- Email: [email protected]
Ceramide Induces Structural Defects into Phosphatidylcholine Bilayers and Activates Phospholipase A2
JOBNAME: BBRC 220#3 PAGE: 1 SESS: 10 OUTPUT: Tue May 7 11:40:09 1996 /xypage/worksmart/tsp000/69829b/45
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 834–838 (1996)
0490
Ceramide Induces Structural Defects into Phosphatidylcholine Bilayers and Activates Phospholipase A2 Hsio-Wen Huang, Edward M. Goldberg, and Raphael Zidovetzki1 Department of Biology, University of California, Riverside, California 92521 Received February 7, 1996 We studied the effects of bovine brain ceramide on the structure of dipalmitoylphosphatidylcholine (DPPC) bilayers and cobra venom phospholipase A2 (PL-A2) activity using 2H NMR and specific enzymatic assays. Addition of ceramide to DPPC at 45°C induces lateral phase separation of the bilayers into regions of gel and liquid crystalline phases. The order parameters of the DPPC acyl chains in the liquid crystalline phase are only slightly affected by the presence of ceramide, indicating that the latter is largely partitioned in the gel phase of DPPC, whereas at 60°C the presence of ceramide induced a large increase of the order parameters of DPPC side chains. The observed structural effects of ceramide correlated with ceramide-induced activation of cobra venom phospholipase A2 (PL-A2). Ceramide activated PL-A2 in a concentration-dependent manner, with a significant effect observed at 5 mol% ceramide, which caused an approximately 3-fold increase in PL-A2 activity. The results showing activation of PL-A2 by ceramide illustrate an additional feature of the biological effects of this second messenger and suggest the possibility of cross-talk between the sphingomyelinase and PL-A2 signal transduction pathways. © 1996 Academic Press, Inc.
Recently, a role for sphingolipids in signal transduction has been suggested by (1). In this signal transduction pathway, external stimulus-induced hydrolysis of sphingomyelin results in the production of ceramide which acts as an intracellular second messenger. To date the intracellular processes known to be modulated by ceramide include phosphatase activation (2,3), protein phosphorylation mediated by a ceramide-activated protein kinase (4,5) and apoptosis (6). Among the external stimuli which activate the ceramide pathway are g-interferon (7), tumor necrosis factor a (7), and interleukin 1b (8) as well as other stimuli (see refs#9–11 for reviews). The structures of ceramide-containing membranes were previously studied in the context of the investigation of the model stratum corneum lipids which are composed primarily of ceramide, cholesterol, and free fatty acids (12–15). In the present work we report that ceramide induces defects in bilayers of dipalmitoylphosphatidylcholine (DPPC), as determined by 2H NMR, and activates cobra venom phospholipase A2 (PL-A2) in a manner similar to that of diacylglycerols (DAGs) (16,17). MATERIALS AND METHODS DPPC, diperdeuteropalmitoylphosphatidylcholine (DPPC-d62) and bovine brain ceramide were purchased from Avanti Polar Lipids (Alabaster, AL). Sphingomyelin and phospholipase A2 (Naja mocambique mocambique) were obtained from Sigma Chemical Co. (St. Louis, MO). Multilamellar lipid dispersions were prepared by dissolving the lipid mixtures in chloroform, and the solvent was evaporated off with dry nitrogen. Lipids were then redissolved in cyclohexane and lyophilized. The dried lipids were hydrated with 25 mM Tris (2-amino-2-(hydroxymethyl)-propane-1,3-diol; pH 7.4) buffer solution with 10 mM Ca2+, prepared in 2H-depleted H2O (Sigma). The samples were always fully hydrated and were typically 1:10 (w/w) in lipid/water. 2 H NMR spectra were acquired at 11.74 T (corresponding to 76.78 MHz 2H frequency) on a General Electric GN500 spectrometer using a high-power, wide line probe (Doty Scientific, Columbia, SC) and the standard quandrupole echo
1
To whom correspondence should be addressed. Fax: (909)-787-4286. E-mail: [email protected] Abbreviations: DPPC, dipalmitoylphosphatidylcholine; DPPC-d62, diperdeuteropalmitoylphosphatidylcholine; PL-A2, phospholipase A2; DAG, diacylglycerol. 834 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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sequence (18). The spectral width was 0.5 MHz, refocusing time 64 ms, 90° pulse 3.5 ms and typically 16,000 scans per spectrum. Phospholipase A2 activity was determined according to the modified method of Stubbs et al., (19) as described in our previous publication (17).
RESULTS 2
H-NMR spectra of DPPC-d62 in the presence or absence of 25 mol% sphingomyelin or ceramide at 45°C are shown in Figure 1. The spectrum of DPPC-d62 (Figure 1A) is typical of the liquid crystalline bilayer lipid phase and is formed by the superposition of Pake doublets contributed by deuterons at different positions along the lipid acyl chains. The distances between the pairs of symmetric peaks, quadrupole splittings (dn), provide information on the order parameters of the corresponding CD2 acyl chains segments according to the equation: Dni 4 4
3
S D e2qQ h
i SCD
2
2
where (e qQ/h) is the static quadruple coupling constant, 170 Hz (20). Addition of 25 mol% sphingomyelin to DPPC-d62 did not significantly change the 2H NMR spectrum (Figure 1B). A dramatic effect on the lipid bilayer structure was observed upon the addition of 25 mol% ceramide: the resulting spectrum had an additional broad component, superimposed with the narrower spectrum that was similar to that of the DPPC-d62 without ceramide (Figure 1C). The broad component of the 2H NMR spectrum is typical of the lipids in gel state, and the superposition of the two types of spectra indicates the presence of ceramide-induced lateral phase separation of the bilayers into gel-like and liquid crystalline domains. Similar effects were previously observed by us upon addition of long-chain saturated DAGs to phosphatidylcholine (16,21) or phosphatidylcholine/phosphatidylserine bilayers (22). As in the case of DAGs, increase in temperature leads to the formation of one lipid phase at 60°C (Figure 2).
FIG. 1. 2H-NMR spectra of DPPC-d62 in the presence or absence of 25 mol% ceramide or sphingomyelin at 45° C. (A) DPPC-d62; (B) DPPC-d62 with sphingomyelin; (C) DPPC-d62 with ceramide. 835
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. 2H-NMR spectra of DPPC-d62 in the presence or absence of 25 mol% ceramide or sphingomyelin at 60° C. (A) DPPC-d62; (B) DPPC-d62 with sphingomyelin; (C) DPPC-d62 with ceramide.
The quadrupole splittings of DPPC-d62 and the corresponding order parameters are unaffected by 25 mol% sphingomyelin at 45° or 60°C (Figure 3). At 45°C only a small increase of the quadrupole splittings was observed in the narrow part of the 2H-NMR spectrum in the presence of ceramide (Figure 3A). The presence of ceramide results in a large increase of the DPPC-d62 quadrupole splittings at 60°C, in the one phase region, which indicates that at this temperature ceramide is only slightly miscible with DPPC-d62 in the liquid crystalline phase (Figure 3B). Because our previous studies suggested that the presence of DAG-induced lateral phase separation of the lipid bilayers on the regions of different fluidities correlates with activation of PL-A2 we measured the effect of the addition of ceramide on the activity of this enzyme. Indeed, the addition of ceramide caused a concentration-dependent increase in the activity of cobra venom PL-A2 (Figure 4). This increase was already significant at 5 mol% ceramide and reached z5-fold increase over the control values at 30 mol% ceramide. No significant effect of sphingomyelin on the PL-A2 activity was observed in this concentration range, except for a small inhibition at high sphingomyelin content, probably due to the surface dilution of DPPC (Figure 4). DISCUSSION The recently identified intracellular second messenger, ceramide, is produced from sphingomyelin in a stimulus-induced manner, by the action of neutral or acidic sphingomyelinases. Similarly with DAGs, ceramide has virtually no headgroup, and was therefore expected to produce similar membrane structure perturbations. Indeed, the perturbation produced by ceramide is similar to that observed by us with long-chain saturated DAGs (21), consistent with the fatty acid composition of the ceramide used where primarily long-chain saturated fatty acids comprise 85% of the total (manufacturer information). As in the case with DAGs, ceramide in the one-phase region (60°C) induces a large increase in 836
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FIG. 3. Plots of quadrupole splittings of DPPC-d62 versus peak number in the presence or absence of 25 mol% ceramide or sphingomyelin. Peak 1 represents approximately seven carbon positions closest to the lipid headgroup. It is the outermost peak of the spectra on Figure 1. Generally the methylene units further from the headgroup have smaller quadrupole splittings, and peak 11, the innermost one, corresponds to the terminal methyl. (M), DPPC-d62; (x), DPPC-d62 with sphingomyelin; (n), DPPC-d62 with ceramide. (A) 45°C; only a few peaks were resolved in the narrower component of the spectrum with ceramide; (B) 60°C.
the order parameters of the DPPC acyl chains, which is a result of the ceramide molecules interspacing the bulky choline headgroups of the DPPC molecules allowing for tighter contact between the lipid acyl chains. At 45°C this leads to a strong interaction between ceramide and DPPC molecules and lateral phase separation of the ceramide-DPPC gel phase-like complexes from the “bulk” DPPC-d62, that remains in the liquid crystalline phase and is little affected by ceramide. In our previous studies we have demonstrated that the long-chain saturated DAGs produced similar perturbations in phosphatidylcholine (21) and phosphatidylcholine/phosphatidylserine (22) bilayers and we correlated the lateral phase separation induced by these DAGs in lipid bilayer structure with activation of phospholipases A2 from different sources, including the cobra venom PL-A2 (17). The present work similarly shows that ceramide-induced lateral phase separation of DPPC-d62 and the corresponding defects in the lipid bilayer structure correlates with activation of PL-A2 This PL-A2 activation is already an approximately 3-fold increase over the control levels at 5 mol% ceramide, which is only a little higher than the 4 mol% global ceramide concentration observed by Okazaki et al., (23) who reported that ceramide levels in 1a,25-dihydroxy-vitamin D3-activated HL-60 cells reach 40 pmol/nmol phospholipids in 2 hours. The local ceramide concentration may be considerably higher depending on the rate of ceramide diffusion in the membrane and rate of its production by sphingomyelinase. In one previous study where the effect of ceramide on PL-A2 activity was measured, no effect was observed (24). However, Franson et al., (24) were primarily addressing the effects of sphingosine and sphingomyelin, and the PL-A2 837
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FIG. 4. Effect of sphingomyelin (n) or ceramide (M) on the activity of phospholipase A2. The assays were performed in triplicates. The error bars correspond to SEM.
assays with ceramide were performed using human disc PL-A2 in the presence of Triton X-100. The presence of detergent would abolish the membrane-perturbing effects of ceramide which we suggest are responsible for PL-A2 activation. Our results showing activation of PL-A2 by ceramide may describe an additional aspect of biological effects of this second messenger molecule and suggest a possibility of cross-talk between the sphingomyelinase and PL-A2 signal transduction pathways. The NMR work was done using a GN500 spectrometer, funded by National Science Foundation Grant DMB840491 and National Institutes of Health Grant BRSG2507. We thank Mr. J. Huang for assistance in preparing samples for the NMR experiments REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076–19080. Dobrowsky, R. T., and Hannun, Y. A. (1992) J. Biol. Chem. 267, 5048–5051. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523–15530. Mathias, S., Dressler, K. A., and Kolesnick, R. N. (1991) Proc. Nat. Acad. Sci. U.S.A. 88, 10009–10013. Kolesnick, R. N. (1991) Prog. Lipid Res. 30, 1–38. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769–1771. Kim, M., Linardic, C., Obeid, L., and Hannun, Y. (1991) J. Biol. Chem. 266, 484–489. Mathias, S., Younes, A., Kan, C.-C., Orlow, I., Joseph, C., and Kolesnick, R. N. (1993) Science 259, 519–522. Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125–3128. Kolesnick, R., and Golde, D. W. (1994) Cell 77, 325–328. Obeid, L. M., and Hannun, Y. A. (1995) J. Cell. Biochem. 58, 191–198. Abraham, W., and Downing, D. T. (1991) Biochim. Biophys. Acta 1068, 189–194. Lieckfeldt, R., Villalain, J., Gomez-Fernandez, J. C., and Lee, G. (1993) Biochim. Biophys. Acta 1151, 182–188. Fenske, D. B., Thewalt, J. L., Bloom, M., and Kitson, N. (1994) Biophys. J. 67, 1562–1573. Hatfield, R. M., and Fung, L. W.-M. (1995) Biophys. J. 68, 196–207. De Boeck, H., and Zidovetzki, R. (1989) Biochemistry 28, 7439–7446. Zidovetzki, R., Laptalo, L., and Crawford, J. (1992) Biochemistry 31, 7683–7691. Davis, J. H., Jeffrey, K. R., Bloom, M., Valic, M., and Higgs, T. P. (1976) Chem. Phys. Lett. 42, 390–394. Stubbs, C. D., Wesley Williams, B., Pryor, C. L., and Rubin, E. (1988) Arch. Biochem. Biophys. 262, 560–573. Burnett, L. J., and Muller, B. H. (1971) J. Chem. Phys. 55, 5829–5831. De Boeck, H., and Zidovetzki, R. (1992) Biochemistry 31, 623–630. Goldberg, E. M., Lester, D. S., Borchardt, D. B., and Zidovetzki, R. (1994) Biophys. J. 66, 382–393. Okazaki, T., Bielawska, A., Bell, R. M., and Hannun, Y. A. (1990) J. Biol. Chem. 15823–15831. Franson, R. C., Harris, L. K., Ghosh, S. S., and Rosenthal, M. D. (1992) Biochim. Biophys. Acta 1136, 169–174.
838
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 834–838 (1996)
0490
Ceramide Induces Structural Defects into Phosphatidylcholine Bilayers and Activates Phospholipase A2 Hsio-Wen Huang, Edward M. Goldberg, and Raphael Zidovetzki1 Department of Biology, University of California, Riverside, California 92521 Received February 7, 1996 We studied the effects of bovine brain ceramide on the structure of dipalmitoylphosphatidylcholine (DPPC) bilayers and cobra venom phospholipase A2 (PL-A2) activity using 2H NMR and specific enzymatic assays. Addition of ceramide to DPPC at 45°C induces lateral phase separation of the bilayers into regions of gel and liquid crystalline phases. The order parameters of the DPPC acyl chains in the liquid crystalline phase are only slightly affected by the presence of ceramide, indicating that the latter is largely partitioned in the gel phase of DPPC, whereas at 60°C the presence of ceramide induced a large increase of the order parameters of DPPC side chains. The observed structural effects of ceramide correlated with ceramide-induced activation of cobra venom phospholipase A2 (PL-A2). Ceramide activated PL-A2 in a concentration-dependent manner, with a significant effect observed at 5 mol% ceramide, which caused an approximately 3-fold increase in PL-A2 activity. The results showing activation of PL-A2 by ceramide illustrate an additional feature of the biological effects of this second messenger and suggest the possibility of cross-talk between the sphingomyelinase and PL-A2 signal transduction pathways. © 1996 Academic Press, Inc.
Recently, a role for sphingolipids in signal transduction has been suggested by (1). In this signal transduction pathway, external stimulus-induced hydrolysis of sphingomyelin results in the production of ceramide which acts as an intracellular second messenger. To date the intracellular processes known to be modulated by ceramide include phosphatase activation (2,3), protein phosphorylation mediated by a ceramide-activated protein kinase (4,5) and apoptosis (6). Among the external stimuli which activate the ceramide pathway are g-interferon (7), tumor necrosis factor a (7), and interleukin 1b (8) as well as other stimuli (see refs#9–11 for reviews). The structures of ceramide-containing membranes were previously studied in the context of the investigation of the model stratum corneum lipids which are composed primarily of ceramide, cholesterol, and free fatty acids (12–15). In the present work we report that ceramide induces defects in bilayers of dipalmitoylphosphatidylcholine (DPPC), as determined by 2H NMR, and activates cobra venom phospholipase A2 (PL-A2) in a manner similar to that of diacylglycerols (DAGs) (16,17). MATERIALS AND METHODS DPPC, diperdeuteropalmitoylphosphatidylcholine (DPPC-d62) and bovine brain ceramide were purchased from Avanti Polar Lipids (Alabaster, AL). Sphingomyelin and phospholipase A2 (Naja mocambique mocambique) were obtained from Sigma Chemical Co. (St. Louis, MO). Multilamellar lipid dispersions were prepared by dissolving the lipid mixtures in chloroform, and the solvent was evaporated off with dry nitrogen. Lipids were then redissolved in cyclohexane and lyophilized. The dried lipids were hydrated with 25 mM Tris (2-amino-2-(hydroxymethyl)-propane-1,3-diol; pH 7.4) buffer solution with 10 mM Ca2+, prepared in 2H-depleted H2O (Sigma). The samples were always fully hydrated and were typically 1:10 (w/w) in lipid/water. 2 H NMR spectra were acquired at 11.74 T (corresponding to 76.78 MHz 2H frequency) on a General Electric GN500 spectrometer using a high-power, wide line probe (Doty Scientific, Columbia, SC) and the standard quandrupole echo
1
To whom correspondence should be addressed. Fax: (909)-787-4286. E-mail: [email protected] Abbreviations: DPPC, dipalmitoylphosphatidylcholine; DPPC-d62, diperdeuteropalmitoylphosphatidylcholine; PL-A2, phospholipase A2; DAG, diacylglycerol. 834 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sequence (18). The spectral width was 0.5 MHz, refocusing time 64 ms, 90° pulse 3.5 ms and typically 16,000 scans per spectrum. Phospholipase A2 activity was determined according to the modified method of Stubbs et al., (19) as described in our previous publication (17).
RESULTS 2
H-NMR spectra of DPPC-d62 in the presence or absence of 25 mol% sphingomyelin or ceramide at 45°C are shown in Figure 1. The spectrum of DPPC-d62 (Figure 1A) is typical of the liquid crystalline bilayer lipid phase and is formed by the superposition of Pake doublets contributed by deuterons at different positions along the lipid acyl chains. The distances between the pairs of symmetric peaks, quadrupole splittings (dn), provide information on the order parameters of the corresponding CD2 acyl chains segments according to the equation: Dni 4 4
3
S D e2qQ h
i SCD
2
2
where (e qQ/h) is the static quadruple coupling constant, 170 Hz (20). Addition of 25 mol% sphingomyelin to DPPC-d62 did not significantly change the 2H NMR spectrum (Figure 1B). A dramatic effect on the lipid bilayer structure was observed upon the addition of 25 mol% ceramide: the resulting spectrum had an additional broad component, superimposed with the narrower spectrum that was similar to that of the DPPC-d62 without ceramide (Figure 1C). The broad component of the 2H NMR spectrum is typical of the lipids in gel state, and the superposition of the two types of spectra indicates the presence of ceramide-induced lateral phase separation of the bilayers into gel-like and liquid crystalline domains. Similar effects were previously observed by us upon addition of long-chain saturated DAGs to phosphatidylcholine (16,21) or phosphatidylcholine/phosphatidylserine bilayers (22). As in the case of DAGs, increase in temperature leads to the formation of one lipid phase at 60°C (Figure 2).
FIG. 1. 2H-NMR spectra of DPPC-d62 in the presence or absence of 25 mol% ceramide or sphingomyelin at 45° C. (A) DPPC-d62; (B) DPPC-d62 with sphingomyelin; (C) DPPC-d62 with ceramide. 835
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FIG. 2. 2H-NMR spectra of DPPC-d62 in the presence or absence of 25 mol% ceramide or sphingomyelin at 60° C. (A) DPPC-d62; (B) DPPC-d62 with sphingomyelin; (C) DPPC-d62 with ceramide.
The quadrupole splittings of DPPC-d62 and the corresponding order parameters are unaffected by 25 mol% sphingomyelin at 45° or 60°C (Figure 3). At 45°C only a small increase of the quadrupole splittings was observed in the narrow part of the 2H-NMR spectrum in the presence of ceramide (Figure 3A). The presence of ceramide results in a large increase of the DPPC-d62 quadrupole splittings at 60°C, in the one phase region, which indicates that at this temperature ceramide is only slightly miscible with DPPC-d62 in the liquid crystalline phase (Figure 3B). Because our previous studies suggested that the presence of DAG-induced lateral phase separation of the lipid bilayers on the regions of different fluidities correlates with activation of PL-A2 we measured the effect of the addition of ceramide on the activity of this enzyme. Indeed, the addition of ceramide caused a concentration-dependent increase in the activity of cobra venom PL-A2 (Figure 4). This increase was already significant at 5 mol% ceramide and reached z5-fold increase over the control values at 30 mol% ceramide. No significant effect of sphingomyelin on the PL-A2 activity was observed in this concentration range, except for a small inhibition at high sphingomyelin content, probably due to the surface dilution of DPPC (Figure 4). DISCUSSION The recently identified intracellular second messenger, ceramide, is produced from sphingomyelin in a stimulus-induced manner, by the action of neutral or acidic sphingomyelinases. Similarly with DAGs, ceramide has virtually no headgroup, and was therefore expected to produce similar membrane structure perturbations. Indeed, the perturbation produced by ceramide is similar to that observed by us with long-chain saturated DAGs (21), consistent with the fatty acid composition of the ceramide used where primarily long-chain saturated fatty acids comprise 85% of the total (manufacturer information). As in the case with DAGs, ceramide in the one-phase region (60°C) induces a large increase in 836
JOBNAME: BBRC 220#3 PAGE: 4 SESS: 10 OUTPUT: Tue May 7 11:40:09 1996 /xypage/worksmart/tsp000/69829b/45
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FIG. 3. Plots of quadrupole splittings of DPPC-d62 versus peak number in the presence or absence of 25 mol% ceramide or sphingomyelin. Peak 1 represents approximately seven carbon positions closest to the lipid headgroup. It is the outermost peak of the spectra on Figure 1. Generally the methylene units further from the headgroup have smaller quadrupole splittings, and peak 11, the innermost one, corresponds to the terminal methyl. (M), DPPC-d62; (x), DPPC-d62 with sphingomyelin; (n), DPPC-d62 with ceramide. (A) 45°C; only a few peaks were resolved in the narrower component of the spectrum with ceramide; (B) 60°C.
the order parameters of the DPPC acyl chains, which is a result of the ceramide molecules interspacing the bulky choline headgroups of the DPPC molecules allowing for tighter contact between the lipid acyl chains. At 45°C this leads to a strong interaction between ceramide and DPPC molecules and lateral phase separation of the ceramide-DPPC gel phase-like complexes from the “bulk” DPPC-d62, that remains in the liquid crystalline phase and is little affected by ceramide. In our previous studies we have demonstrated that the long-chain saturated DAGs produced similar perturbations in phosphatidylcholine (21) and phosphatidylcholine/phosphatidylserine (22) bilayers and we correlated the lateral phase separation induced by these DAGs in lipid bilayer structure with activation of phospholipases A2 from different sources, including the cobra venom PL-A2 (17). The present work similarly shows that ceramide-induced lateral phase separation of DPPC-d62 and the corresponding defects in the lipid bilayer structure correlates with activation of PL-A2 This PL-A2 activation is already an approximately 3-fold increase over the control levels at 5 mol% ceramide, which is only a little higher than the 4 mol% global ceramide concentration observed by Okazaki et al., (23) who reported that ceramide levels in 1a,25-dihydroxy-vitamin D3-activated HL-60 cells reach 40 pmol/nmol phospholipids in 2 hours. The local ceramide concentration may be considerably higher depending on the rate of ceramide diffusion in the membrane and rate of its production by sphingomyelinase. In one previous study where the effect of ceramide on PL-A2 activity was measured, no effect was observed (24). However, Franson et al., (24) were primarily addressing the effects of sphingosine and sphingomyelin, and the PL-A2 837
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FIG. 4. Effect of sphingomyelin (n) or ceramide (M) on the activity of phospholipase A2. The assays were performed in triplicates. The error bars correspond to SEM.
assays with ceramide were performed using human disc PL-A2 in the presence of Triton X-100. The presence of detergent would abolish the membrane-perturbing effects of ceramide which we suggest are responsible for PL-A2 activation. Our results showing activation of PL-A2 by ceramide may describe an additional aspect of biological effects of this second messenger molecule and suggest a possibility of cross-talk between the sphingomyelinase and PL-A2 signal transduction pathways. The NMR work was done using a GN500 spectrometer, funded by National Science Foundation Grant DMB840491 and National Institutes of Health Grant BRSG2507. We thank Mr. J. Huang for assistance in preparing samples for the NMR experiments REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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