Biochem. J. (1977) 164, 389-397 Printed in Great Britain
389
A Comparison of the Effects of Phytohaemagglutinin and of Calcium lonophore A23187 on the Metabolism of Glycerolipids in small Lymphocytes By DAVID ALLAN and ROBERT H. MICHELL Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. (Received 18 October 1976) 1. The effects of phytohaemagglutinin and of a Ca2+ ionophore (A23 187) on glycerolipid metabolism in lymphocytes from pig lymph nodes were compared (a) by studying the incorporation of [32P]Pi and [3H]glycerol, and (b) by following the redistribution of [3H]glycerol among the lipids caused by these agents in pulse-chase experiments. 2. Phytohaemagglutinin only stimulated 32p incorporation into phosphatidylinositol and, to a slight extent, phosphatidate. Removal of most of the extracellular Ca2+ somewhat decreased this response. 3. lonophore A23187 stimulated the labellingof phosphatidateand phosphatidylinositol with 32p to a much greater extent than did phytohaemagglutinin: the increase in phosphatidate labelling, but not that of phosphatidylinositol, was almost abolished by the removal of extracellular Ca2+. 4. The combined effects of phytohaemagglutinin and ionophore appeared to be additive, rather than synergistic. 5. Treatment with ionophore A23187 somewhat decreased the total incorporation of [3H]glycerol into glycerolipids, possibly because it lowered cell ATP content. In these experiments di- and tri-acylglycerol behaved anomalously, triacylglycerol labelling being suppressed completely, whereas that of diacylglycerol was enhanced. The pulse-chase results revealed that triacylglycerol was converted into diacylglycerol in the ionophore-treated cells, and the availability of this diacylglycerol probably led to the enhanced labelling of phosphatidate and phosphatidylinositol in these cells. 6. Thus an increase in intracellular Ca2+ concentration appeared to have three effects on glycerolipid metabolism: (a) slight inhibition of some metabolic step preceding phosphatidate synthesis, (b) inhibition of diacylglycerol acyltransferase and (c) activation of a triacylglycerol lipase. 7. In contrast, it seems likely that the only effect of phytohaemagglutinin is to stimulate phosphatidylinositol breakdown. 8. Pig polymorphonuclear leucocytes treated with ionophore A23187 showed metabolic changes that were similar to those demonstrated with lymphocytes. 9. A possible similarity is suggested between Ca2+-stimulated triacylglycerol lipase in lymphocytes and polymorphonuclear leucocytes and previous observations of enhanced triacylglyerol metabolism in stimulated cells whose metabolic functions involve membrane fusion. A number of plant lectins, including Phaseolus vulgaris (dwarf bean) phytohaemagglutinin, are polyclonal mitogens which stimulate lymphocyte growth and division in much the same way as appropriate antigens (Ling & Kay, 1975). Lymphocytes show an increased permeability to Ca2+ almost immediately after their initial contact with phytohaemagglutinin (Allwood et al., 1971; Whitney & Sutherland, 1972; Freedman et al., 1975) and they can be induced to grow and divide if their intracellular Ca2+ concentration is raised by using the ionophore A23187 (Maino et al., 1974). It thus seems likely that interaction of phytohaemagglutinin or other mitogens with their receptors leads to an increase in cell-surface permeability to Ca2+, and that the resulting rise in the intracellular Ca2+ concentration provides the initial stimulus to cell transformation (Crumpton et al., 1975). Vol. 164
An early change seen in T-lymphocytes stimulated with phytohaemagglutinin or several other polyclonal mitogens (Fisher & Mueller, 1968, 1971; Maino et al., 1975), but maybe not in stimulated B-lymphocytes (Masuzawa et al., 1973; Betel et al., 1974; Maino et al., 1975) or in lymphocytes stimulated with 12-tetradecanoylphorbol 13-acetate (M. J. Crumpton, D. Allan & R. H. Michell, unpublished work), is an increase in the metabolism of phosphatidylinositol, a quantitatively minor anionic membrane glycerophospholipid. It has been stated that the mitogenic Ca2+ ionophore A23187 also brings about an increase in phosphatidylinositol labelling (Maino et al., 1974), but no experimental data have been published in support of this statement. It is thought that the initial reaction in stimulated phosphatidylinositol turnover in a variety of cells is phosphatidylinositol breakdown (MihWll, 1975). The
390 which catalysed this reaction in lymphocytes is exquisitely sensitive to Ca2+ ions at very low concentrations (Allan & Michell, 1974a,b). The obvious inference from these observations is that both the initiation of cell growth and division and the initiation of phosphatidylinositol breakdown in the lymphocytes exposed to either phytohaemagglutinin or ionophore A23187 are direct effects of the increased intracellular concentration of ionized Ca2+. However, two important pieces of information suggest that caution should be exercised before this simple interpretation is accepted. First, although there is an increase in phosphatidylinositol turnover in all cells exposed to extracellular stimuli which, through receptors, control cell-surface Ca2+ permeability (Michell, 1975; Michell et al., 1977b), in several of these situations there is good evidence that the phosphatidylinositol response is not initiated by a change in the intracellular Ca2+ concentration (Trifaro, 1969; Jones & Michell, 1975, 1976; Oron et al., 1975; Jafferji & Michell, 1976). Indeed, it has been suggested that receptor-controlled phosphatidylinositol breakdown, far from being a consequence of Ca2+ influx into cells, may be involved in the coupling between receptors and cell-surface Ca2+ gates (Michell, 1975; Michell et al., 1976a,b, 1977a, b). Secondly, the admission of Ca2+ into erythrocytes with an ionophore leads to an increase in labelling of phosphatidate, the immediate precursor of phosphatidylinositol (Allan & Michell, 1975a,b; Allan et al., 1975, 1976a,b). This labelling of phosphatidate is a secondary result of a Ca2+-stimulated production of diacylglycerol from a pre-existing glycerolipid, which, although not yet definitively identified, is not phosphatidylinositol (Allan et al., 1976b). The experiments described in the present paper were undertaken to try to resolve some of the contradictions which appeared to be present in the existing information. In particular, we wished to describe adequately the changes in lipid metabolism brought about in lymphocytes by an increase in the intracellular Ca2+ concentration and to try to determine whether the effects on lipid metabolism caused by phytohaemagglutinin and by ionophore A23187 were caused by similar or different mechanisms. Having found that they were probably different, we then went on to attempt to identify the source of the diacylglycerol that appeared in lymphocytes treated with the ionophore. Some of these data have been reported in a preliminary form (Allan & Michell, 1977).
enzyme
Materials and Methods Materials were obtained from sources specified previously: cinchocaine, phytohaemagglutinin and radioisotopes (Allan & Michell, 1975a); compound A23187 (Allan et al., 1976b).
D. ALLAN AND R. H. MICHELL Suspensions of small lymphocytes were prepared from pig mesenteric lymph nodes by a modification of the method of Allan et al. (1971). Finely chopped lymph node was dispersed by hand by using two strokes of a loose-fitting (1.5mm clearance) Teflon pestle in a stainless-steel homogenizer tube. Cells were isolated and washed in Hepes*/Ringer solution, pH7.4 (Allan & Michell, 1975a), and were resuspended at 2 x 108 cells/ml in the same buffer. For incorporation studies with [32P]Pj or [3H]glycerol, the radioactive tracer (25 Ci of [32P]PI/ml or 1 Ci of [2-3H]glycerol/ml) was added at the beginning of a 1 h incubation at 37°C. At the end of the incubation period, when the specific radioactivity of cellular [32P]ATP approached that of Pi, either phytohaemagglutinin (in 10,ul of 0.9% NaCl) or ionophore A23187 (in 10,ul of dimethylsulphoxide) was added to 1 ml portions of the cell suspension and incubation was continued for a further period (usually 1 h). Incubation was stopped by addition of methanol/ chloroform (2: 1, v/v; 3.75 ml) and lipids were extracted as described previously (Allan & Michell, 1975a). In some experiments cells were labelled for 2 h with [3H]glycerol (2,pCi/ml) and then centrifuged and washed twice in the presence of unlabelled 10mMglycerol. Owing to the ready permeability of the cells to glycerol the specific radioactivity of lipid precursors was decreased to very low values by this procedure. The cells were then incubated for 1 h with phytohaemagglutinin or ionophore A23187. Experiments were also carried out by using lymphocytes and polymorphonuclear leucocytes purified from pig blood: 200ml of fresh pig blood was collected and mixed rapidly with 10ml of 100mMEDTA as anticoagulant. The leucocyte-rich buffycoat material was isolated and washed by centrifugation in Ca2+-free Hepe3/Ringer solution, pH7.4, and was then resuspended in 6ml of the same medium. Samples (3 ml) of this suspension were carefully layered over step gradients of bovine serum albumin (Sigma Chemical Co., Kingston-upon-Thames, U.K.): these were made up of 2.5 ml of 25% (w/w) albumin and 2.5 ml of 23 % (w/w) albumin in Ca2+free Hepes/Ringer solution in 10ml centrifuge tubes. The tubes were centrifuged at room temperature for 15min at 4000 rev./min (MSE Superminor bench centrifuge). Material from the interface above the 23 % albumin layer was removed with a Pasteur pipette and was found to consist of 90-95% small lymphocytes. The pellet consisted of erythrocytes and leucocytes, over 90% of which were polymorphonuclear. Both fractions were resuspended in Hepes/ Ringer solution containing 2.5mM-Ca2+ at a leucocyte concentration of 107 cells/ml, and erythrocyte numbers were equalized in each fraction by adding *
Abbreviation: Hepes,
azine-ethanesulphonic acid.
4-(2-hydroxyethyl)-1-piper1977
391
MITOGENS AND LYMPHOCYTE GLYCEROLIPlD METABOLISM an appropriate volume of a suspension of erythrocytes to the lymphocytes. Incorporation of [3H]glycerol (5,uCi/ml) into the glycerolipids of these cells was measured as above with and without addition of ionophore A23187 (10,ug/ml). The contaminating erythrocytes were found to have no significant effects on the incorporation of labelled glycerol into the leucocyte suspensions. Phospholipids were separated either (a) on formaldehyde-treated papers run in the upper phase from a mixture of butan-1-ol/water/acetic acid (4:5: 1, by vol.), which separated phosphoinositides from the major phospholipids, or in a similar solvent in which formic acid replaced acetic acid, when phosphatidate alone of the phospholipids ran at the solvent front together with neutral lipids (Allan et al., 1975), or (b) by t.l.c. by the method of Skipski et al. (1964). Neutral lipids were separated by t.l.c. by the procedure of Freeman & West (1966). Radioactivity from the 32P-labelled spots from paper chromatograms was measured by Cerenkov counting after digestion of the spots in 70 % HCl04 (Allan & Michell, 1975a). For counting of 3H radioactivity in spots from thin-layer chromatograms, the spots were scraped off into scintillation vials and 100411 of methanol/water (1:1, v/v) was added, followed by IOml of a 1:1 (v/v) mixture of toluene scintillation fluid [containing 4g of 2,5-diphenyloxazole/l and 0.12g of 1,4-bis-(5-phenyloxazol-2yl)-benzene/1] and Triton X-100. Radioactivity was measured in a Philips scintillation counter. The content of ATP in the cells after incubation was measured as described previously (Allan et al., 1 976b). Results Fig. 1 shows the time-course of the incorporation of 32p into the lipids of lymphocytes to which either phytohaemagglutinin (20,ug/ml) or A23187 (10,ug/ ml) was added after 1 h of preincubation in a 32p_ labelled medium. In agreement with previous workers, we found that phytohaemagglutinin caused a considerable increase in the labelling of phosphatidylinositol and a slight increase in phosphatidate labelling, but did not cause any significant change in the labelling of other phospholipids: in a number of experiments the increase in phosphatidylinositol labelling was from 2 to 7 times the control value. Compound A23187 also caused a marked increase in phosphatidylinositol labelling and its effect was usually larger than that of phytohaemagglutinin, ranging from 3 to 30 times the control values. The ionophore, unlike phytohaemagglutinin, always caused a decrease in the labelling of the major phospholipids (Figs. lb and 2a). However, the most dramatic difference between phytohaemagglutinin and the ionophore lay in their effects on the incorporaVol. 164
4000 0.
*U
3000
o la
MO
2000
o,= .
ooo _
O.
o
_
100 8 00
a600_ 00 0 0
400 o
400
.
2000
-
E 4000 _ 0C .S 0. c,, ,
(c)
A
03000_
0.o
389
A Comparison of the Effects of Phytohaemagglutinin and of Calcium lonophore A23187 on the Metabolism of Glycerolipids in small Lymphocytes By DAVID ALLAN and ROBERT H. MICHELL Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. (Received 18 October 1976) 1. The effects of phytohaemagglutinin and of a Ca2+ ionophore (A23 187) on glycerolipid metabolism in lymphocytes from pig lymph nodes were compared (a) by studying the incorporation of [32P]Pi and [3H]glycerol, and (b) by following the redistribution of [3H]glycerol among the lipids caused by these agents in pulse-chase experiments. 2. Phytohaemagglutinin only stimulated 32p incorporation into phosphatidylinositol and, to a slight extent, phosphatidate. Removal of most of the extracellular Ca2+ somewhat decreased this response. 3. lonophore A23187 stimulated the labellingof phosphatidateand phosphatidylinositol with 32p to a much greater extent than did phytohaemagglutinin: the increase in phosphatidate labelling, but not that of phosphatidylinositol, was almost abolished by the removal of extracellular Ca2+. 4. The combined effects of phytohaemagglutinin and ionophore appeared to be additive, rather than synergistic. 5. Treatment with ionophore A23187 somewhat decreased the total incorporation of [3H]glycerol into glycerolipids, possibly because it lowered cell ATP content. In these experiments di- and tri-acylglycerol behaved anomalously, triacylglycerol labelling being suppressed completely, whereas that of diacylglycerol was enhanced. The pulse-chase results revealed that triacylglycerol was converted into diacylglycerol in the ionophore-treated cells, and the availability of this diacylglycerol probably led to the enhanced labelling of phosphatidate and phosphatidylinositol in these cells. 6. Thus an increase in intracellular Ca2+ concentration appeared to have three effects on glycerolipid metabolism: (a) slight inhibition of some metabolic step preceding phosphatidate synthesis, (b) inhibition of diacylglycerol acyltransferase and (c) activation of a triacylglycerol lipase. 7. In contrast, it seems likely that the only effect of phytohaemagglutinin is to stimulate phosphatidylinositol breakdown. 8. Pig polymorphonuclear leucocytes treated with ionophore A23187 showed metabolic changes that were similar to those demonstrated with lymphocytes. 9. A possible similarity is suggested between Ca2+-stimulated triacylglycerol lipase in lymphocytes and polymorphonuclear leucocytes and previous observations of enhanced triacylglyerol metabolism in stimulated cells whose metabolic functions involve membrane fusion. A number of plant lectins, including Phaseolus vulgaris (dwarf bean) phytohaemagglutinin, are polyclonal mitogens which stimulate lymphocyte growth and division in much the same way as appropriate antigens (Ling & Kay, 1975). Lymphocytes show an increased permeability to Ca2+ almost immediately after their initial contact with phytohaemagglutinin (Allwood et al., 1971; Whitney & Sutherland, 1972; Freedman et al., 1975) and they can be induced to grow and divide if their intracellular Ca2+ concentration is raised by using the ionophore A23187 (Maino et al., 1974). It thus seems likely that interaction of phytohaemagglutinin or other mitogens with their receptors leads to an increase in cell-surface permeability to Ca2+, and that the resulting rise in the intracellular Ca2+ concentration provides the initial stimulus to cell transformation (Crumpton et al., 1975). Vol. 164
An early change seen in T-lymphocytes stimulated with phytohaemagglutinin or several other polyclonal mitogens (Fisher & Mueller, 1968, 1971; Maino et al., 1975), but maybe not in stimulated B-lymphocytes (Masuzawa et al., 1973; Betel et al., 1974; Maino et al., 1975) or in lymphocytes stimulated with 12-tetradecanoylphorbol 13-acetate (M. J. Crumpton, D. Allan & R. H. Michell, unpublished work), is an increase in the metabolism of phosphatidylinositol, a quantitatively minor anionic membrane glycerophospholipid. It has been stated that the mitogenic Ca2+ ionophore A23187 also brings about an increase in phosphatidylinositol labelling (Maino et al., 1974), but no experimental data have been published in support of this statement. It is thought that the initial reaction in stimulated phosphatidylinositol turnover in a variety of cells is phosphatidylinositol breakdown (MihWll, 1975). The
390 which catalysed this reaction in lymphocytes is exquisitely sensitive to Ca2+ ions at very low concentrations (Allan & Michell, 1974a,b). The obvious inference from these observations is that both the initiation of cell growth and division and the initiation of phosphatidylinositol breakdown in the lymphocytes exposed to either phytohaemagglutinin or ionophore A23187 are direct effects of the increased intracellular concentration of ionized Ca2+. However, two important pieces of information suggest that caution should be exercised before this simple interpretation is accepted. First, although there is an increase in phosphatidylinositol turnover in all cells exposed to extracellular stimuli which, through receptors, control cell-surface Ca2+ permeability (Michell, 1975; Michell et al., 1977b), in several of these situations there is good evidence that the phosphatidylinositol response is not initiated by a change in the intracellular Ca2+ concentration (Trifaro, 1969; Jones & Michell, 1975, 1976; Oron et al., 1975; Jafferji & Michell, 1976). Indeed, it has been suggested that receptor-controlled phosphatidylinositol breakdown, far from being a consequence of Ca2+ influx into cells, may be involved in the coupling between receptors and cell-surface Ca2+ gates (Michell, 1975; Michell et al., 1976a,b, 1977a, b). Secondly, the admission of Ca2+ into erythrocytes with an ionophore leads to an increase in labelling of phosphatidate, the immediate precursor of phosphatidylinositol (Allan & Michell, 1975a,b; Allan et al., 1975, 1976a,b). This labelling of phosphatidate is a secondary result of a Ca2+-stimulated production of diacylglycerol from a pre-existing glycerolipid, which, although not yet definitively identified, is not phosphatidylinositol (Allan et al., 1976b). The experiments described in the present paper were undertaken to try to resolve some of the contradictions which appeared to be present in the existing information. In particular, we wished to describe adequately the changes in lipid metabolism brought about in lymphocytes by an increase in the intracellular Ca2+ concentration and to try to determine whether the effects on lipid metabolism caused by phytohaemagglutinin and by ionophore A23187 were caused by similar or different mechanisms. Having found that they were probably different, we then went on to attempt to identify the source of the diacylglycerol that appeared in lymphocytes treated with the ionophore. Some of these data have been reported in a preliminary form (Allan & Michell, 1977).
enzyme
Materials and Methods Materials were obtained from sources specified previously: cinchocaine, phytohaemagglutinin and radioisotopes (Allan & Michell, 1975a); compound A23187 (Allan et al., 1976b).
D. ALLAN AND R. H. MICHELL Suspensions of small lymphocytes were prepared from pig mesenteric lymph nodes by a modification of the method of Allan et al. (1971). Finely chopped lymph node was dispersed by hand by using two strokes of a loose-fitting (1.5mm clearance) Teflon pestle in a stainless-steel homogenizer tube. Cells were isolated and washed in Hepes*/Ringer solution, pH7.4 (Allan & Michell, 1975a), and were resuspended at 2 x 108 cells/ml in the same buffer. For incorporation studies with [32P]Pj or [3H]glycerol, the radioactive tracer (25 Ci of [32P]PI/ml or 1 Ci of [2-3H]glycerol/ml) was added at the beginning of a 1 h incubation at 37°C. At the end of the incubation period, when the specific radioactivity of cellular [32P]ATP approached that of Pi, either phytohaemagglutinin (in 10,ul of 0.9% NaCl) or ionophore A23187 (in 10,ul of dimethylsulphoxide) was added to 1 ml portions of the cell suspension and incubation was continued for a further period (usually 1 h). Incubation was stopped by addition of methanol/ chloroform (2: 1, v/v; 3.75 ml) and lipids were extracted as described previously (Allan & Michell, 1975a). In some experiments cells were labelled for 2 h with [3H]glycerol (2,pCi/ml) and then centrifuged and washed twice in the presence of unlabelled 10mMglycerol. Owing to the ready permeability of the cells to glycerol the specific radioactivity of lipid precursors was decreased to very low values by this procedure. The cells were then incubated for 1 h with phytohaemagglutinin or ionophore A23187. Experiments were also carried out by using lymphocytes and polymorphonuclear leucocytes purified from pig blood: 200ml of fresh pig blood was collected and mixed rapidly with 10ml of 100mMEDTA as anticoagulant. The leucocyte-rich buffycoat material was isolated and washed by centrifugation in Ca2+-free Hepe3/Ringer solution, pH7.4, and was then resuspended in 6ml of the same medium. Samples (3 ml) of this suspension were carefully layered over step gradients of bovine serum albumin (Sigma Chemical Co., Kingston-upon-Thames, U.K.): these were made up of 2.5 ml of 25% (w/w) albumin and 2.5 ml of 23 % (w/w) albumin in Ca2+free Hepes/Ringer solution in 10ml centrifuge tubes. The tubes were centrifuged at room temperature for 15min at 4000 rev./min (MSE Superminor bench centrifuge). Material from the interface above the 23 % albumin layer was removed with a Pasteur pipette and was found to consist of 90-95% small lymphocytes. The pellet consisted of erythrocytes and leucocytes, over 90% of which were polymorphonuclear. Both fractions were resuspended in Hepes/ Ringer solution containing 2.5mM-Ca2+ at a leucocyte concentration of 107 cells/ml, and erythrocyte numbers were equalized in each fraction by adding *
Abbreviation: Hepes,
azine-ethanesulphonic acid.
4-(2-hydroxyethyl)-1-piper1977
391
MITOGENS AND LYMPHOCYTE GLYCEROLIPlD METABOLISM an appropriate volume of a suspension of erythrocytes to the lymphocytes. Incorporation of [3H]glycerol (5,uCi/ml) into the glycerolipids of these cells was measured as above with and without addition of ionophore A23187 (10,ug/ml). The contaminating erythrocytes were found to have no significant effects on the incorporation of labelled glycerol into the leucocyte suspensions. Phospholipids were separated either (a) on formaldehyde-treated papers run in the upper phase from a mixture of butan-1-ol/water/acetic acid (4:5: 1, by vol.), which separated phosphoinositides from the major phospholipids, or in a similar solvent in which formic acid replaced acetic acid, when phosphatidate alone of the phospholipids ran at the solvent front together with neutral lipids (Allan et al., 1975), or (b) by t.l.c. by the method of Skipski et al. (1964). Neutral lipids were separated by t.l.c. by the procedure of Freeman & West (1966). Radioactivity from the 32P-labelled spots from paper chromatograms was measured by Cerenkov counting after digestion of the spots in 70 % HCl04 (Allan & Michell, 1975a). For counting of 3H radioactivity in spots from thin-layer chromatograms, the spots were scraped off into scintillation vials and 100411 of methanol/water (1:1, v/v) was added, followed by IOml of a 1:1 (v/v) mixture of toluene scintillation fluid [containing 4g of 2,5-diphenyloxazole/l and 0.12g of 1,4-bis-(5-phenyloxazol-2yl)-benzene/1] and Triton X-100. Radioactivity was measured in a Philips scintillation counter. The content of ATP in the cells after incubation was measured as described previously (Allan et al., 1 976b). Results Fig. 1 shows the time-course of the incorporation of 32p into the lipids of lymphocytes to which either phytohaemagglutinin (20,ug/ml) or A23187 (10,ug/ ml) was added after 1 h of preincubation in a 32p_ labelled medium. In agreement with previous workers, we found that phytohaemagglutinin caused a considerable increase in the labelling of phosphatidylinositol and a slight increase in phosphatidate labelling, but did not cause any significant change in the labelling of other phospholipids: in a number of experiments the increase in phosphatidylinositol labelling was from 2 to 7 times the control value. Compound A23187 also caused a marked increase in phosphatidylinositol labelling and its effect was usually larger than that of phytohaemagglutinin, ranging from 3 to 30 times the control values. The ionophore, unlike phytohaemagglutinin, always caused a decrease in the labelling of the major phospholipids (Figs. lb and 2a). However, the most dramatic difference between phytohaemagglutinin and the ionophore lay in their effects on the incorporaVol. 164
4000 0.
*U
3000
o la
MO
2000
o,= .
ooo _
O.
o
_
100 8 00
a600_ 00 0 0
400 o
400
.
2000
-
E 4000 _ 0C .S 0. c,, ,
(c)
A
03000_
0.o
