C6-ceramide maintains elevated cytosolic calcium levels in activated platelets

Thrombosis

Pergamon

Research, Vol81, No. 2, pp. 219-229, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in the USA. All rights reserved 0049-3848/96 $12.00 + .I0

0049-3848(95)00239-l

C6-CERAMIDE

MAINTAINS ELEVATED CYTOSOLIC IN ACTIVATED PLATELETS.

CALCIUM

LEVELS

Kenneth Wong, and Xue-Bin Li Dept. of Pharmacology and Therapeutics, Univ. of Calgary, and the Canadian Red Cross Blood Transfusion Centre, Calgary, Alberta, Canada

(Received

19 July 1995 by Editor R.L. Rathbone;

revised/accepted

20 November

1995)

Abstract. The effects of cell-permeable C2 and Cs-ceramides on human platelet responses were investigated. In thrombin-activated platelets, Cs (5-30 uM) potentiated Ca” mobilization and Ca2’ influx, and decreased the rate of removal of Ca*’ from cytosol. The effect of C2 was not significant. Phorbol ester or calyculin A inhibition of thrombin-induced rises in platelet [Ca*‘]i was attenuated by Ca. Assays show that Ca either prolonged the generation, or retarded the metabolism of inositol trisphosphates. Previous studies indicate that protein kinase C (PKC) acts in a negative feedback manner by inhibiting phosphatidylinositol breakdown, accelerating inositol trisphosphate metabolism, and increasing Ca*’ pump activity. Cs may counter these PKC effects indirectly. The synthetic ceramides inhibited platelet aggregation weakly and had no effect on pleckstrin (~47) phosphorylation. Recently we reported that CZ but not Ca inhibits superoxide generation and store-regulated Ca2’ influx in neutrophils at similar concentrations. Cellular differences in ceramide metabolism or ceramide-sensitive enzymes and their substrates may account for the disparate results.

Ceramides arising from the breakdown of sphingomyelin by sphingomyelinase have been shown to act as second messengers. Effecters include proline-directed kinases and serinejthreonine protein phosphatases belonging to the type 2A family (l-3). Recently, we reported that N-acetyl Key words: platelets, calcium, inositol phosphate, ceramide. Abbreviations: C2 or C2-ceramide, N-acetylsphingosine; Cg or Cs-ceramide, N-hexanoylsphingosine; PMA, phorbol 12-myristate 13-acetate; InsP3, inositol trisphosphate; Ins(1,4,5)P3, inositol 1,4,5-t&phosphate; [Ca*‘]i, cytosolic free Ca*’ concentration; PKC, protein kinase C; MezSO, dimethyl sulfoxide. Corresponding author: Kenneth Wong, Research Department, Canadian Red Cross BTS, 737 13th Ave. SW., Calgary, Alberta, Canada T2R 1Jl. Ph. (403) 220 8683; FAX (403) 541 4466. 219

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sphingosine (Cl-ceramide), inhibits some but not all responses induced by phorbol myristate acetate (PMA) in human neutrophils (4). Cz-ceramide (l-20 PM) suppresses PMA-induced superoxide generation dose-dependently, but like PMA, Crceramide inhibits store-regulated Ca” influx in chemotactic peptide-stimulated neutrophils. These results are consistent with the involvement of a ceramide-activated protein phosphatase although alternative models cannot be excluded. A target of this phosphatase may be a protein kinase C (PKC)-phosphorylated component essential for 02- generation. We further propose that PKC and ceramide-activated enzymes downregulate Ca” intlux at different sites and that the latter may inactivate a putative calcium influx factor (4).

Recent studies suggest that store-regulated or capacitative Ca” entry in various cells occurs via the release or activation of a soluble Ca” influx factor that is sensitive to alkaline phosphatase and protected by okadaic acid in vitro (5-7). Since Ca” influx in thrombin-stimulated platelets reportedly occurs by a capacitative entry mode (S), we postulate that Cz-ceramide may similarly block Ca” in&x in platelets. Results obtained in the present study show that this was not the case.

MATERIALS

AND METHODS

Materials Cl-ceramide and N-hexanoyl-sphingosine (C&ceramide) were purchased from Matreya Inc. (Chalfont, PA). Cz-dihydroceramide was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Human thrombin, apyrase, prostacyclin, phorbol myristate acetate @‘MA), and EGTA were purchased from Sigma Chemical Co. (St. Louis, MO). Calyculin A and fUra-2iAM (the acetoxymethyl ester form of fUra-2) were obtained from Calbiochem Biochemicals (San Diego, CA). [3H] myo-inositol, (SO-120 Wmrnol) and [32P] orthophosphate (10 mCi/ml) were from Amersham Canada Ltd. (Oakville, ON). Stock solutions of PMA and synthetic ceramides were dissolved in dimethyl sulfoxide (MelSO) and stored at -80°C. If reagents were added to platelets in combination, the concentration of vehicle was kept below 0.5%.

Preparation ofplatelet suspension Platelets were prepared as described previously (9). Blood was drawn by venipuncture from drug-free volunteers into l/6 volume acid citrate dextrose. Platelet-rich plasma was obtained by centrifuging whole blood at 300 x g at room temperature for 15 min. After careful removal, the plasma was tirther centrifuged at 2500 x g at room temperature for 10 min to pellet the platelets. Platelets were resuspended in Tyrode-Hepes buffer (134 mu NaCl, 12 mu NaHCO3, 2.9 mu KCl, 0.36 mu NaH2P04, 1 mu MgC12, 5 mh4 Hepes, 5 mu glucose; pH 7.2) containing apyrase (0.6 ADPase U/ml) and 0.35% bovine serum albumin. For labeling platelets with [3H] myoinositol, the buffer also contained 1 mu EGTA and 500 @ml prostacyclin (10).

Assay for Inositol phosphates Inositol phosphate formation in platelets was assayed according to procedures described by Lapetina and Siess (9,lO). Briefly, platelets prelabeled with [3H] myo-inositol at a density of 8 x

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CgCERAMIDE

(A)

ELEVATION

(6) C6, +Ca2+

C6, -Ca2+

2 1 T. = 0.53 + % u 0.2. 0.1

21 0.5 c 0.2 I01 -

t thr

t thr

FIG. 1

Ceramide prolonged elevation of [Ca”]i in thrombin-stimulated platelets. Fura- loaded human platelets in Tyrode-Hepes with 1 mM EGTA (Ca*‘-free [A]) or supplemented with 1 mM Ca*’ [B] were incubated for 2 min with MeIS (O.S%), 5, 10, 20, and 30 pM Cgceramide (traces a-e respectively) before thrombin (thr, 0.5 U/ml) was added. [C] C1ceramide effects. Platelets were activated with thrombin in Ca*‘-free (traces a, b) or 1 mM Ca*’ medium (traces c, d). In (b) and (d) the platelets were incubated with 20 uM C2-ceramide for 2 min before the addition of thrombin.

TABLE I.

[Ca*‘]i of Fura- Loaded Platelets Stimulated for 3 Min with Thrombin

Ceramide (added 2 min before thrombin, 0.5 U/ml)

[Ca*+]i, tlM (* SEM, N = 5)

vehicle only control C2-ceramide, 20 1tM Ch-ceramide, 20 uM

17Ok24 220 f 40 310 f 19*

*Cs is significantly different from control and CZ values (Multiple comparisons carried out using the Student-Newman-Keuls method after Kruskal-Wallis one way analysis of variance on ranks).

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IO8 ceils/ml Tyrode-Hepes were incubated with agonists then stopped with chloroform/methanol/HCl. The neutralized aqueous phases were applied to Dowex l-X8 anion exchange columns (formate form) and radio-labeled inositol mono-, bis-, and trisphosphates (InsPJ) were eluted by washing columns with ammonium formate/formic acid. Measurement of [C&]i Platelets (8 x lO”/ml) were loaded with fura- by incubation at 37°C with 1 pM Fura- AM for 45 min (9, I 1). Washed platelets (2.0 ml at 1-3 x lO’/ml) were measured for changes in fluorescence at monochromator settings of 339 nm (excitation) and 505 nm (emission). [Ca”]i was calculated as described previously (9,12).

Aggregation studies Platelet aggregation was measured in a Biomata Model PAP-3 aggregometer at 37’C. The turbidity of the sample cuvette (8.75 x 50 mm) containing 1 x IO* platelets in 0.5 ml was measured against a reference sample with the bulk of cells removed by a low speed centritigation step. Thrombin or PMA was added directly to the stirred sample cuvette. The initial slope of aggregation curves was measured and defined as the rate of aggregation.

Phosphorylation of platelet proteins Platelets suspended at 3 x lO’/ml in phosphate-free Tyrode-Hepes buffer (pH 7.4) with 0.35% bovine serum albumin were incubated with [32P]orthophosphate (0.2 mCi/ml) for 30 min at 37”. After free orthophosphate was removed by washing with buffer, platelets were resuspended at 6 x 106 cells/ml in the same medium and subsequently treated with ceramides and thrombin for varying times. Reactions were stopped by the addition of 0.3 ml 25% cold TCA. The pellet was dispersed in 0.15 ml 4% SDS solubilizing solution, then boiled for 5 min. Proteins from 1 x 106 cell-equivalents were analyzed in 10% SDS gels, the gels dried and autoradiographs made as described previously (13).

RESULTS

Effect of C6-ceramide OH Cd’ mobilization and influx At a dose range of 5-30 )IM, CZ- or Cs-ceramide alone did not significantly alter [Ca*‘]i of resting In accordance with previous studies, thrombin triggered a rapid Ca*’ spike platelets. corresponding to inositol 1,4,5-trisphosphate, [Ins(l,4,5)Pj], mobilization of internal Ca” stores (8,9), followed by a decline of [Ca*‘]i to near basal levels in 3-4 min (fig. 1). Cs-ceramide (S-30 FM) increased the magnitude of the initial spike modestly and prolonged the elevated [Ca2’ji phase dose-dependently, an effect observed in cells suspended in Ca2’-free or Ca*‘-supplemented medium (fig. l,A,B). In Ca*+-free (EGTA) medium, elevation of [Ca*‘]i due only to mobilization of intracellular Ca*’ stores was measured. The results suggest that C,Sslowed the rate of decline of [Ca*‘]i (fig. 1A). In Ca*‘-rich medium, rises in [Ca*‘]i due to Ca*’ influx were superimposed over that mediated by mobilization of internal Ca*+ (fig. 1B). Here, too, Cg prolonged the elevation of [Ca2’]i. MI?’ quench kinetics, an approach in which Mn2’ is acting as a surrogate for Ca”, indicate that enhanced Ca” entry contributed to the increase (fig. 2) (4,9,14). The results

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FIG. 2 MIICL,

Effect of ceramides on Mn*’ influx. Fura- loaded platelets suspended in Tyrode-Hepes were excited at 360 nm, and Mn2’ entry signaled by quenching of the firra-2 fluorescence. In trace (a) platelets were incubated for 2 min with 20 uM Cg before MnC12 (0.2 n&I) was added. In the other traces, cells were incubated for 2 min with Me2SO (b), 30 pM CZdihydroceramide (c), 20 uM CZ (d), and 20 pM C6 (e) before MnC12 and thrombin (0.5 U/ml) were added in the order shown by arrows.

211

Thrombm

’

0

60

I

120

I

160

’

240

seconds

show that C6-ceramide increased the extent rather than the initial rate of cation entry in thrombinstimulated cells.

Results of experiments carried out at the same time with Cz-ceramide showed that CZ had no significant effect on platelet [Ca*‘]i (fig. lC, fig. 2, table I). This is borne out by average [Ca*‘]i values calculated from a single time point from traces obtained in 5 separate experiments (table 1). In all experiments, none showed C2 inhibition of Ca*’ influx.

The combined effects of C6-ceramide with PMA or calyculin A, an inhibitor of serine/threonine protein phosphatases types 1 and 2A, are shown in fig. 3. In [A], the difference between thrombin (con) and thrombin + EGTA curves represents the contribution of Ca*’ influx to elevations in [Ca*‘]i. The effect of PMA confirms the idea that PKC exerts a negative feedback effect on platelet signaling and elevation of [Ca*‘]i (fig. 3A) (14-17). PMA (100 r&I), presumably acting through PKC, decreased the extent of the initial Ca*’ spike and increased the rate of decline of [Ca*‘]i compared with thrombin-only controls. C6-ceramide (20 @I) reversed the PMA inhibition and generated a tiua-2 trace similar to that produced by thrombin+Ch (fig. 3B). A competitive antagonism appears to exist between the two reagents since a higher concentration of PMA (500 nh4) was resistant to the effect of Cg. Results illustrated in fig. 3C,D corroborate recent reports that inhibitors of protein phosphatases 1 and 2A attenuate phosphatidylinositol breakdown and Ca*’ influx in human platelets (l&19). Calyculin A pretreatment for 2 or 10 min before thrombin stimulation decreased the extent of the initial Ca*’ spike. Co-treating platelets with 20 pM C6 slowed the rate of decline of elevated [Ca*‘]; but failed to reverse calyculin A’s effect on the initial Ca*+ transient. Effect on InsPs levels

Results show that relative InsP3 levels over time were unaltered in platelets treated with vehicle or

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FIG. 3

Gceramide alleviated PMA or calyculin A inhibition of Ca*’ transients. Platelets suspended in Tyrode-Hepes were monitored for changes in [Ca2’]i as in tig. 1. [A] Vehicle (control), 100 nM PMA or 2 mM EGTA was added to separate suspensions just before thrombin (thr, 1 U/ml). C6 (20 uM) was added 2 min before thrombin. [B] Platelets were treated with 20 uM C6 for 2 min before 25, 100, or 500 nM PMA and thrombin were added in the order shown. [C] Where indicated, 100 nM calyculin A (CA) was added 10 min, and 20 uM CS added 2 min to platelets before stimulation by thrombin. [D] Same experiment as in the preceding except that cells were treated for 2 min with CA before the addition of thrombin. Direct comparison of traces in [A], [B], and [C] can be made as they were obtained from a single experiment using a single set of cells.

FIG. 4

Effect of C6 on InsP3 formation. InsP3 levels relative to basal radioactivity in platelets were measured as indicated in Methods. Cells were incubated with MelSO vehicle (a); 1 U/ml thrombin (0); 20 uM Cs (m); or 2 min with 20 uM CG(Cl), CZ (A), or CZdihydroceramide (A) before the addition of thrombin (0 time). Results are means * SEM of 3-5 experiments. (*) Significantly different from the corresponding thrombinonly value (p < 0.05, multiple comparisons by the Student-Newman-Keuls method after one way analysis of variance).

20

40

60

time

(seconds)

60

100

120

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C&ERAMIDE

pM

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225

ceramide

FIG. 5

Ceramide modulation of platelet aggregation. Platelets were treated 2 min with Cz, C6, or C2-dihydroceramide before aggregation was induced by 0.1 U/ml thrombin [A], 1 U/ml thrombin [B], or 100 nM PMA [Cl. The rate of aggregation relative to thrombin or PMA-only controls was measured as outlined in Methods. Results are means i SEM of 3-4 experiments. (*) Significantly different from CZdihydroceramide or Cg data (PcO.05) using tests described in fig. 4.

with Cg alone (fig. 4). Thrombin stimulation caused rapid formation of InsP3 that peaked before 20 s and declined more slowly over 2 min in accordance with previous observations (9). The key finding was that in cells pretreated with Cs, InsP3 levels remained elevated over the same period compared with thrombin-only controls. The effects of Cz and Cz-dihydroceramide were not significantly different than those of thrombin alone.

Effect on platelet aggregation The effects of C6, CZ, and Cx-dihydroceramide on platelet aggregation induced by thrombin and PMA were assessed. As results show, at concentrations used in the calcium experiments (5-30 PM), ceramides inhibited platelet aggregation weakly (fig. 5). For example at 20 pM ceramide, the % inhibition ranged between 10 to 30%. When aggregation was induced by PMA or 1 U/ml thrombin, ceramide inhibition appears to be nonspecific as there was no difference between Ce, CZ and C;?-dihydroceramide (fig. 5,B,C). Results diverged when cells were stimulated by 0.1 U/ml thrombin. Initial rates of aggregation induced by this concentration of thrombin were 30-35% of those induced by 1 U/ml thrombin. Under this condition, the dose-response effects of Cs and CZdihydroceramide on aggregation were reduced, while that for CZ remained the same (cf. fig. 5,AP). Effect OFI protein phosphoryiation In thrombin-stimulated platelets, two proteins of molecular mass 20 kDa and 47 kDa are prominently phosphorylated (20) (fig. 6). The former, myosin light chain, is phosphorylated by

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Ca2’-calmodulin activated myosin light chain kinase. Pleckstrin, the 47 kDa protein of unknown function, has been shown to be a substrate for protein kinase C (20). The autoradiogram reproduced in fig. 6 shows that Cg alone has little effect on background labeling compared to control, quiescent platelets. At 20 pM, Cg, C2 and C2-dihydroceramide failed to alter the extent of phosphorylation of the pleckstrin band from thrombin-activated cells. As a positive control, staurosporine, a nonspecific kinase inhibitor, blocked thrombin-induced protein phosphorylation.

DISCUSSION

The results of this study show that cell-permeable ceramides modulated platelet and neutrophil responses differently. Cg increased the duration of elevated [Ca”]i in platelets by potentiating Ca2’ influx and decreasing the rate of Ca” removal from the cytosol (fig. 1,2). The latter effect is more evident in assays performed in Ca”-free medium in which concurrent Ca2’ entry is absent (fig. 1A). Mechanistically, Cg might have interfered with reuptake of Ca” into stores or transport of Ca2’ to the outside. Specificity of action was shown by the finding that CZ and CZdihydroceramide lacked significant effect. By comparison, C2 blocks Ca” influx in neutrophils (4). The different findings do indicate that C2 is not acting as a general inhibitor of Ca2’ entry.

47 kDa

-

C6fhr

20 kDa

976645k

31-

FIG. 6

Effect of ceramides on protein phosphorylation. [“PI labeled platelets (6 x 106/ml) were preincubated for 2 min with Me2S0 (vehicle control), 100 nM staurosporine (stau), or 20 pM ceramides, then activated for 3 min with thrombin. No thrombin was added in the Cs-only sample. Reactions were stopped, and the boiled samples analyzed by SDS polyacrylamide gel electrophoresis and autoradiographs made as outlined in Methods. Densitometry was performed on the scanned image. This result is representative of two separate experiments.

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Results clearly show that, over 2 min, inositol trisphosphate levels remained elevated in platelets treated with a combination of Cg and thrombin compared with thrombin-only controls (fig. 4). The anion ion exchange method used here does not resolve various InsP3 isomers formed in platelets (10,21); therefore, the relative amounts of Ins(1,4,5)Px and other active inositol trisphosphates in this fraction are unknown. This caveat aside, it is possible that G retarded the metabolism of Ins(1,4,5)P3 and prolonged the ability of the latter to release Ca”. According to the capacitative Ca” entry model, mobilization of Ca” from internal stores triggers Ca2+ influx in cells where this pertains (5). Thus, enhanced Ca2’ influx might be a consequence of extended Ca2’ mobilization.

A likely, though not the sole target of Cg action might be PKC since this kinase activates Ca” pumps and modulates inositol phosphate metabolism (15-19). Previous studies on platelets show that Ins(1,4,5)P3 is metabolized by an inositol 3-kinase and various phosphatases to inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-t&phosphate, and inositol bisphosphate (21-23). PKC, activated by endogenous diacylglycerol or PMA behaves in a negative feedback manner by accelerating metabolism of Ins( 1,4,5)P3 and inhibiting phospholipase C (fig. 3A) (15-l 7, 21).

Ceramides do not inhibit PKC directly but may antagonize some PKC effects by activating a type 2A protein phosphatase (3,4). If true, this may explain why Ca did not alleviate calyculin A inhibition of initial Ca” transients (fig. 3C,D). This conjecture derives from Murata and coworkers’ finding (19) that calyculin A blocks Ca2’ influx in platelets by inhibiting protein phosphatase type 1. Similarly, the dephosphorylation of pleckstrin may be mediated by protein phosphatases insensitive to Cg, hence the failure of Cg to inhibit PMA-induced phosphorylation of the 47 kDa protein (fig. 6).

A question raised by present results is why CZ was inactive in modulating platelet [Ca2’]i. In vitro, Cg and C2 are about equally potent in activating a type 2A protein phosphatase isolated from T9 glioma cells (3). This may not be the case with analogous phosphatases in platelets. An alternative hypothesis is that ceramides were metabolized to sphingosine, a PKC inhibitor (24), and that Cg was metabolized more rapidly than C2. The conversion of ceramides to sphingosine would have to be extensive to achieve significant inhibition of p47 phosphorylation since relatively high concentrations (20-40 pM) of sphingosine are required (25). Lower concentrations of sphingosine might be sufIicient to block PMA effects on Ca”’ transients. It is of interest to note that Hashizume et al. (26) working with thrombin-activated rabbit platelets, show that 5-10 uM sphingosine increases Ins(1,4,5)P3 formation and rises in [Ca2’]i independently of PKC inhibition. It remains to be established whether sphingosine mediates similar effects in human platelets.

The effects of ceramides on platelet [Ca”]; failed to correlate with their effects on platelet aggregation (fig. 5). Cg did not potentiate or enhance aggregation significantly. Overall, ceramides were weak inhibitors of this response induced by thrombin or PMA (fig. 5). At the dose range of interest (2 to 30 FM), the inhibition appears to be nonspecific, since there was no difference between the effects of CZ, Cg and Cz-dihydroceramide. For comparison, 20 uM C2 completely blocks 02- generation induced in neutrophils by PMA or a chemotactic peptide while Cg and C2-dihydroceramide are inactive (4).

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The starting premise of present studies was that Ca2’ entry was regulated similarly in neutrophils and platelets and that this similarity may be brought to the fore by using cell-permeable ceramides as pharmacological probes. The contrasting effects of these ceramides on Ca2’ signaling in the two cell types suggest that either Ca2’ entry regulation is different in each system or that ceramide effects are cell-specific. Cellular differences in ceramide metabolism or ceramide-sensitive regulatory components may account for the disparate results. Further studies are needed to shed light on these possible modes of action.

Acknowledgments This study was funded by MRC (Canada), the Canadian Red Cross Society R&D Program and Univ. of Calgary Endowment Funds.

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