Modulation of 32Pi incorporation into phospholipids of polymorphonuclear leukocytes by ionophore A23187

167

Biochimica et Biophysics 0 Elsevier/North-Holland

BBA

Acta,

531 (1978)

Biomedical

167-178

Press

57270

MODULATION OF 32PiINCORPORATION POLYMORPHONUCLEAR LEUKOCYTES

JEN-SIE

TOU

Department of Biochemistry, 70112 (U.S.A.) (Received

INTO PHOSPHOLIPIDS OF BY IONOPHORE A23187

May

18th,

Tulane

University

School

of Medicine,

New Orleans, La

1978)

Summary

The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2’ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca”, but not Mg”‘, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca*‘, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(P-aminoactivity was also ethylether)-N,N’-tetraacetic acid. ATP radiophosphorus enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca” fluxes and perturbation of cellular Ca2’ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.

Introduction

Phagocytosis by polymorphonuclear leukocytes has been shown to stimulate 32Pi and myo [ 2-3H]inositol incorporation into phosphatidic acid and the phosAbbreviation:

EGTA.

ethyleneglycol

bis(P-aminoethylether)-N,N’-tetraacetic

acid.

168

phoinositides [l-3], but the mechanism by which phagocytosis enhances radiotracer incorporation into these phospholipids remains unknown. Smith and Ignaro [4] demonstrated an increased association of external 4sCa2+ with human neutrophils during phagocytosis of zymosan particles. The enzymes responsible for the metabolism of phosphatidic acid, the phosphoinositides and other phospholipids are often modulated by Ca’+, though they have not yet been studied in leukocytes. The availability of the divalent cation ionophore A23187 prompted us to examine the effect of Ca*’ fluxes on phospholipid metabolism of intact polymorphonuclear leukocytes. Ionophore A23187 is a monocarboxylic acid antibiotic from Streptomyces chartreusis and is active against Gram-positive bacteria and fungi. A23187 binds and transfers divalent cations across natural and artifical membranes [5-71 as well as intact leukocytes [4,8]. It has been used as a probe for studying the requirement of Ca*’ and/or Mg2+ for leukocyte functions. Exposure of polymorphonuclear leukocytes from human peripheral blood [4] and guinea pig peritoneal exudates [9] with A23187 resulted in external Ca*’ dependent extrusion of P-glucuronidase from the cells. Studies with A23187 suggest that chemotaxis is both Ca*’ and Mg2’ dependent [lo]. The effect of A23187 on lipid metabolism has been examined in several biological systems. Treatment of human erythrocytes with A23187 in the presence of extracellular Ca2+ enhanced the production of 1,2-diacylglycerol and the labeling of phosphatidic acid by 32Pi [ 111. The ionophore also selectively stimulated fatty acid incorporation into phosphatidylethanolamine in human erythrocytes [12]. In contrast, L-[U-i4C]serine incorporation into phosphatidylserine in cultured brain cells from rat was impaired in the presence of A23187 [13]. In lymphocytes from pig lymph nodes, A23187 enhanced the incorporation of 32Pi into phosp hatidic acid and phosphatidylinositol but slightly reduced the incorporation of [ 2-3H]glycerol into total phospholipids, whereas in polymorphonuclear leukocytes from pig blood, the ionophore increased [ 2-3H]glycerol incorporation into total phospholipids, 1,2-diacylglycerol and monoacylglycerol with a reduction in the labeling of triacylglycerol [14]. The effect of A23187 on 32Pi incorporation into individual phospholipids in polymorphonuclear leukocytes has not been examined, however. In this paper, 32P. incorporation into individual phospholipids and Ca2’ fluxes in polymorphonucl&rr leukocytes in response to A23187 were investigated. In addition, the effect of external divalent cations on the radiophosphorus activity of each phospholipid of polymorphonuclear leukocytes exposed to A23187 was also examined. Materials and Methods

Materials. Ionophore A23187 was a gift from Dr. R.L. Hamill of Eli Lilly and Company, Indianapolis, Ind. A 4 mM stock solution in dimethylsulfoxide was stored at -20°C until use. An aliquot of the stock solution was diluted with 0.9% NaCl to 20 FM, and 0.1 ml 20 ,uM A23187 was added to each incubation tube (final concentration, 1.0 PM). In control experiments, equivalent amount OF dimethylsulfoxide in 0.9% NaCl was used in each incubation tube. Carrierfree 32Pi was purchased from New England Nuclear Corp. Carrier-free 45CaC1,

169

was purchased from ICN Chemical and Radioisotope Division. Phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and sphingomyelin were products of Supelco Co. The diphosphoinositide fraction of Folch containing diphosphoinositide was prepared as described by Lees [ 151. Preparation of polymorphonuclear leukocytes. Polymorphonuclear leukocytes from guinea pig peritoneal exudates were prepared as described previously [3] except that the cells were collected by washing the peritoneal cavity with 0.9% NaCl instead of phosphate-buffered saline containing heparin. The cells were recovered by centrifugation at 250 Xg at room temperature for 10 min and washed twice with 0.9% NaCl. They were then washed once with Krebs-Ringer 15 mM Tris buffer (pH 7.4) [ 161, 1.27 mM CaCl,, 10 mM glucose and resuspended in the same buffer to give a final concentration of 20-25 . lo6 cells/ml and were kept at 37°C. Cell counts were made in hemocytometer and cell viability was measured by trypan blue dye exclusion. Incubations. All incubations were carried out at 37°C in 45 ml siliconized glass-stoppered centrifuge tubes under air with shaking. Each tube contained 0.1 ml 20 /IM ionophore A23187 or 0.1 ml properly diluted dimethylsulfoxide with 0.9% NaCl and an appropriate amount of radiotracer (32 Pi or 45CaC1,) in 0.1 ml 0.9% NaCl. The tubes were preincubated for 10 min at 37”C, then 1.8 ml (40 . lo6 cells) of leukocytes in Krebs-Ringer-Tris buffer (as described above) was added to each tube. They were further incubated for the specified periods of time. Measurement of enzyme release into the incubation medium from leukocytes. At the end of the incubation, the tubes were placed in ice and centrifuged at 250 X g for 5 min, and lactate dehydrogenase and P-glucuronidase in the supernatant solution were measured as described previously [ 17,181. Lipid extraction. At the end of the incubation, the reaction was terminated by the addition of 10 ml 0.1 M HCl/CH,OH, followed by 20 ml CHC13. The homogeneous mixture was partitioned three times each with 5 ml 1 M MgCl, and the washed CHC13 phase was taken to dryness in a rotary evaporator. The residue was redissolved in 0.1 ml of CHCI,/CH30H (2 : 1, v/v) containing 0.01% butylated hydroxytoluene as antioxidant. Chromatographic resolution of phospholipids. Phospholipids were resolved by two-dimensional thin-layer chromatography on Silica gel H (Anatech, Inc.). The glass plates were heated for 1 h in an oven at 100°C before use. 20 ~1 total lipid extract (2.5 pug phosphorus) and 10 d standard phosphatidylglycerol and phosphatidic acid (0.5 ,ug phosphorus each) were applied on the plate. The chromatogram was developed with CHC13-CH30H-28% aqueous NH3 (65 : 25 : 5, v/v) in the first dimension [19] and then with CHC13-CH30H-CH&OOHHz0 (81 : 10 : 45 : 5, v/v) in the second dimension [20]. were separated by oneand triphosphoinositide Diphosphoinositide dimensional thin-layer chromatography on Silica gel H developed in npropanol/4 M ammonia (2 : 1, v/v) [21]. 10 ~1 total lipid extract was co-chromatographed with 10 ~1 (0.54 yg phosphorus) Folch’s diphosphoinositide fraction. After visualization of the chromatogram in iodine vapor, lipid spots were scraped off the plate and placed in a liquid scintillation vial containing 1 ml H,O. After shaking, 10 ml Instagel (Packard) was added to each vial and the

170

radioactivity was measured in a Packard Model 3320 Tri-Carb scintillation “P-labeled lipids were also confirmed by autoradio~aphy on spectrometer. Kodak No-screen X-ray film. Determination of lipid phosphorus. The quantity of each phospholipid was measured by determination of the phosphorus in each spot resolved by twod~ension~ thin-layer chromato~aphy. The silica gel was scraped off the plate and placed into an acid-washed tube (1.4 X 12.5 cm) containing 0.5 ml H20. Lipid phosphorus was measured according to a modification of the method of Chen et al. [22]. Each phospholipid was digested with 0.3 ml 6 M H,S0,/72% HC104 (3 : 2, v/v) at 130°C for 3 h on a heating block (Lab-Line Instruments, Inc.); after cooling, 0.8 ml HzO, 0.3 ml 2.5% ammonium molybdate and 0.3 ml 10% ascorbic acid were added to each tube; each addition was followed by a thorough mixing. The tubes were incubated at 37°C for 90 min and the Silica gel was removed by brief centrifugation. The absorbance was taken at 820 nm. A sample with 0.2 fig phosphorus gave an average absorbance of 0.115. In control tubes, silica gel H up to 2 X 2 cm did not exhibit detectable absorbance at 820 nm. Measurement of radiophosphorus activity and content of cellular ATP. At the end of the incubation, 10 ml Krebs-Ringer-Tris buffer at room temperature was added to each tube which was immediately centrifuged at 250 X g for 5 min. The cell pellet was precipitated with 1.0 ml 0.6 M HC104 and centrifuged at 600 X g for 10 min at 4°C. The HC104 supernatant fraction was neutralized with 5 M K&O3 to pH 6.5. For measurement of ATP radioactivity, an aliquot of the neutralized supernate was co-~hromato~aphed with authentic ATP, ADP and AMP on poly(ethyIene)imine cellulose thin layers developed in 0.85 M KH2P04, pH 3.4, to resolve ATP from Pi and other nucleotides [23]. The nucleotides were visualized under an ultraviolet lamp at 254 nm and the spots were scraped off and counted by liquid scintillation. ATP content was determined by the enzymic method described by Adams [ 241 using phosphoglycerate kinase coupled with glyceraldehyde-3-phosphate dehydrogenase. The glyceraldehyde-3-phosphate formed was converted to dihydroxyacetone phosphate by triose isomerase and dihydroxyacetone phosphate was converted to glycerol 3-phosphate in the presence of NADH and 0.1 ml neutralized HClO~-supernata~lt glycerol-3-phosplla~ dehydrogenase. solution was added to the cuvette containing 1 ml of 0.5 M triethanolamine buffer (pH 7.6), 4 mM MgS04, 6 mM glycerate 3-phosphate, 0.1 ml 2.5 mM NADH, 0.1 ml enzyme mixture (from Boehringer, Mannheim) of 300 units/m1 glycer~dehyde-3-phosphate dehydrogenase, 500 units~ml pllospho~ycerate kinase, 400 units/ml glycerol-3.phosphate dehydrogenase and 300 units/ml triose isomerase. The decreased absorbance at 340 nm after the reaction stopped was recorded by a Beckman Model 25 spectrophotometer. ATP quantity was computed as described by Adams [24-j and expressed as nmol ATP/l O6 cells. Calcium uptake. At the end of the incubation, 10 ml Krebs-Ringer-Tris buffer at room temperature was added to each tube, which was immediately centrifuged at 250 Xg for 5 min. The supernatant fluid was aspirated and the cell pellet was resuspended in 1 ml distilled water. An aliquot of the lysed cell suspension was counted for radioactivity.

171

Calcium and phosphate efflux. Leukocytes were suspended in Krebs-RingerTris buffer to a concentration of 75 - 106/mf. The cell suspension was incubated with 20 &i 45CaCl, in 0.2 ml 0.9% NaCl or 480 000 cpm 32Pi in 10 J 0.01 M HCl for 30 min to allow the cells to take up 45CaC12 or 32Pi* It was then centrifuged for 5 min at room temperature. The supernatant was aspirated and the cell pellet was resuspended in 50 ml buffer, The washed cells were then resuspended in 20 ml buffer, and 9.5 ml labeled cell suspension was incubated with 0.5 ml 20 PM A23187 or 0.5 ml properly diluted dimethysulfoxide. After varying periods (2-30 min) at 37”C, 2 ml cell suspension was removed and centrifuged for 5 min at room temperature. An aliquot of the supernatant fluid was removed and counted by liquid scintillation. A portion of the un~ent~fuged cell suspension was counted for total radioactivity. Ca2’ and Pi efflux was expressed as radioactivity remaining in cells which is the difference between the radioactivity of the uncentrifuged cell suspension and the supernatant solution. It should be noted that the radioactivity measured by this method may not reflect the net radioactivity associated with the cells without additional washings. Since it was noted that cells exposed to A23187 for 20 min or longer became aggregated and some of them adhered to the surface of the incubation tubes, additional washings of cells were not performed. Calcium content was measured with a Perkin-Elmer 306 atomic absorption spe~trophotome~r after digestion of cell pellets with HN03-HC104 (1 : 1, v/v). Cell protein was dete~ined by the method of Lowry et al. 1251 using bovine serum albumin as standard. Results Effect of A23187 on enzyme release from leukocytes into incubation medium. In order to examine the possible cell damage after cells were incubated with 1 PM A23187, the release of lactate dehydrogenase into the incubation medium with or without Ca2’ or Mg2+ was measured, in addition to checking dye-exclusion of the cells. No cell death was detected by trypan blueexclusion after 30 min incubation of cells with 1 PM A23187 in all incubation media and only a slight increase of lactate dehydrogen~e release into the incubation medium was observed (Table I). In contrast, the release of p-glucuronidase induced by A23187 was about 5-fold of the basal activity in medium with both Ca2+ and Mg2+. The data in Table I also indicate the requirement of release from leukocytes external Ca2+, but not Mg2+, for P-glucuronidase induced by A23187. A significant increase in lactate dehydrogenase release into the incubation medium was found when incubation was longer than 30 min or A23187 was above 1 r.tM. Time-course of the labeling of phospholipids by 32Pi in leukocytes in the presence of A23187. As shown in Fig. 1 and 2, the rate of “Pi incorporation into phosphatidic acid and the phosphoinositides of leukocytes was accelerated at all time intervals in the presence of A23187 in comparison with that in control cells. After 30 min incubation of cells with 1 PM A23187 at 37”C, the into phosphatidic acid ionophore resulted in an increased 32P. incorporation (499%), phosphatidylinositiol (947%), hiphosphoinositide (65%), triphospho-

172

TABLE

I

EFFECT

OF A23187

ON ENZYME

RELEASE

FROM

POLYMORPHONUCLEAR

LEUKOCYTES

Polymorphonuclear leukocytes (40 . lo6 cells) in Krebs-Ringer Tris buffer with or wihout Ca2+ or Mg*+ as indicated, were incubated for 30 min at 37°C in the presence or absence of 1 MM A23187. The enzyme activity in the incubation medium was measured. Total activity (100%) in complete medium and in medium with or without Ca*+ or Mg2+ was 98 @g phenophthalein released/IO7 cells per h for &&xuronidase and 1393 absorbance units/l07 cells per h for lactate dehydrogenase. Results represent the averages from two separate experiments, each in duplicate incubations. % of total activity

Enzyme

Medium

-A23187

+A23187

&Glucuronidase

+Ca*+ + Mg2+ -
2.00 1.88 1.70

10.2 2.6 11.0

Lactate dehydrogenase

+Ca*+ + Mg2+ --a*+ -Mg2+

5.50 7.26 3.90

6.90 7.85 5.10

in’ositide (65%), phosphatidylglycero~ (414%), and phosphatidylserine (120%). 32P. incorporation into phosphatidylglyc~rol and phosphatidylse~n~ was rather 10; at incubation times shorter than 30 min. The radioactivity of phosphatidyl-

Phospholldic

mid

-

Control

s..

+A23187

IO TIME

,’ i’ ,’

20 b.VN1

30 TIME

IMINI

Fig. 1. Time-course of 32Pi-iabeling of phosphatidie acid in polymorphonucle~ leukocytes. Leukocytes (40 I 106) were incubated at tge specified period of time with 32Pi (125 MCi) in the presence or absence of 1 /JM A23187. The lipids were extracted and separated. Each point represents the radioactivity from 8.0 . 106 cells. Fig. 2. Time-course of 3ZPi-labeling of the phosphoinositides in poiymorphonucfear leukocytes. The incubation was identical to that described in Fig. 2. The lipids were extracted and separated. Each point represents the radioactivity from 8.0 . 106 cells.

173

choline was, however, slightly decreased (5%) in the presence of A23187. Phosphatidylethanolamine and sphingomyelin were negligibly labeled by 32Pi. The quantity of each resolved phospholipid was determined by measuring the phosphorus content. 1 PM A23187 did not appear to change the quantity of any phospholipid of leukocyte after 5-30 min at 37°C. However, the possibility may exist that A23187 changed the quantity of only certain molecular species of these phospholipids which was not measurable by the techniques used in the present study. Effect of external Ca2+ and Mg2+ on 32Pi incorporation into phospholipids of leukocytes exposed to A23187. Since A23187 causes the transfer of Ca” and

Mg2+ across biological membranes, the effect of external Ca” and Mg2+ on 32Pi incorporation into phospholipids of leukocytes in response to A23187 was examined (Table II). The increased radiophosphorus activity of the acidic phospholipids (except phosphatidylglycerol) provoked by A23187 was significantly suppressed but not eliminated in cells without external Ca2’. The loss of radiophosphorus activity in phosphatidylcholine induced by A23187 in cells without external Ca” became more evident than that in control cells. On the other hand, when cells were incubated in medium without added Mg2+, the

TABLE

II

EFFECT

OF

IONOPHORE

MORPHONUCLEAR Leukocytes, bated were

for

suspended 30

min

extracted

experiment

A23187

ON

LEUKOCYTES

and

in Krebs-Ringer-Tris

at 37°C

with

separated.

and % change

Phospholipid

32Pi

WITH

32Pi 32P

(180

(cpm)

in radioactivity

INCORPORATION

OR

WITHOUT

buffer @Ci)

with

in the

(from

8.0

? S.D.

due

Medium

32Pi

INTO

EXTERNAL or without

presence

. 106

(cpm)/8.0

Ca2+

represent

as indicated.

1 PM

the values

cells

Phosphatidic

acid

Phosphatidylcholine

+Ca2+

+ Mg2’

% change

1779

+414

+

286

650

+108

t

21

-Mg2+

263

+672

+

85

+Ca2+

+ Mgz’

-Mg2+

Phosphatidylserine

Phosphatidylinositol

Diphosphoinositide

Triphosphoinositide

+Ca2+

+ Mg2+

661

-11:

3

314

49*

12

848

800

24

121

-7+

2 + f

75

37

176

+326

-Mgz+

27

151

+420?

62

50

139

+150?

28

+&+

+ Mg2+

-Ca2+

41

-Mgz+

22

+Ca2+

+ MgZ+

50

147

-Mg2+

136

2486

+Ca2+

+ MgZ+

824

1702

778

1234

-Mgz+

684

1786

1326 1008 958

+ MgZ+

=

radioactivity radioactivity

+1055

853

+x5+ +Ca2+

+480

1896

126

in cells

with

in control

50

+15i

152

-c!az+

-Mgz+

in radioactivity

754

-Caz+

qa2+

* % Change

93

620

+375

+504 +1530

f 260 67

? 212 f

25

+141

*

38

2084

+69

?

12

1312

+40

r

10

2334

+156

t

52

A23187 cells

+105

5 2 112 +

+70?

-

100

a typical

in radioactivity

to A23187

301

2242

lipids

3 experiments.

xaz+

-Ca2+ Phosphatidylglycerol

+A23187

incu-

The from

from

due --A23187

POLY-

were

A23187.

the radioactivity

represents

. 106

of

IN

or Mg2+

or Mg2+

or absence

cells)

to A23187

PHOSPHOLIPIDS Ca2+

16

+ S.D.

*

174

percentage of stimulation of radioactivity in phosphatidic acid, the phosphoinositides and phosphatidylserine due to A23187 was further potentiated. These data reflect the involvement of Ca2’ but not Mgzf in the altered 32Pi incorporation into phospholipids of leukocytes exposed to A23187. External Mg2’ may inhibit Ca2’ in eliciting 32Pi incorporation into phospholipids by the ionophore. Effect of ethyleneglycoi bis(@aminoethylether)-N,N’-tetraacetic acid (EGTA) on 32Pi incorporation into phospholipids. Although an increased 32Pi incorporation into acidic phospholipids induced by A23187 was not eliminated in leukocytes without external Ca2+, the involvement of Ca2’ on cell surface cannot be excluded. In this experiment, EGTA was used to chelate the Ca*’ on the cell surface, and the ionophore effect on 32Pi inco~oration into phospholipids WAS examined. Leukocytes were suspended in Krebs-Ringer-Tris buffer without divalent cations and incubated for 10 min at 37°C with 1 mM EGTA. Then the mixture was further incubated with 32Pi and A23187 for 30 min and the lipids were extracted and resolved. It can be seen from the data in Table III that EGTA did not abolish the ionophore effect on 32Pi incorporation into phospholipids in response to A23187 and it was comparable to that in cells without external Ca2* (Table II). EGTA at 1mM did not exhibit any apparent effect on cell viability as checked by dye-exclusion test. A23187 effect on radiophosphorus actiuity and content of cellular ATP. The altered “Pi incorporation into phospholipids of leukocytes in response to A23187 may be secondary to the change in the radiophosphorus activity and/ or content of cellular ATP. In the present study, the radioactivity of ATP was found to be enhanced by A23187. However, a significant increase (50%) in ATP radioactivity was observed only after 20-min incubation with A23187, as shown in Table IV. The presence of external Ca2’ and/or Mg*+ was not found to influence the radioactivity of ATP. Measurement of ATP contknt,in cells exposed to 1 ,uM A23187 revealed no difference from that in control cells at all

TABLE

III

EFFECT OF IONOPHORE A23187 ON 32Pi INCORPORATION MORPHONU~LEAR CELLS IN THE PRESENCE OF EGTA

INTO PHOSPHOLlPIDS

OF POLY-

Polymorphonuclear leukocytes (40 . 106) in Krebs-Ringer-T& buffer without Ca2+ and Mg2+ were incubated for 10 min at 37°C with 1 mM EGTA. Then the mixture was further incubated for 39 min with 32Pi (180 j&i) in the presence or absence of 1 PM A23187. Results represent the average values from two separate incubations. -~ % Change in radioactivity due to Phospholipid cpm/8 . lo6 cells A23187 * -A23187 +A23187 Phosphatidic acid Phosphatidylinositol Diphosphoinositide Triphosphoinositide Phosphatidylglycerol Phosphatidy~e~e Phosphatidyleho~ne

354 144 756 920 31 54 810

* % Change in radioactivity = 100 X

742 1078 935 1202 161 73 255

+110 +649 +24 +31 +414 +35 --69

radioactivity in cells with A23187 radioactivity in control cells

_ loo

175 TABLE IV EFFECT OF A23187

ON INCORPORATION

OF 32Pi INTO ATP

Leukocytes (40 . 106) were incubated with “Pi (3.55. 106 cpm) at the specified period of time in the presence or absence of 1 PM A23198. At the end of incubation. cell pellets were assayed for incorporation Of 32Pi. Into ATP. The counts of ATP are from 0.8 . 106 cells from two separate incubations. Time of incubation (min)

A23187

10

Radioactivity (cpm)

in ATP

100 +

20

+

30 +

108 144 214 182 334

time intervals of incubation (2-30 min). An average value of 1.85 + 0.16 nmol ATP per lo6 cells from three determinations was obtained. A23187 effect on Cu2+ fluxes. An increased 45Ca2+ influx in the presence of 1 WM A23187 was demonstrated in Table V, and the ionophore also rapidly

60

6

Time

(min)

Fig. 3. Effect of A23187 on 4 5 CaZ+ efflux from prelabeled cells. Cells were preincubated with 4 5 CaZ+ for 30 min. After washing, cells were incubated in Krebs-Ringer Tris buffer +_l @M A23187. Efflux is plotted as the percentage of total “SCa2+ counts remaining in the cell pellet at each time point. Each point represents the average value from two experiments.

176

TABLE

V

EFFECT

OF

Leukocytes A23187

A23187 suspended

for

the

ON

45&x2+

UPTAKE

in Krebs-Ringer

indicated

periods

Tris

of time.

buffer Results

were

incubated

represent

the

with average

45Ca2+ vaiues

(7.8

f 10’

cpm)

+ S.D.

from

three

t

1 AIM

separate

incubations. .____.Time

A23187

of incubation

--

45Ca2+

radioactivity

(cpm/mg

(min)

cell protein) ~~

~. 4009

2 10

+

5645

+ 182

-

4606

+ 116

+ 20 30

-f 250

5630

?: 120

5016

+ 150

+

5600

i 108

-

5055

t 193

+

5685

i 145 .--

augmented the release of preloaded 45Cazc when cells were resuspended in fresh medium (Fig. 3). The stimulated uptake and release of label appeared to take place in less than 2-min exposure of cells to the ionophore. The stimulated Ca” efflux by A23187 was not accompanied by 32Pi release into water-soluble fraction. No significant change in cellular calcium was detected after cells were treated with EGTA or exposed to A23187 (2-30 min). An average value of 0.071+ 0.015 fig ca.lcium/mg cell protein was obtained from four determinations. Discussions Using the divalent ionophore, A23187, as a probe, the effect of calcium fluxes on phospholipid metabolism of intact leukocytes was examined in the present study. A23187 selectively stimulated the incorporation of 32Pi into the acidic phospholipids. It also increased the labeling of ATP by 32Pi and promoted a rapid burst of uptake and release of 4sCaz+ by leukocytes. The altered 32Pi inco~orat~on into phosphatidic acid and phosphatidylinositoi induced by A23187 does not appear to be solely derived from the increased radioactivity of ATP; ATP changes may be large enough to account for the increased radioactivity of diphosphoinositide and triphosphoinositide. Time-course studies showed that an increased radioactivity (50%) of ATP was observed after 20-min exposure of cells to A23187, whereas the increased radioactivity of phosphatidic acid and phosphatidylinositol was much greater than that of ATP. The increased 45CaZ’ fluxes may trigger part of the altered 32P. incorporation into phospholipids of leukocytes exposed to the ionophore. i23187 did not induce detectable change in cell associated calcium. There may be a net loss of cellular calcium from cells incubated with’A23187 in medium without added calcium and from cells treated with EGTA but be undetectable by the technique used in the present study. External Ca2’, but not Mg*+, is required for full stimulation by A23187 on 32Pi incorporation into phosphatidic acid and the phosphoinositides. The iono-

177

phore effect on 32Pi incorporation into the acidic phospholipids of leukocytes in medium without added Mg*’ was further potentiated, suggesting the comwith ionophore petition of external MgZf with Ca*’ for complex formation [ 261. However, external Ca*’ was not absolutely required for A23187 to induce “Pi incorporation into phospholipids, and treatment of cells with EGTA did into phospholipids. not abolish the ionophore effect on 32P. incorporation 32Pi incorporation These data suggest that A23187 stimulat;d into phospholipids by perturbing the intracellular Ca2+ distribution. The exact role of cellular Ca2’ on phospholipid metabolism of intact leukocytes cannot be assigned without further studies. The increased labeling of phosphatidic acid, thus of phosphatidylinositol, could be due to an increased production of 1,2-diacylglycerol by lipase action [ 141. The 1,2-diacylglycerol could then be phosphorylated to form phosphatidic acid, but the possibility cannot be excluded that the increased 32Pi incorporation into phospholipids may take place at any or all of the steps leading to phosphatidic acid formation from sn-glycerol 3-phosphate. Studies on the incorporation of labeled glycerol, fatty acid and polar base into phospholipids of leukocytes in response to A23187 are in progress. The pattern of increased 32Pi incorporation into leukocyte phospholipids provoked by A23187 resembles the response of these cells to starch granules [l-3] and cytochalasin B [17]; in each case phosphatidic acid and the phosphoinositides are most responsive to the stimuli. In polymorphonuclear leukocytes during phagocytosis of starch granules and leukocytes treated with cytochalasin B, 32P. incorporations into phosphatidylserine was not significantly altered and the radioactivity of phosphatidylglycerol was not measured. However, the increased radioactivity of phosphatidylserine due to A23187 observed in the present study was much lower than that of phosphatidic acid and phosphatidylinositol (Table II). The presence of phosphatidylglycerol in leukocytes was appreciated for the first time, though it has not been isolated from these cells. The requirement of Ca *+ for full stimulation by A23187 on 32P. incorporation into the acidic phospholipids observed in the present study suggest that the movement of Ca*’ may play a role in the altered phospholipid metabolism during phagocytosis by polymorphonuclear leukocytes. Acknowledgements I thank Dr. W.T. Wu, Department of Clinical Chemistry, Louisiana State University College of Medicine for determination of calcium. This work was supported by Grant CA16483 from the National Institutes of Health. The author is a recipient of Research Career Development Award l-K04 CA00013 from the National Institutes of Health. References 1

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2

Sastry,

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Reed.

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and WaUach, Hokin.

L.E.

Stjemholm,

and

Ignarro.

and

Lardy,

D.F.J. (1966)

R.L. L.J. H.A.

(1961)

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J. Biol.

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J. Biol.

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Biochem.

Natl.

Acad.

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247,

236,

1895-1901

3354-3361 Biophys. Sci.

U.S.

69704977

160. 72,

487-494

108-112

178 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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