©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
N-Acetylsphingosine (C-ceramide) Inhibited Neutrophil Superoxide Formation and Calcium Influx (*)

(Received for publication, October 7, 1994; and in revised form, November 18, 1994)

Kenneth Wong (§) Xue-Bin Li Nicole Hunchuk

From the Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4N1 and the Canadian Red Cross Blood Transfusion Center, Calgary, Alberta, Canada T2R 1J1

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ceramide, a product arising from sphingomyelinase activity, has been shown to act as an intracellular second messenger in effecting growth inhibition, cellular differentiation, and apoptosis. In the present study, the relative effects of cell-permeable ceramides, N-acetylsphingosine (C(2)-ceramide) and N-hexanoylsphingosine (C(6)-ceramide), on neutrophil responses were measured. When cells were activated with fMet-Leu-Phe, C(2)-ceramide both potentiated (<1 µM) and inhibited (>1 µM) superoxide generation. C(2)- and C(6)-ceramide inhibited phorbol ester-induced superoxide release from neutrophils at IC values of 5 and 120 µM, respectively. C(2)-ceramide had no effect on semipurified protein kinase C activity. Neither ceramide affected significantly the general level of phosphorylated proteins in phorbol ester-treated cells. C(2)-ceramide (1-20 µM) alone did not change cytosolic free Ca levels but inhibited Ca and Mn influx in fMet-Leu-Phe-activated neutrophils. In contrast, sphingosine enhanced Ca entry; thus, ceramide conversion to sphingosine was not significant. Unlike C(2)-ceramide, C(2)-dihydroceramide failed to block superoxide generation or Ca influx. Preincubation of cells with 10 nM okadaic acid reversed slightly the effects of C(2)-ceramide. Calyculin A, tautomycin, and much higher concentrations of okadaic acid inhibited agonist-induced Ca influx. We postulate that C(2)-ceramide may inhibit neutrophil superoxide release by activation of type 2A protein phosphatases. Results suggest that protein phosphatase type 1 up-regulates Ca entry, whereas type 2A (or a ceramide-activated subtype) forestalls Ca entry by inactivating a calcium influx factor.


INTRODUCTION

Recent studies have shown that sphingoid lipids function as second messengers in signal transduction(1, 2, 3, 4, 5, 6) . Among other effects, sphingosine inhibits protein kinase C (PKC)(^1)(1, 2) , and sphingosine 1-phosphate mobilizes intracellular Ca stores(6) . The biological effects of 1alpha,25-dihydroxyvitamin D(3), tumor necrosis factor-alpha, interferon-, and interleukin-1 correlate with early activation of a neutral sphingomyelinase and elevations of ceramide levels in target cells(3, 4, 5) . Cell-permeable synthetic ceramides were shown to mimic hormonal effects. For example, C(2)-ceramide, tumor necrosis factor-alpha, or vitamin D(3) each induces differentiation of HL-60 cells (a leukemic cell line) into monocyte-like cells(3, 4) . A downstream effect in each case was the down-regulation of c-myc mRNA. Other studies report that ceramides induce growth arrest, differentiation, and apoptosis in fibroblasts, myeloid, and lymphoid cells(3, 4, 7) . On a biochemical level, Kolesnick and co-workers showed that ceramides activate proline-directed serine/threonine kinases in A431 and HL-60 cells(3, 8) . Hannun and co-workers in turn have advocated a mediator role for protein phosphatases (PPases)(5, 9) . Working with extracts from rat brain and T9 glioma cells, they showed that ceramides activate both heterotrimeric PPase-2A and a related subtype they called ceramide-activated protein phosphatase (CAPP). In vitro, CAPP activity is inhibited by okadaic acid (OA)(5) . The relative primacy of ceramide-activated kinases and PPases remains to be defined in different cell types.

We have reported that exogenous sphingosine mobilizes Ca in human neutrophils (PMNs)(10) . Our results suggest that sphingosine acts indirectly through a metabolite, a likely candidate being sphingosine 1-phosphate. In this study, we address the question of whether the cell-permeable ceramides N-acetylsphingosine (C(2)-ceramide) and N-hexanoylsphingosine (C(6)-ceramide) can modulate cellular activation and Ca homeostasis in PMNs. The results show that C(2)-ceramide effectively dampened specific signaling pathways. We hypothesize that PPases may mediate the observed ceramide effects.


EXPERIMENTAL PROCEDURES

Materials

C(2)-ceramide and C(6)-ceramide were purchased from Matreya Inc. (Chalfont, PA); C(2)-dihydroceramide was obtained from Biomol Research Laboratories (Plymouth Meeting, PA); D-sphingosine, n-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and EGTA were purchased from Sigma. Okadaic acid, calyculin A, tautomycin, and fura-2/AM (the acetoxymethyl ester form of fura-2) were obtained from Calbiochem. Hanks' balanced salt solution (HBSS) was purchased from Life Technologies, Inc. P(i) (in water) and CaCl(2) (36 mCi/mg) was obtained from DuPont NEN. A PKC assay kit was obtained from Amersham Canada Ltd.

Stock solutions of ceramides were made up in dimethyl sulfoxide and stored at -80 °C; when added to aqueous reaction mixtures, the final concentration of the carrier solvent did not exceed 0.5%.

Isolation of Neutrophils

Neutrophils from peripheral venous blood of healthy donors were isolated and purified according to established procedures described previously (10, 11, 12) and involving cell separation in a Ficoll-Paque density gradient followed by lysis of contaminating erythrocytes. Purified neutrophils containing >95% viable cells were normally resuspended in HBSS with 1.26 mM Ca, pH 7.4.

Assay for Superoxide Generation by Human Neutrophils

Superoxide dismutase-inhibitable O released from activated neutrophils was quantitated using the cytochrome c reduction assay(13) . Neutrophils were suspended at 1 times 10 cells/ml in phosphate-buffered saline containing 1.5 times 10M ferricytochrome c and activated by either fMLP or PMA at 37 °C. Absorbance changes at 550 nm were monitored continuously in a double-beam spectrophotometer in which the content of the reference cuvette (1.5-ml volume, polystyrene) was identical to that in the sample cuvette except for the additional presence of superoxide dismutase (0.6 mg/ml).

Fluorometric Measurement of [Ca](i)

Purified neutrophils (1 times 10^7 cells/ml HBSS) were incubated with 1 µM fura-2/AM for 30 min at 37 °C. After that, the cells were washed twice with HBSS and then resuspended at 1 times 10^6 cells/ml HBSS at room temperature. Aliquots (2 ml) were prewarmed for 5 min at 37 °C in disposable fluorometric cuvettes before assays. Fura-2 fluorescence was monitored continuously in an SLM SPF-500C fluorescence spectrophotometer equipped with a thermostatted cuvette compartment. Monochromator settings, unless stated otherwise, were 339 nm (excitation) and 505 nm (emission). The traces generated for each aliquot of cells were calibrated for [Ca] from its F(max) (obtained by lysing the cells with 0.1% (v/v) Triton X-100) and F(min) (obtained by adding 20 mM Tris base and 2-3 mM EGTA to lysed cells) values as described previously(10, 11, 12) .

Quantification of Ca Efflux

Neutrophils (2 times 10^7 cells/ml, in Mg free HBSS) were incubated with 5 µCi/ml Ca for 40 min at 37 °C, washed twice, and resuspended in normal HBSS (with 1.26 mM CaCl(2)) to a density of 2 times 10^6 cells/ml. Upon exposure of cells at 37 °C to agonists for defined periods, 0.58-ml aliquots were removed and layered on top of 0.3 ml of Harwick (SF-1250) silicone oil in microcentrifuge conical tubes. Centrifugation of samples for 1 min in an Eppendorf microcentrifuge separated cells from the supernatant. Radioactivity in supernatants and cell pellets was assayed by standard liquid scintillation counting techniques as outlined previously(10, 11) .

Protein Kinase C Assay

The effect of C(2)- and C(6)-ceramide on PKC activity was measured using a PKC assay kit (Amersham) based on the mixed micelle method of Hannun et al.(14) . Semipurified classical PKC from rat brain was activated with PMA (3 µM) in the presence of Ca (1 mM), phosphatidylserine (0.75 mol%), [-P]ATP (50 µM, 3 µCi/ml), and a specific peptide substrate for 15 min at 25 °C. Labeled substrate bound to binding paper was washed and counted using standard liquid scintillation methods.

Endogenous Protein Phosphorylation

The method used to assess protein phosphorylation in resting and stimulated neutrophils is outlined in a previous study from this group(13) . In brief, neutrophils were incubated in the presence of P(i) for 1 h (37 °C), washed, then treated with vehicle (controls), agonists, or antagonists. P(i) incorporation by proteins was stopped by the addition of 15% trichloroacetic acid; subsequently, the precipitated phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiograms made from dried gels. Protein concentration in samples was determined by the method of Lowry(13) .


RESULTS

Ceramide Inhibited the Respiratory Burst of Neutrophils

We found that C(2)-ceramide inhibited O(2) generation in human neutrophils stimulated by fMLP, a receptor-dependent chemoattractant (Fig. 1). The effect of C(2)-ceramide was time-dependent. Maximal inhibition of the fMLP response occurred after a 1-min preincubation of cells with 10 µM C(2)-ceramide (Fig. 1A). Dose-response studies revealed that C(2)-ceramide exerted a biphasic effect on this response. Concentrations of C(2)-ceramide <1 µM potentiated both the rate and total amount of O(2) released; concentrations >1 µM inhibited this response (Fig. 1, B and C). The IC was estimated to be 6 µM (6 fmol/cell). Cytotoxicity was not a factor since neutrophils incubated for 10 min with the highest concentration of ceramide showed no increase in trypan blue uptake.


Figure 1: Effect of C(2)-ceramide (C-2) on fMLP-induced O(2) generation by human neutrophils. Panel A, effect of incubation time on O(2) release. Superoxide release was measured by ferricytochrome c reduction as described under ``Experimental Procedures.'' Neutrophils (1 times 10^6 cells/ml, at 37 °C) were preincubated with 10 µM C(2)-ceramide for the times indicated in the figure before fMLP (1 µM) was added. Successive traces have been superimposed for better comparison. Panel B, the dose effect of C(2)-ceramide on the kinetics of O(2) generation. Cells were incubated for 1 min with various concentrations of C(2)-ceramide before the addition of fMLP; other conditions were similar to panel A. Panel C, average dose-response effect of a 1-min preincubation with C(2)-ceramide on neutrophil O(2) release. Initial linear rates of O(2) generation were calculated as percentage of control (vehicle-treated cells). Results are means ± S.E. of nine experiments. * indicates significantly difference from control values with p < 0.05 by the Bonferroni t test method after analysis of variance.



Similar results were obtained when neutrophils were stimulated with PMA, a PKC activator. The respiratory burst triggered by PMA is characterized by a lag of about 1 min and prolonged kinetics of O(2) release(13) . When neutrophils were pretreated with C(2)-ceramide (1-10 µM) for 1 min, lag times were increased slightly, and initial linear rates of O(2) release were depressed dose dependently (Fig. 2A). Although not shown, the total amount of O(2) generated after 30 min was also decreased proportionately. In contrast to C(2)-ceramide, C(2)-dihydroceramide, a negative control(4, 9) , was without effect at 50 µM. The duration of incubation was not a factor since cells treated for 30 min with C(2)-dihydroceramide before PMA was added continued to express O(2) rates similar to those of controls. Negative results were also obtained using 80 µM C(2)-dihydroceramide (not shown).


Figure 2: Effect of ceramide on PMA-induced O(2) generation. Panel A, the dose effect of C(2)-ceramide on the kinetics of O(2) release. Neutrophils were preincubated with various doses of C(2)-ceramide for 1 min before PMA (1 µM) was added to cell suspensions. The time course of O(2) release was recorded continuously as in Fig. 1. Similar studies were performed using C(6)-ceramide. Panel B, the initial linear rates of O(2) generation were calculated as a percentage of control rates and the results plotted against ceramide concentration (C-6 = C(6)-ceramide). Results are means ± S.E. of four experiments.



Under similar conditions (1-min incubation before the addition of PMA), C(6)-ceramide was less potent than C(2)-ceramide in blocking free radical formation. The IC at a cell density of 1 times 10^6/ml was estimated to be 5 and 120 µM, respectively for C(2)- and C(6)-ceramide (Fig. 2B). Incubation time influenced C(6)-ceramide effects moderately. Cells treated for 1 min with 30 µM C(6)-ceramide released O(2) at the same rate as controls; if they were treated for 30 min with 30 µM C(6)-ceramide before PMA was added, the expressed rates were 75 ± 3.8% (n = 3) of controls.

Effect of Ceramides on PKC

C(2)-ceramide has been reported not to affect PKC activity(4) . To confirm this, we assayed the effect of C(2)- and C(6)-ceramide on the enzyme activity of Ca and phospholipid-dependent PKC semipurified from rat brain(13) . The results showed that C(2)-ceramide at doses used in the present studies had no effect on classical PKC; C(6)-ceramide at concentrations up to 300 µM inhibited the activity weakly (Fig. 3).


Figure 3: Effect of C(2)- and C(6)-ceramide on Ca/phospholipid-dependent PKC activity. PKC semipurified from rat brain was assayed according to methods described under ``Experimental Procedures.'' P(i) incorporation into a peptide substrate was expressed as a percentage of control radioactivity in which dimethyl sulfoxide vehicle was added instead of ceramide. Staurosporine (10 nM) was used as a positive control. Results shown are means ± S.E. of four experiments.



Effect of Ceramides on Protein Phosphorylation

The effect of C(2)- and C(6)-ceramide on the general protein phosphorylation pattern in neutrophils subjected to prolonged incubation with P(i) was examined (Fig. 4). In resting cells, constitutively active kinases likely mediated incorporation of P(i) into multiple protein bands according to previous studies (lane a)(13, 15) . As expected, the level of phosphorylation and number of bands increased in cells treated for 5 min with PMA (lane b). Incubation of cells for 5 min with calyculin A, C(2)-, or C(6)-ceramide had no noticeable effect on protein labeling in resting cells (lanes c, e, and g). Calyculin A combined with PMA (lane d) caused hyperphosphorylation of protein bands compared with PMA-only controls (lane b). This effect was reported previously for calyculin A, a potent inhibitor of protein phosphatase 1 (PPase-1) and 2A (PPase-2A)(15, 16) . In contrast, C(2)- (20 µM) and C(6)-ceramide (150 µM) neither enhanced nor decreased significantly the amount of protein phosphorylation mediated by PMA (lanes f and h).


Figure 4: Effect of ceramides on protein phosphorylation. Neutrophils prelabeled with P(i) according to the methods described under ``Experimental Procedures'' were incubated for 6 min at 37 °C with vehicle (control, lane a), 50 nM calyculin A (lane c), 10 µM C(2)-ceramide (lane e), or 150 µM C(6)-ceramide (lane g) before reactions were stopped by 15% trichloroacetic acid. About 50 µg of protein/sample was loaded. In lanes b, d, f, and h, the same reagents were added to cells except that 100 nM PMA was added to cells 1 min into the incubation. Total protein was separated by sodium dodecyl sulfate-gel electrophoresis and autoradiograms made from dried gels.



C(2)-ceramide Modulated [Ca](i)

In light of recent reports (6) showing that metabolites of sphingosine such as shingosine 1-phosphate could mobilize intracellular Ca stores and increase [Ca](i), we addressed the question of whether C(2)-ceramide can modulate neutrophil [Ca](i).

We found that 1-20 µM C(2)-ceramide alone had little or no effect on neutrophil [Ca](i) under short term (5-10-min) incubations. This agent did modulate changes in [Ca](i) induced by fMLP (Fig. 5). In panels A-C, control traces show the time course of elevation of [Ca](i) triggered by fMLP. As reported by this group and others(11, 12) , the rise in [Ca](i) was biphasic. Agonist activation of phospholipase C led to the hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)); the latter mobilized internally stored Ca and produced a transient elevation of [Ca](i) to micromolar levels. The primary Ca spike was succeeded by a secondary influx of extracellular Ca which maintained [Ca](i) between 0.2 and 0.5 µM for 6-10 min. The addition of 3 mM EGTA to cells reduced extracellular Ca to submicromolar levels, and, by eliminating Ca influx, isolated the internal Ca release component (Fig. 5B). A similar trace was obtained by treating neutrophils with PMA before the addition of fMLP, a result that is in keeping with a negative feedback role for PKC on Ca influx (Fig. 5A)(11, 12) .


Figure 5: Ceramide inhibition of store-regulated Ca entry. Neutrophils were loaded with fura-2 as described under ``Experimental Procedures,'' and the fluorescence signal was recorded continuously. PMNs from three different donors were used in panels A, B, and C (the control traces illustrate the variability in experiments). In each, traces were recorded consecutively and overlaid afterward for better comparison. In panels A and B, vehicle (control), PMA (100 nM), EGTA (3 mM), C(2)-dihydroceramide (50 µM), or C(2)-ceramide (20 µM or at indicated concentrations) were added to neutrophil suspensions 1 min before fMLP (1 µM). On the right in panel A, the dotted line retraces the control trace shown on the left. In panel C, 10 nM OA was added 30 min and/or 20 µM C(2)-ceramide 2 min before fMLP. Results are representative of at least three experiments.



When PMNs were pretreated with C(2)-ceramide for 1 min before fMLP was added, the ensuing fura-2 traces were similar to those obtained in the presence of EGTA or PMA; that is, C(2)-ceramide dose dependently inhibited the secondary phase of Ca influx (Fig. 5A). By comparison, C(2)-ceramide (20 µM) depressed the peak height of the Ca spike to a lesser extent. As a control, C(2)-dihydroceramide (50 µM) failed to alter fMLP-induced rises in [Ca](i) (Fig. 5B). C(6)-ceramide applied at concentrations similar to those for C(2)-ceramide was also inactive.

C(2)-ceramide inhibition of Ca influx was corroborated by Mn influx studies (Fig. 6). Mn entering fura-2-loaded neutrophils is detected by its quenching of the dye fluorescence and is used as a measure of the permeability of cation channels(10, 11) . Results show that C(2)-ceramide alone failed to change background rates of Mn entry (left panel, Fig. 6). In agreement with previous reports, fMLP increased the rate of Mn influx after a short delay of about 30-60 s (right panel). In contrast, in cell suspensions treated consecutively with C(2)-ceramide and fMLP, Mn entry was blocked completely for several min before resuming at a rate similar to background controls.


Figure 6: Effects of C(2)-ceramide on the kinetics of Mn entry in fura-2-loaded neutrophils. Cell suspensions were excited at 360 nm, the isosbestic wavelength for Ca-fura-2 interactions. Mn influx was indicated by quenching of fura-2 fluorescence. Neutrophils were incubated with C(2)-ceramide for 1 min before MnCl(2) (left panel) or MnCl(2) together with fMLP (right panel) were added at points marked by arrows. Mn interaction with a small amount of extracellular fura-2 caused the abrupt but limited quenching following the addition of MnCl(2). Results are representative of two experiments.



A working model considered in this study is the activation of PPase by ceramides. Since in vitro experiments show that CAPP activation is reversed by OA(5) , we investigated the effect of OA on C(2)-ceramide inhibition of Ca entry (Fig. 5C). We found over three experiments that a 30-min preincubation of cells with 10 nM OA had little or no effect on fMLP-induced secondary Ca influx (the small decrease in the extent of the Ca spike was not reproducible). In cells treated with OA, C(2)-ceramide, and fMLP in the order shown, OA alleviated C(2)-ceramide inhibition of Ca entry by a modest extent (Fig. 3C).

Straightforward interpretations of experiments using higher concentrations of OA were thwarted by the finding that 50-200 nM OA inhibited Ca entry. This echoes previous reports showing that 1 µM OA dampened store-operated Ca entry in neutrophils and HeLa cells (17, 18) . Calyculin A and tautomycin were more potent than OA (Fig. 7). Maximal inhibition of fMLP-triggered Ca influx occurred after a 10-20-min incubation with 2 nM calyculin A or 1 min with 30 nM tautomycin. OA applied under similar conditions had no effect (not shown in Fig. 7).


Figure 7: Effect of PPase-1/PPase-2A inhibitors on fMLP-induced Ca influx. Fura-2-loaded neutrophils were pretreated for 10 min (trace a) or 20 min (trace b) with 2 nM calyculin A before the addition of 1 µM fMLP. In trace c, 30 nM tautomycin was added to cells 1 min before fMLP.



Although ceramides do not inhibit PKC directly, the possibility exists that ceramide were converted to sphingosine, a PKC inhibitor. For comparison, Fig. 8shows the effect of sphingosine pretreatment on fMLP-induced elevations of [Ca](i). As shown, 0.5 and 1 µM sphingosine alone mediated minor but significant elevation of [Ca](i). This was attributed to metabolism of some of the sphingosine to sphingosine 1-phosphate and the mobilization of Ca stores by the latter(6, 10) . When fMLP was added after 2 min, the secondary influx of Ca was enhanced. In this respect sphingosine mediated the same effect as other PKC inhibitors such as staurosporine and auranofin; that is, alleviation of the feedback effect of PKC(11, 12) . In short, sphingosine exerted an effect opposite to that shown for C(2)-ceramide. These results imply that significant conversion of C(2)-ceramide to sphingosine did not occur over the duration of the experiments.


Figure 8: Effect of sphingosine on fMLP-induced Ca influx. Neutrophil [Ca]was monitored as outlined in Fig. 5. Sphingosine (0.5 or 1 µM) and fMLP (0.2 µM) were added to fura-2-loaded neutrophils at points indicated by arrows. The dotted line on the rightmost two traces outlines the superimposition of the fMLP control trace on the left. These results were collected in a Perkin Elmer (MKF-4) fluorescence spectrophotometer.



Accelerated Ca efflux may contribute to apparent declines in [Ca](i). To assess this possibility, Ca-loaded neutrophils were treated with fMLP ± ceramide and Ca release monitored (Fig. 9). As reported previously, the initial rapid phase of release of internal Ca was matched by an equally rapid efflux of Ca in the first 30 s(10, 11) . The results show that the rates of Ca efflux were similar in PMNs stimulated by fMLP with or without C(2)-ceramide preincubation. In all cases, accelerated Ca transport occurred in the first 30 s after fMLP addition. After 30 s, Ca efflux was similar to background. C(2)-ceramide alone had the same effect as dimethyl sulfoxide vehicle controls.


Figure 9: Efflux of Ca from neutrophils treated with sphingosine. Neutrophils were loaded with Ca, incubated with reagents for times shown in the figure at 37 °C, and the supernatant recovered as outlined under ``Experimental Procedures.'' The radioactivity in the supernatant was expressed as a percentage of the total activity in supernatant and pellet fractions. circle, fMLP; bullet, 10 µM C(2)-ceramide added 1 min before fMLP; down triangle, 20 µM C(2)-ceramide added 1 min before fMLP; , 20 µM C(2)-ceramide; box, dimethyl sulfoxide vehicle control. Where fMLP was applied, timing started with the addition of this reagent. Results are representative of two experiments.




DISCUSSION

Present results show that C(2)-ceramide at concentrations >1 µM inhibited O(2) production by human neutrophils in a time- and dose-dependent manner. Below 1 µM, C(2)-ceramide potentiated or primed the O(2) response induced by fMLP (Fig. 1C). This finding raises the possibility that agonists such as tumor necrosis factor-alpha, platelet-activating factor, leukotriene B(4) etc. (20) may prime neutrophils by activation of sphingomyelinase. The linkage of tumor necrosis factor-alpha to the sphingomyelinase pathway has been demonstrated in leukemic cell lines (4) . Whether this occurs in neutrophils remains to be established.

The preceding results differ from those reported by Yanaga and Watson (21) , who found C(2)-ceramide had not affected the formation of reactive oxygen species by neutrophils. In their experiments, PMNs were incubated for 10 min with 30 µM C(2)-ceramide, followed by 10 min with fMLP; cells were then scored for reduction of nitroblue tetrazolium. This is an either-or test and is less sensitive than the continuous cytochrome c reduction assay. In our hands 30 µM C(2)-ceramide completely inhibited O(2) production whether PMNs were preincubated for 2 or 10 min with this agent.

C(2)-ceramide was more potent than C(6)-ceramide in inhibiting O(2) generation. Although C(2)- and C(6)-ceramide were shown to be cell-permeable when applied to other cell types(4, 5) , we have not directly measured the relative rate of entry of these ceramides into human neutrophils. A slower rate of uptake of C(6)-ceramide may partly account for its lower potency. However, it is equally possible that differences between C(2)- and C(6)-ceramide may reside in their dissimilar effects on, or affinities for a putative cellular target.

The mechanism by which C(2)-ceramide inhibited fMLP- and PMA-induced O(2) in neutrophils remains to be established. fMLP signals PMNs by activation of phospholipase C and the generation of Ins(1,4,5)P(3) and diacylglycerol(22) . The finding that C(2)-ceramide inhibited weakly the initial Ca transient induced by fMLP (Fig. 5) suggests that Ins(1,4,5)P(3) formation or Ins(1,4,5)P(3) release of intracellular Ca was not the primary site of action.

Ceramide appears to inhibit processes mediated by PKC, the cellular target of diacylglycerol and PMA. Direct inhibition of PKC is unlikely as C(2)-ceramide failed to inhibit Ca-phospholipid-dependent PKC in vitro (Fig. 2) in agreement with other studies(5) . C(6)-ceramide weakly inhibited PKC at concentrations >50 µM (Fig. 2B), but it is questionable whether such high levels could be attained in intact cells under physiological conditions. As we have argued under ``Results,'' the metabolism of ceramide to sphingosine, a PKC inhibitor, was limited. Substantial sphingosine formation would have enhanced fMLP-induced Ca influx, not inhibited it ( Fig. 5and Fig. 8).

PKC-dependent reactions might be countered by PPase-dependent dephosphorylation of substrate proteins. A possible candidate is CAPP, characterized by Hannun's group(5, 9) . In this study, the effective dose range of C(2)-ceramide and the inactivity of C(2)-dihydroceramide are consistent with a role for CAPP. In vitro studies show that CAPP is a cation-independent, serine/threonine PPase activated by 1-10 µM ceramide. Ceramide activation of CAPP is specific in that C(2)-dihydroceramide is inactive and frequently applied in experiments as a negative control for biological activity. CAPP is related to and regarded as a subtype of the PPase-2A family(5, 9) . More recently, Hannun's group showed that C(2)-ceramide stimulates heterotrimeric forms of PPase-2A up to 3.5-fold. By comparison C(2)-ceramide increases 5.5-fold the activity of CAPP isolated from T9 glioma cells(9) .

In light of analyses of phosphorylated proteins in PMNs, the PPase activation hypothesis must be qualified (Fig. 4). The autoradiograms of total proteins failed to show significant dephosphorylation in cells treated with C(2)-ceramide alone or with concomitant PMA activation (Fig. 4). From this we conclude that C(2)-ceramide did not activate PPases globally. However, this result does not necessarily preclude the involvement of a CAPP that acts on selected substrates. The gel system and labeling approach used in the present study are insufficiently sensitive to discern subtle changes in phosphorylation patterns. Before definitive conclusions can be made, more sensitive methods must be used. Alternative approaches include two-dimensional gel electrophoresis and autoradiography or pulse-chase experiments after labeling electroporated cells with [-P]ATP in the manner described by Lu et al.(15) .

A specific target of putative CAPP may be p47-phox, a component of the O(2)-generating system, NADPH oxidase(23) . Previous studies indicate that the assembly of NADPH oxidase is associated with multiple phosphorylation of p47-phox(24) . In experiments in which neutrophils were stimulated with PMA for 5 min, eight distinct p47-phox phosphoproteins were detected in cytosolic and membrane fractions. The possibility exists that CAPP may inhibit NADPH oxidase assembly and activation by dephosphorylating a limited number of sites on p47-phox. If true, the one-dimensional gel method may not detect such changes as suggested earlier.

C(2)-ceramide inhibition of fMLP-dependent Ca influx resembled that mediated by PMA. The latter finding confirms previous results supporting a negative feedback role for PKC in pathways involving phospholipase C activation and consequent diacylglycerol formation(11, 12) . In neutrophils, the PKC-sensitive components regulating Ca permeability remain to be identified. Diacylglycerol-activated PKC has been shown to enhance plasma membrane Ca pump activity and inhibit phospholipase C(25, 26, 27) . The latter effect is modest, and both effects do not account for the total blockade of unidirectional Mn influx(11, 12, 17) . It is unlikely that C(2)-ceramide follows the same mechanism of action as PKC. C(2)-ceramide neither stimulated significant kinase activity in intact neutrophils (Fig. 4) nor increased Ca efflux (Fig. 9). Paradoxically, ceramide blockade of PKC-mediated events should have enhanced, not depressed Ca entry. We interpreted the C(2)-ceramide effect in the following manner.

Table 1summarizes PKC/PPase regulation of Ca influx in fMLP-activated neutrophils. Present and past results show that PKC inhibitors such as staurosporine, auranofin, and sphingosine potentiate Ca entry by suppressing the negative feedback effect of PKC (Fig. 7)(11, 12) . PPase-1/PPase-2A inhibitors such as calyculin A, tautomycin, and OA cooperate with PKC to tilt the balance in favor of protein phosphorylation thus decreasing Ca influx (Fig. 7). Calyculin A and tautomycin are equally effective inhibitors of PPase-1 and PPase-2A, whereas OA is 50-100 times more effective against PPase-2A (19) . (^2)The greater potency of calyculin A and tautomycin compared with OA suggests that PPase-1 was involved in up-regulating Ca influx. Using these agents, Murata et al.(28) similarly concluded that PPase-1 regulates thrombin-induced Ca influx in human platelets. Initially, we anticipate that ceramides, by stimulating PPase, would follow the nascent pattern and enhance Ca entry. The contrary evidence points to targeting of specific PPases. We propose that PPase-2A or a subtype stimulated by ceramide regulates signaling component(s) different from that targeted by PPase-1.



We speculate that the site affected by C(2)-ceramide may be the putative calcium influx factor recently implicated as a second messenger in Ca homeostasis(29, 30) . According to the capacitative calcium entry model of Putney(31) , the fill state of intracellular Ca stores in many nonexcitable tissues and cells regulates Ca influx. Emptying of such stores generates a physical change or a chemical signal that opens Ca channels on the plasma membrane. Recently several groups have obtained results suggesting that a soluble factor may be responsible for channel opening(29, 30, 32, 33) . Randriamampita and Tsien (29) isolated such a factor from Jurkat cells and characterized it as a small (M(r) < 500) compound requiring a phosphate group for activity. We hypothesize that CAPP may dephosphorylate calcium influx factor and inactivate it. Calcium influx factor with M(r) < 500 would not be detected in the gel system used in this study (Fig. 4). At present we cannot rule out other mechanisms of action for C(2)-ceramide. For example, C(2)-ceramide may interfere with the generation or release of a soluble Ca influx factor. Alternatively, C(2)-ceramide may inhibit the opening or operation of Ca influx channels on the plasma membrane.

Whatever its mechanisms of action, ceramide inhibition of Ca influx may cause or contribute to the onset of cell differentiation, inhibition of cell growth, and repression of inflammatory responses. Ca influx plays a role in sustained directed motility of PMNs, is not needed to trigger the respiratory burst by chemoattractants but does increase the maximum response(34, 35) . In many secretory cells, including PMNs, exocytosis induced by receptor activation is dependent on Ca influx and is eliminated when influx is blocked, in spite of mobilization of intracellularly stored Ca(36, 37) . In fMLP-treated PMNs, the bulk of diacylglycerol generated arises from phospholipase D hydrolysis of phosphatidylcholine, a reaction that is dependent on exogenous Ca(34, 38) . Therefore, reduced Ca influx may be an indirect mechanism by which ceramides inhibit PKC in receptor-activated neutrophils.

To summarize, we found that 1-20 µM C(2)-ceramide inhibited O(2) generation in PMA- and fMLP-activated neutrophils. The same doses inhibited Ca and Mn influx in fMLP-stimulated cells but exerted little effect on Ca efflux. The mechanism of action of C(2)-ceramide remains to be clarified but may involve the activation of more than one type of protein phosphatase.


FOOTNOTES

*
This research was funded by the Canadian Red Cross Society and University of Calgary Endowment Funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: The Canadian Red Cross Society, 737 13 Ave. S.W., Calgary, Alberta Canada T2R 1J1. Tel.: 403-220-8683; Fax: 403-541-4466.

(^1)
The abbreviations used are: PKC, protein kinase C; PMN, polymorphonuclear neutrophil; C(2)-ceramide, N-acetylsphingosine; C(6)-ceramide, N-hexanoylsphingosine; [Ca](i), cytosolic or cytoplasmic free calcium concentration; PPase, protein phosphatase; CAPP, ceramide-activated protein phosphatase; fMLP, n-formyl-methionyl-leucyl-phenylalanine; PMA, phorbol 12-myristate 13-acetate; OA, okadaic acid; HBSS, Hanks' balanced salt solution.

(^2)
Calyculin A, tautomycin, and OA inhibited PPase-1 holoenzyme at IC values of 1.4, 2.2, and 45 nM, respectively, and PPase-2A holoenzyme at IC values of 2.6, 1.8, and 0.5 nM, respectively(19) .


ACKNOWLEDGEMENTS

We thank Bruce Allen for the gift of rat brain PKC.


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