384

Biochimica et Biophysics @ Elsevier/North-Holland

Acta,

575

Biomedical

(1979) Press

384-388

BBA 57469

DECREASED INCORPORATION OF L-[UJ4C]SERINE PHOSPHATIDYLSERINE BY POLYMORPHONUCLEAR DURING PHAGOCYTOSIS

JEN-SIE

TOU

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

(Received

Key

INTO LEUKOCYTES

Tulane

University

School

of Medicine,

New Orleans,

LA

April 3rd, 1979)

words:

Serine

incorporation;

Phosphatidylserine;

Phagocy

tosis; (Leukocyte)

Summary A decreased rate of L-[U-14C]serine incorporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-‘4C]serine uptake. Both uptake and efflux of 45Ca2’ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2’ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.

Introduction Studies from this and other laboratories have shown an increased incorporation of 32Pi into phosp ha t idic acid and the phosphoinsitides and of [2-3H]inositol into the phosphoinositides by polymorphonuclear leukocytes during phagocytosis of inert particles [l-3]. Changes in the specific activity of other phospholipids were not significant. Since not every moiety of the glycerolipid molecule is metabolized at the same rate, the incorporation rate of the phosphorus moiety may not reflect that of the polar base moiety of the lipid molecule. In the present study, data which demonstrate that phagocytosis inhibits the metabolism of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes are presented.

385

Materials and Methods Materials. L-[ U-14C]Serine (159 Ci/mol) was purchased from New England Nuclear Corp. (Boston, MA). Its purity was checked before use by chromatography on Avicel thin-layer plate (250 pm layer, Analtech) developed in nbutanol/acetic/water (14 : 3 : 3, v/v). Carrier-free 45CaC1, was obtained from ICN Chemical and Radioisotope (Irvine, CA). Insoluble starch granules from sweet potato were a gift from Southern Regional Laboratory (New Orleans, LA). Preparation of polymorphonuclear leukocytes. Polymorphonuclear leukocytes from guinea pig peritoneal exudates were prepared as described previously [4], except that Krebs-Ringer/l5 mM phosphate buffer (pH 7.4) was used instead of Krebs-Ringer/l5 mM Tris/HCl buffer. Incubation. All incubations were carried out at 37°C in siliconized glassstoppered tubes under air with shaking. In a final volume of 2.0 ml, each tube contained 0.1 ml fresh autologous serum, 10 mg starch granules in 0.1 ml 0.9% NaCl or 0.1 ml 0.9% NaCl; 2 @i L-[U-‘4C]serine or 195 750 cpm 4sCaC12 in in 0.1 ml 0.9% NaCl, The tubes were preincubated for 10 min at 37”C, then 1.7 ml (35 - 106) leukocyte suspension kept at 37°C was added to each tube. They were further incubated for the specified periods of time. Lipid extraction. Incubation was terminated by the addition of 10 ml 0.1 N HC1/CH30H followed by 20 ml CHCIJ. Total lipid was extracted as previously described [ 41. Chromatographic resolution of phospholipids. Phospholipids were resolved by two-dimensional thin layer chromatography on Silica gel H (Analtech) [41. Measurement of cellular L-[U-‘4C]serine. Incubation was terminated by the addition of 5 ml ice-cold Krebs-Ringer phosphate buffer to each tube which was immediately centrifuged at 1000 X g for 2 min at 4” C and the resulting supematant fraction was aspirated. The cell pellet was washed two additional times each with 5 ml ice-cold buffer, it was then precipitated with 1.0 ml 0.6 M HCI04 and centrifuged at 600 X g for 10 min at 4°C. The HC104 supematant fraction was neutralized with 5 M KzC03 to pH 6.5. An aliquot of the neutralized supernatant fraction was co-chromatographed with 10 pg authentic Lserine on Avicel thin-layer plate (250 pm layer, Analtech) developed in nbutanol/acetic acid/water (14 : 3 : 3, v/v). L-[U-14C]Serine was located by spraying authentic L-serine with 0.2% ninhydrin in n-butanol saturated with water. Its radioactivity was counted by liquid scintillation, after the spot was scraped into a vial containing 1 ml water and 10 ml Instagel (Packard). Calcium uptake. At the end of the incubation, 5 ml ice-cold Krebs-Ringer phosphate buffer was added to each tube, which was immediately centrifuged at 1000 X g for 2 min. The supematant fluid was aspirated. and the cell was washed two additional times each with 5 ml ice-cold buffer. The washed cell pellet was finally resuspended in 1 ml of buffer. An aliquot of cell suspension was counted for radioactivity. In zero time incubation, 45CaC12 was added after ice-cold buffer was delivered to the tube. Cell-associated 45Ca2’ was expressed as cpm/mg protein after it was corrected for zero time incubation. Calcium efflux. Leukocytes were suspended in Krebs-Ringer phosphate

buffer to a concentration of 70 * 106/ml. 3 ml cell suspension was incubated with 8 - lo6 cpm 45CaC12for 60 min at 37°C. It was then centrifuged at 1000 X g for 2 min at room temperature. After the supernatant fraction was aspirated, the cell pellet was washed twice each with 20 ml Krebs-Ringer phosphate buffer. The washed cells were then resuspended in 10 ml buffer, and 4.5 ml labeled cell suspension was incubated with 0.25 ml serum, 0.25 ml starch granules (10 mg/O.l ml) or 0.25 ml 0.9% NaCI. After varying periods (2-30 min) at 37”C, 1 ml cell suspension was removed and centrifuged at 1000 X g for 2 min at 4°C. An aliquot of the supernatant fluid was removed and counted by liquid scintillation. Other methods. Lipid phosphorus and calcium content of leukocytes were measured as described previously [4]. Cell protein was determined by the method of Lowry et al. [ 5 ] using bovine serum albumin as standard. Results Effect of phagocytosis on L-[U-‘4C]serine incorporation into leukocyte phospholipids L-[ U-‘4C]Serine labeling of phosphatidylserine, phosphatidylethanolamine

and spingomyelin was increased with incubation time in the presence and absence of starch granules as shown in Fig. 1. It is apparent that leukocytes with starch granules exhibited reduced L-[ U-14C]serine incorporation into all three phospholipids. Phosphatidylcholine was negligibly labeled by L[LJ-‘~C]serine, suggesting that the formation of phosphatidylcholine via stepwise methylation of phosphatidylethanolamine may be insignificant in leukocytes.

I

.o

30 - phosphotldylserine _ -

t0ntr0, + ,,0rcll

0.5

25 20 4

60

40

60

sphingomyelin

20 Time

40

(min)

Fig. 1. Effect of phagocytosis on L-[ U- 14Clserine incorporation into leukocyte phospholipids as a function of time. Polymorphonuclear leukocytes (35. 106) were incubated at the indicated period of time with L-[U-14C] serine (2 PCi, 159 Ci/mol) + starch granules. Each point represents the average specific radioactivity from two separate experiments each in duplicate incubation.

387

Effect of phagocytosis on L-[U-‘4C]serine uptake by leukocytes The decreased phospholipid labeling by L-[U-14C]serine in cells with starch granules may be secondary to reduced amino acid uptake by leukocytes. L-[Ui4C]Serine uptake by cells with starch granules was found to be inhibited at all time intervals (15-60 min) and the degree of inhibition was comparable to that After 60 min incubation, L-[U-‘4C]serine observed in phosphatidylserine. uptake was reduced approximately 50% in cells with starch granules. Calcium fluxes in leukocytes during phagocy tosis An increased cell-associated 45Ca2’ has been demonstrated in human granulocytes during phagocytosis of zymosan particles [6], while an increased 4sCa2’ efflux has been shown in guinea pig peritoneal granulocytes exposed to zymosan particles [ 71. The biosynthesis of phosphatidylserine has been shown to occur by a calcium-dependent exchange of L-serine with the ethanolamine moiety of phosphatidylethanolamine in mammalian tissues [S]. The present experiment was designed to test whether the decreased L-[U-14C]serine incorporation into phospholipids was also attributed to calcium loss from leukocytes exposed to starch granules. 45Ca2’ uptake was found to be more rapid by cells with starch granules than by control cells. However, in the presence of starch granules, a burst of release of 45Ca2’ by leukocytes was also observed. Measurement of total cellular calcium level in cells after phagocytosis revealed no difference from that in control cells. Calcium level in leukocytes was similar to that previously reported [ 41. Discussion The present study presents the first investigation of phagocytosis effect on the metabolism of the polar base moiety of phosphatidylserine, the major acidic phospholipid of leukocytes. In agreement with the finding of others [9,10], the present study demonstrates that L-[U-14C]serine uptake by leukocytes is impaired during phagocytosis. The decreased labeling of phospholipids in leukocytes exposed to starch granules appears to be secondary to reduced serine uptake. Since leukocytes after phagocytosis may become more fragile, the apparent decreased serine uptake by cells exposed to starch granules could be resulted from the release of label during washings and subsequent centrifugation of labeled cells at 1000 X g for 2 min. However, similar results were obtained for [ U-‘4C]serine uptake or 45CaC12 fluxes when cells were centrifufed at 250 X g for 5 min. The possibility of loss of label from cells after phagocytosis in experiments for measurement of [U-14C]serine incorporation into phospholipids can be excluded, since the incubation was terminated by addition of 0.1 N HCl in methanol without prior separation of cells from incubation medium by centrifugation. In view of the fact that the reduction in specific radioactivity of phosphatidylserine induced by starch granules is parallel to that of L-[U-14C]serine uptake, the availability of L-serine may be a rate-limiting factor in controlling the synthesis of this lipid in leukocytes. However, the synthesis of phosphatidylserine in leukocytes may be modulated by cellular calcium as well. Preliminary experiments showed a calcium-dependent incorporation of radioactive

388

L-serine into phosphatidylserine in leukocyte homogenates (unpublished data). Although measurement of total cellular calcium failed to reveal apparent alteration in calcium levels after phagocytosis, the possibility of redistribution of cellular calcium may exist. Leukocytes exposed to starch granules may release calcium from the intracellular organelle where phosphatidylserine synthesis is located. The rapid 45Ca2+ fluxes in leukocytes exposed to starch granules was also observed in leukocytes incubated with divalent cation ionophore A23187 [4]. The possibility that the altered L-serine uptake by leukocytes may be partly associated with calcium fluxes in phagocytizing leukocytes awaits further study. Acknowledgement This work was supported by Biomedical Research Support Grant 5-507-RR05377-17 from the National Institute of Health. The author is a recipient of Research Career Development Award l-K04 CA00013 from the National Institute of Health. References 1

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Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis.

384 Biochimica et Biophysics @ Elsevier/North-Holland Acta, 575 Biomedical (1979) Press 384-388 BBA 57469 DECREASED INCORPORATION OF L-[UJ4C]S...
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