the association of the cytosolic factor(s) and the plasma membrane (25,36). In the present experiment, we found that NADPH oxidase was synergically activated by hypotonicity and OAG. This synergic activation was dependent on protein kinase C activity, because the NADPH oxidase activity was specifically stimulated by protein kinase C activators (Fig. 3) and inhibited dose-dependently by protein kinase C inhibitors (Fig. 4). Although the exact mechanism(s) associated with hypotonic treatment is not clear, there may be several pos-
Effects of Hypotonicity and OAG on Phospholipid Metabolism in PMNL 32P
Treatment 137 mM Na+-containing isotonicmedium 50 mM Na+-containing hypotonicmedium 137 mM Na+-containing isotonicmedium+ 10 pM OAG 50 mM Na+-containing hypotonicmedium+ 10 pM OAG 137 mMNa+-containing isotonicmedium+ 30 nM fMLP
incorporation(% of control)
187.2 103.2 174.7 322.0
100 + 25.1 + 1.4 f 20.0 z!z 42.3
114.0 129.6 139.7 99.2
100 + 6.9 + 1.0 f 19.1 + 3.0
80.8 101.0 98.0 63.8
100 f 4.4 k 14.2 + 8.9 + 6.5
Note. 32P-prelabeled PMNL were diluted 10 times with 137 mu Na+-containing isotonic or 40 mu Na+-containing hypotonic medium. Immediately after the dilution, 10 pM OAG or 30 nM fMLP was added to the PMNL suspension, and then the cells were incubated for 30 s. [32P]PA and the sum of [e*P]PIP and [szP]PIP2 were measured as described under Materials and Methods. Values, calculated as the percentage of the isotonic treatment level which was about 10,000 cpm/lO’ cells, are the mean _t SE of at least three experiments.
sible factors. We think about one third of the effect of hypotonicity is due to Na+ ion depletion, while the remainder may be caused by physical membrane changes caused by hypotonicity itself (Table I). It is not clear if a decrease in Cl- concentration is involved in the latter effect. In this experiment, we also found an increase in PA formation under hypotonic conditions (Table V). We recently reported that low concentrations of 1,2didecanoyl3-sn-phosphatidate, a short chain cell-permeable PA, enhanced 0; production in PMNL treated concurrently with OAG (37). So PA may be one of the factors in the synergistic activation of 0; production with OAG. Hypotonic conditions may stimulate phospholipase D to produce PA, and accumulation of PA in membranes may cause the change in the membrane charge and/or fluidity, which are important in the activation of NADPH oxidase. But the reversibility of the hypotonic effect suggests that the temporary changes in the membrane structure will bring the resting state of NADPH oxidase to the primed state. Thus it is quite likely that the combination of protein kinase C activation and membrane change(s) will cause the translocation of the cytosolic factor(s) to the membrane (24, 38-40), i.e., the association of all components of NADPH oxidase resulting in the activation of this enzyme. It is our conclusion that hypotonicity prepares the primed state of NADPH oxidase and then synergically activates it with OAG. If OAG is not added, such a primed state can return to the resting state. Several additional experiments are necessary to exactly define the priming mechanism by hypotonicity, but hypotonicity may provide a good model for studying the mechanism of priming in the activation of NADPH oxidase. REFERENCES 1. Babior, B. M., Kipnes, Invest. 52, 741-744. 2. Klebanoff, S. J. (1980) 3. Bennett, Biophys.
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