Veratridine-Induced Depolarization Reduces the Palmitoylation of Brain and Myelin Glycerolipids

Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1998 International Society for Neurochemistry

Veratridine-Induced Depolarization Reduces thePalmitoylation of Brain and Myelin Glycerolipids

Paul Sanchez, Sabine U. Tetzloff, and Oscar A. Bizzozero

Department of Cell Biology and Physiology, University of New Mexico, School of Medicine,Albuquerque, New Mexico, U.S.A.

Abstract: In this study, we have investigated the effectof neuronal depolarization on the palmitoylation of myelinlipids. For this purpose, brain slices from 60-day-old ratswere incubated with [3H]palmitatefor 1 h in the presenceor absence of various drugs. Veratridine (100

1sM) re-duced the incorporation of [

3H]palmitateinto all brainglycerolipids by 40—50%, whereas the labeling of sphin-golipids was unaffected. Similar results were obtainedby using ~H]glycerolas a precursor, demonstrating thatveratridine also causes a reduction in thede novo synthe-sis of glycerolipids. Both tetrodotoxin (1 ~.sM)and ouabain(1 mM) prevented the effect of veratridine, indicating thatit is mediated through the opening of voltage-gated so-dium channels and involves the stimulation of the Na~/K~pump. Decreased levels of both ATP, due to activationof the Na~,K~-ATPase,and the precursor palmitoyl-CoAwere found in the veratridine-treated slices, thus ex-plaining the reduction in lipid synthesis. Neuronal depo-larization also decreased the synthesis of lipids presentin the myelin fraction. The relatively high specific radioac-tivity of myelin lipids and the results from both repeatedpurification experiments and mixing experiments ruledout the possibility that the radioactive lipids present inmyelin could derive from contamination with other sub-cellular fraction(s). Because neither mature oligodendro-cytes nor myelin is known to express voltage-dependentNa~channels, it is conceivable that the effect of veratri-dine on myelin glycerolipid metabolism occurs by an indi-rect mechanism such as an increase in the extracellular[K~].However, the presence of 60 mM KCI in the mediumdid notaffect the acylation of either brain ormyelin lipids.These results raise questions as to the absence of sodiumchannels in myelinating oligodendrocytes and/or myelin.Key Words: Veratridine—Glycerolipids—Depolariza-tion—Myelin lipids—Na~,K~-ATPase.J. Neurochem. 70, 1448—1457 (1998).

Over the past few years, it has become increasinglyclear that CNS myelin has a substantial amount ofmetabolic activity. This view has emerged as the resultof the unambiguous demonstration that myelin con-tains enzymes involved in lipid metabolism, proteindegradation, ion transport, and the dynamic posttrans-lational modifications of proteins (for review, see Led-

een, 1992). Myelin is also endowed with muscariniccholinergic receptors linked to phosphoinositide phos-phodiesterase (Larocca et al., 1987a; Kahnand Morell,1988) and adenylate cyclase (Larocca et al., 1987b),suggesting that the formation and/or maintenance ofthis membrane may require specific neuronal signals.This has been reinforced by the finding that oligoden-drocytes, the CNS myelin-producing cells, expressneuroligand receptors linked to cyclic AMP metabo-lism (Vartanian et al., 1988) and phosphoinositidehydrolysis (Kastritsis et al., 1992), ion channels (Ket-tenmann et al., 1984), and neurotransmitter uptake sys-tems (Reynolds and Herschkowitz, 1986). Further-more, He et al. (1996) have recently shown that axonalcontact and axonal activity contribute to the mainte-nance of functional signaling pathways in oligodendro-cytes.

One of the metabolically active processes occurringin myelin is the phosphorylation and dephosphoryla-tion of myelin basic protein (DesJardins and Morell,1983), which appear to be regulated by axonal depolar-ization (Murray and Steck, 1983). The other dynamicprocess that takes place in myelin is the palmitoylationof the hydrophobic proteolipid protein (PLP) (Bizzo-zero and Good, 1991). Because PLP acylation and my-elm basic protein phosphorylation have many featuresin common (Bizzozero and Good, 1991), we thoughtthat it would be of considerable interest to determinewhether protein palmitoylation is also regulated by ax-onal depolarization. Thus, our laboratory embarked ona study that systematically tested the effect of a number

Received July 22, 1997; revised manuscript received October 10,1997; accepted November 20, 1997.

Address correspondence and reprint requests to Dr. O. A. Bizzozeroat Department of Cell Biology and Physiology, University of NewMexico, School of Medicine, Basic Medical Sciences Building, 914Camino de Salud, Albuquerque, NM 87131-5218, U.S.A.

Abbreviations used: BSA, bovine serum albumin; CER, cerebro-sides; DAG, diacylglycerols; DMEM, Dulbecco‘s modified Eagle‘smedium; FA, fatty acid(s); PA, phosphatidic acid; PC, phosphatidyl-choline; PE, phosphatidylethanolamine; PI, phosphatidylinositol;PLP, proteolipid protein; PS, phosphatidylserine; SPH, sphingomye-lin; TTX, tetrodotoxin.

1448

CHEMICAL DEPOLARIZATION AND LIPID ACYLATION 1449

of depolarizing agents, signal transduction modulators,and neurotransmitters on the palmitoylation of PLP inrat brain slices. In these experiments, the incorporationof tritiated palmitate into brain lipids was also mea-sured to establish whether a change in PLP palmitoyla-tion induced by a particular treatment was indeed spe-cific or it was just the result of drug-induced variationsin the size or specific radioactivity of the precursorpool. This control appeared reasonable because fattyacid (FA) incorporation into both PLP (Bizzozero andLees, 1986) and lipids (Waku, 1992) requires the cellu-lar uptake of FA and their subsequent activation toacyl-CoAs by the long-chain acyl-C0A synthetase.During the course of these studies, we found that mem-brane depolarization induced by the sodium channelagonist veratridine impaired the incorporation of ~3HI-palmitic acid into brain glycerolipids but not into PLP.The present study was therefore undertaken to deter-mine the causes for such an effect. In this article, weshow that the decreased glycerolipid palmitoylation isdue to the reduction of palmitoyl-C0A formation,which results from low levels of ATP. However, some-what to our surprise, veratridine-induced depolariza-tion also affected the acylation of myelin lipids, thusraising questions about the absence of sodium channelsin mature oligodendrocytes and/or myelin.

EXPERIMENTAL PROCEDURES

MaterialsVeratridine, nifedipine, phospholipase A

2 (Crotalouxatrox venom), and FA-free bovine serum albumin (BSA)were purchased from Sigma (St. Louis, MO, U.S.A.). Oua-bain was obtained from ResearchBiochemicals International(Natick, MA, U.S.A.). Tetrodotoxin (TTX) wasfrom Calbio-ehem (La Jolla, CA, U.S.A.). Dulbecco‘s modified Eagle‘smedium (DMEM) was from GIBCO Life Technologies(Grand Island, NY, U.S.A.). All other chemicals were ofthe highest purity available. [9,l0-

3H]Palmitic acid (60 Cilmmol) and [l,2,3-3H]glycerol (200 Ci/mol) were purchasedfrom Du Pont—New England Nuclear (Boston, MA, U.S.A.)and used without further purification.

Incubation of brain slicesSixty-day-old rats of either sex were used throughout.

Housing and handling of the animals as well as the procedurefor killing the animals were in strict accordance with theNIH Guidefor the Care and Use of Laboratory Animals andwere approved by the Institutional Animal Care and UseCommittee. Animals were killed by decapitation; the brainswere rapidly removed and sliced in two directions at a rightangle in sections 400 jim thick. Slices were thoroughlywashed with ice-cold DMEM or Krebs—Ringer bicarbonatebuffer previously equilibrated with 95% 02/5% CO

2. Slicescorresponding to one-fourth brain were transferred to flaskscontaining 4 ml of either DMEM—high glucose buffer orKrebs—Ringer bicarbonate supplemented with 10 mM D-

glucose, and were incubated at 37°Cunder 95% 02/5% CO2.Incubation was started by addition of 100 jiCi [

3H]palmiticacid (60Ci/mmol) dissolved in 100 jil of incubation mediumcontaining 0.1% (wt/vol) BSA. Various drugs were addedat the beginning or during the incubation as indicated in the

figure legends. After incubation, slices were collected bylow-speed centrifugation and homogenized in 6 ml of 0.32M ice-cold sucrose. Aliquots of the homogenate were used toprepare myelin as described by Norton and Poduslo (1973).Finally, myelin membranes were suspended in 1 ml of ice-cold water and stored at —20°C.

Repurification of myelinIn some experiments, myelin was subjected to repeated

purification to ensure the complete removal of possible con-taminants. Myelin membranes isolated as described above(cycle 1) were suspended in 0.32 M sucrose and layered onto0.85 M sucrose. After centrifugation at 100,000 g for 1 h,the material at the interface was suspended in 30 ml of ice-cold water and membranes were collected by centrifugationat 12,000 g for 20 min (cycle 2). This procedure (i.e., centrif-ugation on a sucrose gradient and osmotic shock treatment)was repeated twice (cycles 3 and 4). Aliquots were taken ateach step for protein and lipid analysis.

Lipid analysisAliquots from the brain homogenate or myelin (300—450

jig ofprotein) were extractedwith chloroform/methanol (2:1,vollvol) containing 0.25% 12 M HC1 to ensure the completeextraction of acidic lipids. One-third of the lipid extract(100—150 jig of protein) was used to separate the majorphospholipid classes [phosphatidylcholine (PC), phosphati-dylethanolamine (PE), phosphatidylserine (PS), phosphati-dylinositol (PI), and sphingomyelin (SPH)] by TLC on silicagel G plates developed with chloroform/methanol/aceticacid/water (60:50:1:4, by volume). Another one-third of thelipid extract was used to analyze phosphatidic acid (PA),phosphatidylglycerol, and cerebrosides (CER) by TLC onsilica gel G, using two development steps in the same direc-tion. Plates were first developed with acetone/hexane (1:3,vol/vol) followed by chloroform/methanol/acetic acid/water(80:13:8:0.3, by volume) (Skipski et al., 1967). Neutral lipids[FA; and diacylglycerol (DAG)] were separated from theremaining one-third of the lipid extract by TLC on silica gelG plates, using benzene/ethyl ether/ethyl acetate/acetic acid(80: 10:10:0.2, by volume) as the developing solvent (Stonyand Tuckley, 1967). In all cases, lipids were detected withiodine vapors and were identified by the use of appropriatestandards. Toquantitate radioactivity, the spots were scrapedoff the plate and placed in vials containing 0.2 ml of water.Finally, 10 ml Scinti-Safe(Fischer Scientific, Fair Lawn, NJ,U.S.A.) were added and radioactivity was measured in aPackard-Tricarb 1600CA liquid scintillation analyzer.Counting efficiency for 3H was always better than 45%.Specific radioactivities were calculated as dpm per micro-gram of protein.

To determine theextent of FA interconversion, radioactivephospholipids were dissolved in 1.5 ml of chloroforml0.21M NaOH in methanol (2:1, vol/vol) and the solution wasleft standing overnight at room temperature. After alkalinemethanolysis, 0.3 ml of 0.35 M acetic acid was added andthe upper phase was discarded. The lower phase was washedtwice with 1 ml of methanol/water (1:1, vol/vol) and drieddown under a stream of N

2. The released methyl esters werecombined with 10 ~.tgof a mixture of saturated FA methylesters (C16.0—C24.0) and were separated by reversed-phaseTLC on KC-18 plat6s (Whatman, U.K.) developed twicewith acetone/methanol/water (80:20:10, by volume) (Biz-zozero, 1995). FA methyl esters were detected with iodinevapors, scraped off the plate, and counted.

J. Neurochem., Vol. 70, No. 4, 1998

1450 P. SANCHEZ ET AL.

The percentage of radiolabeled FA present at the sn-2position of PC was assessed by phospholipase A2 hydrolysis(Subbaiah et al., 1992) of the phospholipid isolated by TLC.PC was suspended in 2 ml of ethyl ether and was incubatedwith 100 jil of phospholipase A2 (250 U/mI) in Tris-HC1buffer, pH 8.9, at 25°C.After 30 mm, the sample was driedunder nitrogen, dissolved in 20 jil of chloroform/methanol(1:1, vol/vol) and analyzed by TLC as described for themajor phospholipid classes. Spots were visualized with io-dine vapor, and the bands corresponding to lyso-PC (RF

= 0.11) and FA (RF = 0.98) were scraped off the plate andcounted. The distribution ofradioactivity in CER containingnonhydroxy FA was determined as described by Karlsson(1970).

Fatty acyl-CoAs were extracted from an aliquot of thebrain homogenate with chloroform/methanol (1:1, vol/vol)and were separated from the rest of the lipids by TLC onsilica gel G plates developed twice with chloroform/metha-nol/water (65:25:4, by volume) and once with n-butanol/acetic acid/water (5:3:2, by volume) in the same direction(Olbrich et al., 1981). Spots were detected with 2,7-dichloro-fluorescein, and the band corresponding to fatty acyl-CoAs(RF = 0.44) was scraped off the plate and counted.

Additional proceduresTotal protein was determined by a modification of Low-

ry‘s procedure (Lees and Paxman, 1972), using BSA as stan-dard. L-Glycerol 3-phosphate in the brain homogenate wasenzymatically assayed by the glycerol 3-phosphate dehydro-genase method (Lang, 1988), except that the production ofNADH was measured fluorometrically. ATP was determinedby the luciferïn—luciferase procedure, using the ATP assaykit from Calbiochem (La Jolla, CA, U.S.A.) and a Chem-Glow J4-7441 photometer.

StatisticsResults were analyzed for statistical significance with Sm-

dent‘s unpaired t test and a one-way analysis of variance(ANOVA) using MIN1TAB DataAnalysis Software, release 1.1.

RESULTS

Veratridine reduces the incorporation of[3H]palmitate into brain and myelin glycerolipids

To examine the effect of neuronal depolarization onthe palmitoylation of lipids, brain slices were incubatedwith [3Hlpalmitate for 1 h in the presence or absenceof veratridine. This alkaloid opens the voltage-gatedNa~channels, causing sodium influx down its concen-tration gradient and membrane depolarization (Ul-bricht, 1969). As shown in Table 1, the presence of100 ‚uM veratridine in the incubation media decreasedthe incorporation of [3Hlpalmitate into all brain glycer-olipids, including the precursors DAG and PA, by al-most 50%. The possibility that veratridine could haveimpaired the cellular uptake of the radiolabeled FAwas essentially ruled out because labeling of CER andSPH was unaffected by the drug. Furthermore, the ra-dioactivity in the FA fraction from the stimulated tissuewas not significantly different from that from control.The effect of veratridine was mediated through theopening of voltage-gated sodium channels, because thedrug-induced reduction in lipid acylation was pre-

vented by the addition of 1 ‚uMTTX before the incuba-tion (Table I).

Veratridine-induced depolarization also reduced theincorporation of [3Hlpalmitate into myelin glyceroli-pids, suggesting that the effect of this alkaloid is notrestricted to neuronal lipids (Table 1). The magnitudeof this inhibition was similar to that of brain lipids, and,as before, the effect was prevented by TTX. Because ofthe high specific radioactivity of lipids in myelin rela-tive to the total homogenate, the likelihood that mostof the label in this fraction could originate from con-tamination with other subcellular membranes is rathersmall. Nonetheless, we tested this possibility by sub-jecting the myelin fraction to a four-cycle purificationprocedure, each consisting of an osmotic shock and asucrose-density gradient. As shown in Fig. I, the spe-cific radioactivity of PC and PB (expressed as dpm/jigof protein) increased during the repeatedpurification ofmyelin as protein-rich membranes of nonmyelin originwere removed. However, the difference between con-trol and veratridine-treated samples was preserved, in-dicating that the radioactive lipids in this fraction are,for the most part, of myelin origin. Further supportfor this conclusion came from a mixing experiment inwhich unlabeled myelin membranes were combinedwith a radiolabeled myelin-depleted brain homogenatebefore the subcellular fractionation. The specific radio-activity of lipids in myelin isolated from the mixturewas <20% than those in the original, labeled myelin.

Veratridine-induced depolarization alsodiminishes the synthesis of brain and myelinglycerolipids

In an attempt to clarify why veratridine reducesglycerolipid labeling, the acylation of PC, the majorradioactive lipid, was examined further. After 1 h ofincubation, >94% of the radioactivity associated withphospholipids was identified as [3H]palmitate, indicat-ing that during this period little FA interconversionoccurs. FA can be incorporated into brain glycerolipidsduring de novo synthesis (i.e., by sequential acylationof sn-glycerol 3-phosphate to form PA) or by theLands‘ deacylation—reacylation cycle (MacDonaldand Sprecher, 1991). However, the incorporation ofpalmitate, stearate, and other saturated FA into brainglycerolipids is known to take place mainly by de novosynthesis (Baker and Thompson, 1972). In our experi-ments, [3Hlpalmitic acid was incorporated into brainand myelin phospholipids at both sn-l and sn-2 posi-tions. In myelin PC, the percentage of radioactivity insn-l position (mean ±SE, n = 5) was 53.5 ±1.7 forcontrols, 53.7 ±0.5 for veratridine treated, and 53.2±0.9 for veratridine plus TTX. This suggests that thereduced incorporation of [3H]palmitate into PC in theveratridine-treated slices results from a decrease in thesynthesis of the entire phospholipid, rather than froman impaired acylation of lyso-PC.

As shown in Fig. 2A, the rate of [3H]palmitate incor-poration into brain PC augmented with time. This lag



TABLE 1. Effect of veratridine on the incorporation of [3H]palmitic acid into brain and myelin lipid classes

Myelin Total Homogenate

Lipid Control VeratridineVeratridine

+ TTX Control VeratridineVeratridine

+ ‘FIX

PCPEPSPIPADAGCERSPHFA

90.1 ±8.940.4 ±4.7

5.5 ±0.323.3 ±1.522.1 ±3.517.4 ±0.818.8 ±4.2

1.9 ±0.34,565 ±764

56.8 ±5.5“21.0 ±2.0°

3.5 ±0.2“14.2 ±2.2°12.0 ±1.9“9.4 ±0.7°

28.0 ±8.43.2 ±1.3

5,357 ±473

89.2 ±11.5“39.5 ±4.0“5.4 ±0.8“

22.8 ±3.9“18.8 ±1.9“17.2 ±1.4“

22.1 ±3.81.7 ±0.3

4,476 ±213

151.2 ±13.141.8 ±3.64.3 ±0.2

19.4 ±9.222.2 ±3.920.7 ±0.912.5 ±1.22.3 ±0.2

1,919 ±173

89.1 ±6.4“19.2 ± 1.9°2.2 ±0.8“

13.5 ±6.39.5 ±2.6°9.6 ±0.5“

11.6 ±2.02.2 ±0.3

2,437 ±258

156.0 ±10.5“41.9 ±2.2“4.4 ±0.3“

16.7 ±4.319.1 ±6.8“18.5 ±2.0“9.6 ±1.1°2.1 ±0.1

1,772 ±548

Brain slices were incubated in 4 ml of DMEM with 100 jiCi [3Hlpalmiticacid for I h in the absence or presence of 1001iM veratridine

and 100 jiM veratridine plus I 1jM TTX. After incubation, lipids from the brain homogenate and myelin were analyzed as described inExperimental Procedures. Data are expressed as dpm per microgram of protein and represent mean ±SEM values of four separate experiments.

Significantly different from control (p < 0.05).“Significantly different from veratridine treated (p < 0.05).

likely reflects the time required for the radiolabeledprecursors to attain a constant specific radioactivity,because it was not observed for PA and DAG, whichachieved maximal labeling between 10 and 20 min(not shown). In the presence of veratridine, the rate ofPC palmitoylation was already reduced at 10 min ofincubation, the first time point measured. At longertimes, the extent of the inhibition was approximatelythe same (40—50%), and in all cases the effect wasprevented by TTX. We next examined the possibilitythat the effect of veratridine could be due to an in-creased FA turnover by carrying out pulse—chase ex-periments. For this purpose, slices were incubated with[3H]palmitate for 1 h, after which the medium was

replaced with a new solution containing an excess ofunlabeled palmitate, and incubation was continued foranother hour (Fig. 2B). PC labeling reached a maxi-mum 10—20 min after the addition of the cold sub-strate, and it remained constant during the next 40 mm,

FIG. 1. Specific activity of myelin PC and PE during repeatedpurification of myelin. Myelin from control (U) and veratridine-treated slices (~)was repurified as described in ExperimentalProcedures. Data are the average values from two separate ex-periments.

suggesting that rapid FA exchange does not occur.Similar results were obtained when the chase was per-formed in the presence of either veratridine or veratri-dine plus TTX. Taken together, these data indicate thatveratridine decreases the labeling of PC, and probablythat of many other glycerolipids, by reducing its syn-thesis and not by increasing its deacylation.

We examined directly the hypothesis that decreasedde novo synthesis of glycerolipids accounted for re-duced palmitoylation in the presence of veratridine bydetermining the extent of {3H]glycerol incorporationinto phospholipids. As shown in Table 2, incorporationof this precursor into both myelin and brain phospho-lipids was significantly reduced by veratridine and theeffect was prevented by TTX.

Veratridine-induced depolarization reduces ATPand palmitoyl-C0A levels

The generalized reduction in glycerolipid synthesisindicates that glycerol 3-phosphate levels, palmitoyl-CoA levels, glycerol-3-phosphate acyltransferase ac-tivity, or all three are diminished in the slices incubatedwith veratridine. Because glycerolipid but not sphin-golipid acylation is affected by veratridine, a reducedconcentration of glycerol 3-phosphate was initiallyconsidered as the most likely cause for theeffect. How-ever, tissue levels of glycerol 3-phosphate increasedby C—30% in the presence of veratridine (Table 3),probably reflecting the acceleration of the glycolyticrate and the ensuing rise in the cytosolic [NADHI/[NAD~] ratio. In contrast, veratridine diminished theincorporation of [3Hlpalmitate into the FA-CoA frac-tion by >40%, and this change was prevented, to alarge extent, by TTX (Table 3). Because the half-life ofpalmitoyl-CoA inbrain is ~=7s (Grange et al., 1995), itis safe to assume that isotopic equilibration is achievedin just a few minutes. Therefore, changes in the amountof radioactivity present in the acyl-CoA fraction can

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FIG. 2. A: Time course of incorporation of [3H}palmiticacid intobrain PC. Brain slices were incubated with 100 jiCi [3H]palmiticacid in 4 ml of DMEM as described in Experimental Procedures.Incubation was stopped at the times indicated in the figure, brainlipids were analyzed by TLC, and radioactivity in PC was mea-sured by liquid scintillation counting. Each point represents themean ±SEM of three experiments. B: Pulse—chase experi-ments. Brain slices were incubated with 100 jiCi [3H]palmiticacid as described in Experimental Procedures. After 1 h, themedium was removed and replaced with 4 ml of DMEM con-taining 200 jiM unlabeled palmitic acid and 0.1% (wt/vol) BSA,and incubation continued for up to another 60 min. Radioactivityin brain PC was determined as before. Each point representsthe mean ±SEM of three experiments. °Controlvalues are sig-nificantly different (p <0.05) from veratridine values. bVeratridinevalues are significantly different (p < 0.05) from veratridine pluslix values. “Control values are significantly different (p <z 0.05)from veratridine plus lix values.

be equated to changes in the concentration of the FAdonor. The conclusion to be drawn from these resultsis that low levels of acyl-CoA, and not glycerol 3-phosphate, are responsible for the veratridine-inducedreduction of the de novo synthesis of glycerolipids weobserved before.

In the presence of 100 ‚uM veratridine, the concen-tration of ATPin brain slices was decreased by —~50%and this effect was prevented by TTX (Table 3).In synaptosomes, veratridine-evoked sodium intakecauses a fall in the ATP levels due to the enhancedactivity of the Na~,Kt.ATPase(EreciiIska and Da-gani, 1990). For this reason, we examined whetherinhibition of the Na‘,K~-ATPaseby the glycosideouabain (G-strophantin) could prevent the effect ofveratridine that we observed. In our experiments, a

concentration of ouabain of 1 mM was used to ensurethe complete blockage of the brain Na~/K~pump,which is inhibited by this drug with an IC

50 of — I‚uM (Sweadner, 1985). Addition of ouabain beforeveratridine brought the levels of ATP and the labelingof palmitoyl-C0A to normal values (Table 3). Further-more, this glycoside also prevented the effect of verat-ridine on the palmitoylation of brain (not shown) andmyelin glycerolipids (Table 4). The above results fa-vor the notion that the decrease in ATP levels causedby the intense Na~“/K~pump activity reduces the con-centration of brain palmitoyl-CoA, whose synthesisrequires ATP. Because ouabain, like veratridine, isa depolarizing agent that increases the intracellularsodium activity (Adam-Vizi and Ligeti, 1984), it isclear that the low levels of acyl-CoA and ATP, andneither membrane depolarization nor sodium influxper se, directly cause the reduction in lipid acylation.

The effect of veratridine is not dependent onextracellular Ca

2~Neither the removal of calcium from the incuba-

tion medium nor the presence of 50 ‚uM nifedipine,a voltage-gated L-type Ca2tchannel blocker (Janiset al., 1987), were capable of preventing the effectof veratridine on the palmitoylation of brain (notshown) and myelin glycerolipids (Table 4). Theseresults strengthen the concept that reduction of glyc-erolipid synthesis is not the result of a step that re-quires cellular uptake of calcium ions, such as trans-mitter release or protein phosphorylation, but is dueto a reduction in the concentration of ATP. It isnoteworthy that, although the activity of the Na~“/K~pump can be inhibited by calcium ions (Yingst,1988; Erecidska and Dagani, 1990), in our experi-ments the removal of calcium from the medium didnot enhance the effect of veratridine. Because of thefailure of the calcium-free medium to prevent theveratridine effect, the effect of w-conotoxins, whichblock the synaptosomal N-type Ca2~ channels(Feigenbaum et al., 1988), was not studied.

TABLE 2. Effect of veratridine on the incorporation of[3H]glycerol into myelin and brain phospholipids

Treatment

Specific radioactivity(dpmljig of protein)

Myelin Brain

Control 14.7 ± 1.1 5.3 ±0.5Veratridine (100 jiM)Veratridine (100 jiM)

+ ‘FIX (I jiM)

10.2 ±0.5“ 2.9 ±0.4“

16.3 ±1.2“ 4.7 ±0.4“

Brain slices from 60-day-old rats were incubated with 50 jiCi[3Hlglycerol for 1 h in the absence or presence of various com-pounds. After incubation, the radioactivity present in myelin andbrain phospholipids was determined as described in ExperimentalProcedures. Data are mean ±SEM values of four experiments.

“Significantly different from control (p < 0.05).“Significantly different from veratridine treated (p < 0.05).

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TABLE 3. Effect of veratridine on the levels of ATP and glycerol 3-phosphate and onthe incorporation of [

3H]palmitic acid into brain fatly acyl-CoA

Glycerol 3-phosphate Fatty acyl-CoA ATPTreatment (nmol/mg of protein) (dpmljig of protein) (nmol/mg of protein)

Control 3.98 ±0.08 (4) 4.31 ±0.16 (3) 16.86 ±2.28 (3)Veratridine (100 jiM) 5.27 + 0.43 (4)“ 2.54 ±0.05 (3)“ 8.25 ±1.03 (3)“

+ TTX (I jiM) 3.44 ±0.39 (4)“ 3.69 ±0.13 (3)““ 16.46 ±2.50 (3)“+ Ouabain (I mM) ND 3.74 ±0.18 (3)“ 17.25 ±0.81 (3)“

Brain slices from 60-day-old rats were incubated with 100 jiCi [3Hlpalmitic acid as described inTable I. After incubation, the slices were immediately frozen and aliquots of the homogenate wereused to determine glycerol 3-phosphate, ATP, and incorporation of [3H]palmitic acid into fatty acyl-CoA as described in Experimental Procedures. Data are mean ±SEM values of the number ofexperiments in parentheses. ND, not determined.

Significantly different from control (p < 0.05).“Significantly different from veratridine treated (p < 0.05).

Depolarization with KC1 has no effect on thepalmitoylation of lipids

Finally, the effect of potassium-induced depolariza-tion on lipid acylation was investigated. In these exper-iments, brain slices were incubated in Krebs—Ringerbicarbonate in which 60 mM KC1 replaced a similaramount of NaCl, so that the osmolality of the mediumwas the same as in the control. Incorporation of [3H]-palmitate into myelin PC (Table 5) and the other glyc-erolipids (not shown) was not affected by the elevatedK‘ concentration. The lack of effect of KC1 cannot beattributed to the use of Krebs—Ringer solution insteadof DMEM, because in both media the addition of 100‚uM veratridine reduced lipid acylation to the sameextent (data not shown). Addition of nifedipine or re-moval of calcium from the medium has no additionaleffect. It is noteworthy that incubation of slices in nor-mal medium containing nifedipine or calcium-free me-dium showed a significant decrease in lipid acylation(Table 5).

DISCUSSION

Veratridine-induced depolarization reduces thepalmitoylation of glycerolipids by an indirectmechanism

In this report we have shown that veratridine-in-duced depolarization reduces the incorporation of [3Hj-palmitic acid into all brain and myelin glycerolipids.This effect is caused by a decrease in the levels ofATP, which is necessary for the synthesis of palmitoyl-CoA (Waku, 1992). It has been shown that neuronalenergy metabolism reflects almost exclusively the ac-tivity of the Na~,K“’-ATPase,and that veratridine-in-duced depolarization, despite increasing respirationand ATP synthesis, results in a 30—40% reduction inintrasynaptosomal ATP levels (Ereciñska and Dagani,1990). In our study, veratridine lowered brain ATPconcentration to a comparable extent, suggesting thatthis phenomenon is not limited to synaptic terminals.Because the intracellular concentration of ATP (2 mM)

TABLE 4. Influence of ouabain, nifedipine, and calcium-free medium on veratridine-induced reductionof palmitoylation qf myelin lipids

Lipid ControlVeratridine(100 jiM)

Veratridine +ouabain (I mM)

Veratridine(minus Ca2~)

Veratridine +

nifedipine (50 jiM)

PC 103.1 ±2.5 50.1 ±5.7“ 79.1 ±7.3““’ 55.7 ±3.6° 42.1 ±5.4“FE 35.4 ±1.0 13.9 ±1.5“ 27.4 ±3.2““’ 16.9 ±1.6° 11.8 ±0.9°PS 5.7 ±0.2 2.9 ±0.2° 4.4 ±0.2°“’ 3.6 ±0.3“ 3.0 ±0.4“Pl 13.7 ±0.9 6.2 ±0.7“ 11.9 ±1.1“ 6.9 ±1.3° 5.0 ±0.3°PA 22.3 ±0.8 8.5 ±0.9“ 16.4 ±1.8“’ 13.4 ±1.9““’ 8.3 ±0.1°DAG 14.1 ±1.6 6.7 ±0.4“ 11.7 ±1.7“ 8.2 ±0.6“ 8.7 ±0.7““’CER 15.4 ±1.3 16.1 ±1.0 14.7 ±1.6 26.1 ±2.6““’ 19.2 ±1.9SPH 1.8 ±0.4 1.7 ±0.1 1.8 ±0.3 1.7 ±0.1 1.4 ±0.2FA 4,517 ±244 4,144 ±265 4,174 ±623 6,303 ±640“’ 4,228 ±176

Brain slices from 60-day-old rats were incubated in 4 ml of DMEM with 100 jiCi [3fllpalmitic acid for I h in the absence or presence ofthe various compounds as indicated in the table. Medium without calcium also contained 1 mM EGTA. After incubation, lipids from myelinwere analyzed as described in Experimental Procedures. Data are expressed as dpm per microgram of protein and represent mean ±SEMvalues of three or four experiments.

° Significantly different from control (p < 0.05).“Significantly different from veratridine treated (p < 0.05).



TABLE 5. Effects of high K~and calcium-free mediumon the incorporation of [

3H]palmitic acid into myelin PC

TreatmentSpecific radioactivity

(dpmljig of myelin protein)

Control 99.6 ±5.9 (14)KCI (60 mM) 89.7 ±7.9 (15)

— Ca2* 100.8 ±8.4 (lI)+ Nifedipine (50 jiM) 74.2 ±10.6 (3)

—~ Ca2~ 65.6 ±0.5 (3)“Nifedipine (50 jiM) 52.9 ±12.1 (3)“

Brain slices from 60-day-old rats were incubated with 100 jiCi[3Hlpalmiticacid in 4 ml of Krebs—Ringer bicarbonate buffer con-taining 10 mM glucose for 1 h in the absence or presence of variouscompounds. After incubation, the radioactivity present in myelin PCwas determined as described in Experimental Procedures. Data areexpressed as dpm per microgram of protein and represent the mean±SEM values of the number of experiments in parentheses.

“Significantly different from control (p < 0.05).

is in the same range as the Km of the microsomal long-chain acyl-CoA ligase for ATP (2—4 mM) (Bar-tana etal., 1975), a fall in ATP levels leads to the proportionaldecrease in the concentration of acyl-chain donor thatwe observed. In a similar manner, because the palmi-toyl-CoA concentration in rat brain (5 ‚uM) (Grangeet al., 1995) is close to the Km for acyl-CoA of bothglycerophosphate acyltransferase (10—15 ‚uM) (Abou-Issa and Cleland, 1969) and l-acylglycerophosphateacyltransferase (5—10 ‚uM) (Miki et al., 1977), a de-crease in the levels of the acyl-chain donor is likelyto reduce the formation of PA, and consequently thesynthesis of all glycerolipids. However, because mostacyl-CoA/lysophospholipid acyltransferases displayedKm values for palmitoyl-CoA of 5—10 ‚uM (Baker andThompson, 1973; James et al., 1979; Hasegawa andOhno, 1980), the incorporation of palmitate into phos-pholipid via the Lands‘ cycle is also expected to bereduced in the veratridine-treated slices. This phenom-enon explains why the incorporation [3H]palmitic acidinto all glycerolipids was diminished to almost thesame extent regardless whether they are palmitoylatedduring the de novo synthesis like PC or via FA ex-change as shown for Pl (Baker and Thompson, 1972).It is yet unclear as to why the synthesis of sphingolipidswas notaffected by veratridine-induced depolarization,because acyl-CoAlsphingosine N-acyltransferase has aKm for palmitoyl-CoA of 100—200 ‚uM (Morell andRadin, 1970). It is possible that on the cytoplasmicsurface of the endoplasmic reticulum, where the syn-thesis of ceramide and glycerophospholipids takesplace (Quest et al., 1996), different pools of palmitoyl-CoA with distinct responsiveness to ATP depletionmay exist. In this regard, not only is acyl-CoA hetero-geneously distributed within cells (Den Breejen et al.,1989) but also a significant fraction of it is bound toacyl-CoA binding proteins (Rasmussen et al., 1990).An alternate possibility is that acylation of long-chainbases could have taken place via the reversal of the

reaction catalyzed by ceramidase, that is, using freeFA instead of the CoA derivative (Yavin and Gatt,1969; Sugita et al., 1975).

Palmitoylation of myelin glycerolipids is alsoreduced during veratridine-induceddepolarization

The most important finding described in this studyis that veratridine-induced depolarization reduces thesynthesis of myelin glycerolipids. That membranes ofneuronal origin could have copurified with the isolatedmyelin membranes and comprised some of the radioac-tivity present in that fraction cannot be totally ex-cluded. However, the relatively high specific radioac-tivities of the myelin lipids, together with the resultsfrom repurification and mixing experiments, make thatpossibility very unlikely. The bulk of myelin lipids issynthesized in oligodendrocytes and their processes(Morell and Toews, 1984; Ledeen, 1992), and althoughthere is evidence that some lipids may be transferredfrom the axon (Alberghina et al., 1982; Ledeen andHaley, 1983), the contribution of this process to thesupply of lipids for myelin synthesis is considered tobe quantitatively minor (Gould and Dawson, 1976;Ledeen and Haley, 1983). This assumption is sup-ported because results quantitatively identical to thosedescribed here were observed in actively myelinating20-day-old rats, where the proportion of the myelinlipids derived from transaxonal transport is likely to besmaller than in 60-day-old animals (data not shown).

Oligodendrocytes and/or myelin may containvoltage-gated Na~channels

Because ouabain prevents the veratridine-inducedreduction in palmitoylation of both myelin and brainglycerolipids, it is reasonable to assume (1) that ATPlevels in oligodendrocyte, as in whole brain, are re-duced in the veratridine-stimulated slices, and (2) thatthis reduction was triggered by stimulation of the Na~/K~pump. In this regard, not only has Na~,K~-ATPaseactivity been shown in isolated oligodendrocytes (Zim-merman and Cammer, 1982) but also the enzyme hasbeen immunocytochemically localized to the plasmamembrane of these cells (Wood et al., 1977). However,the mechanism by which oligodendroglial Na~,KtATPase activity is stimulated in response to neuronaldepolarization is not that evident. To the best of ourknowledge, direct communication between neuronsand oligodendrocytes has notbeen found, and althougholigodendrocyte progenitor cells have Na‘ channels(Barres et al., 1990; Barres, 1991), mature oligoden-drocytes in culture express neither voltage-dependentNa~channels nor outwardly rectifying K~channels(Sontheimer et al., 1989). Thus, at first glance, it wouldappear that the enhancement of oligodendrocyte me-tabolism caused by veratridine occurs by an indirectmechanism. In this regard, axonal depolarization byveratridine is known to increase the extracellular con-centration of K‘ by several millimolar (Li and White,1977; Sykova, 1983; Grafe and Bellanyi, 1987), with

.1. Neurochem., Vol. 70, No. 4, 1998


the ensuing activation of the oligodendroglial Na~,KtATPase (Bellanyi and Kettenmann, 1990). However,in our experiments, the presence of 60 mM KC1 in theincubation medium affected the synthesis of neitherbrain nor myelin lipids. It is clear that under theseconditions the activation of the Na~/K~pump was notsufficient to reduce the levels of ATP. This is consis-tent with the notion that the activity of the pump inbrain is determinedby the intracellular Na~concentra-tion rather than by the extracellular concentration ofK~(Erecidska and Dagani, 1990). Veratridine-induceddepolarization also results in the release of a largenumber of neurotransmitters, including acetylcholine,GABA, and glutamate (Adam-Vizi, 1992). The lasttwo neurotransmitters are known to depolarize culturedmouse oligodendrocytes (Kettenmann et al., 1984) byincreasing the intracellular Na~activity, thus leadingto activation of the Na~/K~pump (Bellanyi and Ket-tenmann, 1990). However, depolarization with highK‘, which also triggers extensive release of neuro-transmitters from synaptic terminals (Vargas et al.,1977; Nichols, 1989), had no effect on lipid palmitoy-lation. Thus, it is tempting to speculate that myelinat-ing oligodendrocytes may express functionally activevoltage-dependent Na“ channels. With regard to thelack of channels in cultured oligodendrocytes, it is con-ceivable that in the absence of axonal contact and/orother factors the cells grown inculture may not expressthe same complement of channels that are expressedby glia in vivo.

An alternate, and perhaps not excluding, possibilityis that such voltage-dependent Na~channels are lo-cated in myelin. Although staining with anti-sodiumchannel antibodies is restricted to the nodes of Ranvierand perinodal astrocytes (Black et al., 1989), the highthreshold of sensitivity in this technique (100—200channels/jim2) (Waxman and Black, 1995) does notallow one to rule out conclusively the presence of lowconcentrations of channels in the paranodal oligoden-droglial terminal loops and/or the compacted regionsof the myelin sheath. It is noteworthy that, from thedata of Baumgold et al. (1983), it can be calculatedthat the total binding of [3H]saxitoxin (a specific Na~“channel marker) to rat brain continues to increase be-yond the period of formation of nodes of Ranvier andthroughout myelination, suggesting that myelin mayindeed contain Na‘ channels. In considering a directeffect of veratridine on this membrane, it should benoted that, like the oligodendrocyte plasmalemma, my-elm contains considerable Na~,KtATPase activity(Reiss et al., 1981; Zimmerman and Cammer, 1982),most of which appears to be located at the paranodalloops (Mrsulja et al., 1985). Moreover, myelin has thecomplement of enzymes required for lipid synthesisincluding acyl-CoA ligase and lysophospholipid acyl-transferase, which are believed to be localized withinthe paranodal loops and other uncompacted regions ofthe myelin sheath (Ledeen, 1992). Therefore, if Na~channels are present in these regions, it is very likely

that their activation could lead to a reduction in thelocal synthesis of lipids. It is noteworthy that althoughplasmolipin and PLP may form voltage-dependent cat-ion channels in myelin (Ting-Beall et al., 1979;Fischerand Sapirstein, 1994), they have no structural resem-blance to voltage-dependent Na~“channels and lack thestructural determinants involved in the binding of TTX(Terlau et al., 1991). Thus, it is unlikely that the effectof veratridine, which is prevented by TTX, is mediateddirectly by these hydrophobic proteins. Further studieswill be aimed at determining directly whether oligo-dendrocyte and/or myelin contain voltage-gated Na~channels and, if so, what physiological role they mayplay.

Acknowledgment: This study was supported in part by theNational Multiple Sclerosis Society (RG 2322A), by PHHSgrant S06-GM 08139 from NIH, and by the Dedicated HealthResearch Funds of the University of New Mexico.

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Veratridine-Induced Depolarization Reduces the Palmitoylation of Brain and Myelin Glycerolipids

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