Biochimica et Biophysica Acta, 392 (1975) 1--11

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands

BBA 27657 CYTOCHALASIN B: EFFECT ON PHOSPHOLIPID METABOLISM AND LYSOSOMAL ENZYME RELEASE BY LEUKOCYTES

JEN-SIE TOU and RUNE L. STJERNHOLM Department of Biochemistry Tulane University School of Medicine, New Orleans, La. 70112 (U.S.A.)

(Received November 5th, 1974)

Summary The effect of cytochalasin B on phospholipid metabolism and ~-glucuronidase extrusion by polymorphonuclear leukocytes from guinea pig peritoneal exudates has been studied. Cytochalasin B inhibited the engulfing of starch granules by leukocytes, but it enhanced the incorporation of 32 Pi into phosphatidic acid and the phosphoinositides. It also stimulated the release of /3glucuronidase into the incubation medium in the presence or absence of starch granules. Kinetic studies showed that the effects of cytochalasin B on 32pi incorporation into phosphatidic acid and the phosphoinositides, and the release of ~-glucuronidase into the extracellular medium were comparable. Pulse-chase experiment revealed that cytochalasin B did not stimulate the isotopic decay of prelabeled lipids, indicating that cytochalasin B increased the radiophosphorus activity of phosphatidic acid and the phosphoinositides by increasing the synthesis of these lipids. The incorporation of myo-[2-3H] inositol into the phosphoinositides was also enhanced in the presence of cytochalasin B, but the incorporation of [methyl -I 4 C] choline into phosphatidylcholine and sphingogomyelin was unchanged.

Introduction Cytochalasin B, an alkaloid metabolite of the mold, H e l m i n t h o s p o r i u m d e m a t i o i d e u m [1], inhibits particle uptake by human polymorphonuclear leukocytes [2] but induces the release of ~-glucuronidase into the extracellular medium [3]. In the presence of cytochalasin B, the particles adhere to the plasma membrane but are not engulfed. This compound is therefore useful in studies of metabolic changes during phagocytic release of lysosomal enzymes independent of the engulfment phase of plagocytosis by polymorphonuclear cells.

The metabolism of phospholipids by polymorphonuclear leukocytes is stimulated during phagocytosis of starch granules or polystyrene particles [4--7]. During phagocytosis, the granular (lysosomal) enzymes are released from polymorphonuclear cells into the extracellular medium, a process analogous to that of secretion of amylase and other digestive enzymes from pancreas slices stimulated with pancreozymin or acetylcholine [8]. The pattern of stimulation of phospholipid labeling by 32 Pi during phagocytosis is reminiscent of the pattern obtained for pancreas slices in the presence of pancreozymin or acetylcholine; in each case the incorporation of s 2Pi into phosphatidic acid and phosphatidylinositol was more rapid in stimulated cells than in resting cells. We have recently reported the stimulation of the incorporation of 32pi and myo[2-3H]inositol into phosphatidylinositol, diphosphoinositide and triphosphoinositide in polymorphonuclear cells during phagocytosis of starch granules [9]. Now we are interested to know whether the increased isotope incorporation into phospholipids accompanies the extrusion of lysosomal enzymes. In the present study, we used cytochalastin B to block particle uptake and measured the incorporation of isotope into phosphatidic acid and the phosphoinositides. The results suggest that the increased incorporation of isotope into these acidic phospholipids during phagocytosis by polymorphonuclear cells may be associated with the process of lysosomal enzyme extrusion.

Materials and Methods

Materials Cytochalasin B, a product of ICI Ltd, Cheshire, U.K. was purchased from Aldrich Chemical Company, Milwaukee, Wis. It was dissolved as a stock solution in dimethylsulfoxide at a concentration of 3 mg per ml and stored at --20°C until use. An aliquot of the stock solution was diluted with 0.9% NaC1 to 1--10/~g/10 pl. The highest cytochalasin B concentration used contained 0.165% dimethylsulfoxide in the incubation medium. In control experiments it was shown that this concentration of dimethylsulfoxide had no apparent effect on the cells. Phosphatidylinositol, phosphatidic acid, and sphingomyelin were purchased from Supelco Co. The diphosphoinositide fraction of Folch containing diphosphoinositide and triphosphoinositide was prepared as described by Lees

[10].

Carrier-free 32P_labeled sodium phosphate for injection was obtained from Mallinckrodt. myo-[2 -3 H]Inositol "(2.8 Ci/mmol, 98% pure), [methyl14 C] choline chloride (30 Ci/mol), and [carboxyl-I 4C] inulin (3.2 mCi/g) were purchased from New England Nuclear Corp. The insoluble starch granules from sweet potatos were a gift from the Southern Regional Laboratory, New Orleans, La.

Methods Preparation o f leukocytes. Polymorphonuclear leukocytes from guinea pig

peritoneal exudates were prepared as described previously [9]. The cells were suspended in Krebs-Ringer phosphate buffer (pH 7.4), modified to contain 1.5 mM CaC12 and 10 mM glucose to give a final concentration of 20--30 × 106 cells/ml. Cell counts were made in a h e m o c y t o m e t e r , and cell viability was measured by trypan blue dye-exclusion. The cell preparations contained more than 95% of polymorphonuclear leuckocytes. Incubations. Separate aliquots of cell suspensions were used for measurements of particle uptake, isotope incorporation into phospholipids, and the release of/3-glucuronidase. All incubations were performed in siliconized glassstoppered tubes of 45 ml capacity at 37°C with continuous shaking. For measurement of particle uptake, 1.6 ml of cell suspension and 0.1 ml of autologous serum were preincubated for 10 min with varying concentrations of cytochalasin B. Then 0.2 ml (5 pCi) [carboxyl-z 4C] inulin was added and followed immediately by 10 mg of starch granules in 0.1 ml of saline; 0.1 ml of saline was added to the control tubes. After incubation for 30 min, phagocytosis was stopped by adding 5 ml of ice-cold Krebs-Ringer phosphate buffer containing 0.5 mg of unlabeled inulin per ml. The cells were recovered by centrifugation of the tubes at 250 × g for 5 min. The pellet was then washed twice each with 5 ml of inulin-containing buffer. It was finally dissolved in 1.0 ml of 1% (v/v) Triton X-100. A volume of 0.1 ml of this suspension was added to 20 ml of scintillation phosphor made up of 2 vol. toluene and ! vol. Triton X-100 with 0.5% 2,5-diphenyloxazole and 0.005% 1,4-bis-(2-(5-phenylloxazolyl))benzene. The radioactivity was measured in a liquid scintillation spectrometer. Uptake of starch particles was expressed as cpm per mg of cell protein, determined by assaying appropriate aliquots of cell suspension by the method of Lowry et al. [11]. ~-Glucuronidase was measured at 2 h of incubation with phenolphthalein glucuronidate as substrate [12]. Lactate dehydrogenase was determined according to Bergmeyer et al. [13]. After the cells {30--35 × 106 in 1.6 ml) and 0.1 ml of autologous serum were preincubated with 4 pg (in 10 pl) of cytochalasin B for 10 min, 10 mg of starch granules (in 0.1 ml saline} or 0.1 ml of saline, was added to each tube. The tubes were further incubated for the specified periods of time. They were then placed in ice and centrifuged at 250 × g for 5 min. /~-Glucuronidase and lactate dehydrogenase in the supernate were measured. Total enzyme activity was determined by diluting 1 vol. of cell suspension with 9 vol. of 0.1% Triton X-100. Lipid extraction and chromatographic resolution of phospholipids. The incorporation of 32Pi and myo-[2-3H]inositol into phospholipids was measured as described before [9], except 15 pg phosphorus of Folch's diphosphoinositide fraction was used as carrier for lipid extraction. No carrier was added when the radioactivity of [ 14 C] phosphatidylcholine and [ 14 C] sphingomyelin was measured. Phosphatidic acid and the phosphoinositides were resolved by silicic acidimpregnated glass fiber paper (Gelman) chromatography as described before [9]. Phosphatidylcholine and sphingomyelin were separated on silica gelimpregnated glass fiber paper. The chromatogram was developed in a solvent consisting of 100 ml of chloroform and 11 ml of a mixture of methanol/acetic acid/water (90 : 5 : 5.5, v/v/v).

Results

Effect o f cytochalasin B on particle uptake by polymorphonuclear leukocytes. In this experiment the uptake of [~4C]inulin into cells in the absence and presence of starch particles was used as an index of phagocytosis as described by Berger and Karnovsky [14] and Skosey et al. [15]. As shown in Fig. 1, the process of particle uptake was completely blocked by cytochalasin B at a concentration of approx. 2.0 pg/ml. Cell death at all concentrations of cytochalasin B used did not exceed 2%, when the total incubation period was 40 min. The release o f fi-glucuronidase and lactate dehydrogenase in the presence o f cytochalasin B. We found that there was significant increase in the release of lactate dehydrogenase into the extracellular medium, when cells were challenged with starch particles for 60 rain in the absence of cytochalasin B, though there was no apparent cell death. Therefore, we elected to use 30 min incubation for all experiments. Fig. 2 shows the time course of enzyme release in the absence and presence of cytochalasin B. In control cells (not treated with cytochalasin B), a phagocytic release of ~-glucuronidase was increased with incubation time; a slight b u t significant increase of lactate dehydrogenase was observed at 30 min of incubation in the presence of starch particles. Cytochalasin B selectively

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Fig. 1. E f f e c t of c y t o c h a l a s i n B o n the u p t a k e o f s t a r c h granules b y p o l y m o r p h o n u c l e a r l e u k o c y t e s . Cell s u s p e n s i o n s (1.6 ml) and 0.1 m l of a u t o l o g o u s s e r u m w e r e p r e i n c u b a t e d for 10 rain w i t h v a r y i n g c o n c e n t r a t i o n s o f c y t o c h a l a s i n B. T h e n 0.2 m i (5 #Ci) of [ c a r b o x y l - 1 4 C ] i n u l i n was a d d e d a n d f o l l o w e d i m m e d i a t e l y b y 1 0 m g o f s t a r c h granules ( e ) in 0.1 m l of saline; 0.1 ml o f saline was a d d e d to t h e c o n t r o l t u b e s (o). A f t e r i n c u b a t i o n for 3 0 rain the r a d i o a c t i v i t y p e r m g o f cell p r o t e i n was m e a s u r e d as d e s c r i b e d u n d e r Methods. Fig. 2. T i m e - c o u r s e of release of /3-glucuronidase a n d l a c t a t e d e h y d r o g e n a s e ( L D H ) in the p r e s e n c e of c y t o c h a l a s i n B (CB). P o l y m o r p h o n u c l e a r cells (1.6 m l ) a n d 0.1 m l of a u t o l o g o u s s e r u m w e r e p r e i n c u b a t e d with 4 / z g ( 1 0 #1) of c y t o c h a l a s i n B; 10 /zl of 1 3 . 3 % d i m e t h y l s u l f o x i d e waS a d d e d t o t h e c o n t r o l tubes. A f t e r 1 0 m i n p r e i n c u b a t i o n , the cells w e r e e x p o s e d to s t a r c h (e) or saline (o) for the specified p e r i o d s of t i m e . The e n z y m e a c t i v i t y in the i n c u b a t i o n m e d i u m was m e a s u r e d as d e s c r i b e d u n d e r M e t h o d s . T o t a l a c t i v i t y ( 1 0 0 % ) of ~-glucuronidase waS 1 7 0 +- 21 pg of p h e n o l p h t h a l e i n p e r 107 cells p e r 2 h; of l a c t a t e d e h y d r o g e n a s e waS 2 0 2 5 -+ 159 a b s o r b a n c e u n i t s p e r 107 cells. R e s u l t s r e p r e s e n t m e a n values f r o m t h r e e e x p e r i m e n t s ; s t a n d a r d d e v i a t i o n s are i n d i c a t e d .

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Fig. 4. ~ - G l u c u r o n i d a s e release a n d t h e i n c o r p o r a t i o n of 3 2 p i i n t o p h o s p h a t i d i c acid a n d the p h o s p h o inositides in r e s p o n s e t o d i f f e r e n t c o n c e n t r a t i o n s of c y t o c h a l a s i n B. P o l y m o r p h o n u c l e a r l e u k o c y t e s ( 3 0 × 105 in 1.7 m l ) a n d 0.1 m l of s e r u m w e r e i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of c y t o c h a l a s i n B. 3 2 p i ( 1 1 2 pCi in 0.2 ml) was a d d e d 3 0 s a f t e r c y t o c h a l a s i n B. T h e s y s t e m was i n c u b a t e d f o r 30 rain. E a c h p o i n t r e p r e s e n t s the r a d i o a c t i v i t y f r o m 3.0 X 106 p o l y m o r p h o n u c l e a r cells. Values are t h e a v e r a g e s f r o m d u p l i c a t e i n c u b a t i o n s . PA = p h o s p h a t i d i c acid; PI = p h o s p h a t i d y l i n o s i t o l ; DPI = d i p h o s p h o i n o s i t i d e ; TPI = triphosphoinositide.

be analogous to that of the secretion of amylase from pancreas in response to pancreozymin. In the latter process, the concentration of pancreozymin that required to elicit phospholipid response greatly exceeded that necessary to elicit maximum enzyme extrusion [16]. In order to examine the dose-related stimulation of cytochalasin B on/]-glucuronidase release and the incorporation of 32pi into the phosphoinositides, varying concentrations of cytochalasin B were incubated with polymorphonuclear leukocytes for 30 min, then the 32p radioactivity of phospholipids and the release of ~-glucuronidase into the incubation medium were measured. The results are shown in Fig. 4. In response to cytochalasin B in concentrations of 0.5--1.0 pg/ml, /3-glucuronidase extrusion from polymorphonuclear cells is parallel to the increased radioactivity of phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide. The responses of enzyme extrusion and phospholipid metabolism to cytochalasin B in the concentrations above 1 pg/ml are not parallel, b u t comparable. When the cells were incubated for 30 min with cytochalasin B at a concentration of 2 pg/ml, ~-glucuronidase release was enhanced 1.77-fold and the radioactivity of phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide, was increased, respectively, 2.0-, 2.0-, 1.63-, and 1.64-fold. These data suggest that/3-glucuronidase extrusion and phospholipid effect may occur concurrently in the presence of cytochalasin B.

Effect of cytochalasin B on myo-[2-ZH] inositol incorporation into the phosphoinositides. We have recently demonstrated that the incorporation of myo-[ 2- 3H] inositol into phosphatidylinositol, diphosphoinositide and triphosphoinositide was stimulated during phagocytosis by polymorphonuclear cells [9]. In the present experiment, the effect of cytochalasin B on m y o - [ 2 - 3 H ] inositol labeling of the inositides was examined. After the cells were incubated for 30 rain with cytochalasin B (2 pg/ml), the radioactivity of phosphatidylinositol, diphosphoinositide, and triphosphoinositide was increased 1.43-, 1.53-,

released fi-glucuronidase from polymorphonuclear cells both in the absence and presence of starch granules. When particle uptake was arrested by preincubating the cells for 10 min with cytochalasin B, the presence of starch particles still potentiated fi-glucuronidase extrusion. The release of lactate dehydrogenase from polymorphonuclear cells was also enhanced in the presence of cytochalasin B, but to a lesser extent. Starch particles did not significantly further increase the release of lactate dehydrogenase into the incubation medium from cytochalasin B-treated cells. Effect of cytochalasin B on 32Pi incorporation into phosphatidic acid and the phosphoinositides. Hokin and Hokin [8] demonstrated an increased 32P i incorporation into phosphatidic acid and phosphatidylinositol during the secretion of enzymes from pancreas slices stimulated with pancreozymin or acetylcholine. In the present study, cytochalasin B was found to induce ~-glucuronidase secretion from polymorphonuclear cells. We therefore reasoned that lysosomal enzyme release from polymorphonuclear cells may be accompanied by an increased 3 :Pi incorporation into phosphatidic acid and the phosphoinositides. Accordingly, cytochalasin B is expected to accelerate the labeling of these phospholipids by 32 Pi. As we anticipated, cytochalasin B stimulated 32 Pi incorporation into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide in the presence and absence of starch particles (Fig. 3). The increased radioactivity of phospholipids in the presence of starch particles was more evident after 30 min continuous incubation with cytochalasin B. The radioactivity of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin was not influenced by cytochalasin B (data not shown). Effect of increasing concentrations of cytochalasin B on fl-glucuronidase extrusion and 32Pi incorporation into phospholipids. The process of enzyme extrusion from polymorphonuclear cells elicited by cytochalasin B appears to 150-•~ PA ,-, I00 -

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TIME (MIN.) Fig. 3. E f f e c t of c y t o c h a l a s i n B o n 3 2 p i i n c o r p o r a t i o n into phosphatidic a c i d a n d t h e phosphoinositides. P o l y m o r p h o n u c l e a r l e u k o c y t e s ( 3 8 X 106 in m l ) a n d 0.1 m l o f s e r u m w e r e p r e i n c u b a t e d w i t h or w i t h o u t c y t o c h a l a s i n B (4 # g ) f o r 1 0 rain, then 0.2 ml of 3 2 p i ( 8 9 p C i ) w a s a d d e d to e a c h t u b e f o l l o w e d i m m e d i a t e l y b y s t a r c h g r a n u l e s as i n d i c a t e d . A f t e r t h e t u b e s w e r e f u r t h e r i n c u b a t e d a t t h e s p e c i f i e d t i m e p e r i o d , t h e lipids w e r e e x t r a c t e d a n d s e p a r a t e d as d e s c r i b e d u n d e r M e t h o d s . E a c h p o i n t r e p r e s e n t s t h e r a d i o a c t i v i t y f r o m 3.8 X 106 p o l y m o r p h o n u c l e a r cells. V a l u e s are t h e a v e r a g e s f r o m d u p l i c a t e i n c u b a t i o n s , C = c o n t r o l ; S = s t a r c h g r a n u l e s ; CB = c y t o c h a l a s i n B; PA = phosphatidic acid; PI = phosphatidylinositol; D P I = d i p h o s p h o i n o s i t i d e ; T P I = t r i p h o s p h o i n o s i t i d e .

TABLE I E F F E C T O F C Y T O C H L A S I N B ON T H E I N C O R P O R A T I O N O F M Y O - [ 2 - 3 H ] I N O S I T O L I N T O T H E PHOSPHOINOSITIDES P o l y m o r p h o n u c l e a r cells ( 4 5 × 1 0 6 ) , 0.1 m l of s e r u m , a n d 50 #Ci of m y o - [ 2 - 3 H ] i n o s i t o l in a final v o l u m e of 2.0 m l w e r e i n c u b a t e d f o r 30 m i n in t h e p r e s e n c e a n d a b s e n c e of c y t o c h a l a s i n B (4 pg in 1 0 pl). C y t o c h a l a s i n B was a d d e d 3 0 s a f t e r [ 3 H ] i n o s i t o l . The i n c o r p o r a t i o n of m y o - [ 2 - 3 H ] i n o s i t o l into the p h o s p h o i n o s i t i d e s was m e a s u r e d as d e s c r i b e d u n d e r M e t h o d s . Additions

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and 1.82-fold, respectively (Table I). The labeling pattern is similar to that of phagocytizing polymorphonuclear cells [9]. Effect of cytochalasin B on [methyl -14C]choline chloride incorporation into phosphatidylcholine and sphingomyelin. The incorporation of 32P i into phosphatidylcholine and sphingomyelin was not influenced by the process of phagocytosis [4]. We have found that cytochalasin B did not change the incorporation of 32P i into phosphatidylcholine and sphingomyelin (data not shown). Table II shows that the incorporation of [ 14 C] choline into these two lipids was not altered by the presence of starch particles or cytochalasin B. Sphingomyelin was appreciably labeled by [14C] choline, though its radioactivity was much lower than that of phosphatidylcholine. Effect of cytochalasin B on the loss of radioactivity from 32Pi-labeled phosphatidic acid and the phosphoinositides. The increased incorporation of 32 Pi into phosphatidic acid and the phosphoinositides could be due to an increased synthesis or increased turnover of the lipid molecules. If cytochalasin B stimulates the turnover of these phospholipids, the radioactivity of prelabeled lipids should be lost at a greater rate in cells treated with cytochalasin B than in untreated cells. If cytochalasin B increases the incorporation of 32pi into these lipids by increasing their synthesis, the isotopic decay of phosphatidic acid and the inositides should not be altered by the presence of cytochalaT A B L E II E F F E C T OF C Y T O C H A L A S I N B ON T H E I N C O R P O R A T I O N OF [ M E T H Y L - 1 4 C ] C H O L I N E C H L O R IDE INTO P H O S P H A T I D Y L C H O L I N E AND S P H I N G O M Y E L I N P o l y m o r p h o n u c l e a r cells ( 4 0 X 106 in 1.6 m l ) a n d 0.1 m l of s e r u m w e r e p r e i n c u b a t e d f o r 10 m i n w i t h c y t o c h a l a s i n B (4 p g in 10 pl). T h e n 2.2 #Ci of [ m e t h y l - 1 4 C ] choline c h l o r i d e (in 0.2 m l of K r e b s - R i n g e r p h o s p h a t e b u f f e r ) a n d 10 m g of s t a r c h granules (0.1 m l ) w e r e a d d e d to e a c h t u b e . T h e t u b e s w e r e f u r t h e r i n c u b a t e d for 3 0 rain. P h o s p h a t i d y l c h o l i n e a n d s p h i n g o m y e l i n w e r e r e s o l v e d as d e s c r i b e d u n d e r M e t h o d s . Additions

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TIME (MIN) Fig. 5. E f f e c t of c y t o c h a l a s i n B on the i s o t o p i c d e c a y of 3 2 p - l a b e l e d p h o s p h a t i d i c acid a n d the p h o s p h o i n o s i t i d e s in p o l y m o r p h o n u c l e a r cells a f t e r a chase in u n l a b e l e d m e d i u m . P o l y m o r p h o n u c l e a r cells ( 1 3 6 × 1 0 6 ) in 5 m l of K r e b s - R i n g e r p h o s p h a t e b u f f e r w e r e p r e i n c u b a t e d w i t h 3 2 p i ( 4 5 0 #Ci in 3.0 ml), 0.5 m l of s e r u m , a n d c y t o c h a l a s i n B (16 p g in 4 0 #l) f o r 30 rain. A t the e n d of the i n c u b a t i o n , the cells were s e d i m e n t e d b y c e n t r i f u g a t i o n for 3 rain at 2 5 0 X g, the s u p e r n a t a n t r a d i o a c t i v e m e d i u m was r e m o v e d , a n d t h e cell p e l l e t w a s h e d t w i c e e a c h with 20 ml of n o n - r a d i o a c t i v e K r e b s - R i n g e r p h o s p h a t e b u f f e r . T h e l a b e l e d cells w e r e t h e n r e s u s p e n d e d in t h e s a m e b u f f e r in a t o t a l v o l u m e of 12 ml. This s u s p e n s i o n was e q u a l l y d i s t r i b u t e d o v e r six t u b e s , e a c h o f w h i c h c o n t a i n e d 0.1 m l o f s e r u m and, w h e r e i n d i c a t e d , 4 # g (in 10 /zl) of c y t o c h a l a s i n B ( e ) or 1 0 p l of 1 3 . 3 % d i m e t h y l s u l f o x i d e (o). A f t e r the t u b e s w e r e i n c u b a t e d at the specified t i m e , the lipids w e r e e x t r a c t e d a n d resolved as d e s c r i b e d u n d e r M e t h o d s . PA = p h o s p h a t i d i c acid; PI = p h o s p h a t i d y l i n o s i t o l ; DPI = d i p h o s p h o i n o s i t i d e ; T P I = t r i p h o s p h o i n o s i t i d e .

sin B. In this experiment, polymorphonuclear cells were incubated with 32pi for 30 rain in the presence of cytochalasin B (2 pg/ml) at 37 °C and then chased with non-radioactive Krebs-Ringer phosphate buffer. The labeled cells were then incubated with and without cytochalasin B for 0--30 min. At the end of each incubation, the lipids were extracted by the methods described above. As shown in Fig. 5, after the chase the decay of radioactivity of phosphatidic acid, diphosphoinositide and triphosphoinositide proceeded at a similar rate in cells with and without cytochalasin B. The results indicate that the increased 32pi incorporation into these lipids in the presence of cytochalasin B is due to increased synthesis rather than increased turnover of these phospholipids. However, 32P_labele d phosphatidylinositol can not be effectively chased with unlabeled phosphate, its radioactivity was continuously increased with incubation time in the absence and presence of cytochalasin B. This may be caused by the rapid loss of label from phosphatidic acid, diphosphoinositide, and triphosphoinositide. It could also be due to the possibility that phosphatidylinositol did not reach isotopic equilibration with 32pi during 30 min preincubation, therefore the isotopic decay was not demonstrable. Since the time period of cell viability and functionality in the presence of cytochalasin B is limited, preincubation of cells longer than 30 min was not attempted. We have preincubated cells with 32 Pi, but without cytochalasin B, for 4 h at 37°C. The cells were chased with non-radioactive Krebs-Ringer phosphate buffer and then incubated in the absence and presence of cytochalasin B. The pattern of isotopic decay of these phospholipids was similar to that preincubated for 30 min in the presence of cytochalasin B (data not presented).

Discussion In the present study, we have demonstrated that the selectively stimulated incorporation of 32pi into phosphatidic acid and the phosphoinositides during phagocytosis is associated with the process of lysosomal enzyme extrusion. We have found that cytochalasin B at a final concentration of 2 pg/ml (10 min) completely blocked the uptake of starch granules by polymorphonuclear cells. The concentration of cytochalasin B required to inhibit phagocytosis appears to be dependent on the incubation condition, such as time period of incubation and concentration of serum in the medium. Lin et al. [17] recently reported that in the presence of serum, the amount of cytochalasian B bound to bovine platelets was decreased. Cytochalasin B accelerated the release of ~-glucuronidase from polymorphonuclear cells into the incubation medium in the absence and presence of starch granules. This observation is in agreement with that reported by Davies et al. [18] using rabbit peritoneal exudate polymorphonuclear cells challenged by [3H]uridine Escherichia coll. Zurier et al. [3], however, reported that cytochalasin B (5 pg/ml, 15 rain) enhanced the release of ~-glucuronidase into the surrounding medium from human polymorphonuclear cells only when the cells encountered zymosan particles, not in the absence of particles. The difference in ~-glucuronidase release from peritoneal exudate polymorphonuclear cells and human polymorphonuclear cells to the action of cytochalasin B may be attributed to the difference in the origin of the cells. The release of lactate dehydrogenase is significantly increased in cytochalasin B-treated cells. Although other investigators [3,18] claimed that cytochalasin B selectively induced the release of lysosomal enzyme, ~-glucuronidase, without affecting the cytoplasmic enzyme, lactate dehydrogenase, we have repeatedly detected more lactate dehydrogenase activity in the medium from cytochalasin B-treated cells. After 30 min incubation of polymorphonuclear cells with cytochalasin B, the activity of lactate dehydrogenase in the incubation medium was increased a b o u t 50%, b u t cell death was no more than 2% of the total cells as checked by dye-exclusion. It appears that measurement of lactate dehydrogenase released into extracellular medium does not correlate with the dye-exclusion test. As pointed o u t by Woodin [19], the dye-exclusion test could well measure only complete cessation of physiological responsiveness and a small reduction in cell motility could be unnoticed. Although cytochalasin B inhibited phagocytosis by polymorphonuclear cells, it stimulated the incorporation of 32pi into phosphatidic acid and the phosphoinositides. Kinetic studies show that the effect of cytochalasin B on phospholipid metabolism and ~-glucuronidase secretion are comparable. The phospholipid response to cytochalasin B in polymorphonuclear cells is analogous to the phospholipid effect produced by pancreozymin in pancreas. However, a higher concentration of pancreozymin was required to elicit 32 Pi incorporation into phosphatidic acid and phosphatidylinositol (diphosphoinositide and triphosphoinositide were not examined) than to induce amylase extrusion [16]. It is suggested in the latter study that the incorporation of 32P i into phospholipids may represent a second, adaptive response to the hormone. In the present study the phospholipid effect and ~-glucuronidase extrusion seemed to take place concurrently.

10 The increased radiophosphorus activity of phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triposphoinositide in cytochalasin B-treated cells does not appear to be due to a higher radiospecific activity of ATP or higher ATP content, since the incorporation of 3 ~Pi and [methyl-14C] choline into phosphatylcholine was not influenced by cytochalasin B. In addition, Skosey et al. [15] have shown that cytochalasin B decreased zymosan-stimulated glycolysis in human polymorphonuclear cells. The incorporation of myo[2-3H]inositol into the inositides was augmented by the presence of cytochalasin B, indicating that the increased radiophosphorus activity of phosphatidylinositol was not solely derived from that of phosphatidic acid but also at the step of inositol incorporation. Pulse-chase experiment showed that cytochalasin B did not stimulate the loss of radioactivity from previously labeled lipids. The data suggested that the increased 32pi was attributed to a greater rate of synthesis of the lipid molecules in cytochalasin B-treated cells. The mechanism whereby cytochalasin B augments the synthesis of phosphatidic acid and the phosphoinositides is not known. It has been suggested that cytochalasin B may exert its biological actions by direct binding with the cell membranes [20]. The enzymes involved in the synthesis of phosphatidic acid and the phosphoinositides may be activated when cytochalasin B binds cell membranes and changes the membrane conformation. Harwood and Hawthorne [21] found an active phosphatidylinositol kinase in the surface membrane fraction from rabbit polymorphonuclear cells. Hokins-Neaverson's recent work [22] suggests that acetylcholine stimulates an initial hydrolysis of phosphatidylinositol in mouse pancreas with the formation of diacylglycerol. The increased labeling would then be due to resynthesis of this phosphatidylinositol. However, in the present study we did not observe a breakdown of prelabeled phosphatidylinositol in response to cytochalasin B. This argues against the involvement of an initial hydrolysis of phosphatidylinositol. The mechanism by which cytochalasin B induces fl-glucuronidase release is not clear. Davies et al. [18] proposed that cytochalasin B interferes with contractile microfilament functions, these latter including phagocytosis and the maintenance of cellular structure. Cytochalasin B disrupts microfilaments thereby allowing easier access of lysosomes to the plasma membrane. In cytochalasin B-treated polymorphonuclear cells, starch particles presumably as zymosan particles [3] and bacteria [18] are not internalized, but attached to specific receptors on the plasma membrane. The fusion of these specific receptors with lysosomes would result in lysosomal enzyme release. Cytochalasin B selectively stimulates the metabolism of phosphatidic acid and the phosphoinositides accompanying fl-glucuronidase release from polymorphonuclear cells. This observation presents another example of selective control of phospholipid metabolism. The phosphoinositides have been found in both cytoplasmic and plasma membranes of polymorphonuclear cells from rabbit peritoneal exudates [23]. Their specific biological function in leukocytes is not yet fully understood. Hawiger et al. [34] reported that liposomes composed of phosphatidylinositol activated fi-glucuronidase release from polymorphonuclear granules, and the cationic protein fraction isolated from granules reduced the activating effect of liposomes. Thus, the increased synthesis of the phosphoinositides in cytochalasin B-treated cells may interact with cationic

11

protein and facilitate the fusion of lysosomes with the plasma membrane. Recent studies on the mechanisms of lysosomal enzyme release from human leukocytes [25--27] have indicated that the release of lysosomal enzymes requires intact microtubules and may be modulated by adrenergic and cholinergic agents which appear to provoke changes in concentrations of cyclic nucleotides. It would be helpful to further understand the relationship between phospholipid metabolism and enzyme secretion from polymorphonuclear cells by studying the effect of cyclic nucleotides and agents that affect the integrity of microtubules on phospholipid metabolism. Acknowledgements This investigation was supported by a research grant from the Cancer Association of Greater New Orleans Inc., United States Public Health Service Grant CA-13214 from the National Cancer Institute, and the Edward G. Schlieder Educational Foundation. One of the authors (JST) was a recipient of United States Public Health Service Special Research Fellowship 1-F03-CA5394-02. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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