Biochimica et Bioplryvica Acta, 1136(1992) 169-174

169

6~ 19~,2El,,t'vicrScicncc Publishers B.V. All rights reserved 0167-4889/92/S05.(H|

Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine Richard C. Franson

~, L e s l e y K. H a r r ! s ~, S.S. G h o s h

" and Miriam D. Rosenthal

h

° Del~rlmoff$ of Biochemistry and M~decular Bioph~ics. Vitgimu Commwmcallh Unic.,r~ty. Ri~.hn~tuld. VA fUSA) and I, D:"parlment of Biochemisw~: F.a~tero Firginia Medwal St'l;otd. ,%'or],;ik. V.4 I,L'~: ~9

IRecc/vcd 20 Ntr,,cmhcr 1991)

IG.'y~ords: Sphingc,sinc;Sphingoid base:.Phosphulipas¢ A 2; Phospholipasc D; Inhibition; Substratc Jilution Sphingosinc inhibiLs protein kina~c C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosinc dircclly inhibits phospholipascs A z and D. Sphingnsinc inhibits Ca2 ÷-dependent phospholipases A., from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrnphil in a dose-dependent manner with IC,;~ values ranging from 5-40 # M using [l-I~C]oleate-labclled autocla ::d E. coli (20 #M) as substrate. Inhibition is comparable using the same concentrations (20 p.M) of [i-14C]olcate-labclled C. clbicans or E. coli, or aqueous dispersions of l-acyl-2-[iJaC]linolcoylglyccrophosphocthanolamineor -choline..~;phinganinc ana: ste;,rylaminc are as inhibitory as sphingosioc: monoolein ;~ less i ~h~itoo (IC.~t = 70/.tM), while octylamir,c, N'-acctylsphin~osine, sP~ingomyelin and ccramide have no effect. Inhibition =~rcli~'cd :W increasing concentration~ of substrate phospholipid. The mol;'r ratio of sphingosinc to phospholipid required for 5I~; inhibition ."an~cs from 0.5 to 1.0 wi:h 2-100 IzM E. coli phospholipid. :~ contrast, sphingosine has a biphasic effect on the hv:'rolysis of E. c J i by ¢3. chromo~zst'us phospholipasc [". conecnt ration~ ~ 25 p.M stimulate activity while concentrations > 25 #M are inhibitory. Addition of Triton X-100 elimi~-~;;.cs :;z:;.'. t!lc shmulatory and inhibitory cffccl~ of ~phingosinc on ph~pholipasc D activj:y.

Intreduction Interest in sphingolipid metabolism has been stimulated by reports that sphingosine and related compounds inhibited protein kinasc C activity and binding of phorbol dibutyratc in vitro [1]. The major structural requirements for i n h ~ i t i o n was a free amino group and a long aliphatic chain [2]. Sphingosine and related sphingoid bases blocked thrombin- and diglyceride-depep~.eot protein phosphowlation in human platelets [1], phorbol-ester-dcpcndent differentiation of H L 60 cells [3], and the oxidative burst, P A F production, arachidonic acid release and its fu~, cr metabolism in human ncutro~I:ils [4,5]. Thus, sphingosine, a naturally o c c u ~ h ' g by-produ, t of sphingolipid metabolism, and related sphingoid Uases, were thought to function as physiologic, negative mGdifiers of protein kinase C in

Correspondence: R.C. Franson. Departmer,l of BiochemLsu3' and Molecular biophysic~ Box 614 MCV Station. Richmond. VA !~,29.'i0614. USA. Abbreviations: Esdwriclda c o K E ¢otk Candidc ~:i,.i~ar,.¢ t~ albicans: phospholipa.~ A 2. PLA2: phospholipase D. PLD: l,hosphalid~lethanolamine. PFA plmsphatidykholine. PC': phorbol myrislate acetate. PMA.; protein Idnase C. PKC.

t r a n s m e m b r a n e signalling (for reviews, see Ref. 6-8). Consequently, the cellular effects of sphingosine noted above were attributed to inhibition of protein kinase C, presumably at the level of the cell membrane. In support of this hypothesis, it has been shown that most of the free sphingosine in rat liver is associated with plasma membrane, and incubation of those membranes at 37°C resulted in the hydrolysis of spimIgomyelin and increased levels of membrane-associated sphingosine [9]. Recent studies, however, indicate that sphingusine has multiple enzymic effects. In addition to inhibition of protein kinase C, sphingosine inhibits CTP-phosphocholine cytidyitransferase [10], stimulates or inhibits otber protein kinases [11,12], and inhibits phosphatide phosphohydrolase [13]. The later f n d i n g would depress diglyccride release from phosphatidic acid and may provide an alternative explanation of reports that sphingoid bases activate cellular phospholipase D (PLD) [14,15]. Thus, sphingosine as an amphipath affects a variety of enzymic activities in vitro, and these recent fit~dings suggest alternative interpretations of previous cell studies using sphingosine [1418]. Because sphingosine has been used to implicate protein kinase C d e p e n d e n t activation of phospho-

170 lipas':, A_, (PLA,) in arachidonatc r. ~bilization [19}, wc c.~.:nined ~he effe~ of sphingosinc on in-vitro PLA, activity. Cur results show that sph'ngosinc and sphingold bases inhibit PLA 2 activity in a concentration-dependent manner, inhibition i~. sphingosinc appears to be competitive with, but independe, tt of. the physical form of thc substrate used. Interesti:~gly. in-vitro PLD activity was first stimulated and t~.en inhibited by sphing(,sine in the absence of Triton X-100.

activity is expressed as percent of conlrol:cpm fatty a~-id released/total phospholipid cpm × 100 minus background which was ~ 1.8% in all experiments. All data are the average of at least duplicatc determinations from a single experiment and are representative of at least 2 experiments. Phosphollpld phosphorus was measured by the method of Bartlett [26] and protein by the method of Bradford [27] using BSA as a standard.

Assay of phospholipase D Materials PLA 2 from bee venom (ApLs mellifera). Naja naja venom, Crotalus adamanteus, and porcine pancreas were obtained from Sigma (St. Louis, MO) as was PLD Type V! from Streptonzyces chromofilscus and sphingosine (bovine brain), sphinganine (dih.vdrosphingosine), mono- and di-olein, stcarylamine, and ceramide. PLA ,s were isolated from degenerated human discs, and human neutrophils by acid extraction and cation cxchange chromatography as prcvionsly described [20,21 ]. Sphingomyelin wag purchased from Matrcya (Pleasant Gap, PA), octylamine from Aldrich (Milwaukee, WI) and phnrbol myristate acet3:." from C~lbiochem (La Jolla, CA). N-acetyi~phingo~in . ~as preparer! from sphingo~inc and ate:tic anhydride [22]: ,L[I-a~C]linoleoyI-PE (55.6 mCi/mmol) and -PC (59 mCi/mmol) were purchased from Amersham (Arlington Heights, IL) and [I-mC]oleat~ (~6 mCi/mmol) from American Radiolabeled Chemicals (St. Louis, MO). All solvents and chemicals were at least reagent grad:. Methods

PLD actici~, was measured using [l-n4C~:eatelabelled autoclaved E. colt [28] a,kd 2.[i-+~C]linoleos"iphosphatidylcholine [291 as substrates. Unless otherwise indicated, reaction mixtures contained 10 nmol J~C-E. colt or -PC 110110 cpm/nmol), 5 mM CaCI,, 50 mM Hcpes buffer (pH 7.0) + I).05% Triton X-100, and 200 ng PLD from S. chromofiL~CltS. Reaction mixtures containing the indicated concentrations of sphingosine in DMSO were incubated for 20 rain at 37°(7. in a shaking water bath and stopped by the addition of CHCi3/CH3OH (2: I. v/v). Lipids extracted by the method of Bligh ;rod Dyer [25] were subjected to thin-layer chromatography on silica G plates using a solvent system consisting of C H C I 3 / C H 3 O H / N H 3 / H _-O (qq: 54 : 53 : 5..5, :'/v) to separate [ '4C]phosphatidic acid (R a = 0.14) and diglyceride (Rf = 0.95, from phospholinids (Rfs 0.48-0.60). Hydrolysis was quantified by liquid scintillation counting after in:line vapor localization of appropriate lipids. Activity is expressed as percent of control: cpm pho~phatidic acid/total phospholipid cpm × 100 minus background which was ~< 1.6%. All data are the average of dnplicate determinations from a single experiment anti are representative of 2 experiments.

Assay of phos.nholipase A , PLA: was measurcd by established methods using sul~stratcs that were labelled with ~C-fa'.ty acid in the 2-acyl position. The substrates were [I-14C]oleatelabelled autoclaved 1=:colt (bacterial) or autoclaved C. albicuil~ (yeast) prepared as previously dcscribe,I [23,24], and 2-[I-a~C]linoleoylphosphatidyl-ethanolamine or -choline. In a tolal volume of 0.5 ml. standard reaction mixtures contained 10 nmol substrate phospholipid phosphorus (1000 cpm/nmol), 5 mM CaCI:, and 50 mM buffer +_150 mM NaCI a n d / o r Triton X-100 and the indicated concentration of sphingosine (or related compound) in DMSO, such that the final DMSO concentration in all reactions was ~ 5% and did not influence control enzymic activity. Protein and time of incubation at 37°C in a shaking water bath were adjusted to limit hydrolysis to the linear range. Reactions were stopped by the addition of 3 mi of CHCi3/CH3OH ( i : L v/v). Lipids were extracted by the method of Bligh and Dyer [25}. separated by thinlayer chromatography, and quantitated by liquid scintillation counting as previously described [23,24]. PLA:

Results

luhibilion of PLA 2 by Sphingosine The effect of sphingosine on various PLAes hydrolyzing [i-4C]oleate-labelled autoclaved E. colt is shown in Fig. I. PLA:s from group ! (N. naja and porcine pancreas) and group II (C. adamanteus, human disc, and human neutrophil) were inhibited in a dose-dependent manner with IC.~ls ranging from 5 to 40/zM. The activity of bee venom PLA2, an enzyme cle~,s,.'~ed neither group i or IL was modestly stimulated at concentrations of sphingosine < 25 izM, and was inhibited only 30% by 50 p.M sphingosine. Sphingosine inh~ited the hydrolysis of membranous. liposomal, and micellar substrates. Fig. 2 shows that inhibition of the human disc PLA 2 was comparable using [I-'4C]oleate-labeiled autoclaved C albicans or E. colt, or 2-[l-I~C]linoleoyi-PE; the ICs0s were 6, 15 and 5/~M, respectively. The inset demonstrates that sphingosine also inhibited the hydrolysis of 2-[l'4C]linoleoyI-PC by C adamanteus PLA 2 in the pres-

171

12°tk !

120 100

~

r

0

~o

2

20

• 0

10

20

30

40

50

Sphingosine(uM) Fig. I. Effect of sphingosine on the hydrolysis of II-'~CJoleatc labelled autoclaved F,. coL/ by ~.~r/ous low-molecular-weight. C a : ' dependent, phospholipases A z. Standard react/on mixtures in a total ,,~olumc of 0.5 mi contained 10 nmo! E. co// phospholipid phosphoros. P L A : . 50 mM Hc-pt~ buffer (p;~ 7.5). 150 mM NaCI. and were incubated at 3 T C for 10 rain. Sphiogosim: in D M S O was added such that tho final concentration of DMSO was 5c,~ (v/v) which had no effect on enzymic acth,i ~ . ActP.ity is expressed as percent of control (control = I 0 0 ~ ) : the control values as percent hydrolysis/lO rain vdth 5 ~ D M S O were: bee ~noq:.. liB. 4 ng) 21.1_+12~. porcine pancreas re. 216 noo) 8.9+_ l.b~. N. naja ( • , t3 ng) 8.6+_0.9e,~, C. adamameus ( v . I Aug) 21.6_+ 1.0. and human neutrophil = human disc (@. 14 rig) lh.O+_l.l,~. Each data point is the average of duphcate detcrminat/oos from one experiment, and is representative of at least two such :xperiments unless indicated otherw/s¢. All ,,~lucs arc corrected for nonenzymatic hydrolysis which was ~ 1.8c~ in all experiwents.

.

Sphingosine (uM) Fig. 3. Inhibilion of phospholipase A ; activity by sphingosine as a function of substrate concentration. Standard reaction mixtures contained 14.8 ng human disc PLA z. the indicated concentrations of sphingosine, v,ith 2 (el. 20 ( • ) or 100 ( • ) ~ M E. coil phospholipid phosphorus and were incubated for ! 10. or 38 rain. respectb.'ely. Activity. expressed as percent of control hydrolysis/indicated time (control = 100%) was: 22.4+_3.4c,~-/2 mio with 2 p.M E. co/i whose radiospecific activity was 27770 cpm/nmol. 28.2 ± 0.f~C~/10 rain with 20 /zM s~andard E. coli, and 2 4 . 8 + ( l . 3 ~ / 3 ~ mio V.lth 100 /.~M standard E. coil Each data point is the average of duplicate determinations from a single experiment corrected for nonenzymatic hydrolysis ( ~< 1.8%).

ence or absence of micellar concentrations of Triton X-100. Thus, the inhibition of PLA 2 activity by sphingosine, with the possible exception of the bee venom en .zyme, is largely independent of the low-molecularweight PLA~ used as well as the physical and chemical properties of the aggregate substrate.

1 2 0 ~ 120

160

-~

100

120

~

4o

160

4O

0

2o

Sphingosine (uM) Fig. 2. Effect of sT,ilingosine on the hydrolysis of various substraleS by human disc ~.,d IV. naja phospholipases ." :. Standard reaction mLx::.-;,~ ,':ontaired 10 nmol c f [I-'aCJoleate-lat-:.lied autoclaved E. coil ( • ) or yeast (eL or 241-t4Clliooleoyl-PE ( • ) as an aqueous dispersion, :,rid 14 ng human disc P L A : . Sphingosine in D M S O was added ~ desoa'bnd in the legend to Fig. 1. In the inset, hydrolysis of 2-;,I-t4C]linocoyI-PC by N. rmja P L A : w/lh (0, lower) and without ( • ) 0.05 ,c,c Triton X-IO0 is shown. Activity is expressed as percent of control hydrolysis/10 rain {control = 100%) which was: E. co//, 16.0 + I.! ,%; yeast, 19.2+_ ! I,C,e;PE, 16.8+4.5,%. Inset: PC a/one, 8. 9± 1.6: detergent-PC 15.1 _+ 1.6. Each data point is the average of duplicate determinations corrected for nonenzymatic hydrolysis ( < 1.8,~ ).

'i]. 0

10

2o

3o

4o

5o

Inhibitor (uM) Fig. 4. Effect of sphingoid bases and related compounds on human disc phospholipase A : activity. Standard n" Jction mixtures contained 10 nmol E. cuff phospholipid phosphorus, 14 ng human disc PLA. and the indicated concentration of sphingosioe (el. stearylamioe (,~), sphi,,anine (o); and the inset: ceramide (A), Nacewhphingosine(O), diolein ( • ) . and I -monoolcio ( v ) in DMSO. Tile results obtained with octylamine, sphingomyelin, and ceramide were comparable (A). Activity is expressed as percent of control hyJrolysis/l'J rain (see legend to Fig. l). Each data point is the aven.ge of duplicate determinations from one experiment correcled for noncn;p/matic hydrolysis ( < 1.8%).

Sphingosine's inhibition of in-vitro and collular enzymic activity and cell function is known to be competitive with added phuspholipid [I-3,18]. Therefore, we tested the PLA 2 inhibitory activity of sphingosine as a function of substrate concentration. As shown in Fig. 3, the IC~ 0 for inhibition by sphingosine is directly related to E colt phospholipid phosphorus content. Increasing concentration of substratc competes with sphingusine and relieves inhibition; or, the less substrate used in the reaction, the lower the IC_~t and the greater the apparent sensitivity. An ICso is obsetwed when the molar ratio of s p h i n g o s i n e / p h o s p h o l i p i d is 0.5-1.0. Similar results were obtained using C. albicans as substrate (data not shown). Sphingoid bases are reported to have inhibitory effects comparable to sphingosine [2]. Fig. 4 illustrates that comp~mnds related to sphingosine, i.e., amphipaths with a long aliphatie chain and a free amine function, inhibit i~LA2 activ.;ty similarly. H u m a n disc P E A 2 activity was inhibited by sphinganine (dihydrosphingosine) and stearylamine, but not by oct~lamine or N-acetylsphingosinc. Activity was moderately inhibited by monoolein (IC_~ = 7 0 / . t M ) but not by diolein. PMA, sphingomyelin, or ceramide. Inhibition o f P L D by sphingosine Recent studies have reported that sphingosine stimulates cellular PLD activity [14,15]. This finding was thought to bc secondary to an inhibition of phosphatide phosphohydrolase [13]. Since sphingosine pre250 ,

~

~5o 1GO



5

0

25

50 Sphingosine

75

100

(uM)

Fig. 5. Effect of sphingosinc on the hydroh,~is of [I-s4C]oleatelabelled E. ccdi or 2-[I-t4CIlinoleoyl PC by S. chromoftt~ru.s pho~pholipas¢ D in the pre~ence and absence of 0.05% Triton X-I~). Standard reaction mixtures in a total volume of 11.5 ml c~mtained 10 nrn,~l E. co//(o) or PC ( v ) phospholipid phosphorus in the prew,:nce(open s)'mbol~)or absence Icluscd sajtn~oi~)of 0.05£; T;iton X-lgO. and 2(Xl ng S. chrcmlofttsclts PLD. Reactions were termin~,;:.; .Jfter 15 rain ( F coli~ or 7.5 rain (PC) at 3"PC. PLD activity, cxpres~:d as pc[cent or" conffol h)drolysLs/time, was 12.4+ 1.2r~/15 rain E. coli ak~:;e. 34.3_+3.2%/15 mitt E. colt +Triton. 8.4+ 1.3%/7.5 rain PC alone, and 2.5_'~_+2.6~/7.5 rain PC+Triton. Each data point is the average of duplicate determinations from two experiments corrected for nonen~'rnatic hydrolys*s( p.M resulted in dose-dependent inhibition of activit:,,; the I C ~ s for E: colt and PC were 50 and 70 p.M, resp=-ctively. Inhibition of PLD activity, like that of P L A , . was relieved by increasing substrate concentrations i d a t a not shown). Interestingly, unlike PLA 2 (Fig. irL~ct), both the stimulatory and inhibitory effects of spb~.,:gosine were barely detectable in the presence of 0.0:,.-~[ Triton X-IO0. Discussion O u r results demonstrate that sphingosine directly modulates phospholipid-dependent enzymatic reaclions, in this case. low-molecular-weight, Ca2÷-depen dent P L A , s from various sources, and Ca-'÷-depcn dent, microbial PLD. Sphingosine has been widely used as a probe of cell signal transduct/on because it inhibits both in-vitro and in-s'.'lu PKC activity [1,2], and events that are thought to be at least in part mediated by PKC [3-5,30]. In some instances that inhibition was overcome by phorbol esters [5,30], further suggesting an effect of sphingosinc at the level of PKC. However, recent studies have reported that sphingosine inkibits enzymic activities other than PKC; these include other kinascs [16.17L phosphatidic acid phosphohydrolase [13] and CTP phosphocholine cytidyltransferasc [18]. These findings, and o m i,.~ullo of inhibition of PLA 2 and PLD activities, demonstrate that sphiugosine has direct effects on a variety of lipid-dependent enzymes, many of which contribute to cell signal transduction. The mechanism by which sphingosine inhibits invitro PLA z or PLD activiW is complex. The observation that all of the PLA2s tested (groups ! and II) with the exception of bcc venom, were inhibited vJP.h ,.oreparable IC~ts (Fig~ 1) suggests that differences in primary and secondary structur¢ of these closely related proteins do not significantly influence inhibition. Bee venom P L A , , rel~.tive to group I and l i enzymes, is unique in its structure and surface recognition and penetration properties [31,32]. These differences may accoun:, for its diminished sensitivity to sphingosinc (Fig. 1). Comparable ICsos were obtained using widely different substrates, including atttoclaved E . . c o l t or yeast, P E or PC, with and without Triton-X 100 (Fig. 2), indicating that inhibition of PLA2 activity by sphingosinc is not markedly affected by the physical structure of the lipid aggregate. By contrast, hydrolysis of E. coli membrane, and to a lesser extent PC, by S. cltromo~i~scus PLD, was stimulated up to 200% by low

173 concentrations of sphingosine ( ~ 25 /LM). Activity toward both substrates was inhibited in a dose-dependent manner by concentrations of sphingosinc > ,,xM (Fig. 5). Interestingly, both the stimulatory aad inhibitory effects were abo!ishcd by the addition of 0.05% Triton X-100, a detergent concentration which optimizes in-vitro PLD activity in the absence of sphingosine [27,28]. The lack of effect in the presence of detergent demonstrates that sphinqosine's inhibition of PLD is influenced by the physical nature of the substrate. These results demonstrate that the response of different lipolytic enzymes to sphingosine is not uniform, but rather is enzyme- and assay-dependent. The ability of added phospholipid to overcome inhibition of both P L A , and PLD activities (Fig. 3; data not shown) suggest that sphlngosine competes with substrate to mediate inhibition. This competition was also observed for sphingosine's inhibition of protein kinasc C and cellular responses thought to be dependent on PKC [!-3,18]. Both protein kinasc C and PLA, are inhibited by stearyi-, but not octyl-amine [2] (Fig. 4) suggesting that a long alkyl chain with increased hydrophobicity may promote interaction with the activator (PKC) or substrate ( P L A , ) pbospholipid. Furthermore. it has been reported the' sphingosine neither interacts directly with protein kinase C nor does it effect the binding of PKC to its activator lipids. Rather, sphingosinc appea:s to associate with activator phospholipids resulting in a nonproductive interaction with protein kinase C [2]. It is not known at this time whether sl~hingosine binds to the isolated P L A , or affects the binding of P L A , to a sphingosine-substreZe aggregate. Studies with PKC [2] and our results demonstrate that a long aliphatic side chain and an ionizable amino group are necessary for inhibition of enzymic activity by sphingosine. Addition of one N-methyl group (NCH.~] to sphingosine had little effect on enzymic activit3', a second methyl group "~-N-(CH3),) diminished ~nhibition, while a third (-N-(CH.O.0 was stimulatory. Similar structural requiremen'.s have been reported for inhibition of pbosphatidate phosphohydrolase by sphingosine [13]. Our finding that N-acetylsphingosine is not inhibitory (Fig. 4) is consistent with these results. In addition, that sphinganine and stearylamine arc as inhibitory as sphingosinc indicates that the 4-5-trans double bond and the hydroxyl functions most likely play a minor role with respect to inhibition in vitro. Although hydroxyl groups do not appear to play a major role in inhibition of P L A , activity, they may regulate enzymic activity by influencing membrane partitioning of sphingnsine. Hydrox,/I groups are reported to stabilize sphingosine stereoisomers by hydrogen bonding and modulate the pK,. by up to 2 pH units [2]. In this way hydroxyl groups may impar', flexibib'ty in the molecular charge of sphingnsine at physiologic pH

that can promote partitioning of sphingosine from aqueous (polar) to membrane (uncharged) compartments. Stearyltrimctbylamine, which lacks the hydroxyl functions of sphingosine, inhibits in vitro, but not in situ, PKC activity presumably due to a charge inappropriate to penetrate the cell membrane [2]. The contribution of hydroxyl groups to the pK~ might also explain the fact that stearylamine (pK~ = 8.0) and sphingosine (pK, = 6.7) are equally effective inhibitors of phosphatidate phosphohydrolase [13]. PKC [2] and PLA,_ activities. We found that human disc PLA, activity was inhibited to the same extent by sphingosinc when assayed at pHs 6.7 and 7.5. If inhibition is a function of cationic charge and a fixed pK~ of 6.7, then sphingosine should have been more inhibitory at pH 6.7. Sphingosine is thought to function as a cationic amphiphi:e, intercalating itself into membranes to inhibit enzymic activities and cellular functions by membrane charge neutralization [33,34]. Our results of inhibition of PLA 2 activity by sphingosine are consistent with such a mechanism. We [351 and others [36,371 have previously demonstrated that the low-molecularweight, Ca2+-dcpendent PLA 2 exhibits an electrokinetic requirement for optimal hydrolysis; a net negative zeta potential or an anionic phospholipid was preferred. PLA 2 activity was modulated by the surface charge of the liposome and the charge of the double layer, and these pioperties ate affected by cationic amphlpaths, such as sphingosinc, as well as Ca 2". Tile fact that comparable ICs,s were obtained for PLA2s hydrolyzing substrates of varying surface charge and shape implies that sphingosine may affect other biophysical parameters of interracial kinetics such as surface packing, curvature, defects, or phase transition. Our results suggest that sphingosine may stimulate or inhibit cellular, lipid-dependent enzymic activity in a concentration-and/or enzyme-dependent manner. Studies with cationic amphipilic drugs, such ,~s chlorpromazine, chloroquine, and propranolol, have demonstrated that these amphipaths inhibit lysosomal [38] as well as neutral-active PLAs [39]. Inhibition by these drugs was thought to be mediated by substrate interaction, but some studies have reported direct interaction of the amphipath with enzyme [39,40]. Sphingosine blocked arachidonic acid release and PLAz activity in neutrophils [5] and smooth muscle cells [19] and stimulated PLD activity in neuroblastoma and 3"I"3 cells [14,15]- in these studies, the effect of sphingosine was attributed to inhibition of PKC (PLA 2) or phosphatidic acid phosphol:ydrolase activity (PLD), but the molar ratio of sphingosine-to-cell phospholipid ranged from 0.2-1.0. Our re:~ults demonstrate that membrane-mediated effects of sphingosine alter in-vitro PLA 2 or PLD activities at comparable molar ratios of sphingosineto-phospholipid. These results suggest that sphingosine

174 has e f f e c t s o n l i p o l y t i c e n z y m e s i n v o l v e d in cell s i g n a l

transduction that are i n d e p e n d e n t of PKC. S p h i n g o s i n e m a y act as a p h y s i o l o g i c , as well a s pharmacologic, modulator of cellular phospholipases In t h i s r e g a r d , it is k n o w n t h a i c e l l u l a r u p t a k e o f 3 H - s p h i n g o s i n e t o a m o u n l s lO-fold h i g h e r t h a n end o g e n o u s l e v e l s (approx. 5 0 p m o ! / 1 0 7 cells) c o m p l e t e l y i n h i b i t s P K C activity, a n d t h a t e n d o g e n o u s levels o f s p h i n g o s i n e u n d e r g o r e l a t i v e l y slow, a g o n i s t - d e p e n d c n t c h a n g e s t h a t a r e n o t d u e t o d e - n o v o s y n t h e s i s [42]. Sphingosine prevented ionopbore-induccd arachidon a t e r e l e a s e f r o m h u m a n P M N s w h i c h s~,as r e s t o r e d by PMA, but activation of PKC itself was not sufficient to induce arachidonatc mobilization and PAF or LTb4 r e l e a s e [5]. Clearly, c a t a b o l i s m o f s p h i n g o l i p i d t o s p h i n gosine and related bases generates e n d o g e n o u s amp h i p a t h s c a p a b l e o f i n f l u e n c i n g m u h i p l e p o i n t s in cell s i g n a l t r a n s d u c t i o n . C o n s e q u e n t l y , it is d i f f i c u l t t o ascribe Sl~Cificity t o s p h i n g o s i n e ' s c e l l u l a r cffccLs. Acknowledgement T h i s r e s e a r c h was s u p p o r t e d by g r a n t D K 42615 from the National Institutes of Health. References I Hannun, Y.A., Le~n'ais.C.R_ Merrill. A.H. and I},=11.R.M. (191161 J. Biol. Chem. 261, 12604-12609. 2 Merrill. A.H.. Nimkar. S, Menaldino. D.. Hannun. Y.A.. Loomis. C_ B¢11, R.M_ Tya~i. S R . !ambeth. J.D., Steveus. V., Hunter. I l and Liotta. D.C. (|QSQ) Bioch~',~fisl~'28. 3138-3145. 3 Merrill. A.II., SercnL A.M, St¢.-.'ens, V.L. Hannun, Y.A.. Bell. R.M. and Kinkadc. J.M. IIW66)J. Biol. Chem. 261. 12610-126|5. 4 Wilson, E.. Olcen. M.C~ Bell. R.M.. Merrill. A.H. and Lambeth. J.D. (1986) J. Bio'.. Cht:m. 26.1. 12616-12623. 5 Mclnq,'re, T.M_ Reinhold. S.L. Prescott. S.M. aud Zimmcrman. G.A. ~.1987)J. Biol. Chem. 26.L 15370-1¢,'~76. 6 Merrill. A.H. and Sic,ecru. VL_ (1~89) Biochim. Biopby~,. Acta 1010. 131-13t). 7 Hannun. Y.A. and Ek:ll. R.M. (1981~)Science 243. 500-5U7. 8 Merrill. A.H. and Jones, D.D. 119911) Biochim. Bu~phys. Aria 10.14. 1-17 9 Slife, C.W., War.g.E.. Hunter. R., Wang, S.. Bu*g~,~,. C., Liona, D.C. and Merrill. A H (1989). J. BIOL Chem. 26,4. U)371-U|3T,r. 10 Johnson. J.A. and Oark. R.B. (19~1) Bi(~chern. J. 268. 507-511. II Fiore. S.. Nicolaou. K.C., Caulfield. T.. katao~ka. II. and Serhan. C.N. (19911}Bh~chcm. J. 266. 2_5-31. 12 Kirshnamurthi. S.. Patel. Y. :rod Kakkar. V.V. (l~89~ Biochim. Bioph}s. Acta 1010. ?~itL264.

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Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine.

Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report...
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