Báo cáo khoa học: Signaling events mediating activation of brain ethanolamine plasmalogen hydrolysis by ceramide pot

Signaling events mediating activation of brain ethanolamine plasmalogen hydrolysis by ceramide Eduardo Latorre 1 , M. Pilar Collado 1 , Inmaculada Ferna ´ ndez 1 , M. Dolores Aragone ´ s 1 and R. Edgardo Catala ´ n 2 1 Departamento de Bioquı ´ mica y Biologı ´ a Molecular I, Facultad de Quı ´ micas, Universidad Complutense de Madrid, Madrid, Spain; 2 Departamento de Biologı ´ a Molecular, Centro de Biologı ´ a Molecular ‘Severo Ochoa’, Universidad Auto ´ noma de Madrid, Madrid, Spain Ceramide is a lipid second messenger that acts on mul- tiple-target enzymes, some of which are involved in other signal-transduction systems. We have previously demon- strated that endogenous ceramide modifies the metabolism of brain ethanolamine plasmalogens. The mechanism involved was studied. On the basis of measurements of breakdown products, specific inhibitor effects, and previ- ous findings, we suggest that a plasmalogen-selective phospholipase A 2 is the ceramide target. Arachidonate- rich pools of the diacylphosphatidylethanolamine subclass were also affected by ceramide, but the most affected were plasmalogens. Concomitantly with production of free arachidonate, increased 1-O-arachidonoyl ceramide formation was observed. Quinacrine (phospholipase A 2 inhibitor) and 1-O-octadecyl-2-O-methyl-rac-glycerol-3- phosphocholine (CoA-independent transacylase inhibitor) prevented all of these ceramide-elicited effects. Therefore, phospholipase and transacylase activities are tightly cou- pled. Okadaic acid (phosphatase 2A inhibitor) and PD 98059 (mitogen-activated protein kinase inhibitor) modified basal levels of ceramide and sphingomyelinase- induced accumulation of ceramide, respectively. Therefore, they provided no evidence to determine whether there is a sensitive enzyme downstream of ceramide. The evidence shows that there are serine-dependent and thiol-dependent enzymes downstream of ceramide generation. Further- more, experiments with Ac-DEVD-CMK (caspase-3 speci- fic inhibitor) have led us to conclude that caspase-3 is downstream of ceramide in activating the brain plasmalo- gen-selective phospholipase A 2 . Keywords: brain ethanolamine plasmalogens; caspase-3; ceramide; phospholipase A 2 ; plasmalogen-selective phos- pholipase A 2 . It is well known that messengers derived from sphingolipid and glycerolipid, and their target enzymes, establish mul- tiple relationships leading to the formation of complicated networks for the effective transduction of signals. In the last few years, the regulatory role of ceramide (Cer) generated by the sphingomyelin cycle has received increasing attention. It is known to activate multiple serine/threonine protein kinases and protein phosphatases [1], leading to the tissue- specific downstream regulation of several target enzymes, some of which are involved in other lipid signaling pathways. In this context, we have previously reported [2] that exogenous sphingomyelinase (EC 3.1.4.12) treatment brought about alterations in brain ethanolamine (Etn) plasmalogen metabolism. The role of plasmalogens as a source of second messengers in lipid signal-transduction systems [3–5] and as ubiquitous endogenous antioxidants [6] has been investigated. Plasmalogens are phospholipids characterized by the presence of a vinyl ether substituent at the sn-1 position of the glycerol backbone. They are especially abundant in electrically active tissues, such as brain, where most of them are Etn-phosphoglycerides. The latter have the propensity to facilitate membrane fusion, strongly suggesting their involvement in synaptic transmission [3]. In addition, Etn plasmalogens have been reported to be involved in the vulnerability to oxidative stress associated with aging and pathological conditions [6]. Evidence is accumulating on age-related changes in the quantities [7] and fatty acid profile of these phospholipids [7,8]. On the other hand, significant and selective deficiencies in brain Etn plasmalogens have been reported at the site of neurodegeneration in Alzheimer’s disease [9], brain peroxisomal disorders [8] and Down’s syndrome [10]. In some instances, the decreased Etn plasmalogen levels are accompanied by a marked increase in the concentration of the degradation metabolites or their derivatives, such as PEtn [11] or prostaglandins [3]. Therefore, the evidence suggests that several phospholipase types may be involved Correspondence to R. E. Catala ´ n, Departamento de Biologı ´ a Molecular, Centro de Biologı ´ a Molecular ‘Severo Ochoa’, Universidad Auto ´ noma de Madrid, E-28049 Madrid, Spain. Fax: + 34 91 3974870, Tel.: + 34 91 3974869, E-mail: ecatalan@cbm.uam.es Abbreviations: BSS, balanced salt solution; Cer, ceramide; C 2 -Cer, N-acetylsphingosine; Etn, ethanolamine; ET-18-OCH 3 ,1-O-octa- decyl-2-O-methyl-rac-glycerol-3-phosphocholine; MAPK, mitogen-activated protein kinase; PLA 2 , phospholipase A 2 ; PtdEth, phosphatidylethanolamine. Enzymes: phospholipase A 2 (EC 3.1.1.4); sphingomyelinase (EC 3.1.4.12); CoA-independent transacylase (EC 2.3.1.147). (Received 2 August 2002, revised 16 October 2002, accepted 7 November 2002) Eur. J. Biochem. 270, 36–46 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03356.x in the metabolism of brain Etn plasmalogens in physio- pathological states. The existence of a plasmalogen-select- ive phospholipase A 2 (PLA 2 , EC 3.1.1.4) which selectively, but not exclusively, acts on 1-alk-1¢-enyl-2-acyl-sn-glycero- 3-PEtn has been reported [4,5,12]. This enzyme has been purified from bovine brain and shown to be specific for neural tissues and distinct from other non-neuronal plasmalogen-specific PLA 2 enzymes and brain PLA 2 enzymes [3,4]. There is evidence that Etn plasmalogen degradation by a PLA 2 plays an important role in neutrophil activation by agonists [13]. From the latter study and others [14–16], the idea has emerged that Etn plasmalogen hydrolysis may be coupled with the formation of acyl Cers, eicosanoids and/or platelet-activating factor. Thus, PLA 2 in the presence of a suitable acceptor molecule possesses a dual enzymatic function, i.e. PLA 2 and CoA- independent transacylase, generating: (a) free arachidonate, which can be converted into eicosanoids [13,15], and (b) an acyl derivative, mainly the arachidonoyl derivative [16]. On the other hand, Etn plasmalogens can be resynthesized from the lyso-plasmenylEtn released by a CoA-independ- ent transacylase from 1-radyl-2-arachidonoylGroPCho, generating lyso-platelet-activating factor derivatives, which can lead to formation of platelet-activating factor by transacetylation [15,16]. We would like to emphasize that all this experimental evidence has been obtained in non- neural cell-free systems or isolated cells. Taking into account that the following have been reported, (a) a brain plasmalogen-selective PLA 2 [4,5], (b) abrainPLA 2 acting on Etn phosphoglyceride with trans- acylase activity [16], and (c) a Cer-elicited decrease in brain Etn plasmalogen levels concomitant with 1-O-acylCer formation [2], the aim of this study was to clarify the mechanism by which Cer regulates brain Etn plasmalogen metabolism. First, we investigated the type of enzymatic activities involved and, secondly, the involvement of poten- tial downstream Cer target enzyme(s). Materials and methods Materials Staphylococcus aureus sphingomyelinase [180 UÆ(mg pro- tein) )1 ], N-acetylsphingosine (C 2 -Cer), Cer type III (from brain sphingomyelin containing primarily stearic and ner- vonic acids), phenylmethanesulfonyl fluoride, quinacrine hydrochloride, ganglioside type II (from bovine brain containing 15% N-acetylneuraminic acid) were purchased from Sigma, St Louis, MO, USA. Bromoenol lactone was from Alexis Biochemicals, La ¨ ufelfingen, Switzerland. [1- 14 C]Arachidonic acid (55 mCiÆmmol )1 )wasfrom American Radiolabeled Chemicals Inc., St Louis, MO, USA. [c- 32 P]ATP (3000 CiÆmmol )1 )wasfromNuclear Iberica, Madrid, Spain. [2- 14 C]Ethan-1-ol-2-amine hydro- chloride (55 mCiÆmmol )1 ) was purchased from Amersham. Escherichia coli diacylglycerol kinase (EC 2.7.1.107), 2¢-amino-3¢-methoxyflavone (PD 98059), okadaic acid, Ac-DEVD-chloromethylketone (Ac-DEVD-CMK) and a-iodocetamide were from Calbiochem, San Diego, CA, USA. 1-O-Octadecyl-2-O-methyl-rac-glycerol-3-PCho (ET-18-OCH 3 ) was from Bachem AG, Budendorf, Switzer- land. High-performance TLC plates were obtained from Merck, Darmstadt, Germany. All other reagents were of the highest analytical grade available. 1-O-AcylCer standard was synthesized as described previously [2]. Tissue preparation and incubation of slices Experiments were carried out with male Wistar rats (180– 200 g). The animals were maintained at 22–24 °Cand given free access to standard laboratory diet and water ad libitum. Rat care, handling and all the experimental procedures were in accordance with internationally accep- ted principles concerning the care and use of laboratory animals. The rats were killed [2], and their brains were removed. Pial vessels and white matter were carefully discarded, and cerebral cortex was obtained. Slices (dimensions: 350 · 350 lm) were prepared with a MacIl- wain tissue chopper, as previously reported [17]. They were equilibrated in a balanced salt solution (BSS): 135 m M NaCl, 4.5 m M KCl, 1.5 m M CaCl 2 ,0.5m M MgCl 2 , 5.6 m M glucose, 10 m M Hepes, pH 7.4, equilibrated with 95% O 2 /5% CO 2 for 1 h. Aliquots (300 lL) of gravity- packed slices were transferred to glass tubes containing BSS and then sphingomyelinase (unless otherwise indica- ted, the final concentration was 0.38 UÆmL )1 , as described previously [2]) dissolved in 50 m M phosphate buffer, pH 7.4, with 50% (v/v) glycerol, or C 2 -Cer dissolved in dimethyl sulfoxide (10–100 l M ), or diluents alone were added and the mixture incubated for 30 min at 37 °C[2]. In one set of experiments, slices were treated with 0.1 l M endothelin-1 for 30 min [18]. In experiments in which different inhibitors were tested, the slices were preincubated in their absence or presence before the addition of sphingomyelinase or C 2 -Cer. When the inhibitors used were dissolved in dimethyl sulfoxide or ethanol, the final concentration of diluent was never higher than 1%. The incubation mixtures were continuously gassed with 95% O 2 /5% CO 2 . The incubations were stopped by removal of the medium and replacement with 0.38 mL BSS containing 10 m M EDTA and 1 mL chloroform/methanol/13 M HCl (100 : 100 : 1, v/v/v). Lipids were immediately extracted as described below. As we used an inhibitor (ET-18-OCH 3 )withlow diffusion through slices, some experiments with homogen- ates were performed. Homogenates of cerebral cortex were prepared in BSS equilibrated as described above. Previous comparative experiments showed that cerebral slices and homogenates exhibited the same responsiveness to the sphingomyelinase treatment [2]. Experiments with labeled precursors In some experiments, labeled precursors were used. Slices from 8–10 brains were preincubated in the presence of labeled precursors: 4 lCi (0.2 lCiÆmL )1 )[1- 14 C]arachidonic acid [2] or 50 lCi (2 lCiÆmL )1 )[2- 14 C]ethan-1-ol-2-amine hydrochloride [19] at 37 °C in BSS for 120 or 30 min, respectively. The preincubations were continuously gassed with 95% O 2 /5% CO 2 . Then, the incubation medium was removed, and the slices were washed three times with cold BSS. Aliquots of slices were taken for incubation with sphingomyelinase or C 2 -Cer; incubations were stopped as described above. Ó FEBS 2003 Activated plasmalogen hydrolysis by Cer (Eur. J. Biochem. 270)37 Extraction of total lipids; separation of sphingolipids Lipids were extracted as described previously [20]. The organic phases were dried under a N 2 atmosphere, and total lipids were weighed and dissolved in chloroform/methanol (2 : 1, v/v). Lipids were separated by TLC. 1-O-AcylCer was resolved by sequential 1D TLC in: (a) ethyl ether; (b) chloroform/methanol/acetic acid/water (25 : 15 : 4 : 1.5, v/v/v/v), and (c) chloroform/methanol/acetic acid (65 : 2.5 : 4, v/v/v). The first solvent system was developed through the plate, the second reached 7 cm from the bottom of the plate, whereas the third reached 13 cm from the bottom. This sequential TLC also resolves the nonesterified fatty acid fraction and the Etn phospholipid subclasses, as stated below. To determine Cer levels, one aliquot of total lipid was subjected to alkaline hydrolysis in 0.1 M metha- nolic KOH at 37 °C for 1 h to remove glycerolipids, as previously described [2]. Cer was resolved by sequential 1D TLC using solvent systems (a) and (c) described above, but the former reached 3 cm from the top of the plate, whereas the latter was developed through the plate, as described previously [2]. Lipid standards were cochromatographed with samples. Lipids were visualized with iodine vapor, and the bands of 1-O-acylCer and nonesterified fatty acids were scraped from the plates to quantitate the radioactivity incorporated by liquid scintillation. The bands correspond- ing to Cers were scraped from the plates and extracted with chloroform/methanol (4 : 5, v/v) and dried under a N 2 atmosphere for subsequent quantitation. Analysis of the subclasses of Etn phospholipids Etn plasmalogen levels were determined as described previously [2]. In some experiments with [ 14 C]arachidonic acid as precursor, three further subclasses of Etn phospholipids were separated, as previously described [21]. First, total Etn phospholipids were obtained from the total lipids by sequential 1D TLC as described above. After extraction with chloroform/methanol (2 : 1, v/v), the dry residue was incubated with 40 U phospholipase C per sample for 16 h. The resulting diacylglycerols were extracted three times with ether/hexane (1 : 1, v/v). Once the extracts had been dried, acetylated derivatives were prepared by incubation for 3 h in pyridine/acetic anhydride (1 : 5, v/v). The solution was dried and extracted twice with ether/hexane (1 : 1, v/v). The final dried residue was fractionated by TLC using sequen- tially: (a) hexane/ether/methanol/acetic acid (90 : 20 : 3 : 2, v/v/v/v), and (b) toluene as solvents [22]. Phospholipids were visualized with iodine vapor and identified from the respective standards and reported R f values. Once scraped from the plate, the radioactivity in each fraction was measured by liquid scintillation. Radioenzymatic determination of Cer levels Extracted Cer was phosphorylated in the presence of diacylglycerol kinase, as described previously [2]. Cers were solubilized and phosphorylated in the presence of 5 lgof theenzymeand10m M [c- 32 P]ATP for 10 min. After incubation, phosphorylated derivatives of Cer were extrac- ted, fractionated by TLC, visualized by autoradiography using Kodak X-Omat film and quantitated by liquid- scintillation counting. Calibration curves were constructed using known amounts of Cer. Radioenzymatic determination of diacylglycerol mass Aliquots of total lipids were phosphorylated in the presence of diacylglycerol kinase, as described previously [23]. Aliquots of total lipids were evaporated under N 2 and the dried lipids were solubilized and phosphorylated in the presence of 5 lg enzyme and 10 m M [c- 32 P]ATP for 30 min. Then, samples were spotted on silica gel TLC plates and developed with chloroform/methanol/acetic acid/acetone/ water (40 : 13 : 12 : 15 : 8, v/v/v/v). Spots corresponding to phosphatidic acid were visualized by autoradiography using Kodak X-Omat film and quantitated by liquid-scintillation counting. Calibration curves were constructed using known quantities of 1-stearoyl-2-arachidonoylglycerol. Analysis of water-soluble products of hydrolysis of Etn phospholipids In experiments with [ 14 C]Etn, the upper phases from the lipid extraction (see above) containing the water-soluble metabolites were analyzed by TLC [24]. The upper phases were lyophilized and the residue was then dissolved in 50% ethanol, and Etn, PEtn and CDP-Etn tracers were added as carriers. Water-soluble products were separated by TLC using methanol/0.5% NaCl/NH 4 OH (50 : 50 : 5, v/v/v) as solvent. Bands were detected with 1% ninhydrin in ethanol. Spots were scraped from the plate and analyzed for radioactivity counting. Determination of 1- O -alkenyl-2-lysoGro P Etn radioactivity Aliquots of total lipids from experiments performed with [ 14 C]Etn were subjected to alkaline hydrolysis and separated using TLC. The system used was chloroform/methanol/ acetic acid (65 : 25 : 4, v/v/v). After development, spots were visualized with ninhydrin and identified from respect- ive standards. Spots were scraped from the plates, and their mass determined by measurement of phosphorus content [25]. The radioactivity incorporated was quantitated by liquid-scintillation counting. Statistical analysis Student’s t test was used for paired observations. P <0.05 was considered to be significant. Results Sphingomyelinase and C 2 -Cer affect brain Etn plasmalogen metabolism We have previously reported that Etn plasmalogen meta- bolism is specifically affected by sphingomyelinase treatment [2]. Here we first studied the effect of different concentrations of sphingomyelinase on Etn plasmalogen and Cer levels (Fig. 1). At a concentration of 0.38 UÆmL )1 , sphingomye- linase significantly (P < 0.05) decreased Etn plasmalogens 38 E. Latorre et al.(Eur. J. Biochem. 270) Ó FEBS 2003 to 65% (Fig. 1A). Concomitantly, a significant (P < 0.05) increase in Cer levels was observed (100% over control value), in agreement with our previous data [2]. Higher sphingomyelinase concentrations further increased Cer levels, but had no further effect on Etn plasmalogen levels (Fig. 1A). A concentration of 0.19 UÆmL )1 sphingo- myelinase had a slight, but not significant, effect (data not shown). As many effects evoked by sphingomyelinase treatment are mimicked by short-chain cell-permeable Cer analogs, we also tested the effect of C 2 -Cer on Etn plasmalogen levels. The concentration range of C 2 -Cer was chosen on the basis of previous evidence [26,27]. A concentration of 50 l M was the lowest capable of decreasing Etn plasmalogen levels by 55% of the control value (P < 0.05) (Fig. 1B). Higher concentrations did not produce further variation in Etn plasmalogen levels. To determine the mechanism by which sphingomyelinase and C 2 -Cer affect Etn phosphoglyceride metabolism, we carried out labeling studies with [1- 14 C]arachidonic acid and [1- 14 C]Etn (Table 1). Sphingomyelinase and C 2 -Cer both significantly (P < 0.05) reduced labeling in the plasmalo- gen fraction but scarcely affected that in the acid-resistant fraction. Interestingly, the most noticeable result was the low radioactivity from [1- 14 C]arachidonic found in the plasmalogen fraction ( 10% of the control value) when slices were treated with sphingomyelinase. Experiments to separate the Etn phosphoglycerides into their three subclasses, i.e. diacyl, alkylacyl and plasmalo- gens, were also performed. In the light of the above data (Table 1), we used [ 14 C]arachidonate as the labeled precur- sor. These results are presented in Table 2. Both diacyl and plasmalogen fractions exhibited significantly (P < 0.05) reduced radioactivity ( 30% of the control value) after treatment with sphingomyelinase or C 2 -Cer. Lipids were extracted [20] from one aliquot of incuba- tion medium, and 14 C radioactivity was determined. This provides a measure of activation of secretory PLA 2 .Results in Table 3 show that extracellular [1- 14 C]arachidonate release was not affected, but the cell-associated 14 C Fig. 1. Dose–response relationship of sphingomyelinase-induced and C 2 -ceramide-induced changes in brain Etn plasmalogen levels. (A) Cerebral cortex slices were exposed to sphingomyelinase (SMase) for 30 min, and levels of Etn plasmalogens (PlsEtn; left axis; filled bars) and ceramide (right axis; striped bars) were measured. (B) Cerebral cortex slices were exposed to C 2 -ceramide for 30 min, and Etn plas- malogens were measured. Data represent mean ± SE and are from two experiments performed in triplicate. Values significantly different from their respective controls are indicated: *P < 0.05. Table 1. Variations in [ 14 C]arachidonic acid-labeled and [ 14 C]Etn-labeled Etn phospholipids evoked by sphingomyelinase and C 2 -Cer. Slices were labeled with 0.2 lCiÆmL )1 [ 14 C]arachidonic acid for 120 min or 2 lCiÆmL )1 [ 14 C]Etn for 30 min. After removal of the labeled precursor, slices were exposedto0.38UÆmL )1 sphingomyelinase or two different C 2 -Cer concentrations for 30 min. Total lipids were split into two aliquots: one was untreated, and the other was exposed to HCl fumes. Radioactivity in plasmalogen was obtained by subtracting the acid-resistant fraction from that obtained in the total Etn phospholipids. Data are expressed as the percentage of radioactivity incorporated in each fraction with respect to that incorporated in total lipid. They represent mean ± SD from one representative experiment of two experiments performed in quintuplicate. ND, Not determined. Treatment Radioactivity incorporated into Etn phospholipids [ 14 C]Arachidonic acid [ 14 C]Etn Acid-resistant fraction Plasmalogen fraction Acid-resistant fraction Plasmalogen fraction Control 1.78 ± 0.32 0.62 ± 0.09 70.8 ± 1.7 9.20 ± 2.71 Sphingomyelinase 2.06 ± 0.10 0.06 ± 0.02* 67.3 ± 2.1 5.32 ± 1.50* C 2 -Cer 100 l M 1.65 ± 0.40 0.42 ± 0.04* 76.3 ± 3.1 4.62 ± 0.93* 50 l M 1.62 ± 0.52 0.34 ± 0.06* ND ND * P < 0.05 compared with respective control. Ó FEBS 2003 Activated plasmalogen hydrolysis by Cer (Eur. J. Biochem. 270)39 radioactivity had increased by nearly 30% after treatment with sphingomyelinase or C 2 -Cer. Identification of the phospholipase type involved in the Cer-elicited decrease in Etn plasmalogen levels To determine the type of enzymatic activity involved, we next measured the levels of the breakdown products released by phospholipase type D or C, i.e. Etn, PEtn, and diacylglycerol. In addition, an intermediary of their biosynthesis, CDP-Etn, was measured (Fig. 2A,B). It is evident that no significant alterations were elicited by sphingomyelinase treatment. The involvement of a PLA 2 was tested by examining potential alterations in levels and [ 14 C]Etn radioactivity in the lyso form of Etn plasmalogens evoked by sphingomyelinase or C 2 -Cer (Fig. 2C,D, respect- ively). Surprisingly, no significant changes were found in the presence of 0.38 UÆmL )1 sphingomyelinase (Fig. 2C). How- ever, in a dose–response study with C 2 -Cer as agonist, a significant increase in the level of and radioactivity in lyso-Etn plasmalogens could only be observed in the presence of 100 l M C 2 -Cer (Fig. 2D). Before definitely establishing whether a PLA 2 was the Cer target, we next examined the effect of the widely used nonspecific PLA 2 inhibitor quinacrine [4] on the sphingo- myelinase-elicited effect (Fig. 3). Quinacrine alone (25 l M ) did not alter the 14 C radioactivity from [1- 14 C]arachidonic acid in the Etn plasmalogens, but, in the presence of sphingomyelinase, it not only prevented the decrease caused by sphingomyelinase, but also evoked a significant (P < 0.05) increase in the 14 C radioactivity found in Etn plasmalogens. This led us to hypothesize that the target enzyme for Cer action may be the 39 kDa plasmalogen-selective PLA 2 described and characterized previously [3,5,12]. The enzyme is specifically and markedly inhibited by sialic acid, glucos- aminoglucans, gangliosides and sialoglycoproteins [3,5]. In contrast, the brain 110 kDa cytosolic PLA 2 , acting prefer- entially on PtdEtn, has been reported to be much less sensitive to these inhibitory effects [3,5,12]. These differences in behavior prompted us to test the effect of sphingomye- linase on slices pretreated with a brain ganglioside mixture. The ganglioside mixture did not itself evoke significant variation in either 14 C radioactivity or levels of PtdEtn, but did prevent the decrease in radioactivity in, and levels of, Etn plasmalogens caused by sphingomyelinase (Fig. 3A,B). We also tested the effect of bromoenol lactone, a specific and potent inhibitor of myocardial Ca 2+ -independent plasmalogen-specific PLA 2 [28] devoid of effect on the brain plasmalogen-selective PLA 2 [4,9]. Pretreatment with bromoenol lactone did not block the effect of sphingo- myelinase on Etn plasmalogen levels (Fig. 3B). Therefore, our results are in agreement with those reported for the brain enzyme [4,9]. A first attempt was made to establish whether the sphingomyelinase-sensitive PLA 2 acting on Etn plasmalo- gens also shows CoA-independent transacylase activity. For this, we used ET-18-OCH 3 , a specific inhibitor [29]. ET-18- OCH 3 (25 l M ) itself did not modify either 14 C radioactivity in, or levels of, Etn plasmalogens (Fig. 3A,B, respectively). However, when ET-18-OCH 3 was added before sphingo- myelinase, the effect of sphingomyelinase on the Etn plasmalogens was prevented (Fig. 3A,B). In agreement with our previous report [2], we first observed a significant (P < 0.05) sphingomyelinase-elicited increased production of 1-O-[1- 14 C]acylCer (Table 4), which can be used as an index of transacylase activity [16]. It is also evident that an increase in the level of 14 C radioactivity in the nonesterified fatty acid fraction was concomitantly evoked by sphingo- myelinase. Interestingly, the ganglioside mixture (0.26 gÆL )1 ) and ET-18-OCH 3 (25 l M ) both completely prevented both these sphingomyelinase-evoked effects. Mechanism by which Cer decreases Etn plasmalogens levels Cer has been reported to activate okadaic acid-sensitive protein phosphatase 2A. To test whether this protein phosphatase is involved in the Cer effect, we treated brain Table 2. Variations in [ 14 C]arachidonic acid-labeled Etn phospholipid subclasses evoked by sphingomyelinase and C 2 -Cer. Slices were labeled with 0.2 lCiÆmL )1 [ 14 C]arachidonic acid for 120 min. After removal of the labeled precursor, slices were exposed to 0.38 UÆmL )1 sphingo- myelinase or 100 l M C 2 -Cer for 30 min. Total Etn phospholipids were hydrolyzed with phospholipase C. The resulting diacylglycerols were extracted and the acetylated derivatives were prepared. After their fractionation by TLC, the radioactivity in each was measured. Data are expressed as radioactivity incorporated (d.p.m.) in each subclass per mg of total lipids. They represent mean ± SD from one repre- sentative experiment of two experiments performed in triplicate. Treatment Radioactivity incorporated into Etn phospholipid subclasses Alkenylacyl Alkylacyl Diacyl Control 54.8 ± 7.2 49.3 ± 1.7 52.5 ± 7.2 Sphingomyelinase 38.8 ± 3.2* 43.8 ± 3.6 39.4 ± 5.1* C 2 -Cer 33.1 ± 4.1* 48.9 ± 0.8 30.1 ± 4.1* * P < 0.05 compared with their respective controls. Table 3. Variations in the extracellular and cell-associated radioactivity from [ 14 C]arachidonic acid evoked by sphingomyelinase and C 2 -Cer. Slices were labeled with 0.2 lCiÆmL )1 [ 14 C]arachidonic acid (AA) for 120 min. After removal of the labeled precursor, slices were exposed to 0.38 UÆmL )1 sphingomyelinase or 100 l M C 2 -Cer for 30 min. Aliquots (50 lL) from the incubation medium were taken for radioactivity measurement. Tissue total lipids were extracted, dissolved, and aliqu- ots (10 lL) were taken for radioactivity measurement. Extracellular arachidonic acid is expressed as d.p.m. per aliquot and cell-associated arachidonic acid as d.p.m. per mg total lipids. Data represent mean ± SD from one representative experiment of two experiments performed in triplicate. Treatment Radioactivity incorporated Extracellular AA Cell-associated AA Control 1030 ± 181 24266 ± 5488 Sphingomyelinase 892 ± 158 31061 ± 2575* C 2 -Cer 942 ± 206 29611 ± 2455* * P < 0.05 compared with their respective controls. 40 E. Latorre et al.(Eur. J. Biochem. 270) Ó FEBS 2003 slices with okadaic acid (2.5 and 25 n M ) before sphingo- myelinase treatment (Fig. 4). Okadaic acid alone produced no change in Etn plasmalogen levels (Fig. 4A) but did prevent the effect of sphingomyelinase treatment on Etn plasmalogen levels. Data on Cer levels are shown in Fig. 4B. Sphingomye- linase increased the level of endogenous Cer by nearly 100% (P < 0.05). Okadaic acid by itself did not alter Cer levels. However, when slices were pretreated with okadaic acid, the sphingomyelinase-elicited increase was prevented. There- fore, okadaic acid was acting as a modulator of Cer metabolism but not of the Cer-evoked effect. On the other hand, Cer has been reported to induce mitogen-activated protein kinase (MAPK) activity, which in turn phosphorylates and activates cytosolic PLA 2 [1]. PD 98059 has been widely used as a specific inhibitor to study whether p42/p44 MAPK is downstream of Cer generation. Experiments with PD 98059 were therefore performed (Fig. 5). At concentrations ranging from 10– 100 l M , PD 98059 significantly (P < 0.05) increased Cer levels in a dose-dependent manner (Fig. 5B). Concomit- antly, Etn plasmalogen levels decreased by about 75%, in a dose-independent manner (Fig. 5A). Unexpectedly, PD 98059 was able to prevent the sphingomyelinase-elicited increase in Cer levels (Fig. 5B) and partially reverse the Cer- evoked reduction in Etn plasmalogen levels (Fig. 5A). It has been shown that specific protease activation is a pivotal element in Cer-regulated processes. Thus, Cer acts downstream of caspase-8 but upstream of caspase-3 [27]. In addition, a serine proteolytic enzyme is also a Cer target [30]. Therefore, iodoacetamide (as a thiol-specific inhibitor) and phenylmethanesulfonyl fluoride (as a blocking agent of serine enzymes) were tested (Fig. 6). Neither iodoacetamide nor phenylmethanesulfonyl fluoride by themselves affected basal Cer (Fig. 6B) or Etn plasmalogen (Fig. 6A) levels. However, both inhibitors were able to prevent the Cer effect on Etn plasmalogen levels (Fig. 6A) without modifying the enhanced Cer levels (Fig. 6B). In view of these results, we next explored whether caspase-3 is involved in the regulation of plasmalogen- selective PLA 2 . Experiments with the cell-permeable caspase-3-specific tetrapeptide inhibitor Ac-DEVD-CMK were performed. The Ac-DEVD-CMK concentration used has been shown to inhibit apoptosis induced by 30 l M C 2 -Cer and caspase-3 activity [27]. The results obtained are shown in Table 5. The caspase-3 inhibitor by itself did not produce any effect, but partially prevented the sphingo- myelinase-elicited decrease in Etn plasmalogen levels with- out affecting Cer levels. Etn plasmalogen hydrolysis can also be elicited by endogenous agonists We have previously reported that the neuropeptide endo- thelin-1 is able to evoke Cer production in cerebral cortex [18]. Therefore, we next hypothesized that Etn plasmalogen hydrolysis may occur concomitantly with endogenous Cer production evoked by a natural agonist. Table 6 shows that treatment with 0.1 l M endothelin-1 for 30 min (conditions under which maximum Cer production is evoked by Fig. 2. Variations in breakdown products of Etn phospholipid evoked by sphingomyelinase (SMase) and C 2 -ceramide. (A) Sphingomyelinase-evoked variations in [ 14 C]Etn-labeled water-soluble metabolites; (B) sphingomyelinase-evoked variations in levels of total diacylglycerols; (C) sphingo- myelinase-evoked variations in [ 14 C]Etn radioactivity (left axis; open bars) and in levels of 1-O-alkenyl-2-lyso-GroPEtn (right axis; filled bars); (D) C 2 -ceramide-evoked variation in [ 14 C]Etn radioactivity (left axis; open bars) and in levels of 1-O-alkenyl-2-lyso-GroPEtn (right axis; filled bars). Cerebral cortex slices were prelabeled with 2 lCiÆmL )1 [ 14 C]Etn for 30 min (A, C and D) and then exposed to either 0.38 UÆmL )1 sphingomyelinase or different C 2 -ceramide concentrations for 30 min. Levels of, and the radioactivity in, the metabolites were determined. Data represent mean ± SE from two separate experiments performed in quintuplicate. Values significantly different from their respective controls are indicated: *P<0.05. Ó FEBS 2003 Activated plasmalogen hydrolysis by Cer (Eur. J. Biochem. 270)41 endothelin-1) resulted in a significant (P < 0.05) decrease ( 35%) in Etn plasmalogen levels, concomitantly with an increase of 60% in the Cer level. Discussion Involvement of the brain plasmalogen-selective PLA 2 in the Cer-elicited decrease in Etn plasmalogen levels We have previously shown that sphingomyelinase decreases the levels of brain Etn plasmalogens [2]. To rule out the possibility that Etn plasmalogens are directly hydrolyzed by sphingomyelinase, experiments with C 2 -Cer were per- formed. The effect of sphingomyelinase on Etn plasmalogen levels was mimicked by C 2 -Cer. Although several differen- tial effects of sphingomyelinase and Cer analogs have been described [26], we conclude that the decrease in Etn plasmalogen levels is a response, at least in part, to endogenous Cer accumulation. The complete prevention of the Cer effect caused by quinacrine and gangliosides, combined with the lack of effect exhibited by bromoenol lactone, led us to think that the enzyme involved is the 39 kDa plasmalogen-selective PLA 2 [4,9,12]. That other Etn phospholipids besides plasmalogens are affected is consistent with the specificity shown by the brain 39 kDa plasmalogen-selective PLA 2 [12]. In addition, we also observed that there was no loss of sphingomyelinase-elicited extracellular arachidonate or its derivatives, which precludes the involvement of a secretory PLA 2 . Two additional findings are noteworthy. First, the arachi- donate-rich pool of Etn plasmalogens is appreciably affected by Cer (Tables 1 and 2). The docosahexaenoate-rich pool of Etn plasmalogens is the other major pool of brain Etn plasmalogens [7]andthereforeit wouldbeinterestingtostudy it further. Secondly, plasmalogen hydrolysis by PLA 2 is coupled with CoA-independenttransacylaseactivity, as these processes are blocked in parallel by inhibitors of each (gangliosides and ET-18-OCH 3 ). This coupling has also been observed in other PLA 2 enzymes acting on alkenylacylglyc- erophospholipids of Etn, such as the 14 kDa PLA 2 present in monocytes [31], or on diacylglycerophospholipids of Etn, such as the 40 kDa PLA 2 of brain [16]. In fact, the latter enzyme is a single polypeptide chain with a molecular mass of  40 kDa, similar to that of the plasmalogen-selective PLA 2 described previously [12]. An interesting picture begins to emerge from the present evidence. However, it is necessary to consider several facts. Fig. 3. Effect of quinacrine (Q), ganglioside mixture (G), ET-18-OCH 3 (E) and bromoenol lactone (B) on sphingomyelinase (SMase)-induced alterations in brain Etn plasmalogens (PlsEtn). (A) Radioactivity from [ 14 C]arachidonic acid in Etn plasmalogens is expressed as the per- centage of radioactivity incorporated into these phospholipids with respect to that incorporated into total lipids. (B) Levels of Etn plas- malogens. Cerebral cortex slices were labeled with 0.2 lCiÆmL )1 [ 14 C]arachidonic acid for 120 min. After removal of the labeled pre- cursor, slices were incubated with 250 l M quinacrine for 25 min, 0.26 gÆL )1 ganglioside mixture for 2 min, or 10 l M bromoenol lactone for 10 min. Cerebral cortex homogenates were labeled as described above and exposed to 25 l M ET-18-OCH 3 for 2 min. They were then treated with 0.38 UÆmL )1 sphingomyelinase for 30 min. Respective controls were performed by incubating slices or homogenates in the presence of the respective solvents. Radioactivity and/or levels of Etn plasmalogens were measured. Data represent mean ± SE and are from two experiments performed in quintuplicate. Values significantly different from the control are indicated: *P <0.05. Table 4. Effect of plasmalogen-selective PLA 2 and CoA-independent transacylase inhibitors on the formation of 1-O-acylCer and release of free arachidonic acid evoked by sphingomyelinase. Slices were labeled with 0.2 lCiÆmL )1 [ 14 C]arachidonic acid for 120 min. After removal of the labeled precursor, slices were incubated with 0.26 gÆL )1 ganglioside mixture (G) for 2 min or with 25 l M ET-18-OCH 3 (E) for 2 min. Then, 0.38 UÆmL )1 sphingomyelinase was added for 30 min. Total lipids were fractionated by TLC, and the radioactivity in 1-O-acylCer and nonesterified fatty acid fractions was measured. Data are expressed as radioactivity incorporated (d.p.m.) in each fraction per mg total lipids. Data represent mean ± SD from one representative experiment of two experiments performed in quintuplicate. Treatment Radioactivity incorporated 1-O-AcylCer Nonesterified fatty acid Control 516 ± 107 9155 ± 609 Sphingomyelinase 732 ± 139* 13134 ± 2918* G 597 ± 85 9209 ± 1609 G + sphingomyelinase 383 ± 200 6354 ± 503 E 686 ± 148 9884 ± 1177 E + sphingomyelinase 472 ± 95 8364 ± 807 * P < 0.05 compared with their respective controls. 42 E. Latorre et al.(Eur. J. Biochem. 270) Ó FEBS 2003 First, in the plasma membrane of eukaryotic cells, the Etn-containing phospholipids reside in the inner leaflet whereas sphingomyelin is located in the outer leaflet. There is evidence that, during the early stages of apoptosis, this asymmetric distribution is lost, resulting in exposure of PtdEtn on the cell surface [32]. Thus, the potential activation of a membrane-associated neutral sphingomyelinase by apoptosis inducers would generate Cers that initiate a cascade of events, including the hydrolysis of Etn plasma- logens, which may be suitably positioned by a previous traslocation event. Secondly, Cer has been shown to be involved in oxidative stress through the production of mitochondrial oxygen-free radicals [33]. On the other hand, Etn plasmalogens are antioxidant molecules that protect cells from oxidative stress [6]. Cer-elicited hydrolysis of Etn plasmalogens could produce an increase in susceptibility to oxidative agents, leading to apoptosis. Thus, our data may indicate a new role for Cer in apoptosis. Caspase-3 is involved in the Cer-elicited decrease of Etn plasmalogen levels It has been previously reported that Cer does not affect purified plasmalogen-selective PLA 2 [12]. Therefore, our next experiments were designed to identify enzyme(s) downstream of Cer capable of regulating plasmalogen PLA 2 . We unexpectedly found that okadaic acid and PD 98059, used as inhibitors of protein phosphatase and MAPK, respectively, were able to modify the sphingomyelinase- enhanced or basal endogenous Cer levels. Consistent with this, complex modulation of Cer levels evoked by okadaic acid has been reported [34]. Although we cannot rule out the possibility that okadaic acid itself affects sphingomyelinase, it is very likely that the metabolic fate of Cer is affected. As the okadaic acid effect is evoked by concentrations as low as 2.5 n M , a protein phosphatase 2A may regulate Cer metabolism. Fig. 4. Effect of okadaic acid on basal and sphingomyelinase-altered Etn plasmalogen levels and ceramide accumulation. (A) Etn plasmalogen (PlsEtn) levels; (B) ceramide levels. Cerebral cortex slices were incu- bated in the absence or presence of 2.5 or 25 n M okadaic acid (OKA) for 10 min and then exposed to 0.38 UÆmL )1 sphingomyelinase (SMase) for 30 min. Etn plasmalogen and ceramide levels were obtained from the same tissue sample. Data represent mean ± SE from three experiments performed in quintuplicate. Values signifi- cantly different from the control are indicated: *P <0.05. Fig. 5. PD 98059-evoked effect on basal and sphingomyelinase-altered Etn plasmalogens levels and ceramide accumulation. (A) Etn plasma- logen (PlsEtn) levels; (B) ceramide levels. Cerebral cortical slices were incubated in the absence or presence of several concentrations of PD 98059 for 30 min and then exposed to 0.38 UÆmL )1 sphingo- myelinase (SMase) for 30 min. Etn plasmalogen and ceramide levels were obtained from the same tissue sample. Data represent mean ± SE from three experiments performed in quintuplicate. Val- ues significantly different from control are indicated: *P <0.05. Ó FEBS 2003 Activated plasmalogen hydrolysis by Cer (Eur. J. Biochem. 270)43 The effects of PD 98059 are even more complex, as the inhibitor increased the basal levels of Cer but prevented the sphingomyelinase-induced increase. This may be explained in terms of some MAPK members being upstream and/or downstream of Cer generation. As the decrease in Etn plasmalogens was still observed in its presence, it is likely that a Cer metabolite is also regulating the plasmalogen- selective PLA 2 . Studies are currently being carried out to clarify this point. Despite the complex mechanism of action of PD 98059, it is clear that the Cer-elicited activation of plasmalogen- selective PLA 2 is not mediated by the activation of p42/p44 MAPK. This is a characteristic that is not shared by the cytosolic PLA 2 from many cell types, including that from rat cerebral cortex [35]. Nevertheless, the possibility of the involvement of p38 MAPK remains open, as it is also a Cer target [1], and the regulation of cytosolic PLA 2 by this MAPK subfamily has been reported in other biological systems [36]. The potential involvement of some proteolytic step in the regulation of plasmalogen PLA 2 by Cer was also tested. First, we studied the action of thiol protease and serine protease inhibitors. Neither class of inhibitors was able to modify both basal and sphingomyelinase-enhanced Cer levels, but they did prevent the Cer-elicited lowering effect on Etn plasmalogen levels. One possible explanation is that there are proteases (or other enzymes) that contain serine or cysteine in their active center downstream of Cer that mediate the activation of the plasmalogen PLA 2 .This hypothesis is supported by evidence on the regulation of other types of PLA 2 by proteolytic cleavage phenomena [37]. Alternatively, it is possible that serine and cysteine residues are functionally important and/or are present in the catalytic site of the plasmalogen-selective PLA 2 . Consistent with this, it is well known that many esterases, including PLA 2 , are sensitive to the action of iodoacetate and phenylmethanesulfonyl fluoride. Furthermore, in studies with the purified plasmalogen-selective PLA 2 , preliminary evidence on its sensitivity to iodoacetate has been reported [3]. In contrast, it has been reported that any serine residue is essential for the transacylase reaction of the 40 kDa brain Fig. 6. Preventive effect of iodoacetamide and phenylmethanesulfonyl fluoride on the sphingomyelinase-elicited alterations in Etn plasmalogen levels and ceramide accumulation. (A) Etn plasmalogen (PlsEtn) levels; (B) ceramide levels. Cerebral cortex slices were pretreated with 10 m M iodoacetamide (I) or 2 m M phenylmethanesulfonyl fluoride (PMSF) for 60 min and then exposed to 0.38 UÆmL )1 sphingomyelinase (SMase) for 30 min. Etn plasmalogen and ceramide levels were obtained from the same sample. Data represent mean ± SE from two experiments performed in quintuplicate. Values significantly different from the control are indicated: *P < 0.05. Table 5. Effect of caspase-3 inhibitor on Etn plasmalogen hydrolysis and Cer accumulation evoked by sphingomyelinase. Cerebral cortex slices were preincubated with 50 l M Ac-DEVD-CMK for 60 min, then treated with 0.38 UÆmL )1 sphingomyelinase for 30 min. Total lipids were split into three aliquots: one was untreated, another was exposed to HCl fumes, and the other was hydrolyzed by alkali. They were fractionated by TLC, and the levels of Etn plasmalogens and Cer were measured. Data are expressed as nmol each fraction per mg total lipids. They represent mean ± SD from one representative experiment of two experiments performed in quintuplicate. Treatment Lipid fraction level Etn plasmalogens Cer Control 179.6 ± 26.3 96.4 ± 19.0 Sphingomyelinase 64.7 ± 10.3* 202.4 ± 37.2* Ac-DEVD-CMK 213.7 ± 44.5 145.7 ± 28.0 Ac-DEVD-CMK+ sphingomyelinase 132.9 ± 10.3* 227.3 ± 21.9* * P < 0.05 compared with their respective controls. Table 6. Effect of endothelin-1 (ET-1) on brain Etn plasmalogen and Cer levels. Slices were incubated with 0.1 l M endothelin-1 for 30 min. After total lipid extraction, Etn plasmalogen and Cer levels were measured. Data are expressed as nmol per mg total lipids. They are mean ± SD from one experiment performed in triplicate. Treatment Lipid fraction level Etn plasmalogens Cer Control 125.1 ± 31.1 96.4 ± 15.0 ET-1 77.5 ± 15.5* 154.2 ± 23.0* * Significantly different (P < 0.05) from the control value. 44 E. Latorre et al.(Eur. J. Biochem. 270) Ó FEBS 2003 transacylase. These contradictory observations remain to be clarified. Of more interest was the fact that the caspase-3-like protease-specific inhibitor Ac-DEVD-CMK could partially abolish Cer-elicited Etn plasmalogen hydrolysis without altering sphingomyelinase-elicited Cer accumulation. Sev- eral PLA 2 enzymes are substrates for caspase-3, but, depending on the type of PLA 2 , this cleavage leads to their inactivation (in the case of the cytosolic PLA 2 ,typeIV, without arachidonate-phospholipid remodeling activity) or activation (in the case of the Ca 2+ -independent PLA 2 ,type VI, with arachidonate-phospholipid remodeling activity) [37]. As the brain plasmalogen-selective PLA 2 is Ca 2+ - independent [4], and has been shown here to have CoA- independent transacylase activity, its potential activation by caspase-3 is consistent with the available evidence. Further- more, it has been suggested that activation of hydrolysis of Etn phospholipids by PLA 2 results from the covalent modification of the enzyme [38]. In an attempt to establish the potential pathophysiolo- gical significance of the present findings, we made a preliminary study to determine whether a natural Cer-generating agonist, such as the neuropeptide endothe- lin-1 [18], can modify Etn plasmalogen levels in brain tissue. The positive evidence obtained suggests that the present findings can be extrapolated to in vivo conditions. Further studies in this field are currently being performed in our laboratory. We may tentatively conclude that the findings reported here are relevant to the knowledge of some processes in Alzheimer’s disease and cerebral ischemia, despite the fact that some aspects still remain unclear. Activated caspase-3 has been in situ-immunodetected in only a small subpopulation of hippocampal neurons, but not in the cortex in patients with Alzheimer’s disease [39]. In addition, amyloid beta peptide can activate caspase-3 and induce neuronal apoptosis in vitro [39].Furthermore,ithas been suggested [9] that stimulation of the Ca 2+ -independent plasmalogen-selective PLA 2 may account for the decreased levels of Etn plasmalogens found in the affected regions of the brain in Alzheimer’s disease, such as the cerebral cortex. It is feasible that an early and reversible activation of caspase-3 by endogenous Cer may be sufficient to produce an irreversible loss of Etn plasmalogens. The picture in other pathological states is clearer. Brain sections from patients with neuropathological evidence of apoptosis secondary to stroke, seizure or trauma exhibit neuronal-activated caspase-3 immunoreactivity [39]. Acti- vation of a brain Ca 2+ -independent PLA 2 acting on PtdEtn [38] and a decrease in Etn plasmalogen levels [6,40] have been reported to occur in ischemia. 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Pilar. downstream of Cer that mediate the activation of the plasmalogen PLA 2 .This hypothesis is supported by evidence on the regulation of other types of PLA 2 by proteolytic