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Specific stimulation of phospholipase activities with phosphatidylethanolamine in transformed cells
238
BBA 51783
SPECIFIC STIMULATION OF PHOSPHOLIPASE AC~~~ITIE~ ~~~~ PHOSPHATIDYLETHANOLAMINE IN TRANSFORMED CELLS
(Received February (Revised manuscript
13th, 1984: received July 9th, 1984)
Phospholipase A,, A, and C activities with phosphatidylethanolamine were enhanced in C6 celDisrelative to primary astrocytic cultures, Enhancement was a function of sell density, Phospholipase activities with phosphatidylcholine were unchanged as a function of cell density, while phospholipase C activity with phosphatidylinosito1 was reduced. All acid phospholipas~ activities measured were low or essentia& absent in the three transformed cell lines examined. These results suggest that arachidonate release upon ~~~f~~e~~~ Is mainly from phosphatidylethanolamine.
The metabolism of glycerophospholipids is involved in responses to a variety of stimuli [1,2], the regulation of membrane fluidity [3], the control of the asymmetric distribution of g~ycerophospholipids in some membranes [4] and in the provision of polyunsaturated fatty acids for cyclooxygenase and lipoxygenase pathways 151. Hn addition, the physicochemical potentials of phospholipids in bilayer systems are different /6-81. A variety of metabolic pathways may be necessary in order to diffcre~tially express the respective function sf each phospholipid class. Evidence for separate pools of arachidonate release from phospholipids in macrophages has been recently presented [9,10].
* To whom correspondence should be addressed c/o Louis Freysz, Centre de Neurochimie du CNRS, 5 rue Blake Pascal, 67084 Strasbourg, Cedex, France. Abbreviations: PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; Mops, 4-morpholinepropanesutfonic acid. ~55-2760/84/$03.~0
@ 1984 Ekevier
Science Publishers
I?.‘?.
Arachidonate release is generally via ~hos~ho~~pase AZ and/or the coupled action of phosphohpase C and diglyceride lipase. IIn the central nervous system several phosphoiipases have been investigated and in many cases the characteristics differ somewhat from similar enzymes in bob-nervous tissue [IF;-IT]. A major problem encountered in characterization of brain phospholipases is the heterogeneity of ihe tissue with regard to celll types. Apparently, nauronal phospholipases are more active than gilal. phospholipases jt3J. Here we describe several of the pbospholipid catabol~zi~g enzymes an primary astrocytic cultures and C6 tumoral cells. Materials and Methods [~-‘4~]Arachido~ic acid and ~9~~0~~~-3~~ palmitic acid were obtained from ~~d~ochern~~a~ Centre, Amersham, U.K. Fetal calf serum and Dulbecco’s modified Eagle’s medium -were purchased from the Grand Island Biological @o. Grand Island, NY, U.&A. Phospholipase AI and
239
A, activities were measured by the release of radiolabelled fatty acid from l-[ 3Hlpalmitoyl-2-acylsn-glycerophospholipids (prepared by the method of Brockerhoff et al. [18]) and I-acyl-2[‘4C]arachidonoyl-sn-glycerophospholipids (prepared by the method of Woelk et al. [19], respectively. Similar results were obtained if the release of radiolabelled lysophospholipids was measured using the former substrate for A, and the latter substrate for A, activity. Phospholipase C activities were measured by the release of radiolabelled diacylglycerol from the above substrates. Substrate (final concentration of 150 PM, with a specific activity of 0.1 Ci/mol) in the appropriate buffer was briefly sonicated and added to 0.4-2.0 mg protein in a final volume of 1 ml with 0.08% sodium deoxycholate. Buffers used were citrate (pH 3.0-4.0), acetate (pH 4.1-6.0), Mops (pH 6.1-8.0), Tris-HCl (pH 8.1-10.0). Co-sonication of radiolabelled substrate and tissue sample indicated that in the presence of detergent exogenous and endogenous phospholipids competed equally. The samples, in triplicate, were incubated for 60 min (20 min for phospholipase C) at 37°C in a shaking-water bath; the reactions were stopped by the addition of 4 ml of chloroform/methanol, (2 : 1, v/v) and lipids extracted by the method of Folch et al. [20]. Fatty acids, phosphoglycerides and lysophospholipids were separated by TLC on 0.5mm silica Gel G plates by the solvent system chloroform/methanol/water (70 : 20 : 2, v/v) and visualized by iodine vapors. Fatty acids, diacylglycerols and phosphatides were separated by hexane/diethyl ether/acetic acid (80 : 20 : 1, v/v). Corresponding areas were scraped into scintillation vials with the addition of 0.5 ml water and 10 ml scintillator. Rates were calculated following subtraction of blank values for sample without protein. Protein was determined by the method of Lowry et al. [21] with bovine serum albumin as standard. The standard deviation of these assays was routinely less than 5%. Pure primary astrocytic cultures were prepared from l-day-old rat cerebral hemispheres [22] and harvested on day 21 in 0.9% saline with the aid of a rubber policeman. C6 [23] and NN cells [24] were subcultured after mild trypsinization and harvested at low density (lo5 cells/cm*), moderate density ((3-5). lo5 cells/cm2)) or confluency (8 .
10’ cells/cm2) in the case of C6 cells or 14. lo5 cells/cm2 for NN and HTC cells. Cells were lysed by homogenization for 25 s with a Polytron (Kinematics GmBH, Lucern, Switzerland). Membrane and soluble fractions were then prepared by centrifugation at 100000 X g for 1 h. CDPcholine phosphotransferase [25] served as a marker of the membrane fraction, while lactate dehydrogenase [26] was a marker of the soluble fraction. Results Phospholipase A 2 activities measured with phoshatidylethanolamine (PE) did not share the same pH optima as those measured with phosphatidylcholine (PC) in either primary astrocytic cultures or C6 cells (Fig. 1). In addition, no detectable phospholipase A 2 activity could be measured at acidic pH in C6 cells with either PC or PE. NN cells had a phospholipase A, profile similar to C6 cells in that there was an alkaline optima for PE (at pH 8.0) and no acid activity. Additionally, the acid phospholipase A, activities in the hepatoma cell line HTC were only 30% and 10% that of the alkaline activities with PC and PE, respectively
P”
Fig. 1. The pH profile of phospholipase A2 activity, measured via [‘4C]arachidonate release, in primary astrocytic and C6 cultures. 1-acyl-2-[‘4C]arachidonoyl-sn-glycero-3-phosphoethanolamine (left hand scale) was incubated for 1 h with homogenates of primary astrocytic cultures (o- -0) or C6 cells (W=). In a similar manner, l-acyl-2[‘4C]arachidonoyl-sn-glycero-3-phosphochohne (right hand scale) was incubated with homogenates of primary astrocytic cultures (0- - - - - -0) or C6 cell (O0). See text for additional assay conditions.
(FeSUh§ not shown). Acid phospholipase A, activrties with PC and PE were nearly absent in C6 cells, while in primary astrocytic cultures the acid phospholipase activities were greater than alkaline activites (Fig. 2). Removai of detergent, or substitution of sodium deoxycholate with Triton X-400 or Tween 20 did not result in an enhancement in the acid phospholipase activities. The phospholipase A, and A, activities with PC and PE did not require divalent cations. Concentrations of EDTA higher than 4 mlki stimulated alkaline phosphoiipase activity though this was attributable to the increase in ionic strength, while Ca*+ concentrations up to 1 mM had no effect on activity (results not shown). The cell density had a marked effect on phospholipase activities with PE in C6 cells while activities with PC were not affected. Phospholipase A, activity at pH 7-7.5 with PC was greater in C6 cells than in primary astrocytic cultures though the activity in C6 cells was not density dependent (results not shown). The phospholipase A1 activity with PE in low- to medium-density C6 cells ((l-2) . HO’ cells/cm2) and primary astrocytic cultures was maximal at approx. pH 9.5. As C6 cells reached confluency there was a specific increase in the phospholipase A, activity with PE, corre-
Fig 3. The pH profile of phospholipase A2 activity measured via !!4C]arachidonate release from phosphatidyiethanolamine in low- and high-density C6 cells. 7-Acyl-2-i’4C]arachidonoy~sn-glycero-3-phosphoethanolamine was incubated with homogenates of (m- - -6) low-density ((l-2).10’ cells/cm’) or (t---o) high-density (8.105 cells/cm2) C6 cultures. *$ i C.005 vs. low-density cuitures. With respect to the high-density pH profile, the phospholipase activities at pH 8 and 9.5 were significantly higher than the value at pH 9, P < 0.05.
sponding with the appearance second optimum at pH 7.5-8 an increase in the activity at pase A, activities with PE
q--85
PH Fig. 2. The pH profile of phospholipase Al activity measured via [ 3Hlpalmitate release in primary astrocytic and C6 cuhures. l-[ 3 H]Palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine was incubated for 1 h with homogenates of primary astrocytic cultures (O------O) or C6 celis (M-M). Similarly, I[ 3Hlpalmitoyl-2-acyl-sn-glycero-3-phosphocholine was incubated with homogenates of primary astrocytic cultures (0- - - - -0) or C6 cells (0 -0). See text for additional assay conditions.
_-a-___ 70
-0
____
7s
and dominance of a (Fig. 3) along with pH 9.5. Phospholi(but not PC) were
~~-~~~~~_~~~_-J~~~~~_~
BD
8.5
SW3
9.5
PH Fig. 4. The pH profile of the release of diacylglycerol from phosphatidylethanolamine in soluble and particulate fractions of low- and high-density C6 cells. l-[3H]Palmitoyl-2i’4C]arachidonoyl-sn-glycero-3-phosphoethano~amine was incubated with either the soiuble (- - -) or the particulate ) fraction of low-density @ q) ((l-2).10’ ceils/cm’) (or (00) high-density (8*10’ ce!ls/cm’) C6 cells. “P < 0.005 for ail high-density phospholipase activities vs. low-densaty control.
241
similarly affected by cell density (results not shown). In C6 cells, phospholipase C activity could be observed for PE but not PC. In low-density cells the activity of this enzyme for PE was essentially membrane bound. A considerable increase in this activity was observed in high-density cultures along with the appearance of activity in the soluble fraction as well (Fig. 4). It is currently unclear whether these represent two distinct enzymes or soluble and membranous forms of the same enzyme. Primary astrocytic cultures contained substantial Ca’+-dependent phospholipase C activity with phosphatidylinositol (PI) though, in contrast to PE, this activity was barely detectable in C6 cells. Discussion C6 glial cells exhibited a density dependent, highly specific increase in alkaline phospholipase activities with PE and a virtual loss of acid phospholipases as compared to nontransformed primary astrocytic cultures. Alkaline phospholipase activities with PC were unchanged as a function of C6 cell density, while the phospholipase C activity with PI was decreased. Collectively this suggests that enhanced arachidonate release in confluent C6 cells is mainly a property of PE metabolism, specifically the alkaline phospholipases. While primary astrocytes have at least two routes for arachidonate release, C6 cells exhibited a reduction in the phospholipase C hydrolysis of PI and an increase in phospholipase activity with PE. A characteristic feature of many cell lines and tumors is enhanced levels of E and F type prostaglandins (27-29). The results presented here, in the case of three cell lines, strongly implicate PE turnover as a source of enhanced arachidonate release. The augmentation in phospholipase activity does not appear to be a property of the transformed cell per se but rather a product of the high cell density of the confluent state. This is in contrast to the loss of acid phospholipase activity which does appear to follow transformation. The increase in PE phospholipase activity in C6 cells relative to astrocytic cultures is reflected in an increase in the percent phospholipid composition
of PE from 10.2% for astrocytes to 18% for C6 cells. PI content, on the other hand, decreased from 9.0 to 3.8% in parallel with the decrease in phospholipase C activity, while PC decreased only slightly from 52.2 to 48.3% [30]. Based on the pH optima, the results also suggest the existence in astrocytes of separate phospholipases catabolizing PC and PE. This is in lieu of a difference in pH optima being due to, for example, the preference of one enzyme for only a positively or negatively charged substrate [31]. These phospholipase activities are not CaZf-requiring, in contrast to the phospholipase C activity with PI. References 1 Michell, R.H. (1975) Biochim. Biophys. Acta 415, 81-147 2 Horrocks, L.A., Ansell, G.B. and Porcellati, G. (1982) Phospholipids in the Nervous System, Vol I, Metabolism, Raven Press, New York M.J., Kleinfeld, A.M., Hoover, R.L. and 3 Karnovsky, Klausner, R.D. (1982) J. Cell Biol. 94, l-6 4 Op den Camp, J.A.F. (1979) Annu. Rev. Biochem. 48, 47-71 5 Flower, R.F. and Blackwell, G.J. (1976) Biochem. Pharmacol. 25, 285-291 6 Cullis, P.R. and De Kruijff, B. (1978) Biochim. Biophys. Acta 513, 31-42 P.H.J. (1978) 7 Cullis, P.R., Verkleij, A.J. and Ververgaert, Biochim. Biophys. Acta 513, 11-20 8 Verkleij, A.J., De Maagd, R., Leunissen, J. and De Kruijff, B. (1982) Biochim. Biophys. Acta 684, 255-262 F., Lamb, R. and 9 Hsueh, W., Desai, U., Gonzalez-Crussi, Chu, A. (1981) Nature 290, 710-713 10 Humes, J.L., Sadowski, S., Galavage, M., Gordenberg, M.. Subers, E.. Bouney, R.J. and Kuehl, F.A. (1982) J. Biol. Chem. 257, 1591-1594 11 Williams, D.J., Spanner, S. and Ansell, G.B. (1973) Biothem. Sot. Trans. 1, 466-467 12 Rooke, J.A. and Webster, G.R. (1976) J. Neurochem. 27, 613-620 13 Woelk, H., Goracci, G., Arienti, G. and Porcellati, G. (1978) in Advances in Prostaglandin and Thromboxane Research (Galli, G. and Porcellati, G. eds.), pp. 77-83, Vol. 3, Raven Press, New York 31 14 Irvine, R.F. and Dawson, R.M.C. (1978) J. Neurochem. (1427-1434) 15 Sun, G.Y., Su, K.L., Der, O.M. and Tang, W. (1979) Lipids 14, 229-235 F.J. and Rowe, C.E. (1980) Brain Res. 197, 16 Doherty, 113-122 17 Gray, N.C.C. and Strickland, K.P. (1982) Can. J. Biochem. 60, 108-117 18 Brockerhoff, H., Schmidt, P.C.. Fong, J.W. and Tirri, L.J. (1976) Lipids 11, 421-424
19 Woelk, Hoppe 20 Folch, Chem.
H., Goracci, 6.: Gaiti, A. and Porcellati, 6. (1973) Seyler’s Z. Physio!. Chem. 354, 729-736 P.J., Lees, M. and Sloane-Stanley, J.H. (2957) 9. Biol. 226, 497-509
21 Lowry, O.K., Rosebrough, N.J., Fur, A.&. and Randall, W.J. (1951) J. Biol. Chem. 193, 265-275 22 Booher, J. and Sensenbrenner, M. (1972) Neurobiology 2, 97-105 23 Benda, P., Lightbody, J., Sara, G. and Sweet, W. (1968) Science 161, 370-371 24 Shein, S.H., Bitra, A., Hess, M.M. and Selkoe, D.J. (1970) Brain Res. 19, 497-501
25 Dreyfus, H., Ha&, S., Urban, P.F., Mande! ?. 2nd F-e~sz. E. (1978) J. Neurochem. II. 1157-1162 26 Swanson, P.D. (1967) J. Netirochem. 14, 343-356 27 Honn, K.V., Bockman, R.S. and Marnet;, L.J. (‘;%I) i”costaglandios 21. 833-864 28 Tan, W.C., Privett, OS. and Goldyne, M.E. (1974) Cancer Res. 34. 3229-3231 29 Hammarstrom, S. (1977) Eur. J. Biochem. 74, 7-12 30 Robert, J. (1978) These, U. Louis Pasteur de Strasbourg 31 Waite, M. and Sisson, P. (1971) Biochem. IO, 2377-2383
BBA 51783
SPECIFIC STIMULATION OF PHOSPHOLIPASE AC~~~ITIE~ ~~~~ PHOSPHATIDYLETHANOLAMINE IN TRANSFORMED CELLS
(Received February (Revised manuscript
13th, 1984: received July 9th, 1984)
Phospholipase A,, A, and C activities with phosphatidylethanolamine were enhanced in C6 celDisrelative to primary astrocytic cultures, Enhancement was a function of sell density, Phospholipase activities with phosphatidylcholine were unchanged as a function of cell density, while phospholipase C activity with phosphatidylinosito1 was reduced. All acid phospholipas~ activities measured were low or essentia& absent in the three transformed cell lines examined. These results suggest that arachidonate release upon ~~~f~~e~~~ Is mainly from phosphatidylethanolamine.
The metabolism of glycerophospholipids is involved in responses to a variety of stimuli [1,2], the regulation of membrane fluidity [3], the control of the asymmetric distribution of g~ycerophospholipids in some membranes [4] and in the provision of polyunsaturated fatty acids for cyclooxygenase and lipoxygenase pathways 151. Hn addition, the physicochemical potentials of phospholipids in bilayer systems are different /6-81. A variety of metabolic pathways may be necessary in order to diffcre~tially express the respective function sf each phospholipid class. Evidence for separate pools of arachidonate release from phospholipids in macrophages has been recently presented [9,10].
* To whom correspondence should be addressed c/o Louis Freysz, Centre de Neurochimie du CNRS, 5 rue Blake Pascal, 67084 Strasbourg, Cedex, France. Abbreviations: PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; Mops, 4-morpholinepropanesutfonic acid. ~55-2760/84/$03.~0
@ 1984 Ekevier
Science Publishers
I?.‘?.
Arachidonate release is generally via ~hos~ho~~pase AZ and/or the coupled action of phosphohpase C and diglyceride lipase. IIn the central nervous system several phosphoiipases have been investigated and in many cases the characteristics differ somewhat from similar enzymes in bob-nervous tissue [IF;-IT]. A major problem encountered in characterization of brain phospholipases is the heterogeneity of ihe tissue with regard to celll types. Apparently, nauronal phospholipases are more active than gilal. phospholipases jt3J. Here we describe several of the pbospholipid catabol~zi~g enzymes an primary astrocytic cultures and C6 tumoral cells. Materials and Methods [~-‘4~]Arachido~ic acid and ~9~~0~~~-3~~ palmitic acid were obtained from ~~d~ochern~~a~ Centre, Amersham, U.K. Fetal calf serum and Dulbecco’s modified Eagle’s medium -were purchased from the Grand Island Biological @o. Grand Island, NY, U.&A. Phospholipase AI and
239
A, activities were measured by the release of radiolabelled fatty acid from l-[ 3Hlpalmitoyl-2-acylsn-glycerophospholipids (prepared by the method of Brockerhoff et al. [18]) and I-acyl-2[‘4C]arachidonoyl-sn-glycerophospholipids (prepared by the method of Woelk et al. [19], respectively. Similar results were obtained if the release of radiolabelled lysophospholipids was measured using the former substrate for A, and the latter substrate for A, activity. Phospholipase C activities were measured by the release of radiolabelled diacylglycerol from the above substrates. Substrate (final concentration of 150 PM, with a specific activity of 0.1 Ci/mol) in the appropriate buffer was briefly sonicated and added to 0.4-2.0 mg protein in a final volume of 1 ml with 0.08% sodium deoxycholate. Buffers used were citrate (pH 3.0-4.0), acetate (pH 4.1-6.0), Mops (pH 6.1-8.0), Tris-HCl (pH 8.1-10.0). Co-sonication of radiolabelled substrate and tissue sample indicated that in the presence of detergent exogenous and endogenous phospholipids competed equally. The samples, in triplicate, were incubated for 60 min (20 min for phospholipase C) at 37°C in a shaking-water bath; the reactions were stopped by the addition of 4 ml of chloroform/methanol, (2 : 1, v/v) and lipids extracted by the method of Folch et al. [20]. Fatty acids, phosphoglycerides and lysophospholipids were separated by TLC on 0.5mm silica Gel G plates by the solvent system chloroform/methanol/water (70 : 20 : 2, v/v) and visualized by iodine vapors. Fatty acids, diacylglycerols and phosphatides were separated by hexane/diethyl ether/acetic acid (80 : 20 : 1, v/v). Corresponding areas were scraped into scintillation vials with the addition of 0.5 ml water and 10 ml scintillator. Rates were calculated following subtraction of blank values for sample without protein. Protein was determined by the method of Lowry et al. [21] with bovine serum albumin as standard. The standard deviation of these assays was routinely less than 5%. Pure primary astrocytic cultures were prepared from l-day-old rat cerebral hemispheres [22] and harvested on day 21 in 0.9% saline with the aid of a rubber policeman. C6 [23] and NN cells [24] were subcultured after mild trypsinization and harvested at low density (lo5 cells/cm*), moderate density ((3-5). lo5 cells/cm2)) or confluency (8 .
10’ cells/cm2) in the case of C6 cells or 14. lo5 cells/cm2 for NN and HTC cells. Cells were lysed by homogenization for 25 s with a Polytron (Kinematics GmBH, Lucern, Switzerland). Membrane and soluble fractions were then prepared by centrifugation at 100000 X g for 1 h. CDPcholine phosphotransferase [25] served as a marker of the membrane fraction, while lactate dehydrogenase [26] was a marker of the soluble fraction. Results Phospholipase A 2 activities measured with phoshatidylethanolamine (PE) did not share the same pH optima as those measured with phosphatidylcholine (PC) in either primary astrocytic cultures or C6 cells (Fig. 1). In addition, no detectable phospholipase A 2 activity could be measured at acidic pH in C6 cells with either PC or PE. NN cells had a phospholipase A, profile similar to C6 cells in that there was an alkaline optima for PE (at pH 8.0) and no acid activity. Additionally, the acid phospholipase A, activities in the hepatoma cell line HTC were only 30% and 10% that of the alkaline activities with PC and PE, respectively
P”
Fig. 1. The pH profile of phospholipase A2 activity, measured via [‘4C]arachidonate release, in primary astrocytic and C6 cultures. 1-acyl-2-[‘4C]arachidonoyl-sn-glycero-3-phosphoethanolamine (left hand scale) was incubated for 1 h with homogenates of primary astrocytic cultures (o- -0) or C6 cells (W=). In a similar manner, l-acyl-2[‘4C]arachidonoyl-sn-glycero-3-phosphochohne (right hand scale) was incubated with homogenates of primary astrocytic cultures (0- - - - - -0) or C6 cell (O0). See text for additional assay conditions.
(FeSUh§ not shown). Acid phospholipase A, activrties with PC and PE were nearly absent in C6 cells, while in primary astrocytic cultures the acid phospholipase activities were greater than alkaline activites (Fig. 2). Removai of detergent, or substitution of sodium deoxycholate with Triton X-400 or Tween 20 did not result in an enhancement in the acid phospholipase activities. The phospholipase A, and A, activities with PC and PE did not require divalent cations. Concentrations of EDTA higher than 4 mlki stimulated alkaline phosphoiipase activity though this was attributable to the increase in ionic strength, while Ca*+ concentrations up to 1 mM had no effect on activity (results not shown). The cell density had a marked effect on phospholipase activities with PE in C6 cells while activities with PC were not affected. Phospholipase A, activity at pH 7-7.5 with PC was greater in C6 cells than in primary astrocytic cultures though the activity in C6 cells was not density dependent (results not shown). The phospholipase A1 activity with PE in low- to medium-density C6 cells ((l-2) . HO’ cells/cm2) and primary astrocytic cultures was maximal at approx. pH 9.5. As C6 cells reached confluency there was a specific increase in the phospholipase A, activity with PE, corre-
Fig 3. The pH profile of phospholipase A2 activity measured via !!4C]arachidonate release from phosphatidyiethanolamine in low- and high-density C6 cells. 7-Acyl-2-i’4C]arachidonoy~sn-glycero-3-phosphoethanolamine was incubated with homogenates of (m- - -6) low-density ((l-2).10’ cells/cm’) or (t---o) high-density (8.105 cells/cm2) C6 cultures. *$ i C.005 vs. low-density cuitures. With respect to the high-density pH profile, the phospholipase activities at pH 8 and 9.5 were significantly higher than the value at pH 9, P < 0.05.
sponding with the appearance second optimum at pH 7.5-8 an increase in the activity at pase A, activities with PE
q--85
PH Fig. 2. The pH profile of phospholipase Al activity measured via [ 3Hlpalmitate release in primary astrocytic and C6 cuhures. l-[ 3 H]Palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine was incubated for 1 h with homogenates of primary astrocytic cultures (O------O) or C6 celis (M-M). Similarly, I[ 3Hlpalmitoyl-2-acyl-sn-glycero-3-phosphocholine was incubated with homogenates of primary astrocytic cultures (0- - - - -0) or C6 cells (0 -0). See text for additional assay conditions.
_-a-___ 70
-0
____
7s
and dominance of a (Fig. 3) along with pH 9.5. Phospholi(but not PC) were
~~-~~~~~_~~~_-J~~~~~_~
BD
8.5
SW3
9.5
PH Fig. 4. The pH profile of the release of diacylglycerol from phosphatidylethanolamine in soluble and particulate fractions of low- and high-density C6 cells. l-[3H]Palmitoyl-2i’4C]arachidonoyl-sn-glycero-3-phosphoethano~amine was incubated with either the soiuble (- - -) or the particulate ) fraction of low-density @ q) ((l-2).10’ ceils/cm’) (or (00) high-density (8*10’ ce!ls/cm’) C6 cells. “P < 0.005 for ail high-density phospholipase activities vs. low-densaty control.
241
similarly affected by cell density (results not shown). In C6 cells, phospholipase C activity could be observed for PE but not PC. In low-density cells the activity of this enzyme for PE was essentially membrane bound. A considerable increase in this activity was observed in high-density cultures along with the appearance of activity in the soluble fraction as well (Fig. 4). It is currently unclear whether these represent two distinct enzymes or soluble and membranous forms of the same enzyme. Primary astrocytic cultures contained substantial Ca’+-dependent phospholipase C activity with phosphatidylinositol (PI) though, in contrast to PE, this activity was barely detectable in C6 cells. Discussion C6 glial cells exhibited a density dependent, highly specific increase in alkaline phospholipase activities with PE and a virtual loss of acid phospholipases as compared to nontransformed primary astrocytic cultures. Alkaline phospholipase activities with PC were unchanged as a function of C6 cell density, while the phospholipase C activity with PI was decreased. Collectively this suggests that enhanced arachidonate release in confluent C6 cells is mainly a property of PE metabolism, specifically the alkaline phospholipases. While primary astrocytes have at least two routes for arachidonate release, C6 cells exhibited a reduction in the phospholipase C hydrolysis of PI and an increase in phospholipase activity with PE. A characteristic feature of many cell lines and tumors is enhanced levels of E and F type prostaglandins (27-29). The results presented here, in the case of three cell lines, strongly implicate PE turnover as a source of enhanced arachidonate release. The augmentation in phospholipase activity does not appear to be a property of the transformed cell per se but rather a product of the high cell density of the confluent state. This is in contrast to the loss of acid phospholipase activity which does appear to follow transformation. The increase in PE phospholipase activity in C6 cells relative to astrocytic cultures is reflected in an increase in the percent phospholipid composition
of PE from 10.2% for astrocytes to 18% for C6 cells. PI content, on the other hand, decreased from 9.0 to 3.8% in parallel with the decrease in phospholipase C activity, while PC decreased only slightly from 52.2 to 48.3% [30]. Based on the pH optima, the results also suggest the existence in astrocytes of separate phospholipases catabolizing PC and PE. This is in lieu of a difference in pH optima being due to, for example, the preference of one enzyme for only a positively or negatively charged substrate [31]. These phospholipase activities are not CaZf-requiring, in contrast to the phospholipase C activity with PI. References 1 Michell, R.H. (1975) Biochim. Biophys. Acta 415, 81-147 2 Horrocks, L.A., Ansell, G.B. and Porcellati, G. (1982) Phospholipids in the Nervous System, Vol I, Metabolism, Raven Press, New York M.J., Kleinfeld, A.M., Hoover, R.L. and 3 Karnovsky, Klausner, R.D. (1982) J. Cell Biol. 94, l-6 4 Op den Camp, J.A.F. (1979) Annu. Rev. Biochem. 48, 47-71 5 Flower, R.F. and Blackwell, G.J. (1976) Biochem. Pharmacol. 25, 285-291 6 Cullis, P.R. and De Kruijff, B. (1978) Biochim. Biophys. Acta 513, 31-42 P.H.J. (1978) 7 Cullis, P.R., Verkleij, A.J. and Ververgaert, Biochim. Biophys. Acta 513, 11-20 8 Verkleij, A.J., De Maagd, R., Leunissen, J. and De Kruijff, B. (1982) Biochim. Biophys. Acta 684, 255-262 F., Lamb, R. and 9 Hsueh, W., Desai, U., Gonzalez-Crussi, Chu, A. (1981) Nature 290, 710-713 10 Humes, J.L., Sadowski, S., Galavage, M., Gordenberg, M.. Subers, E.. Bouney, R.J. and Kuehl, F.A. (1982) J. Biol. Chem. 257, 1591-1594 11 Williams, D.J., Spanner, S. and Ansell, G.B. (1973) Biothem. Sot. Trans. 1, 466-467 12 Rooke, J.A. and Webster, G.R. (1976) J. Neurochem. 27, 613-620 13 Woelk, H., Goracci, G., Arienti, G. and Porcellati, G. (1978) in Advances in Prostaglandin and Thromboxane Research (Galli, G. and Porcellati, G. eds.), pp. 77-83, Vol. 3, Raven Press, New York 31 14 Irvine, R.F. and Dawson, R.M.C. (1978) J. Neurochem. (1427-1434) 15 Sun, G.Y., Su, K.L., Der, O.M. and Tang, W. (1979) Lipids 14, 229-235 F.J. and Rowe, C.E. (1980) Brain Res. 197, 16 Doherty, 113-122 17 Gray, N.C.C. and Strickland, K.P. (1982) Can. J. Biochem. 60, 108-117 18 Brockerhoff, H., Schmidt, P.C.. Fong, J.W. and Tirri, L.J. (1976) Lipids 11, 421-424
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