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.. Taking
Trang 1Signaling events mediating activation of brain ethanolamine
plasmalogen hydrolysis by ceramide
Eduardo Latorre1, M Pilar Collado1, Inmaculada Ferna´ndez1, M Dolores Aragone´s1
and R Edgardo Catala´n2
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 A2 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 A2
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 A2
Keywords: brain ethanolamine plasmalogens; caspase-3; ceramide; phospholipase A2; plasmalogen-selective phos-pholipase A2
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)
Trang 2in the metabolism of brain Etn plasmalogens in
physio-pathological states The existence of a
plasmalogen-select-ive phospholipase A2(PLA2, 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 PLA2 enzymes and brain PLA2
enzymes [3,4] There is evidence that Etn plasmalogen
degradation by a PLA2 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, PLA2in the presence of a suitable acceptor molecule
possesses a dual enzymatic function, i.e PLA2 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 PLA2[4,5], (b)
a brain PLA2acting 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 (C2-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
[1-14C]Arachidonic acid (55 mCiÆmmol)1) was from
American Radiolabeled Chemicals Inc., St Louis, MO,
USA [c-32P]ATP (3000 CiÆmmol)1) was from Nuclear
Iberica, Madrid, Spain [2-14C]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-OCH3) 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 22–24C and 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 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, 10 mM Hepes, pH 7.4, equilibrated with 95% O2/5% CO2 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 mM phosphate buffer,
pH 7.4, with 50% (v/v) glycerol, or C2-Cer dissolved in dimethyl sulfoxide (10–100 lM), or diluents alone were added and the mixture incubated for 30 min at 37C [2]
In one set of experiments, slices were treated with 0.1 lM 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 C2-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%
O2/5% CO2 The incubations were stopped by removal of the medium and replacement with 0.38 mL BSS containing
10 mMEDTA and 1 mL chloroform/methanol/13MHCl (100 : 100 : 1, v/v/v) Lipids were immediately extracted as described below
As we used an inhibitor (ET-18-OCH3) with low diffusion through slices, some experiments with homogen-ates were performed Homogenhomogen-ates 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) [1-14C]arachidonic acid [2] or 50 lCi (2 lCiÆmL)1) [2-14C]ethan-1-ol-2-amine hydrochloride [19] at 37C in BSS for 120 or 30 min, respectively The preincubations were continuously gassed with 95% O2/5% CO2 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 C2-Cer; incubations were stopped as described above
Trang 3Extraction of total lipids; separation of sphingolipids
Lipids were extracted as described previously [20] The
organic phases were dried under a N2atmosphere, 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 0.1M
metha-nolic KOH at 37C 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 N2
atmosphere for subsequent quantitation
Analysis of the subclasses of Etn phospholipids
Etn plasmalogen levels were determined as described
previously [2]
In some experiments with [14C]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 Rfvalues 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 lg of
the enzyme and 10 mM [c-32P]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 N2and the dried lipids were solubilized and phosphorylated in the presence of 5 lg enzyme and 10 mM[c-32P]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 [14C]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/NH4OH (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-lysoGroPEtn radioactivity
Aliquots of total lipids from experiments performed with [14C]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 C2-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
Trang 4to 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 C2-Cer on Etn plasmalogen levels The concentration range of C2-Cer was chosen on the basis
of previous evidence [26,27] A concentration of 50 lMwas 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 C2-Cer affect Etn phosphoglyceride metabolism, we carried out labeling studies with [1-14C]arachidonic acid and [1-14C]Etn (Table 1) Sphingomyelinase and C2-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 [1-14C]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 [14C]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 C2-Cer
Lipids were extracted [20] from one aliquot of incuba-tion medium, and 14C radioactivity was determined This provides a measure of activation of secretory PLA2 Results
in Table 3 show that extracellular [1-14C]arachidonate release was not affected, but the cell-associated 14C
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[14C]arachidonic acid for 120 min or 2 lCiÆmL)1[14C]Etn for 30 min After removal of the labeled precursor, slices were exposed to 0.38 UÆmL)1sphingomyelinase 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
Acid-resistant fraction
Plasmalogen fraction
Acid-resistant fraction
Plasmalogen fraction
C 2 -Cer
* P < 0.05 compared with respective control.
Trang 5radioactivity had increased by nearly 30% after treatment
with sphingomyelinase or C2-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 PLA2
was tested by examining potential alterations in levels and
[14C]Etn radioactivity in the lyso form of Etn plasmalogens
evoked by sphingomyelinase or C2-Cer (Fig 2C,D,
respect-ively) Surprisingly, no significant changes were found in the
presence of 0.38 UÆmL)1sphingomyelinase (Fig 2C) How-ever, in a dose–response study with C2-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 lMC2-Cer (Fig 2D)
Before definitely establishing whether a PLA2was the Cer target, we next examined the effect of the widely used nonspecific PLA2 inhibitor quinacrine [4] on the sphingo-myelinase-elicited effect (Fig 3) Quinacrine alone (25 lM) did not alter the14C radioactivity from [1-14C]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 the14C radioactivity found in Etn plasmalogens
This led us to hypothesize that the target enzyme for Cer action may be the 39 kDa plasmalogen-selective PLA2 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 PLA2, 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 either14C 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 Ca2+-independent plasmalogen-specific PLA2 [28] devoid of effect on the brain plasmalogen-selective PLA2[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 PLA2 acting on Etn plasmalo-gens also shows CoA-independent transacylase activity For this, we used ET-18-OCH3, a specific inhibitor [29] ET-18-OCH3(25 lM) itself did not modify either14C radioactivity
in, or levels of, Etn plasmalogens (Fig 3A,B, respectively) However, when ET-18-OCH3was 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-[1-14C]acylCer (Table 4), which can be used as an index of transacylase activity [16] It is also evident that an increase in the level of14C radioactivity in the nonesterified fatty acid fraction was concomitantly evoked by sphingo-myelinase Interestingly, the ganglioside mixture (0.26 gÆL)1) and ET-18-OCH3(25 lM) 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 [14C]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
Sphingomyelinase 38.8 ± 3.2* 43.8 ± 3.6 39.4 ± 5.1*
* 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[14C]arachidonic acid (AA) for
120 min After removal of the labeled precursor, slices were exposed to
0.38 UÆmL)1sphingomyelinase 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
* P < 0.05 compared with their respective controls.
Trang 6slices with okadaic acid (2.5 and 25 nM) 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 PLA2 [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 lM, 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 PLA2 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 lM
C2-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 lMendothelin-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 [14C]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[14C]Etn for 30 min (A, C and D) and then exposed to either 0.38 UÆmL)1sphingomyelinase
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.
Trang 7endothelin-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 PLA2
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 C2-Cer were per-formed The effect of sphingomyelinase on Etn plasmalogen levels was mimicked by C2-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 PLA2 [4,9,12] That other Etn phospholipids besides plasmalogens are affected is consistent with the specificity shown by the brain 39 kDa plasmalogen-selective PLA2 [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 PLA2
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] and therefore it would be interesting to study
it further Secondly, plasmalogen hydrolysis by PLA2 is coupled with CoA-independent transacylase activity, as these processes are blocked in parallel by inhibitors of each (gangliosides and ET-18-OCH3) This coupling has also been observed in other PLA2enzymes acting on alkenylacylglyc-erophospholipids of Etn, such as the 14 kDa PLA2present in monocytes [31], or on diacylglycerophospholipids of Etn, such as the 40 kDa PLA2 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 PLA2 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
[14C]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)1ganglioside 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)1sphingomyelinase 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[14C]arachidonic acid for 120 min After removal of the labeled precursor, slices were incubated with 0.26 gÆL)1ganglioside mixture (G) for 2 min or with 25 l M ET-18-OCH 3 (E) for 2 min Then, 0.38 UÆmL)1sphingomyelinase 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
G + sphingomyelinase 383 ± 200 6354 ± 503
* P < 0.05 compared with their respective controls.
Trang 8First, 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 PLA2 [12] Therefore, our next experiments were designed to identify enzyme(s) downstream of Cer capable of regulating plasmalogen PLA2
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 nM, 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.
Trang 9The 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 PLA2 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 PLA2is not mediated by the activation of p42/p44
MAPK This is a characteristic that is not shared by the
cytosolic PLA2from 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 by this
MAPK subfamily has been reported in other biological systems [36]
The potential involvement of some proteolytic step in the regulation of plasmalogen PLA2 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 PLA2 This hypothesis is supported by evidence on the regulation of other types of PLA2 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 PLA2 Consistent with this, it is well known that many esterases, including PLA2, are sensitive to the action of iodoacetate and phenylmethanesulfonyl fluoride Furthermore, in studies with the purified plasmalogen-selective PLA2, 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)1sphingomyelinase 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
Ac-DEVD-CMK+
sphingomyelinase
* 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
* Significantly different (P < 0.05) from the control value.
Trang 10transacylase 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 PLA2 enzymes are substrates for caspase-3, but,
depending on the type of PLA2, this cleavage leads to their
inactivation (in the case of the cytosolic PLA2, type IV,
without arachidonate-phospholipid remodeling activity) or
activation (in the case of the Ca2+-independent PLA2, type
VI, with arachidonate-phospholipid remodeling activity)
[37] As the brain plasmalogen-selective PLA2 is Ca2+
-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 PLA2 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, it has
been suggested [9] that stimulation of the Ca2+-independent
plasmalogen-selective PLA2may 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 Ca2+-independent PLA2acting on PtdEtn
[38] and a decrease in Etn plasmalogen levels [6,40] have
been reported to occur in ischemia
It is noteworthy that other Ca2+-independent PLA2
enzymes acting on plasmalogens from non-neural sources
have been described [3] Whether these are also regulated by
Cer remains an open question; the answer may provide
evidence for cross-talk phenomena between the
sphingo-myelin cycle and PLA2-mediated arachidonate metabolism
[30]
In summary, our data show, for the first time, that brain
Ethanolamine plasmalogen hydrolysis is regulated by the
endogenous level of Cer, and a caspase-3-like protease is a
downstream Cer effector
Acknowledgements
We are indebted to Mrs M V Mora Gil and Mrs Belinda Benhamu´ This work was funded by grants from the DGICYT and the Fundacio´n
‘Ramo´n Areces’.
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