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Phenobarbital competes with diacylglycerol for protein kinase C
Life Sciences, Vol. 40, pp. 89-93 Printed in the U.S.A.
Pergamon Journals
PHENOBARBITAL COMPETES WITH DIACYLGLYCEROL FOR PROTEIN KINASE C
Ved P.S. Chauhan and Hans Brockerhoff New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314 (Received in final form September 29, 1986) Summary Phenobarbital inhibits protein kinase C of rat brain by competitively displacing the effector of the enzyme, diacylglycerol. The drug appears to occupy the triple hydrogen bonding site which bonds diaeylglycerol - and also phorbol esters - to the enzyme. It remains to be seen if the effect is responsible for the pharmaceutical activity of the drug; even so, it provides an example of a restructuring of lipid-protein hydrogen bonding, in the hydrogen belt of the membrane, in a manner postulated as a mechanism of anesthesia. The phosphorylating enzyme, protein kinase C, is attached to membranes, and activated, by diacylglycerol (DG) (1-3). Enzyme - DG bonding is achieved by triple hydrogen bonding (evidence collected in ref. 4): both H-receiving ester CO groups and the H-donating 3-OH group of DG are involved. Sterically similar H-bonding group arrangements are found in some tumor promotors, e.g., phorbol esters, which compete with DG and also activate the enzyme (5). A search for DG-displacing but deactivating compounds led us to consider phenobarbital, which has three CO groups (though no COH group) in a pattern which can be superposed on the three hydrogen-bonding oxygens of DG (Fig. I). The neuropharmacological properties of the drug together with the involvement of protein kinase C in synaptic transmission (6-9) add interest to the investigation. We find that phenobarbital is, indeed, an inhibitor of the kinase by virtue of competition with DG. Materials and Methods Chemicals: [~-32p]ATP of specific activity 3,000 Ci/mmol was from New England Nuclear, MA. All other chemicals were from Sigma. Diacylglycerol was prepared by enzymatic hydrolysis of egg phosphatidylcholine (I0). Phenobarbital was synthesized (11). Protein Kinase-C: Extracted essentially by the method of Boni and Rando (12). Rat brains were removed and washed in ice-cold extraction buffer (20 mM Tris-HCl, pH 7.5 containing 0.3M sucrose, 2 mM EDTA, 10 mM EGTA, 2 mM dithiothreitol, 2 mMphenylmethylsulfonyl fluoride, 10 ~g/ml pepstain, I0 ~g/ml soybean trypsin inhibitor, I0 ~g/ml leupeptin and 25 Bg/ml aprotinin). The brains were then homogenized with 4 volumes of the same buffer with glass Teflon pestle homogenizer and centrifuged at I00,000 X g for 1 h. The supernatant was used as the source of the enzyme.
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
90
Phenobarbital Inhibits Protein Kinase C
m
Vol. 40, No. i, 1987
mm
PB
FIG. I Space-filling models of diacylglycerol (DG) and phenobarbital (PB; oxygen atoms are slotted; the phenyl group is eclipsed in this perspective). The three oxygens of PB (on CO 2, 4, and 6) appear congruent with the I- and 2-C0 oxygens and with the 3-OH group of diacylglycerol; the hydrogen bond donating OH proton missing in PB may be supplied by the I-NH group. This is demonstrated by the O-ring which stands for a hypothetical oxo group of the enzyme which can be reached, by hydrogen bonding, from either the 3- OH of DG or the I- NH of PB. Refer (4) for further details. Enz~we Assay: Activity was assayed by measuring the incorporation of 32p from [7-~P]ATP into lysine-rich histone, type III-S (Sigma) in 20 mM Tris-HCI, pH 7.65, 0.4 mM CaClg, 0.2 mM EDTA, 1.0 mM EGTA, i mM ATP, 3.1 mM MgCI 2, 40 ~g/ml leupeptin, 800 ~g/ml histone and i00 ~M lipid. The mixture, 0.2 ml in plastic tubes, was incubated for 5 min. at 37 C. The reaction was stopped by the addition of 2 ~ trichloroacetic acid, followed by collection of the acid precipitate on a Millipore HA type filter, and counting in Filteron-X scintillation fluid (National Diagnostics. NJ). Vesicle Preparation: Phosphollplds and diacylglycerol were mixed in organic solvent which was removed by drying on a rotary evaporator at 70°C for i hr with repeated flushing with argon. Lipids were hydrated in 20 mM Tris HCI, pH 7.5 containing 2 mM dithiothreitol and 2 mM phenylmethyl sulfonylfluoride and sonicated until clear, under argon.
Vol. 40, No. i, 1987
Phenobarbital Inhibits Protein Kinase C
91
Soluble Fraction (SF): Protein kinase C was extracted in 20 mM Tris-Cl, pH 7.5 containing 0.3M sucrose, 2 mM EDTA, I0 mM EGTA and 2 mM dithiothreitol. The extracted enzyme (5 mg protein) was incubated for 5 min. at 37°C with 17 or 34 ~g of trypsin (14). The reaction was stopped by the addition of 340 ~g of trypsin inhibitor. Protein kinase C activity and the effect of phenobarbital were measured after trypsinization. Partition Coefficient: One mg/ml phenobarbital was dissolved in 20 mM Tris-HCL pH 7.65. Four ml of octanol and 4 ml of phenobarbital solution were mixed and shaken for 30 and 60 min.; optical density at 254 ran was then measured in octanol phase and buffer after centrifugation for 15 min. The partition coefficient (P) is the ratio of OD's. This value was divided by 5.0 since the partition coefficient in octanol/water has been found to be regularly 5.0 times higher than that in biological membrane/water (13).
Mole % Diacylglycerol
oL ,~
SF
i
250
i! ,°° o
-PB +PB]
F
E 150
'
•~ ~ lOO
1
E 5O o
~
0
.23
.33
0
ii
Mole % Phenobarbital/Lipid
FIG. 2 Activation of protein kinase C by diacylglycerol (DG) and inhibition by phenobarbital (PB). Results arranged in groups of equal concentrations of diacylglycerol (2, 4 and 6 mole% of total lipid in-membrane); columns are for different PB concentrations (in-membrane O, 0.23 and 0.33 mole%) which are calculated from aqueous concentration of PB times partition coefficien~ P = 0.31. Groups 1 and 3 represent 3 sets each, group 2 represents 2 sets of experiments. The PB concentrations in water are 8.75 mM and 12.5 mM, in the membrane 2.72 mM and 4.89 mM. The last two columns represent kinase activities of the soluble fraction, SF, i.e., the kinase activity (in the absence of DG, calcium, or phosphatidylserine) liberated by digestion with trypsin. PB at 12.5 mM does not influence the activity in SF. Bars represent standard deviation.
92
Phenobarbital
Inhibits Protein Kinase C
Vol. 40, No. i, 1987
Results and Discussion Figure 2 shows how the kinase is activated by diacylglycerol, then inhibited by phenobarbital. A double reciprocal plot (Fig. 3) shows that DG and PB act competitively. While other compounds have been reported to act on the active center of the kinase (15) or on the phospholipid matrix of the assay system (16), phenobarbital appears to inhibit protein kinase C by binding to the site otherwise occupied by the natural enzyme activator, diacylglycerol. This conclusion is confirmed by the behavior of the soluble fragment (SF) obtained
,~ ¢
12
0.33
rnol%
"=
~
/
~o
/ /
~_N 0.23 mot%
/'
m ,. 0.
0.5
0.4
0.3
0.2
A~//
6
0.1
0
O.1
,
•
Phenobarbital
0
n
J
i
0.2
0.3
0.4
1/s(mol%DG/lipid)
0.5 -~
FIG. 3 Double reciprocal plot of diacylglycerol concentration (mole~) against velocity (protein phosphorylation), at 0, 0.23, and 0.33 mole% phenobarbital in membrane. Slopes were calculated by linear regression analysis. Two additional sets of experiments gave similar results. The result is expected for the case of competition between PB and DG. by proteolysis of the enzyme (17). SF has full kinase activity but does no longer depend on diacylglycerol, nor is it inhibited by PB (Fig. 2). There are other compounds known which bind to the DG-receiving site of the kinase; in particular, tumor promotors such as phorbol esters or aplysiatoxin (18,19); also possibly some others (20,21). The tumor promotors possess the same arrangement of two hydrogen bond accepting oxo groups and one donating hydroxyl group (4). In PB, the hydroxyl group is missing (there is no evidence for an enol tautomer at physiological pH) (22); however, there are two potentially donating NH groups in barbital-I and -3 positions. It can be shown in a molecular model (Fig. I) that, when two oxo atoms each of DG and PB are superposed, it is possible for the 3-OH proton of DG as well as for the I-NH proton of PB to contact - with hydrogen bonding - the same hypothetical 0 atom of the DG-receiving site of the enzyme. In Fig. l, this 0 atom is shown as a ring. The triple hydrogen bonding between effectors (or inhibitor) can thus be realized with DG, tumor promotors, or PB. In the inhibitor, however, the angle of incidence of the hydrogen bonds would be different. A resulting change in enzyme conformation might explain the inhibition. Alternatively, there may be improper reconstruction of the DG-bonding site because of supernumerary hydrogen bonding (PB has five against DG's three bonding sites).
Vol. 40, No. i, 1987
Phenobarbital Inhibits Protein Kinase C
93
Is the inhibition of the kinase by PB related to the anticonvulsive and hypnotic properties of the drug? That remains unproven, but not unlikely. Favorable evidence is supplied by reports on the involvement of the enzyme in nerve conductance (6-9); also, the inhibitor acts in a range (3-6 mM in membrane) where anesthetics are active (13). In any case, our results furnish an example for the theory of anesthesia which holds that the drugs act by restructuring the "hydrogen belts", i.e., those regions of the neuronal plasma membrane that contain the hydrogen bonding lipid and protein CO and OH groups (23-25). Acknowledgement The study was supported by NIH grant GM21875 . Brockerhoff assisted in the experimental work.
J. Zingoni and S.
References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Y. TAKAI, A. KISHIMOTO, U. UIKKAWA, T. MORI and Y. NISHIZUKA, Biochem. Biophys. Res, Commun. 91 1218-1224 (1979). N.A. SHARKEY, K.L. LEACH, P.M. BLUMBERG, Proc. Natl. Acad. Sci. USA 81 607-610 (1984). B.R. GANONG, C.R. LOOMIS, Y.A. HANNUN and R.M. BELL, Proc. Natl. Acad. Sci. USA 83 1184-1188 (1986). H. BROCKERHOFF, FEBS Lett. 201 1-4 (1986). M. CASTAGNA, Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA and Y. NISHIZUKAS, J. Biol. Chem. 257 7847-7851 (1982). K.M.M. MURPHY, R.J. GOULD, M.L. OSTER-GRANITE, J.D. GEARHART and S.H. SNYDER, Science 222 1036-1038 (1983). S.A. DeRIEMER, P. GREENGARD and L.K. KACZMAREK, J. Neurosci. 5 2672-2676 (1985). S.A. DeRIEMER, J.A. STRONG, K.A. ALBERT, P. GREENGARD and L.K. KATZMAREK, Nature 313 313-316 (1985). D.V. MADISON, R.C. MALENKA and R.A. NICOLL, Nature 321 695-697 (1986). L.D. BERGELSON, Lipid Biochemical Preparations, p. 118-119, Elsevier, Amsterdam (1980). B.S. FUMISS, A.J. HANNAFORD, V. ROGERS, P.W.G. SMITH and A.R. TATCHELL, Vogel's Textbook of Practical Organic Chemistry, 4th ed., p. 907-909, Longman, London (1978) . L.T. BONI and R.R. RAN-DO, J, Biol. Chem. 260 10819-10825 (1985). P. SEEMAN, Pharmacol. Rev. 24 583-655 (1972). A. KISHIMOTO, N. KAJIKAWA, M. SHIOTA and Y. NISHIZUKA, J. Biol. Chem. 258 1156-1164 (1983). S. KAWAMOTO and H. HIDAKA, Biochem. Biophys. Res. Commun. 125 258-264 (1984). T. MORI, Y. TAKAI, R. MINAKUCHI, B. YU and Y. NISHIZUKA, J. Biol. Chem. 255 8378-8380 (1980). M. INOUE, A. KISHIMOTO, Y. TAKAI and Y. NISHIZUKA, J. Biol. Chem. 252 7610-7616 (1977). U. KIKKAWA, Y. TAKAI, Y. TANAKA, R. MIYAKE and Y. NISHIZUKA, J. Biol. Chem. 258 1142-1145 (1983). T. SUGIMURA, Gann 73 499-507 (1982). M. GSCHWENDT, F. HORN, W. KITTSTEIN, G. FURSTENBERGER, E. BESEMFELDER, and F. MARKS, Biochem. Biophys. Res. Commun. 124 63-68 (1984). K. KITAGAWA, H. NISHINO, and A. IWASHIMA, Oncology 43 127-130 (1986). A.V. BOGATSKII, L.Ya. GLINSKAYA and A.I. GREN, J. Gen. Chem. USSR 39 2508-2514 (1969). H. BROCKERHOFF, Lipids 17 1001-1003 (1982). V.P.S. CHAUHAN, L.S. RAMSAMMY and H. BROCKERHOFF, Biochim. Biophys. Aeta 772 239-243 (1984) H. BROCKERHOFF, S.'BROCKERHOFF and L.L. BOX, Lipids 21 405-408 (1986).
Pergamon Journals
PHENOBARBITAL COMPETES WITH DIACYLGLYCEROL FOR PROTEIN KINASE C
Ved P.S. Chauhan and Hans Brockerhoff New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314 (Received in final form September 29, 1986) Summary Phenobarbital inhibits protein kinase C of rat brain by competitively displacing the effector of the enzyme, diacylglycerol. The drug appears to occupy the triple hydrogen bonding site which bonds diaeylglycerol - and also phorbol esters - to the enzyme. It remains to be seen if the effect is responsible for the pharmaceutical activity of the drug; even so, it provides an example of a restructuring of lipid-protein hydrogen bonding, in the hydrogen belt of the membrane, in a manner postulated as a mechanism of anesthesia. The phosphorylating enzyme, protein kinase C, is attached to membranes, and activated, by diacylglycerol (DG) (1-3). Enzyme - DG bonding is achieved by triple hydrogen bonding (evidence collected in ref. 4): both H-receiving ester CO groups and the H-donating 3-OH group of DG are involved. Sterically similar H-bonding group arrangements are found in some tumor promotors, e.g., phorbol esters, which compete with DG and also activate the enzyme (5). A search for DG-displacing but deactivating compounds led us to consider phenobarbital, which has three CO groups (though no COH group) in a pattern which can be superposed on the three hydrogen-bonding oxygens of DG (Fig. I). The neuropharmacological properties of the drug together with the involvement of protein kinase C in synaptic transmission (6-9) add interest to the investigation. We find that phenobarbital is, indeed, an inhibitor of the kinase by virtue of competition with DG. Materials and Methods Chemicals: [~-32p]ATP of specific activity 3,000 Ci/mmol was from New England Nuclear, MA. All other chemicals were from Sigma. Diacylglycerol was prepared by enzymatic hydrolysis of egg phosphatidylcholine (I0). Phenobarbital was synthesized (11). Protein Kinase-C: Extracted essentially by the method of Boni and Rando (12). Rat brains were removed and washed in ice-cold extraction buffer (20 mM Tris-HCl, pH 7.5 containing 0.3M sucrose, 2 mM EDTA, 10 mM EGTA, 2 mM dithiothreitol, 2 mMphenylmethylsulfonyl fluoride, 10 ~g/ml pepstain, I0 ~g/ml soybean trypsin inhibitor, I0 ~g/ml leupeptin and 25 Bg/ml aprotinin). The brains were then homogenized with 4 volumes of the same buffer with glass Teflon pestle homogenizer and centrifuged at I00,000 X g for 1 h. The supernatant was used as the source of the enzyme.
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
90
Phenobarbital Inhibits Protein Kinase C
m
Vol. 40, No. i, 1987
mm
PB
FIG. I Space-filling models of diacylglycerol (DG) and phenobarbital (PB; oxygen atoms are slotted; the phenyl group is eclipsed in this perspective). The three oxygens of PB (on CO 2, 4, and 6) appear congruent with the I- and 2-C0 oxygens and with the 3-OH group of diacylglycerol; the hydrogen bond donating OH proton missing in PB may be supplied by the I-NH group. This is demonstrated by the O-ring which stands for a hypothetical oxo group of the enzyme which can be reached, by hydrogen bonding, from either the 3- OH of DG or the I- NH of PB. Refer (4) for further details. Enz~we Assay: Activity was assayed by measuring the incorporation of 32p from [7-~P]ATP into lysine-rich histone, type III-S (Sigma) in 20 mM Tris-HCI, pH 7.65, 0.4 mM CaClg, 0.2 mM EDTA, 1.0 mM EGTA, i mM ATP, 3.1 mM MgCI 2, 40 ~g/ml leupeptin, 800 ~g/ml histone and i00 ~M lipid. The mixture, 0.2 ml in plastic tubes, was incubated for 5 min. at 37 C. The reaction was stopped by the addition of 2 ~ trichloroacetic acid, followed by collection of the acid precipitate on a Millipore HA type filter, and counting in Filteron-X scintillation fluid (National Diagnostics. NJ). Vesicle Preparation: Phosphollplds and diacylglycerol were mixed in organic solvent which was removed by drying on a rotary evaporator at 70°C for i hr with repeated flushing with argon. Lipids were hydrated in 20 mM Tris HCI, pH 7.5 containing 2 mM dithiothreitol and 2 mM phenylmethyl sulfonylfluoride and sonicated until clear, under argon.
Vol. 40, No. i, 1987
Phenobarbital Inhibits Protein Kinase C
91
Soluble Fraction (SF): Protein kinase C was extracted in 20 mM Tris-Cl, pH 7.5 containing 0.3M sucrose, 2 mM EDTA, I0 mM EGTA and 2 mM dithiothreitol. The extracted enzyme (5 mg protein) was incubated for 5 min. at 37°C with 17 or 34 ~g of trypsin (14). The reaction was stopped by the addition of 340 ~g of trypsin inhibitor. Protein kinase C activity and the effect of phenobarbital were measured after trypsinization. Partition Coefficient: One mg/ml phenobarbital was dissolved in 20 mM Tris-HCL pH 7.65. Four ml of octanol and 4 ml of phenobarbital solution were mixed and shaken for 30 and 60 min.; optical density at 254 ran was then measured in octanol phase and buffer after centrifugation for 15 min. The partition coefficient (P) is the ratio of OD's. This value was divided by 5.0 since the partition coefficient in octanol/water has been found to be regularly 5.0 times higher than that in biological membrane/water (13).
Mole % Diacylglycerol
oL ,~
SF
i
250
i! ,°° o
-PB +PB]
F
E 150
'
•~ ~ lOO
1
E 5O o
~
0
.23
.33
0
ii
Mole % Phenobarbital/Lipid
FIG. 2 Activation of protein kinase C by diacylglycerol (DG) and inhibition by phenobarbital (PB). Results arranged in groups of equal concentrations of diacylglycerol (2, 4 and 6 mole% of total lipid in-membrane); columns are for different PB concentrations (in-membrane O, 0.23 and 0.33 mole%) which are calculated from aqueous concentration of PB times partition coefficien~ P = 0.31. Groups 1 and 3 represent 3 sets each, group 2 represents 2 sets of experiments. The PB concentrations in water are 8.75 mM and 12.5 mM, in the membrane 2.72 mM and 4.89 mM. The last two columns represent kinase activities of the soluble fraction, SF, i.e., the kinase activity (in the absence of DG, calcium, or phosphatidylserine) liberated by digestion with trypsin. PB at 12.5 mM does not influence the activity in SF. Bars represent standard deviation.
92
Phenobarbital
Inhibits Protein Kinase C
Vol. 40, No. i, 1987
Results and Discussion Figure 2 shows how the kinase is activated by diacylglycerol, then inhibited by phenobarbital. A double reciprocal plot (Fig. 3) shows that DG and PB act competitively. While other compounds have been reported to act on the active center of the kinase (15) or on the phospholipid matrix of the assay system (16), phenobarbital appears to inhibit protein kinase C by binding to the site otherwise occupied by the natural enzyme activator, diacylglycerol. This conclusion is confirmed by the behavior of the soluble fragment (SF) obtained
,~ ¢
12
0.33
rnol%
"=
~
/
~o
/ /
~_N 0.23 mot%
/'
m ,. 0.
0.5
0.4
0.3
0.2
A~//
6
0.1
0
O.1
,
•
Phenobarbital
0
n
J
i
0.2
0.3
0.4
1/s(mol%DG/lipid)
0.5 -~
FIG. 3 Double reciprocal plot of diacylglycerol concentration (mole~) against velocity (protein phosphorylation), at 0, 0.23, and 0.33 mole% phenobarbital in membrane. Slopes were calculated by linear regression analysis. Two additional sets of experiments gave similar results. The result is expected for the case of competition between PB and DG. by proteolysis of the enzyme (17). SF has full kinase activity but does no longer depend on diacylglycerol, nor is it inhibited by PB (Fig. 2). There are other compounds known which bind to the DG-receiving site of the kinase; in particular, tumor promotors such as phorbol esters or aplysiatoxin (18,19); also possibly some others (20,21). The tumor promotors possess the same arrangement of two hydrogen bond accepting oxo groups and one donating hydroxyl group (4). In PB, the hydroxyl group is missing (there is no evidence for an enol tautomer at physiological pH) (22); however, there are two potentially donating NH groups in barbital-I and -3 positions. It can be shown in a molecular model (Fig. I) that, when two oxo atoms each of DG and PB are superposed, it is possible for the 3-OH proton of DG as well as for the I-NH proton of PB to contact - with hydrogen bonding - the same hypothetical 0 atom of the DG-receiving site of the enzyme. In Fig. l, this 0 atom is shown as a ring. The triple hydrogen bonding between effectors (or inhibitor) can thus be realized with DG, tumor promotors, or PB. In the inhibitor, however, the angle of incidence of the hydrogen bonds would be different. A resulting change in enzyme conformation might explain the inhibition. Alternatively, there may be improper reconstruction of the DG-bonding site because of supernumerary hydrogen bonding (PB has five against DG's three bonding sites).
Vol. 40, No. i, 1987
Phenobarbital Inhibits Protein Kinase C
93
Is the inhibition of the kinase by PB related to the anticonvulsive and hypnotic properties of the drug? That remains unproven, but not unlikely. Favorable evidence is supplied by reports on the involvement of the enzyme in nerve conductance (6-9); also, the inhibitor acts in a range (3-6 mM in membrane) where anesthetics are active (13). In any case, our results furnish an example for the theory of anesthesia which holds that the drugs act by restructuring the "hydrogen belts", i.e., those regions of the neuronal plasma membrane that contain the hydrogen bonding lipid and protein CO and OH groups (23-25). Acknowledgement The study was supported by NIH grant GM21875 . Brockerhoff assisted in the experimental work.
J. Zingoni and S.
References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Y. TAKAI, A. KISHIMOTO, U. UIKKAWA, T. MORI and Y. NISHIZUKA, Biochem. Biophys. Res, Commun. 91 1218-1224 (1979). N.A. SHARKEY, K.L. LEACH, P.M. BLUMBERG, Proc. Natl. Acad. Sci. USA 81 607-610 (1984). B.R. GANONG, C.R. LOOMIS, Y.A. HANNUN and R.M. BELL, Proc. Natl. Acad. Sci. USA 83 1184-1188 (1986). H. BROCKERHOFF, FEBS Lett. 201 1-4 (1986). M. CASTAGNA, Y. TAKAI, K. KAIBUCHI, K. SANO, U. KIKKAWA and Y. NISHIZUKAS, J. Biol. Chem. 257 7847-7851 (1982). K.M.M. MURPHY, R.J. GOULD, M.L. OSTER-GRANITE, J.D. GEARHART and S.H. SNYDER, Science 222 1036-1038 (1983). S.A. DeRIEMER, P. GREENGARD and L.K. KACZMAREK, J. Neurosci. 5 2672-2676 (1985). S.A. DeRIEMER, J.A. STRONG, K.A. ALBERT, P. GREENGARD and L.K. KATZMAREK, Nature 313 313-316 (1985). D.V. MADISON, R.C. MALENKA and R.A. NICOLL, Nature 321 695-697 (1986). L.D. BERGELSON, Lipid Biochemical Preparations, p. 118-119, Elsevier, Amsterdam (1980). B.S. FUMISS, A.J. HANNAFORD, V. ROGERS, P.W.G. SMITH and A.R. TATCHELL, Vogel's Textbook of Practical Organic Chemistry, 4th ed., p. 907-909, Longman, London (1978) . L.T. BONI and R.R. RAN-DO, J, Biol. Chem. 260 10819-10825 (1985). P. SEEMAN, Pharmacol. Rev. 24 583-655 (1972). A. KISHIMOTO, N. KAJIKAWA, M. SHIOTA and Y. NISHIZUKA, J. Biol. Chem. 258 1156-1164 (1983). S. KAWAMOTO and H. HIDAKA, Biochem. Biophys. Res. Commun. 125 258-264 (1984). T. MORI, Y. TAKAI, R. MINAKUCHI, B. YU and Y. NISHIZUKA, J. Biol. Chem. 255 8378-8380 (1980). M. INOUE, A. KISHIMOTO, Y. TAKAI and Y. NISHIZUKA, J. Biol. Chem. 252 7610-7616 (1977). U. KIKKAWA, Y. TAKAI, Y. TANAKA, R. MIYAKE and Y. NISHIZUKA, J. Biol. Chem. 258 1142-1145 (1983). T. SUGIMURA, Gann 73 499-507 (1982). M. GSCHWENDT, F. HORN, W. KITTSTEIN, G. FURSTENBERGER, E. BESEMFELDER, and F. MARKS, Biochem. Biophys. Res. Commun. 124 63-68 (1984). K. KITAGAWA, H. NISHINO, and A. IWASHIMA, Oncology 43 127-130 (1986). A.V. BOGATSKII, L.Ya. GLINSKAYA and A.I. GREN, J. Gen. Chem. USSR 39 2508-2514 (1969). H. BROCKERHOFF, Lipids 17 1001-1003 (1982). V.P.S. CHAUHAN, L.S. RAMSAMMY and H. BROCKERHOFF, Biochim. Biophys. Aeta 772 239-243 (1984) H. BROCKERHOFF, S.'BROCKERHOFF and L.L. BOX, Lipids 21 405-408 (1986).