Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet MARIANA N. NIKOLOVA-KARAKASHIAN, NADJAJ. GAVRILOVA, DIANAH. PETKOVA,AND MILKAS. SETCHENSKA Central Laboratory of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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Received November 25, 1991 M. N., GAVRILOVA, N. J., PETKOVA, D. H., and SETCHENSKA, M. S. 1992. SphingomyelinNIKOLOVA-KARAKA~HIAN, metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterolsupplemented diet. Biochem. Cell Biol. 70: 613-616. The effect of cholesterol-supplementeddiet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcho1ine:ceramide-phosphocholinetransferase, and phosphatidylethanolamine:ceramide-phosphoethanolaminetransferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10,20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcho1ine:ceramide-phosphocholinetransferase, and r = 0.998 for protein kinase C and phosphatidylethano1amine:ceramide-phosphoethanolaminetransferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide. Key words: protein kinase C, sphingomyelin-metabolizing enzymes, cholesterol, plasma membranes.
NIKOLOVA-KARAKASHIAN, M. N., GAVRILOVA, N. J., PETKOVA, D. H., et SETCHENSKA, M. S. 1992. Spingomyelinmetabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterolsupplemented diet. Biochem. Cell Biol. 70 : 613-616. Nous avons ktudik l'effet d'un regime enrichi de cholestkrol sur l'activitt des enzymes mttabolisant la sphingomytline et de la protkine kinase C dans la membrane plasmique de foie de rat. L'activitt de la prottine kinase C, de la phosphatidy1choline:ckramide-phosphocholinetransftrase et de la phosphatidylkthanolamine:ckramide-phosphotthanolamine transftrase augmente de facon continue et presque paralltle durant la ptriode exptrimentale avec le regime riche en cholestkrol Cjours 10,20 et 30). L'analyse de la rkgression linkaire montre une relation positive entre ces activitts avec des coefficients de corrdation r = 0,959 entre la prottine kinase C et la phosphatidylcholine:ckramide-phosphocholine transftrase et r = 0,998 entre la protkine kinase C et la phosphatidyl~thanolamine:c~ramide-phosphotthanolamine transftrase. D'autre part, I'activation de la protkine kinase C ne correspond pas aux changements d'activitt de la sphingomytlinase. Ces donntes suggkent que l'activation de la prottine kinase C obsewie dans les membranes plasmiques riches en cholestkrol est due B la production augmentte de diacylglyctrol et A l'acylation accrue de la sphingosine en ckramide. Mots elks : prottine kinase C, enzymes mttabolisant la sphingomykline, cholesttrol, membranes plasmiques. [Traduit par la rtdaction] Introduction Sphingolipids have emerged as a major class of lipid molecules with an important role in cell growth and differentiation (Hannun and Bell 1989a, 1989b; Merrill and Stevens 1989). Recently sphingolipid-derived products have been discussed as cell regulatory molecules (Hampton and Morand 1989; Hannun and Bell 1987, 1989b; Hannun et al. 1986). The sphingomyelin cycle, involving hydrolysis and regeneration of sphingomyelin with concomitant accumulation of ceramide, shares many features similar t o t h e phosphoinositide cycle (Majerus et al. 1986). However, the steps in the sphingomyelin cycle have not been confirmed experimentally t o the same extent as that in the phosphoinositide cycle. In a review by Hannun and Bell (1989b), a hypothesis has been discussed that reactions involved in sphingolipid breakdown may regulate protein kinase C activity through the generation of protein kinase C inhibiting metabolites: sphingosine and lysosphingolipids. Hampton and Morand (1989) have suggested an important addition to this hypothABBREVIATIONS: PC:Cer-PCh, phosphatidylcho1ine:ceramidephosphocholine; PE:Cer-PEt, phosphatidylethanolamine:ceramidephosphoethanolamine; DAG, diacylglycerol. Printed in Canada / Imprime au Canada
esis: the reaction catalyzed by PC:Cer-PCh transferase (sphingomyelin synthase). This enzyme transfers the phosphorylcholine head group from phosphatidylcholine to ceramide yielding sphingomyelin and diacylglycerol. Since the latter is a known activator of protein kinase C (Bell 1986), the action of sphingomyelin synthase provides a mechanism by which the metabolism of sphingolipids can result in stimulation of protein kinase C through diacylglycerol production and removal of sphingosine (Hampton and Morand 1989; Merrill and Jones 1990). Malgat et al. (1986) have demonstrated the synthesis of a sphingomyelin analog (ceramide-phosphoethanolamine) by PE:Cer-PEt transferase and its further methylation to sphingomyelin in liver and brain plasma membranes. Since this metabolic pathway, similar t o the PC:Per-PCh transferase, generates diacylglycerol and utilizes sphingosine, we assume that it could also be involved in protein kinase C regulation. Recent data indicate association between cholesterol and sphingomyelin metabolism (Merrill and Jones 1990). It was of interest to examine the protein kinase C activity in plasma membranes with altered sphingomyelin metabolism. In the present work, we have measured neutral sphingomyelinase, PE:Cer-PEt transferase, and PC:Cer-PCh transferase activ-
BIOCHEM. CELL BIOL. VOL. 70, 1992
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TABLE1. Cholesterol content of liver plasma membrane from control and cholesterol-fed rats pg .mg protein -
Cholesterol feeding (days)
'
Control 10 20 30
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**", P < 0.001; mean
* SD ( n = 5). T
Protein Kinase C activity (pmol min-l mg p i 1 )
*
10
20 30
days
FIG. 1. Changes in plasma membrane PC:Cer-PCh transferase ( a ) , PE:Cer-PEt transferase (o), sphingomyelinase(A), and protein kinase C (A) activities with time in rats fed a cholesterolenriched diet. The enzyme activities are expressed as nmol .h - mg Pr - for the PC:Cer-PCh and PE:Cer-PEt transferases, as nmol - min - .mg Pr - for sphingomyelinase, and as pmol min - .mg Pr - for protein kinase C. The activities were measured as described in Materials and methods. The results are the means k SEM of five different determinations. The asterisk represents the control group of animals fed a standard diet without cholesterol.
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'.
.
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'
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ities in parallel with the protein kinase C activity in plasma membranes from cholesterol-fed animals. The data showed a positive correlation between the activities of sphingomyelin-synthesizing enzymes and protein kinase C activity in these membranes.
Methods Animals and diet Male Wistar rats, body weight 200 15 g, were randomly divided into four groups of 10 animals. The control group was fed a standard laboratory diet and the other three groups were on the same diet plus 3% (by weight) cholesterol.
*
Isolation of rat liver plasma membranes Liver plasma membranes were isolated according to the procedure described by Wisher and Evans (1975). The method included membrane flotation through discontinuous sucrose gradient under
FIG. 2. Linear correlations between the activities of sphingomyelin-synthesizingenzymes and protein kinase C in liver plasma membranes from rats fed a cholesterol-supplementeddiet. (m) Protein kinase C and PC:PCh-PCh transferase activities (correlation coefficient, r = 0.959); (0) protein kinase C activity and PE:Cer-PEt transferase activities (correlation coefficient, r = 0.998). Each point is mean of five determinations. Pr, protein. centrifugation at 96 000 x g for 3 h. The purity of membranes was assessed by electron microscopy and by the specific activities of marker enzymes.
Assay of PC:Cer-PCh and PE:Cer-PEt transferase activities PC:Cer-PCh and PE:Cer-PEt transferases were assayed as described by Malgat et al. (1986). The reaction mixtures contained 132 nmol ['4~]ethanolamine-labeleddioleoyl phosphatidylethanolamine or 100 nmol ['4~]choline-labeleddipalmitoyl phosphatidylcholine, 50 mM Tris-HC1 (pH 7.4), 0.24 M sucrose, 0.15 mM KCI, 20 pg Triton X-100. m~ and 0.250 mg membrane protein in a total volume of 0.350 mL. When phosphatidylethanolamine was used as a precursor, 8-hydroxyethylhydrazine (a methylation inhibitor) was added (Jaiswal et al. 1983). Incubations continued for 3 h at 37OC in a shaking water bath. Reactions were terminated by addition of 2 mL chloroform-methanol (2:l). To separate sphingomyelin or its analog ceramidephosphoethanolamine, the total lipid extract was subjected to mild alkaline methanolysis in 0.3 N NaOH - methanol at 37°C for 1 h. The solution was cooled and neutralized with HCI. The chloroformextractable products (sphingomyelin or its analog) were recovered, washed with 50 mM KCl, and counted. Specific activities are expressed as nanomoles ceramide-phosphoethanolamine or sphingomyelin produced per hour per milligram protein.
-'
Assay of sphingomyelinase activity The sphingomyelinase activity was measured according to Hostetler and Yasaki (1979). The incubation medium contained 267 nmol of [methyl-'4~]cholinesphingomyelin, 10 mM Tris-HC1 (pH 7.4), 40 mM MgCI,, 2.5 mg Triton X - 1 0 0 - m ~ - ' , and 0.2 mg of membrane protein in a total volume of 0.2 mL. Incubations were carried out for 60 min at 37°C. The reaction was terminated by adding 100 rnM EDTA. The phosphorylcholine produced was extracted with ether and counted. The activity was expressed as nanomoles of sphingomyelin hydrolyzed per minute per milligram of protein.
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Phosphatidyl-
f
choljne
ethanolomine
4Protein Kinase C FIG. 3. Regulation of protein kinase C by the products of sphingomyelin metabolism. The hydrolysis of membrane sphingolipids results in formation of sphingosine, a negative effector of protein kinase C. Its utilization by acylation and subsequent head group transfer in PC:Cer-PCh and PE:Cer-PEt transferase reactions leads to accumulation of DAG, the positive effector of protein kinase C. The competition between sphingosine and DAG molecules for binding to the regulatory domain of protein kinase C allows for a bimodal mechanism of the enzyme regulation.
Protein kinase C assay Protein kinase C activity was measured as described by Palfrey and Wassem (1985). The reaction mixture of 0.1 mL contained 50 mM Tris-HC1, 5 mM MgSO,, 1 mM Ca-EGTA buffer (pH 7.4), 5 mM free c a Z + , 25 pg phosphatidylserine-m~-', 20 pg 1,2-dioleoyl sn-glycerol mL-I, 0.2 mg histone I11 S .mL -I, 40 pg leupeptin. mL - , 20 pM [^I-3 2 ~(500] cpm ~ .pmol ~ -~'), and aliquots of membrane preparations (50 pg protein). The reaction continued for 10 min at 30°C in a shaking water bath and was stopped by adding 2 mL of 10% trichloracetic acid. Samples were poured onto glass-fibre filters, washed five times with 5% trichloracetic acid, dried, and counted. The protein kinase C activity was calculated as picomoles [Y-~'P]ATP incorporated per minute per milligram membrane protein.
'
Analytical methods Lipids were extracted from membranes by the method of Folch et al. (1957) and cholesterol content was determined according to Sperry and Webb (1950). The protein content of membranes was estimated by the method of Lowry et al. (1951). Linear regression analysis was used for determination of the correlation coefficients.
Results Experiments proving the plasma membrane purity showed that the activity of 5'-nucleotidase (a marker enzyme for plasma membranes) increased from 36.4 + 2.5 to 546.0 k 27.3 nmol min - mg - 'for homogenate and plasma membranes, respectively; i.e., the purification factor was 15. Assay of galactosyltransferase activity demonstrated almost a fivefold decline in the plasma membrane fraction as compared with the values in the homogenate, showing that the contamination with Golgi membranes was not significant. The data in Table 1 show that cholesterol content of rat liver plasma membranes increased after 10 and 20 days feeding of animals with a cholesterol-supplemented diet: 18 and 27070 over the control, respectively. By day 30 of the diet, however, cholesterol level tended to return to normal values.
'
Protein kinase C, PC:Cer-PCh transferase, and PE:CerPEt transferase activities increased continuously and almost in parallel during the experimental period (Fig. 1). Linear regression analysis showed a strong positive correlation between these activities with correlation coefficients r = 0.998 for protein kinase C and PE:Cer-PEt transferase and r = 0.959 for protein kinase C and PC:Cer-PCh transferase (Fig. 2). The activity of neutral sphingomyelinase upon cholesterol feeding declined up to day 20 and returned to the control value on day 30 (Fig. 1). Discussion Originally protein kinase C activation by endogenous DAG was thought to occur through the phosphoinositide cycle 1986; Berridge 1987)3 but the (Nishizuka possibility for generation of DAG molecules through other phospholipid Precursors such as ~hos~hatid~lcholine was not excluded (Bishop and Bell 1988; Hannun and Bell 1989a). Similar to the phosphoinositide cycle, the sphingomyelin cycle also generates one of the second messengers, DAG molecules (Majerus et al. 1986). It is shown that PC:Cer-PCh and PE:Cer-PEt transferase reactions generate DAGs and utilize cerarnide (Malgat et al. 1986; Merrill and Jones 1990), which in turn is hydrolyzed by membrane-bound ceramidase to sphingosine (Spence et al. 1986). The latter is a potent and reversible inhibitor of protein kinase C activity in vitro (Hannun et al. 1986). The elevation of PC:Cer-PCh and PE:Cer-PEt transferase activities observed by us in cholesterol-enriched plasma membranes would cause net loss of the protein kinase C inhibitor (sphingosine) and gain of the protein kinase C activator (DAG). Thus the changes in the balance between these two competitive effectors of protein kinase C provides a possibility for its activation. The positive correlations between the changes in PC:Cer-PCh and PE:Cer-PEt transferase activities and the change in protein kinase C activity under
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our experimental conditions suggest that the metabolites of sphingomyelin synthesis could be involved in regulation of protein kinase C in vivo. On the other hand, the observed inhibition of neutral sphingomyelinase resulting in less sphingosine accumulation may also contribute to the activation of protein kinase C in cholesterol-enriched membranes. Furthermore, in membranes with elevated synthesis and decreased hydrolysis of sphingomyelin, the phosphoinositide cycle is probably suppressed, as sphingomyelin is shown to be an inhibitor of phospholipase C (MomchilovaPankova et al. 1991). Activation of protein kinase C is not a direct result of diet-induced enrichment of plasma membranes with cholesterol, since the latter is ineffective in the alteration of protein kinase C activity in vitro (Kishimoto et al. 1980). In Fig. 3 we present a scheme for protein kinase C regulation by the products of sphingomyelin metabolism. The participation of sphingomyelinase in this model of regulation was proposed by Hannun and Bell (19896). Hampton and Morand (1989) and Merrill and Jones (1990) included the PC:Cer-PCh transferase reaction, which produces DAG and utilizes sphingosine. Now we are adding the analogous reaction catalyzed by PE:Cer-PEt transferase. According to this model, sphingosine is removed by acylation to ceramide, with subsequent transformation to sphingomyelin by PC:Cer-PCh and PE:Cer-PEt transferase reactions and accompanied with simultaneous generation of DAG molecules. Despite the increasing amount of evidence that suggests a role of sphingomyelin cycle metabolites in signal transduction events, there is not yet direct proof for this attractive theory. Our results indicate that such a mechanism for protein kinase C regulation may operate in vivo in rat liver plasma membranes under elevated cholesterol nutritional status and may have physiologically important consequences. There are convincing data for short term regulation of cholesterol-metabolizing enzymes in the liver by reversible phosphorylation-dephosphorylation (Ghosh and Grogan 1989). Bell, R.M. 1986. Protein kinase C activation by diacylglycerol second messengers. Cell, 45: 63 1-632. Berridge, M.J. 1987. Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56: 159-193. Bishop, W., and Bell, R. 1988. Functions of diacylglycerol and glycerolipid metabolism, signal transduction and cellular transformation. Oncog. Res. 2: 205-218. Folch, J., Lees, M., and Sloane-Stanley, G.H. 1957. A simple method for isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226: 497-507. Ghosh, S., and Grogan, W.M. 1989. Activation of rat liver cholesterol ester hydrolase by CAMP-dependent protein kinase and protein kinase C. Lipids, 24: 733-736.
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Hampton, R.Y., and Morand, O.H. 1989. Sphingomyelin synthase and PKC activation. Science (Washington, D.C.), 246: 1050. Hannun, Y.A., and Bell, R.M. 1987. Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses. Science (Washington, D.C.), 235: 670-674. Hannun, Y.A., and Bell, R.M. 1989a. Regulation of protein kinase C by sphingosine and lysosphingolipids. Clin. Chim. Acta, 189: 333-346. Hannun, Y.A., and Bell, R.M. 1989b. Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science (Washington, D.C.), 243: 500-507. Hannun, Y.A., Loomis, C.R., Merrill, A.H., and Bell, R.M. 1986. Sphingosine inhibition of protein kinase C activity and phorbol dibutirate binding in vitro and in human platelets. J. Biol. Chem. 261: 12 604 - 12 609. Hostetler, K.Y., and Yasaki, P.J. 1979. The subcellular localization of neutral sphingomyelinase in rat liver. J. Lipid Res. 20: 456-463. Jaiswal, R.K., London, E.Y., and Sastry, B.V.R. 1983. Methylation of phospholipids in microsomes of the rat aorta. Biochim. Biophys. Acta, 735: 367-379. Kishimoto, A., Takai, Y., Mori, T., et al. 1980. Activation of calcium and phospholipid-dependent protein kinase C by diacylglycerol, its possible relation to phosphatidylinositol turnover. J. Biol. Chem. 255: 2273-2276. Lowry, O.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J. 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Majerus, P.W., Connolly, T.M., Deckmyn, H., et al. 1986. The metabolism of phosphoinositide-derived messenger molecules. Science (Washington, D.C.), 234: 1519-1526. Malgat, M., Maurice, A., and Baraud, J. 1986. Sphingomyelin and ceramide-phosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain. J. Lipid Res. 27: 251-260. Merrill, A.H., and Jones, D.D. 1990. An update of the enzymology and regulation of sphingomyelin metabolism. Biochim. Biophys. Acta, 1044: 1-12. Merrill, A.H., and Stevens, V.L., Jr. 1989. Modulation of protein kinase C and diverse cell functions by sphingosine-a pharmacologically interesting compound linking sphingolipids and signal transduction. Biochim. Biophys. Acta, 1010: 131-139. Momchilova-Pankova, A., Markovska, T., Koshlukova, S., and Koumanov, K. 1991. Phospholipid dependence of phospholipase C in rat liver plasma membranes. J. Lipid Mediators, 3: 215-223. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science (Washington. D.C.), 233: 305-3 11. Palfrey, H.C., and Wassem, A. 1985. Protein kinase C in human erythrocyte. J. Biol. Chcm. 260: 16 021 - 16 029. Spence, M.W.. Beed, S.. and Cook, H.W. 1986. Acid and alkdine ceramidases o f rat tissues. Biochm, Cell Biol. 64: 400-404. Sperry, W.M., and Webb, M. 1950. A revision of SchoenheimerSperry method for cholesterol determination. J. Biol. Chem. 187: 97-106. Wisher, M.H., and Evans, H. W. 1975. Functional polarity of rat liver hepatocyte surface membrane. Biochem. J. 146: 375-388.
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Received November 25, 1991 M. N., GAVRILOVA, N. J., PETKOVA, D. H., and SETCHENSKA, M. S. 1992. SphingomyelinNIKOLOVA-KARAKA~HIAN, metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterolsupplemented diet. Biochem. Cell Biol. 70: 613-616. The effect of cholesterol-supplementeddiet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcho1ine:ceramide-phosphocholinetransferase, and phosphatidylethanolamine:ceramide-phosphoethanolaminetransferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10,20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcho1ine:ceramide-phosphocholinetransferase, and r = 0.998 for protein kinase C and phosphatidylethano1amine:ceramide-phosphoethanolaminetransferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide. Key words: protein kinase C, sphingomyelin-metabolizing enzymes, cholesterol, plasma membranes.
NIKOLOVA-KARAKASHIAN, M. N., GAVRILOVA, N. J., PETKOVA, D. H., et SETCHENSKA, M. S. 1992. Spingomyelinmetabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterolsupplemented diet. Biochem. Cell Biol. 70 : 613-616. Nous avons ktudik l'effet d'un regime enrichi de cholestkrol sur l'activitt des enzymes mttabolisant la sphingomytline et de la protkine kinase C dans la membrane plasmique de foie de rat. L'activitt de la prottine kinase C, de la phosphatidy1choline:ckramide-phosphocholinetransftrase et de la phosphatidylkthanolamine:ckramide-phosphotthanolamine transftrase augmente de facon continue et presque paralltle durant la ptriode exptrimentale avec le regime riche en cholestkrol Cjours 10,20 et 30). L'analyse de la rkgression linkaire montre une relation positive entre ces activitts avec des coefficients de corrdation r = 0,959 entre la prottine kinase C et la phosphatidylcholine:ckramide-phosphocholine transftrase et r = 0,998 entre la protkine kinase C et la phosphatidyl~thanolamine:c~ramide-phosphotthanolamine transftrase. D'autre part, I'activation de la protkine kinase C ne correspond pas aux changements d'activitt de la sphingomytlinase. Ces donntes suggkent que l'activation de la prottine kinase C obsewie dans les membranes plasmiques riches en cholestkrol est due B la production augmentte de diacylglyctrol et A l'acylation accrue de la sphingosine en ckramide. Mots elks : prottine kinase C, enzymes mttabolisant la sphingomykline, cholesttrol, membranes plasmiques. [Traduit par la rtdaction] Introduction Sphingolipids have emerged as a major class of lipid molecules with an important role in cell growth and differentiation (Hannun and Bell 1989a, 1989b; Merrill and Stevens 1989). Recently sphingolipid-derived products have been discussed as cell regulatory molecules (Hampton and Morand 1989; Hannun and Bell 1987, 1989b; Hannun et al. 1986). The sphingomyelin cycle, involving hydrolysis and regeneration of sphingomyelin with concomitant accumulation of ceramide, shares many features similar t o t h e phosphoinositide cycle (Majerus et al. 1986). However, the steps in the sphingomyelin cycle have not been confirmed experimentally t o the same extent as that in the phosphoinositide cycle. In a review by Hannun and Bell (1989b), a hypothesis has been discussed that reactions involved in sphingolipid breakdown may regulate protein kinase C activity through the generation of protein kinase C inhibiting metabolites: sphingosine and lysosphingolipids. Hampton and Morand (1989) have suggested an important addition to this hypothABBREVIATIONS: PC:Cer-PCh, phosphatidylcho1ine:ceramidephosphocholine; PE:Cer-PEt, phosphatidylethanolamine:ceramidephosphoethanolamine; DAG, diacylglycerol. Printed in Canada / Imprime au Canada
esis: the reaction catalyzed by PC:Cer-PCh transferase (sphingomyelin synthase). This enzyme transfers the phosphorylcholine head group from phosphatidylcholine to ceramide yielding sphingomyelin and diacylglycerol. Since the latter is a known activator of protein kinase C (Bell 1986), the action of sphingomyelin synthase provides a mechanism by which the metabolism of sphingolipids can result in stimulation of protein kinase C through diacylglycerol production and removal of sphingosine (Hampton and Morand 1989; Merrill and Jones 1990). Malgat et al. (1986) have demonstrated the synthesis of a sphingomyelin analog (ceramide-phosphoethanolamine) by PE:Cer-PEt transferase and its further methylation to sphingomyelin in liver and brain plasma membranes. Since this metabolic pathway, similar t o the PC:Per-PCh transferase, generates diacylglycerol and utilizes sphingosine, we assume that it could also be involved in protein kinase C regulation. Recent data indicate association between cholesterol and sphingomyelin metabolism (Merrill and Jones 1990). It was of interest to examine the protein kinase C activity in plasma membranes with altered sphingomyelin metabolism. In the present work, we have measured neutral sphingomyelinase, PE:Cer-PEt transferase, and PC:Cer-PCh transferase activ-
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614
TABLE1. Cholesterol content of liver plasma membrane from control and cholesterol-fed rats pg .mg protein -
Cholesterol feeding (days)
'
Control 10 20 30
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**", P < 0.001; mean
* SD ( n = 5). T
Protein Kinase C activity (pmol min-l mg p i 1 )
*
10
20 30
days
FIG. 1. Changes in plasma membrane PC:Cer-PCh transferase ( a ) , PE:Cer-PEt transferase (o), sphingomyelinase(A), and protein kinase C (A) activities with time in rats fed a cholesterolenriched diet. The enzyme activities are expressed as nmol .h - mg Pr - for the PC:Cer-PCh and PE:Cer-PEt transferases, as nmol - min - .mg Pr - for sphingomyelinase, and as pmol min - .mg Pr - for protein kinase C. The activities were measured as described in Materials and methods. The results are the means k SEM of five different determinations. The asterisk represents the control group of animals fed a standard diet without cholesterol.
'
'.
.
'
'
'
'
ities in parallel with the protein kinase C activity in plasma membranes from cholesterol-fed animals. The data showed a positive correlation between the activities of sphingomyelin-synthesizing enzymes and protein kinase C activity in these membranes.
Methods Animals and diet Male Wistar rats, body weight 200 15 g, were randomly divided into four groups of 10 animals. The control group was fed a standard laboratory diet and the other three groups were on the same diet plus 3% (by weight) cholesterol.
*
Isolation of rat liver plasma membranes Liver plasma membranes were isolated according to the procedure described by Wisher and Evans (1975). The method included membrane flotation through discontinuous sucrose gradient under
FIG. 2. Linear correlations between the activities of sphingomyelin-synthesizingenzymes and protein kinase C in liver plasma membranes from rats fed a cholesterol-supplementeddiet. (m) Protein kinase C and PC:PCh-PCh transferase activities (correlation coefficient, r = 0.959); (0) protein kinase C activity and PE:Cer-PEt transferase activities (correlation coefficient, r = 0.998). Each point is mean of five determinations. Pr, protein. centrifugation at 96 000 x g for 3 h. The purity of membranes was assessed by electron microscopy and by the specific activities of marker enzymes.
Assay of PC:Cer-PCh and PE:Cer-PEt transferase activities PC:Cer-PCh and PE:Cer-PEt transferases were assayed as described by Malgat et al. (1986). The reaction mixtures contained 132 nmol ['4~]ethanolamine-labeleddioleoyl phosphatidylethanolamine or 100 nmol ['4~]choline-labeleddipalmitoyl phosphatidylcholine, 50 mM Tris-HC1 (pH 7.4), 0.24 M sucrose, 0.15 mM KCI, 20 pg Triton X-100. m~ and 0.250 mg membrane protein in a total volume of 0.350 mL. When phosphatidylethanolamine was used as a precursor, 8-hydroxyethylhydrazine (a methylation inhibitor) was added (Jaiswal et al. 1983). Incubations continued for 3 h at 37OC in a shaking water bath. Reactions were terminated by addition of 2 mL chloroform-methanol (2:l). To separate sphingomyelin or its analog ceramidephosphoethanolamine, the total lipid extract was subjected to mild alkaline methanolysis in 0.3 N NaOH - methanol at 37°C for 1 h. The solution was cooled and neutralized with HCI. The chloroformextractable products (sphingomyelin or its analog) were recovered, washed with 50 mM KCl, and counted. Specific activities are expressed as nanomoles ceramide-phosphoethanolamine or sphingomyelin produced per hour per milligram protein.
-'
Assay of sphingomyelinase activity The sphingomyelinase activity was measured according to Hostetler and Yasaki (1979). The incubation medium contained 267 nmol of [methyl-'4~]cholinesphingomyelin, 10 mM Tris-HC1 (pH 7.4), 40 mM MgCI,, 2.5 mg Triton X - 1 0 0 - m ~ - ' , and 0.2 mg of membrane protein in a total volume of 0.2 mL. Incubations were carried out for 60 min at 37°C. The reaction was terminated by adding 100 rnM EDTA. The phosphorylcholine produced was extracted with ether and counted. The activity was expressed as nanomoles of sphingomyelin hydrolyzed per minute per milligram of protein.
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Phosphatidyl-
f
choljne
ethanolomine
4Protein Kinase C FIG. 3. Regulation of protein kinase C by the products of sphingomyelin metabolism. The hydrolysis of membrane sphingolipids results in formation of sphingosine, a negative effector of protein kinase C. Its utilization by acylation and subsequent head group transfer in PC:Cer-PCh and PE:Cer-PEt transferase reactions leads to accumulation of DAG, the positive effector of protein kinase C. The competition between sphingosine and DAG molecules for binding to the regulatory domain of protein kinase C allows for a bimodal mechanism of the enzyme regulation.
Protein kinase C assay Protein kinase C activity was measured as described by Palfrey and Wassem (1985). The reaction mixture of 0.1 mL contained 50 mM Tris-HC1, 5 mM MgSO,, 1 mM Ca-EGTA buffer (pH 7.4), 5 mM free c a Z + , 25 pg phosphatidylserine-m~-', 20 pg 1,2-dioleoyl sn-glycerol mL-I, 0.2 mg histone I11 S .mL -I, 40 pg leupeptin. mL - , 20 pM [^I-3 2 ~(500] cpm ~ .pmol ~ -~'), and aliquots of membrane preparations (50 pg protein). The reaction continued for 10 min at 30°C in a shaking water bath and was stopped by adding 2 mL of 10% trichloracetic acid. Samples were poured onto glass-fibre filters, washed five times with 5% trichloracetic acid, dried, and counted. The protein kinase C activity was calculated as picomoles [Y-~'P]ATP incorporated per minute per milligram membrane protein.
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Analytical methods Lipids were extracted from membranes by the method of Folch et al. (1957) and cholesterol content was determined according to Sperry and Webb (1950). The protein content of membranes was estimated by the method of Lowry et al. (1951). Linear regression analysis was used for determination of the correlation coefficients.
Results Experiments proving the plasma membrane purity showed that the activity of 5'-nucleotidase (a marker enzyme for plasma membranes) increased from 36.4 + 2.5 to 546.0 k 27.3 nmol min - mg - 'for homogenate and plasma membranes, respectively; i.e., the purification factor was 15. Assay of galactosyltransferase activity demonstrated almost a fivefold decline in the plasma membrane fraction as compared with the values in the homogenate, showing that the contamination with Golgi membranes was not significant. The data in Table 1 show that cholesterol content of rat liver plasma membranes increased after 10 and 20 days feeding of animals with a cholesterol-supplemented diet: 18 and 27070 over the control, respectively. By day 30 of the diet, however, cholesterol level tended to return to normal values.
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Protein kinase C, PC:Cer-PCh transferase, and PE:CerPEt transferase activities increased continuously and almost in parallel during the experimental period (Fig. 1). Linear regression analysis showed a strong positive correlation between these activities with correlation coefficients r = 0.998 for protein kinase C and PE:Cer-PEt transferase and r = 0.959 for protein kinase C and PC:Cer-PCh transferase (Fig. 2). The activity of neutral sphingomyelinase upon cholesterol feeding declined up to day 20 and returned to the control value on day 30 (Fig. 1). Discussion Originally protein kinase C activation by endogenous DAG was thought to occur through the phosphoinositide cycle 1986; Berridge 1987)3 but the (Nishizuka possibility for generation of DAG molecules through other phospholipid Precursors such as ~hos~hatid~lcholine was not excluded (Bishop and Bell 1988; Hannun and Bell 1989a). Similar to the phosphoinositide cycle, the sphingomyelin cycle also generates one of the second messengers, DAG molecules (Majerus et al. 1986). It is shown that PC:Cer-PCh and PE:Cer-PEt transferase reactions generate DAGs and utilize cerarnide (Malgat et al. 1986; Merrill and Jones 1990), which in turn is hydrolyzed by membrane-bound ceramidase to sphingosine (Spence et al. 1986). The latter is a potent and reversible inhibitor of protein kinase C activity in vitro (Hannun et al. 1986). The elevation of PC:Cer-PCh and PE:Cer-PEt transferase activities observed by us in cholesterol-enriched plasma membranes would cause net loss of the protein kinase C inhibitor (sphingosine) and gain of the protein kinase C activator (DAG). Thus the changes in the balance between these two competitive effectors of protein kinase C provides a possibility for its activation. The positive correlations between the changes in PC:Cer-PCh and PE:Cer-PEt transferase activities and the change in protein kinase C activity under
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our experimental conditions suggest that the metabolites of sphingomyelin synthesis could be involved in regulation of protein kinase C in vivo. On the other hand, the observed inhibition of neutral sphingomyelinase resulting in less sphingosine accumulation may also contribute to the activation of protein kinase C in cholesterol-enriched membranes. Furthermore, in membranes with elevated synthesis and decreased hydrolysis of sphingomyelin, the phosphoinositide cycle is probably suppressed, as sphingomyelin is shown to be an inhibitor of phospholipase C (MomchilovaPankova et al. 1991). Activation of protein kinase C is not a direct result of diet-induced enrichment of plasma membranes with cholesterol, since the latter is ineffective in the alteration of protein kinase C activity in vitro (Kishimoto et al. 1980). In Fig. 3 we present a scheme for protein kinase C regulation by the products of sphingomyelin metabolism. The participation of sphingomyelinase in this model of regulation was proposed by Hannun and Bell (19896). Hampton and Morand (1989) and Merrill and Jones (1990) included the PC:Cer-PCh transferase reaction, which produces DAG and utilizes sphingosine. Now we are adding the analogous reaction catalyzed by PE:Cer-PEt transferase. According to this model, sphingosine is removed by acylation to ceramide, with subsequent transformation to sphingomyelin by PC:Cer-PCh and PE:Cer-PEt transferase reactions and accompanied with simultaneous generation of DAG molecules. Despite the increasing amount of evidence that suggests a role of sphingomyelin cycle metabolites in signal transduction events, there is not yet direct proof for this attractive theory. Our results indicate that such a mechanism for protein kinase C regulation may operate in vivo in rat liver plasma membranes under elevated cholesterol nutritional status and may have physiologically important consequences. There are convincing data for short term regulation of cholesterol-metabolizing enzymes in the liver by reversible phosphorylation-dephosphorylation (Ghosh and Grogan 1989). Bell, R.M. 1986. Protein kinase C activation by diacylglycerol second messengers. Cell, 45: 63 1-632. Berridge, M.J. 1987. Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56: 159-193. Bishop, W., and Bell, R. 1988. Functions of diacylglycerol and glycerolipid metabolism, signal transduction and cellular transformation. Oncog. Res. 2: 205-218. Folch, J., Lees, M., and Sloane-Stanley, G.H. 1957. A simple method for isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226: 497-507. Ghosh, S., and Grogan, W.M. 1989. Activation of rat liver cholesterol ester hydrolase by CAMP-dependent protein kinase and protein kinase C. Lipids, 24: 733-736.
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Hampton, R.Y., and Morand, O.H. 1989. Sphingomyelin synthase and PKC activation. Science (Washington, D.C.), 246: 1050. Hannun, Y.A., and Bell, R.M. 1987. Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses. Science (Washington, D.C.), 235: 670-674. Hannun, Y.A., and Bell, R.M. 1989a. Regulation of protein kinase C by sphingosine and lysosphingolipids. Clin. Chim. Acta, 189: 333-346. Hannun, Y.A., and Bell, R.M. 1989b. Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science (Washington, D.C.), 243: 500-507. Hannun, Y.A., Loomis, C.R., Merrill, A.H., and Bell, R.M. 1986. Sphingosine inhibition of protein kinase C activity and phorbol dibutirate binding in vitro and in human platelets. J. Biol. Chem. 261: 12 604 - 12 609. Hostetler, K.Y., and Yasaki, P.J. 1979. The subcellular localization of neutral sphingomyelinase in rat liver. J. Lipid Res. 20: 456-463. Jaiswal, R.K., London, E.Y., and Sastry, B.V.R. 1983. Methylation of phospholipids in microsomes of the rat aorta. Biochim. Biophys. Acta, 735: 367-379. Kishimoto, A., Takai, Y., Mori, T., et al. 1980. Activation of calcium and phospholipid-dependent protein kinase C by diacylglycerol, its possible relation to phosphatidylinositol turnover. J. Biol. Chem. 255: 2273-2276. Lowry, O.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J. 195 1. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Majerus, P.W., Connolly, T.M., Deckmyn, H., et al. 1986. The metabolism of phosphoinositide-derived messenger molecules. Science (Washington, D.C.), 234: 1519-1526. Malgat, M., Maurice, A., and Baraud, J. 1986. Sphingomyelin and ceramide-phosphoethanolamine synthesis by microsomes and plasma membranes from rat liver and brain. J. Lipid Res. 27: 251-260. Merrill, A.H., and Jones, D.D. 1990. An update of the enzymology and regulation of sphingomyelin metabolism. Biochim. Biophys. Acta, 1044: 1-12. Merrill, A.H., and Stevens, V.L., Jr. 1989. Modulation of protein kinase C and diverse cell functions by sphingosine-a pharmacologically interesting compound linking sphingolipids and signal transduction. Biochim. Biophys. Acta, 1010: 131-139. Momchilova-Pankova, A., Markovska, T., Koshlukova, S., and Koumanov, K. 1991. Phospholipid dependence of phospholipase C in rat liver plasma membranes. J. Lipid Mediators, 3: 215-223. Nishizuka, Y. 1986. Studies and perspectives of protein kinase C. Science (Washington. D.C.), 233: 305-3 11. Palfrey, H.C., and Wassem, A. 1985. Protein kinase C in human erythrocyte. J. Biol. Chcm. 260: 16 021 - 16 029. Spence, M.W.. Beed, S.. and Cook, H.W. 1986. Acid and alkdine ceramidases o f rat tissues. Biochm, Cell Biol. 64: 400-404. Sperry, W.M., and Webb, M. 1950. A revision of SchoenheimerSperry method for cholesterol determination. J. Biol. Chem. 187: 97-106. Wisher, M.H., and Evans, H. W. 1975. Functional polarity of rat liver hepatocyte surface membrane. Biochem. J. 146: 375-388.
