307
Biochimmicaet Biophyrice: Ada, 672 (1979) 307-313 0 ~v~r~Nor~-~o~~d 3kmedicai Pms
BBA 67318
FATTY ACID AND GLYCEROL LABELING OF GLY~RO~~S LEUKOCYTES IN RESPONSE TO IONOPHORE A23187
OF
3EN-SIE TOU ~@~~entpf Bi~hern~~, 70112 (U.S.A.)
Tulane Uni~~i~
School of l#&xWne, New Orleans, LA
(Received July 24th, 1978)
The effect of divalent cation ionophore, A23187, on the ~~~~tion of [I-“C]palmitic a&l, [l-i4C]llnoleic acid and [U-*‘C]glycerol into glycerolipids of polymorphonuclear leukocytes was examined. Ionophore A23537 stimulated the labeling of pbosphatidic acid,’ phospbatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. fl-“‘C]Palmitic acid and [l-‘4Cflinoleic acid ~co~o~tion into ~~h~yl~o~e and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [W4C]glycerol labeling of these lipids was not significantly changed under identical conditions. These datareflect that the acylation of snglycerol 3-phparphate is a&iv&d, and the aeylations of ly~~~hatidylcholine and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23137. External calcium was not requlred for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the rne~~~ of the fatty acid and glycerol moieties of ~y~ro~pids by changing the distribution of intracellular calcium of leukocytes. IlltrOdUCtiOXl
An increased 3iP, incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides of polymorphonuclear leukocytes exposed to ionqphore A23187 has recently been demonstrated [l]. Incubation of ~olym~honu~e~ leukocytes with A231S7 did not rest& in calcium accumulation, though a’rapid burst uptsttre and release of *%a2+ by leukocytes was observed. Extracellular calcium was required for full stimula-
tion by A23187 on 32Pl-labeling of acidic glyc~opho~ho~pi~ of leukocytes, but the increased radiophosphorus activity ~88 not abolished in EGTA&eated leukocytes. Since not every moiety of the glycerolipid moleculi? is metabolized at the same rate, the stimulation by A23187 on the phosphorus moiety incorporation into the lipid molecule may not reflect the incorporation rate of other moieties of the molecule. In the present study, the effect of A23187 on fatty acid and glycerol incorporation into leukocyte glycerolipids is presented. Materials and Methods
~u~e~~. The source of ionophore A23187 and preparation of the ionophore working solution were described previously [I]. [I-“C]Palmitic acid (65.26 Ci/mol), [ l-“Cllinoleic acid (50.60 Ci/mol) and [U-14C]glycerol (130.9 Ci/mol) were purchased from New England Nuclear Corp. Individual glyceropho~ho~p~ standards, sn-1,2diolein and triolein were products of Supelco. Fatty acid free bovine serum albumin was obtained from Miles Laboratories. Preparation of polymorphonuclear leukocytes. Polymorphonuclear leukocytes from guinea pig peritoneal exudates were prepared as described previou6ly [l]. Prepamdion of ulbumin-bound fatty acid. Albumin-bound fatty acid was prepared by the method similar to that described by Hawitiy and Gordon [2] but with modification. 0.23 mg [l-14C]Palmitic acid (SO &i) or 0.28 mg [l-“Cflinoleic acid (60 &!i) in 1.6 ml hexane was added to 200 mg fatty acidfree albumin in 5 ml 0.9% NaCl in a ~~~~p~~ tube. The mixture was flushed with Nz and shaken at 37’6 for 3 h. The tube was #en centrifuged at 600 X g for 10 min at room temperature and the hexane layer was removed by apiration. The resulting albumin soluti,on contained. about 95% of the total radioactivity. ~nc~b~t~n$. Au ~ubatio~ were carried out at 37-C in siliconized glassstoppered tubea under air with shakingb Each tube (final volume 2.0 ml) contained 0.1 ml 20 &i ionophore A28187 or 0.1 ml properly diluted dimethylsulfoxide with 0.9% NaCl and an appropriate amotit of radiotracer ([l-‘3C]palmitic acid, [ l-l’C]~~e~ acid or ~U-i4C]~~ro~). The tubes were preincubated for 10 min at S’fC, then 1.8 ml (40 * IS4 cells) of leukocyte8 was added to each tube. They were further incubated for the specified periods of time. Lipid extraction. The incubation wasstopped by the addition of 10 ml 0.1 N HCl/CHsOH followed by 20 ml CRC&. The homogeneous mixture was partitioned three times each with 6 ml 1 M MgC12,and the washed CHCl3 layer was taken to dryness in a rotary evaporator. The residue was rediiolved in 0.1 ml CHClJ/CH30H (2 : 1, v/v) containing 0.01% butylated hydroxyytoluene aa antioxidant. Ch~rnato~ph~ re~lu~n of ~lyce~lipids. Glyceropho~ho~pids were resolved by twodimeusional thin-layer chromatography on Silica gel H (Analtech) as described previously [ 11. Neutral glyceroliplda were resolved by onediiensional thin-layer chromatography on airica gel H developed in petroleum ether/die&y1 ether/acetic acid (80 : 20 : 1, v/v) f3]. The radioactivity of each lipid wan measured as described previously fl]. Measurement of cellular 14C-labeled fatty acid and acidgoiuble mdioactiuity
309
from [U-14C]g&erol. The incubation was stopped by the addition of 10 ml Krebs-Ringer-Tris buffer to each tube which was immediately centrifuged at 260 Xg for 5 min and the resulting supernatant was aspirated. For measurement of radioactivity of cell* 14C-labeled fatty acid, 1.5 ml distilled water was added to the cell pellet and total lipid was extracted as described above. Fatty acid was resolved from other lipids by thin-layer chromatography on Silica gel H developed in petroleum ether/diethyl ether/acetic acid (80 : 20 : 1, v/v) [ 31. When [U-“Clglycerol was used as a tracer, acid-soluble radioactivity (including glycerol and glycerol phosphate) was measured, the cell pellet was precipitated with 1.0 ml 0.6 N HClO,, and centrifuged at 600 X g for 10 min at 4’C. The supematant was neutralized with 5 M KZCOJ to pH 6.5. An aliquot of the neutralized supematant was counted for radioactivity. Results and Discussion [I-“C]Palmitic acid and [1-“C]linoleic acid were used as radiotracers to examine the effect of A23187 on the metabolic activity of the a@ groups in
the glycerolipid molecules, since the phospholipid fraction from rabbit peritoneal exudates polymorphonuclear leukocytes was found mostly linoleic acid (40%) and pahnitic acid (20%) [4]. The incorporation of labeled fatty acids into leukocyte glycerolipids as a function of time in control cells and in cells exposed to A23187 is shown in Figs. 1 and 2. At all time intervals of incubation (5-30 mm), the ionophore induced a significant incorporation of both [1-“C]palmitic acid and [1-“Cllinoleic acid into phosphatidic acid, phosphatidylglycerol, phosphatidyhnositol, and diacylglycerol. In contrast, the labeling of phosphatidylcholine and triacylglycerol was depressed by the presence of A23187. Phosphatidylcholine labling by [l-“Cjpalmitic acid was less A23187 sensitive than by [l-“Cllinoleic acid, its radioactivity derived from [l-“Clpahnitic acid was only reduced 11% with P values of 93.9% from four experiments in cells with the ionophore after 30 min of incubation. Also revealed in Figs. 1 and 2 is the paratlel time-course of laMed fatty acid incorporation into phosphatidic acid and diacylglycerol. The observed decline in the labeling,of phosphatidic acid and diacylglycerol in cells incubated with fatty acids longer than 10 mm appears to be consequences of an exhaustion of the labeled fatty acids and a rapid metabolism of the acyl moiety of the glycerolipids, since it could be restored by a further addition of labels. The increased fatty acid incorporation into phosphatidic acid of leukocytes in the presence of A23187 revealed an increased mation of sn-glycerol 3phosphate. A23187 has been shown to stimulate j2Pi incorporation into phosphatidic acid of leukocytes [l], but the ionophore affe&ed Wosyntbetic step(s) leading to phosphatidic acid formation has not been defined. Results from the present study suggest that at least part of the increased radiophosphorus &ivity of phosphatidic acid in ceils with A23187 is der&ed from sn-glycerol 3-phosphate. However, the possibility cannotbe el$minxted t&at a stimulated pbosphorylation of endo@nous diacylglyoerol may aleo cornz&ute to part of the increased phosphatidic acid labeling by 32Pi. Unlike “Pi labeling of glycerophospholipids in leukocytes, the incorporation of labeled fatty acids into glycerolipids induced by A23187 was not affected
limo
(min)
Fig. 1. Effect of AM187 on Cl-‘%IIMUW add incorpor~tton trto leukocyte t&caoWda as. funotioa of time. ~o~~morphoauaar ~~ulrocyte~ (40.106) were incubrted at the indicated peztod of tima aiti~ [1-14CIpaltnttlc add (0.884 pCi, IS.!438 Ci/mol) u albumin complex iu the Fmwnce or abaeace of 1 jM A28187. Each point nPramnt8 the avera~ rulioactivity from 8.0 * 104 aelb from two aepurte ineubationa. PA, Pho&&ld&3 &d; PC. Pho~h&dylcholinO: PG. Pho&&id~ycerol: Pi, Pho&Uldyiinodtol; control cella; 0 DG, dkcylslycerol; TO. trlacrl@‘~cerol. -, 0, + A23187.
by external calcium (Table I). The response of leukocytes to A23187 monitored by fatty acid incorporation demonstratedin the presentstudy is different from that of human eqthrocytes [5]. Incubation of human erythrocytes with A23187 selectively rem$.ed in a calcium4ependent increase in fbe incorporation of pahuitic acid and liwleic. a&l into phosphatidyletha&amine but not phosphqtidylcholiw In the present study, f/&y a&d hwo~ into phosphatidylethanolamkww&snot responsiveto A!Bl&7 (TabIe I). The dtice may reflect the rwponse of Mfemnt cells to A2&187. The redwtlon by A23187 of fatty acid incorpwvtion into phosph&ldyluholine and trlacylglycerol observed in the @ent study appears to refkct a decmawd acylation of 1ysophosphatidylchoUneand endogenous diacylglycerol, respectively. It is less likely caused by enhanced hydrolysis of these glycw&pids, &ce therate of isotopic decay of .preWeIed glycerotipidawas not alteredin cells inrmbated with,A23187.
311
PG
E
1.0
r
IO
OG
0
5
K)
20
4L
r
TO
SoObIO
20
30
Tima(mlnI FU. 2. Effect of A13187 on [l- “Cllinoleic
add incorpomtion into leukocyte &~erolipidr u l function of time. PoWmorphonueleu leukocyter (40 - 104) were ina~bated at the indicated period of time with ~0~ add (0.364 /.K%,30.60 Cilmol) u albumin complex in the presenceor ebmnee of 1 @d A23187. Each point ~~~aenta tbe average radioactivity from 8.0 * 106 W& from two rpemte iaeubatioae. The + A23187. abbreviationaused are the ume as those in Fig. 1. -, control cells. -,
When [U-14C]glycerol was used a a tracer, A23187 markedly potentiated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and diacylglycerol as illustrated in Fig. 3. These findinga tend to suggest an activa~ tion of de nova ~qyntheait~ of these lipids in leukocytes by the presence of A23187. [U-“‘C]Olycerol labeling of phoaphatidykholine and tHacyl&werol was not chau@ Ggnificantiy ‘by the prewnce of A23187 (Fig. 3); wggwting the reduced fatty acid incorporation into these lipids to be att;ribssezl-to an inhibition of the acylation of lysophosphatidylcholine and endogenotw &cylglycerol by fatty acyl COA. External calcium did not affect [U-%)g#ycer& incorporation of leukocytes in response to A23187. In addition, n&t&r the cellu.W.labeled fhtty acid nor acidsoluble radioactivi@ WBSir&uencwl by the ionophore. Allan and Michell [6] rep&& that A23187 stimuiated ~[2ipw]j&wrol incorporation into total phcxpholipids and c%cyl@yCernawith a reduction in the. EabeWg of tziacylglyce~~~lin polymozph~ear ~b0ukwyW fwn pig blood. In agreement with the& find&J& the pmuent study dem_,an
312 TABLE I EFFECT OF EGTA ON LEUKOCYTES EKP?SRD
11-14ClPALMITIC ACID INCORPORATION TO THE IONOPHORE A2Sl87
INTO
GLYCEBOLIPIDS
OF
Leukocytes (40 * lOa) in KrebcRinger Tris buffer without CaZ+ were Incubated for 10 min at 37*C with and without EGTA. The mixture’ was then further incubated for 6 min with [ 1-14C1Fahnitic acid (0.384 pCi, 65.26 Ci/mol) and 1 uhi A28187. Glvcerolipids were resolved and the rem&e represent the average from two separate incubations. LiFid
Phos&atidic
EGTA
A23187
acid + + +
+
FborphatIdyIlnositol + + +
+
Phorphatidylchohne + + f
+
Pho~~~dy~~~l + + +
*
DiacYltzlYcerOl
+ + +
+
TriacyIgWcerol 4
+ +
+
BhO8phddYl&lW30-
+ + +
4
Radioactivity fdFm/S.O - lo6 eeIIsI 4 10 4 8
782 079 187 902
608 1668 326 1 673 7 6 b 6
072 769 866 013 188 239 227 297
17 896 21602 17 264 22 006 21104 16 094 22 298 16 660 780 806 706 722
increa6ed #$lycerol labeliug Qf the aMic ~y~pb~h~l~~ and diacyl&werol in leukwytes iruxb&d with A2318-7, However, the change in triacyl@yces~l lab&ix@by glycerol induced by A23187 was not meawrable in tbe preiwnt tiy. Tbe pxw& study presented the fir& in-W of fatty acid metabolism into @ycerolipids o# IcukcwyW in response to AZ3187. The labeiingaf &ycerolipids by fa#iy tici& msembled tbat by 32Pi;in each caw phwphatk& acid ad
313
s
10
20
30
Mlnutoo Fi6.3. Effect of A23187 on [U-14C161~cerolinco~oreUon into leukocyte glycerolinideesefunctionof time.Polymoxpbonucleuleukocytee(4O~l06) were incubeted et the indicatedperkA of timewitb[UofljrMA23187.Eeck poi&nwreeeiktethe "Cldy~mf~.2~CL180~Cymd)10~~re#~or~ lve%e@redtoeetIvtt~ fzom6.0.104 callr~mtwo~puclkIPcubetiolu,The~b~~~~tbe contro1$!elle:-. +A28187. nmeesthomi.n~lu.1.-.
@do glycerolipids was not abolished by treatment of intact cells with 1 mM
EGTA, which probably is unable to extract the internalmembrane-boundcalcium. .If the ionophore is only responsible for transferringdivalent,cations across biological membranes,the external calcium independent stimulationby A23187 on the incorporation of fatty acid and glycerol into glycerolipids observed in the present 8krdy can only be interpreteda8 the redistributionof cellular calcium induced by the ionophore. A detailed study on the effect of calcium on the enzymes involved in the synthesis of leukocyte glycerobpids may be helpful to understandthe ionophore effect on lipid metabolism. Acknowledgement
This work was supported by Grant CA16483 from the National hsititute of Heal$h. The author is a recipient of Research Career Development Award l-K04 CA00013 from the Natiqnal Institute of Health. References Tou.J.8.(1078) Biocbim.Biophyr.Acte 681.167-178 Hawley. H.P.endGordon.G.B.(1076) Leb.Inveet.34.218-222 h~.R.,P$~.P.~d~h.P.(1974)J.~~ Rer.lK.380-388 Elabecb.P.(1969)J.ExMLMed.110.969-980 Dtee.C.G..kLe.W.C..Good~.D.B.P.~d~~ursn.H.(1976)J.Btol.C~m.2bl.416~164 Allen,D.mdMIche~.R.H.(1077)Biocbem.J.164.399--397
Biochimmicaet Biophyrice: Ada, 672 (1979) 307-313 0 ~v~r~Nor~-~o~~d 3kmedicai Pms
BBA 67318
FATTY ACID AND GLYCEROL LABELING OF GLY~RO~~S LEUKOCYTES IN RESPONSE TO IONOPHORE A23187
OF
3EN-SIE TOU ~@~~entpf Bi~hern~~, 70112 (U.S.A.)
Tulane Uni~~i~
School of l#&xWne, New Orleans, LA
(Received July 24th, 1978)
The effect of divalent cation ionophore, A23187, on the ~~~~tion of [I-“C]palmitic a&l, [l-i4C]llnoleic acid and [U-*‘C]glycerol into glycerolipids of polymorphonuclear leukocytes was examined. Ionophore A23537 stimulated the labeling of pbosphatidic acid,’ phospbatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. fl-“‘C]Palmitic acid and [l-‘4Cflinoleic acid ~co~o~tion into ~~h~yl~o~e and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [W4C]glycerol labeling of these lipids was not significantly changed under identical conditions. These datareflect that the acylation of snglycerol 3-phparphate is a&iv&d, and the aeylations of ly~~~hatidylcholine and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23137. External calcium was not requlred for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the rne~~~ of the fatty acid and glycerol moieties of ~y~ro~pids by changing the distribution of intracellular calcium of leukocytes. IlltrOdUCtiOXl
An increased 3iP, incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides of polymorphonuclear leukocytes exposed to ionqphore A23187 has recently been demonstrated [l]. Incubation of ~olym~honu~e~ leukocytes with A231S7 did not rest& in calcium accumulation, though a’rapid burst uptsttre and release of *%a2+ by leukocytes was observed. Extracellular calcium was required for full stimula-
tion by A23187 on 32Pl-labeling of acidic glyc~opho~ho~pi~ of leukocytes, but the increased radiophosphorus activity ~88 not abolished in EGTA&eated leukocytes. Since not every moiety of the glycerolipid moleculi? is metabolized at the same rate, the stimulation by A23187 on the phosphorus moiety incorporation into the lipid molecule may not reflect the incorporation rate of other moieties of the molecule. In the present study, the effect of A23187 on fatty acid and glycerol incorporation into leukocyte glycerolipids is presented. Materials and Methods
~u~e~~. The source of ionophore A23187 and preparation of the ionophore working solution were described previously [I]. [I-“C]Palmitic acid (65.26 Ci/mol), [ l-“Cllinoleic acid (50.60 Ci/mol) and [U-14C]glycerol (130.9 Ci/mol) were purchased from New England Nuclear Corp. Individual glyceropho~ho~p~ standards, sn-1,2diolein and triolein were products of Supelco. Fatty acid free bovine serum albumin was obtained from Miles Laboratories. Preparation of polymorphonuclear leukocytes. Polymorphonuclear leukocytes from guinea pig peritoneal exudates were prepared as described previou6ly [l]. Prepamdion of ulbumin-bound fatty acid. Albumin-bound fatty acid was prepared by the method similar to that described by Hawitiy and Gordon [2] but with modification. 0.23 mg [l-14C]Palmitic acid (SO &i) or 0.28 mg [l-“Cflinoleic acid (60 &!i) in 1.6 ml hexane was added to 200 mg fatty acidfree albumin in 5 ml 0.9% NaCl in a ~~~~p~~ tube. The mixture was flushed with Nz and shaken at 37’6 for 3 h. The tube was #en centrifuged at 600 X g for 10 min at room temperature and the hexane layer was removed by apiration. The resulting albumin soluti,on contained. about 95% of the total radioactivity. ~nc~b~t~n$. Au ~ubatio~ were carried out at 37-C in siliconized glassstoppered tubea under air with shakingb Each tube (final volume 2.0 ml) contained 0.1 ml 20 &i ionophore A28187 or 0.1 ml properly diluted dimethylsulfoxide with 0.9% NaCl and an appropriate amotit of radiotracer ([l-‘3C]palmitic acid, [ l-l’C]~~e~ acid or ~U-i4C]~~ro~). The tubes were preincubated for 10 min at S’fC, then 1.8 ml (40 * IS4 cells) of leukocyte8 was added to each tube. They were further incubated for the specified periods of time. Lipid extraction. The incubation wasstopped by the addition of 10 ml 0.1 N HCl/CHsOH followed by 20 ml CRC&. The homogeneous mixture was partitioned three times each with 6 ml 1 M MgC12,and the washed CHCl3 layer was taken to dryness in a rotary evaporator. The residue was rediiolved in 0.1 ml CHClJ/CH30H (2 : 1, v/v) containing 0.01% butylated hydroxyytoluene aa antioxidant. Ch~rnato~ph~ re~lu~n of ~lyce~lipids. Glyceropho~ho~pids were resolved by twodimeusional thin-layer chromatography on Silica gel H (Analtech) as described previously [ 11. Neutral glyceroliplda were resolved by onediiensional thin-layer chromatography on airica gel H developed in petroleum ether/die&y1 ether/acetic acid (80 : 20 : 1, v/v) f3]. The radioactivity of each lipid wan measured as described previously fl]. Measurement of cellular 14C-labeled fatty acid and acidgoiuble mdioactiuity
309
from [U-14C]g&erol. The incubation was stopped by the addition of 10 ml Krebs-Ringer-Tris buffer to each tube which was immediately centrifuged at 260 Xg for 5 min and the resulting supernatant was aspirated. For measurement of radioactivity of cell* 14C-labeled fatty acid, 1.5 ml distilled water was added to the cell pellet and total lipid was extracted as described above. Fatty acid was resolved from other lipids by thin-layer chromatography on Silica gel H developed in petroleum ether/diethyl ether/acetic acid (80 : 20 : 1, v/v) [ 31. When [U-“Clglycerol was used as a tracer, acid-soluble radioactivity (including glycerol and glycerol phosphate) was measured, the cell pellet was precipitated with 1.0 ml 0.6 N HClO,, and centrifuged at 600 X g for 10 min at 4’C. The supematant was neutralized with 5 M KZCOJ to pH 6.5. An aliquot of the neutralized supematant was counted for radioactivity. Results and Discussion [I-“C]Palmitic acid and [1-“C]linoleic acid were used as radiotracers to examine the effect of A23187 on the metabolic activity of the a@ groups in
the glycerolipid molecules, since the phospholipid fraction from rabbit peritoneal exudates polymorphonuclear leukocytes was found mostly linoleic acid (40%) and pahnitic acid (20%) [4]. The incorporation of labeled fatty acids into leukocyte glycerolipids as a function of time in control cells and in cells exposed to A23187 is shown in Figs. 1 and 2. At all time intervals of incubation (5-30 mm), the ionophore induced a significant incorporation of both [1-“C]palmitic acid and [1-“Cllinoleic acid into phosphatidic acid, phosphatidylglycerol, phosphatidyhnositol, and diacylglycerol. In contrast, the labeling of phosphatidylcholine and triacylglycerol was depressed by the presence of A23187. Phosphatidylcholine labling by [l-“Cjpalmitic acid was less A23187 sensitive than by [l-“Cllinoleic acid, its radioactivity derived from [l-“Clpahnitic acid was only reduced 11% with P values of 93.9% from four experiments in cells with the ionophore after 30 min of incubation. Also revealed in Figs. 1 and 2 is the paratlel time-course of laMed fatty acid incorporation into phosphatidic acid and diacylglycerol. The observed decline in the labeling,of phosphatidic acid and diacylglycerol in cells incubated with fatty acids longer than 10 mm appears to be consequences of an exhaustion of the labeled fatty acids and a rapid metabolism of the acyl moiety of the glycerolipids, since it could be restored by a further addition of labels. The increased fatty acid incorporation into phosphatidic acid of leukocytes in the presence of A23187 revealed an increased mation of sn-glycerol 3phosphate. A23187 has been shown to stimulate j2Pi incorporation into phosphatidic acid of leukocytes [l], but the ionophore affe&ed Wosyntbetic step(s) leading to phosphatidic acid formation has not been defined. Results from the present study suggest that at least part of the increased radiophosphorus &ivity of phosphatidic acid in ceils with A23187 is der&ed from sn-glycerol 3-phosphate. However, the possibility cannotbe el$minxted t&at a stimulated pbosphorylation of endo@nous diacylglyoerol may aleo cornz&ute to part of the increased phosphatidic acid labeling by 32Pi. Unlike “Pi labeling of glycerophospholipids in leukocytes, the incorporation of labeled fatty acids into glycerolipids induced by A23187 was not affected
limo
(min)
Fig. 1. Effect of AM187 on Cl-‘%IIMUW add incorpor~tton trto leukocyte t&caoWda as. funotioa of time. ~o~~morphoauaar ~~ulrocyte~ (40.106) were incubrted at the indicated peztod of tima aiti~ [1-14CIpaltnttlc add (0.884 pCi, IS.!438 Ci/mol) u albumin complex iu the Fmwnce or abaeace of 1 jM A28187. Each point nPramnt8 the avera~ rulioactivity from 8.0 * 104 aelb from two aepurte ineubationa. PA, Pho&&ld&3 &d; PC. Pho~h&dylcholinO: PG. Pho&&id~ycerol: Pi, Pho&Uldyiinodtol; control cella; 0 DG, dkcylslycerol; TO. trlacrl@‘~cerol. -, 0, + A23187.
by external calcium (Table I). The response of leukocytes to A23187 monitored by fatty acid incorporation demonstratedin the presentstudy is different from that of human eqthrocytes [5]. Incubation of human erythrocytes with A23187 selectively rem$.ed in a calcium4ependent increase in fbe incorporation of pahuitic acid and liwleic. a&l into phosphatidyletha&amine but not phosphqtidylcholiw In the present study, f/&y a&d hwo~ into phosphatidylethanolamkww&snot responsiveto A!Bl&7 (TabIe I). The dtice may reflect the rwponse of Mfemnt cells to A2&187. The redwtlon by A23187 of fatty acid incorpwvtion into phosph&ldyluholine and trlacylglycerol observed in the @ent study appears to refkct a decmawd acylation of 1ysophosphatidylchoUneand endogenous diacylglycerol, respectively. It is less likely caused by enhanced hydrolysis of these glycw&pids, &ce therate of isotopic decay of .preWeIed glycerotipidawas not alteredin cells inrmbated with,A23187.
311
PG
E
1.0
r
IO
OG
0
5
K)
20
4L
r
TO
SoObIO
20
30
Tima(mlnI FU. 2. Effect of A13187 on [l- “Cllinoleic
add incorpomtion into leukocyte &~erolipidr u l function of time. PoWmorphonueleu leukocyter (40 - 104) were ina~bated at the indicated period of time with ~0~ add (0.364 /.K%,30.60 Cilmol) u albumin complex in the presenceor ebmnee of 1 @d A23187. Each point ~~~aenta tbe average radioactivity from 8.0 * 106 W& from two rpemte iaeubatioae. The + A23187. abbreviationaused are the ume as those in Fig. 1. -, control cells. -,
When [U-14C]glycerol was used a a tracer, A23187 markedly potentiated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and diacylglycerol as illustrated in Fig. 3. These findinga tend to suggest an activa~ tion of de nova ~qyntheait~ of these lipids in leukocytes by the presence of A23187. [U-“‘C]Olycerol labeling of phoaphatidykholine and tHacyl&werol was not chau@ Ggnificantiy ‘by the prewnce of A23187 (Fig. 3); wggwting the reduced fatty acid incorporation into these lipids to be att;ribssezl-to an inhibition of the acylation of lysophosphatidylcholine and endogenotw &cylglycerol by fatty acyl COA. External calcium did not affect [U-%)g#ycer& incorporation of leukocytes in response to A23187. In addition, n&t&r the cellu.W.labeled fhtty acid nor acidsoluble radioactivi@ WBSir&uencwl by the ionophore. Allan and Michell [6] rep&& that A23187 stimuiated ~[2ipw]j&wrol incorporation into total phcxpholipids and c%cyl@yCernawith a reduction in the. EabeWg of tziacylglyce~~~lin polymozph~ear ~b0ukwyW fwn pig blood. In agreement with the& find&J& the pmuent study dem_,an
312 TABLE I EFFECT OF EGTA ON LEUKOCYTES EKP?SRD
11-14ClPALMITIC ACID INCORPORATION TO THE IONOPHORE A2Sl87
INTO
GLYCEBOLIPIDS
OF
Leukocytes (40 * lOa) in KrebcRinger Tris buffer without CaZ+ were Incubated for 10 min at 37*C with and without EGTA. The mixture’ was then further incubated for 6 min with [ 1-14C1Fahnitic acid (0.384 pCi, 65.26 Ci/mol) and 1 uhi A28187. Glvcerolipids were resolved and the rem&e represent the average from two separate incubations. LiFid
Phos&atidic
EGTA
A23187
acid + + +
+
FborphatIdyIlnositol + + +
+
Phorphatidylchohne + + f
+
Pho~~~dy~~~l + + +
*
DiacYltzlYcerOl
+ + +
+
TriacyIgWcerol 4
+ +
+
BhO8phddYl&lW30-
+ + +
4
Radioactivity fdFm/S.O - lo6 eeIIsI 4 10 4 8
782 079 187 902
608 1668 326 1 673 7 6 b 6
072 769 866 013 188 239 227 297
17 896 21602 17 264 22 006 21104 16 094 22 298 16 660 780 806 706 722
increa6ed #$lycerol labeliug Qf the aMic ~y~pb~h~l~~ and diacyl&werol in leukwytes iruxb&d with A2318-7, However, the change in triacyl@yces~l lab&ix@by glycerol induced by A23187 was not meawrable in tbe preiwnt tiy. Tbe pxw& study presented the fir& in-W of fatty acid metabolism into @ycerolipids o# IcukcwyW in response to AZ3187. The labeiingaf &ycerolipids by fa#iy tici& msembled tbat by 32Pi;in each caw phwphatk& acid ad
313
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10
20
30
Mlnutoo Fi6.3. Effect of A23187 on [U-14C161~cerolinco~oreUon into leukocyte glycerolinideesefunctionof time.Polymoxpbonucleuleukocytee(4O~l06) were incubeted et the indicatedperkA of timewitb[UofljrMA23187.Eeck poi&nwreeeiktethe "Cldy~mf~.2~CL180~Cymd)10~~re#~or~ lve%e@redtoeetIvtt~ fzom6.0.104 callr~mtwo~puclkIPcubetiolu,The~b~~~~tbe contro1$!elle:-. +A28187. nmeesthomi.n~lu.1.-.
@do glycerolipids was not abolished by treatment of intact cells with 1 mM
EGTA, which probably is unable to extract the internalmembrane-boundcalcium. .If the ionophore is only responsible for transferringdivalent,cations across biological membranes,the external calcium independent stimulationby A23187 on the incorporation of fatty acid and glycerol into glycerolipids observed in the present 8krdy can only be interpreteda8 the redistributionof cellular calcium induced by the ionophore. A detailed study on the effect of calcium on the enzymes involved in the synthesis of leukocyte glycerobpids may be helpful to understandthe ionophore effect on lipid metabolism. Acknowledgement
This work was supported by Grant CA16483 from the National hsititute of Heal$h. The author is a recipient of Research Career Development Award l-K04 CA00013 from the Natiqnal Institute of Health. References Tou.J.8.(1078) Biocbim.Biophyr.Acte 681.167-178 Hawley. H.P.endGordon.G.B.(1076) Leb.Inveet.34.218-222 h~.R.,P$~.P.~d~h.P.(1974)J.~~ Rer.lK.380-388 Elabecb.P.(1969)J.ExMLMed.110.969-980 Dtee.C.G..kLe.W.C..Good~.D.B.P.~d~~ursn.H.(1976)J.Btol.C~m.2bl.416~164 Allen,D.mdMIche~.R.H.(1077)Biocbem.J.164.399--397
