Lecithin) Biosynthesis in Rat Brain Neurons (Phospholipid/N-Methylation/Synaptosomes/Catecholamines/Norepinephrine) CAROL E
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Proc. Natl Acad. Sci. USA Vol. 80, pp. 2063-2066, April 1983 Neurobiology Dopamine stimulation of phosphatidylcholine (lecithin) biosynthesis in rat brain neurons (phospholipid/N-methylation/synaptosomes/catecholamines/norepinephrine) CAROL E. LEPROHON, JAN K. BLUSZTAJN, AND RICHARD J. WURTMAN* Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Communicated by Julius Axelrod, October 18, 1982 ABSTRACT Rat brain synaptosomes contain enzymes, phos- nuclei. The supernatant was layered on top of 4 ml of 1.2 M phatidylethanolamine N-methyltransferase(s) (EC 2.1.1.17), that sucrose and centrifuged at 165,000 X -g for 15 min in a Beck- catalyze the methylation of endogenous phosphatidylethanol- man SW 41 Ti rotor. The resulting pellet contained mito- amine to form its mono-, di-, and trimethyl (i.e., phosphatidyl- chondria; the material at the interface of the gradient (con- choline) derivatives. We observe that the activity of these en- taining the synaptosomes and myelin) was diluted to obtain zymes is enhanced when synaptosomes are incubated with cat- approximately 0.32 M sucrose, layered on top of 4 ml of 0.8 echolamines: 0.1 mM dopamine increases incorporation of [3H]- M sucrose, and centrifuged at 165,000 X g for 15 min (17). methyl groups into monomethylphosphatidylethanolamine, di- The material at the interface, containing myelin, and the pel- methylphosphatidylethanolamine, and phosphatidylcholine by let, shown to contain synaptosomes by electron factors of 1.7, 1.3, and 2.1, respectively, and 0.1 mM norepi- microscopy, nephrine increases [3H]methyl incorporation into monomethyl- were collected. In some cases, the postnuclear supernatant phosphatidylethanolamine and dimethylphosphatidylethanol- was centrifuged at 20,000 x g for 20 min to obtain the P2 pel- amine by factors of 1.6 and 2.1, respectively. Stimulation by let (containing synaptosomes, mitochondria, and myelin). The dopamine, which is observed at concentrations as low as 1 ,IM, post-P2 supernatant was centrifuged at 100,000 X g for 60 min is blocked by haloperidol. to obtain a microsomal pellet. PtdEtnMeTase Assay. PtdEtnMeTase activity was assayed Synaptosomal membranes from rat (1-3) and bovine (4, 5) brain by a modification of the method described previously (4). The contain an enzyme, or enzymes, phosphatidylethanolamine N- standard assay medium was changed to 50 mM Tris HCl, pH methyltransferase(s) (PtdEtnMeTase; EC 2.1.1.17) that en- 7.5/5 mM MgCl2/0.2 mM EDTA/0. 1 mM ATP and tissue ables them to synthesize phosphatidylcholine (PtdCho) by the preparation (0.2-0.4 mg of protein) in a final volume of 120 stepwise methylation of phosphatidylethanolamine (PtdEtn), 1.l. The effect on phospholipid [3Himethyl incorporation of using S-adenosylmethionine (AdoMet) as the methyl donor. omitting various components of this medium was examined Part of this newly synthesized PtdCho is hydrolyzed to lib- (see Table 1). Norepinephrine-HCI (Sigma), dopamineHCl erate free choline (1), which might be available for acetyl- (Sigma), haloperidol lactate (5 mg/ml, containing 1.8 mg of choline synthesis; part may be retained as a constituent of cel- methylparaben and 0.2 mg of propylparaben; a gift from lular membranes. PtdEtnMeTases in various tissues have been McNeil, Fort Washington, PA), were added to some tubes shown to be activated by such receptor agonists as catechol- before incubation. Reactions were started by addition of 2.5 amines [rat reticulocytes (6, 7), C6 glioma astrocytoma (8), jCi of S-adenosyl[methyl-3H]methionine (New England Nu- lymphocytes (9)], lectins [lymphocytes (10), mast cells (11)], clear; 13-15 Ci/mmol; 1 Ci = 37 GBq) to a final AdoMet con- immunoglobulins [mast cells (12), leukemic basophils (13)], centration of 1.2-1.6 ,uM. Samples were incubated for 30 min glucagon [hepatocytes (14)]., vasopressin [pituitary extracts (15), at 37TC. Reactions were stopped by addition of 3 ml of chlo- hepatocytes (16)], and angiotensin [hepatocytes (16)]. This roform/methanol/HCI, 100:50:1 (vol/vol). Water-soluble ra- process is rapid and not dependent on cAMP synthesis. Little dioactive. materials were extracted by washing the incubation information has been available on the control of PtdEtn- mixture with two 2-ml portions of 0.75% KCI in 50% meth- MeTase activity within brain neurons. We report here that anol. catecholamine neurotransmitters, especially dopamine, en- The chloroform phase was taken to dryness in a Savant Speed- hance the activities of phosph6lipid methylating enzymes in Vac lyophilizing centrifuge, and the [3H]phospholipids were rat brain synaptosomes. suspended in 50 p.1 of chloroform/methanol, 1:1 (vol/vol). The [3H]phospholipids, together with phosphatidylmono- METHODS methylethanolamine (PtdMeEtn), phosphatidyldimethyletha- Preparation of Subcellular Fractions. Male Sprague-Daw- nolamine (PtdMe2Etn), and PtdCho standards, were applied ley rats (200-300 g; Charles River Breeding Laboratories) were to silica gel TLC plates (Silica gel G; Absorbosil Plus, Applied housed under a 12-hr/12-hr light/dark schedule (lights on, Science Division, State College, PA) and the chromatogram 0900-2100 hr) and had free access to water and food (Charles was developed in chloroform/propionic acid/n-propanol/water, River chow; 22% protein, 0.018% choline). The animals were 2:2:3:1 (vol/vol) (4). Compounds were visualized with di- decapitated at 1000 hr, and the brains were quickly dissected on ice and homogenized in 10 vol of 0.32 M sucrose in a Pot- Abbreviations: PtdCho, phosphatidylcholine; PtdEtn, phosphatidyl- ter-Elvehjem homogenizer; the homogenate was then cen- ethanolamine; PtdEtnMeTase, phosphatidylethanolamine N-methyl- transferase; PtdMe2Etn, phosphatidyldimethylethanolamine; Ptd- trifuged at 1,000 x g for 15 min to remove tissue debris and MeEtn, phosphatidylmonomethylethanolamine; AdoMet, S-adenosyl- methionine; Bt2cAMP, N6,02-dibutyryladenosine 3',5'-cyclic mono- The publication costs of this article were defrayed in part by page charge phosphate. payment. This article must therefore be hereby marked "advertise- *To whom reprint requests should be addressed at: Room E25-604, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. M.I.T., Cambridge, MA 02139. 2063 Downloaded by guest on October 2, 2021 2064 Neurobiology: Leprohon et al. Proc. Natl. Acad. Sci. USA 80 (1983) phenylhexatriene (10 mg/100 ml of petroleum ether; ref. 18) PtdEtnMeTase activity by dopamine (Table 1); tubes were in- and the silica was then scraped into scintillation vials. The newly cubated with or without dopamine, Mg2+, ATP, or phospho- formed [3H]phospholipids were eluted with 1 ml of methanol, lipid substrates. In most situations, dopamine markedly en- 15 ml of Betafluor (National Diagnostics, Somerville, NJ) was hanced the incorporation of [3H]methyl into PtdMeEtn, added to each vial, and the radioactivity of the samples was PtdMe2Etn, and PtdCho. Mg2+ alone increased both basal and determined. Efficiency of assay for 3H was =50%. Recovery dopamine-stimulated [3H]phospholipid accumulation. ATP, of [3H]phospholipids was =70%. added alone or with Mg2+, lacked a consistent effect on [3H]- To ensure that the methods used for assessing [31H]methyl phospholipid synthesis; in some experiments the combination incorporation correctly identified the various [3H]phospholip- of ATP and Mg2+ enhanced basal Ptd[3H]MeEtn and Ptd[3H]- id fractions, we analyzed both the fractions themselves (from Me2Etn syntheses while in others it did not. Addition of phos- basal and catecholamine-stimulated samples) and their [3H]base pholipid substrates tended to enhance the accumulation of all contents after hydrolysis. The [3H]phospholipids were sepa- three [3H]phospholipids, both in the presence and in the ab- rated by two-dimensional TLC using chloroform/methanol/ sence of dopamine. Since the phospholipids added did not acetic acid, 65:30:5 (vol/vol) in the first dimension and chlo- include PtdEtn, the enhanced accumulation of Ptd[3H]MeEtn roform/methanol/2-propanol/0.25% KCI/triethylamine, 30: probably reflected a slowing in its further metabolism. The 9:25:6:18 (vol/vol) (19) in the second dimension; they were addition of phospholipid substrates to the incubation mixture then scraped from the TLC plate and their radioactivities were did not further enhance determined. Although the triethylamine system results in bet- the effect of dopamine on Ptd- ter separation of the [3H]phospholipids than the propionic acid EtnMeTase. In subsequent experiments, we assayed Ptd- system, the dopamine metabolites homovanillic acid and 4-hy- EtnMeTase activity in the presence of Mg2+ and ATP and droxy-3-methoxyphenylethanol were found to comigrate with without PtdMeEtn and PtdMe2Etn to maximize the dopamine PtdMeEtn and PtdMe2Etn, respectively. Hence, triethylamine effect. was not the TLC system of choice for unidirectional chroma- Both dopamine and norepinephrine stimulated PtdEtn tography. Both these dopamine metabolites migrate with the methylation in a dose-dependent fashion (Table 2). The effects solvent front in the propionic acid system. The bases hydrolyzed of dopamine were detectable at concentrations as low as 1 uM from the [3H]phospholipids (by incubation of phospholipid frac- and, in most studies, could be seen with PtdMeEtn, Ptd- tions, separated by routine TLC, in 6 M HCl at 80'C for 1 hr) Me2Etn, and PtdCho; norepinephrine required higher (10 ,uM) were separated by HPLC on a