Rat brain synaptosomes contain enzymes, phosphatidylethanolamine N-methyltransferase(s) (EC 2.1.1.17), that catalyze the methylation of endogenous phosphatidylethanolamine to form its mono-, di-, and trimethyl (i.e., phosphatidylcholine) derivatives. We observe that the activity of these enzymes is [hepatocytes (16)]. This process is rapid and not dependent on cAMP synthesis. Little information has been available on the control of PtdEtnMeTase activity within brain neurons. We report here that catecholamine neurotransmitters, especially dopamine, enhance the activities of phosph6lipid methylating enzymes in rat brain synaptosomes. METHODSPreparation of Subcellular Fractions. Male Sprague-Dawley rats (200-300 g; Charles River Breeding Laboratories) were housed under a 12-hr/12-hr light/dark schedule (lights on, 0900-2100 hr) and had free access to water and food (Charles River chow; 22% protein, 0.018% choline). The animals were decapitated at 1000 hr, and the brains were quickly dissected on ice and homogenized in 10 vol of 0.32 M sucrose in a Potter-Elvehjem homogenizer; the homogenate was then centrifuged at 1,000 x g for 15 min to remove tissue debris and nuclei. The supernatant was layered on top of 4 ml of 1.2 M sucrose and centrifuged at 165,000 X -g for 15 min in a Beckman SW 41 Ti rotor. The resulting pellet contained mitochondria; the material at the interface of the gradient (containing the synaptosomes and myelin) was diluted to obtain approximately 0.32 M sucrose, layered on top of 4 ml of 0.8 M sucrose, and centrifuged at 165,000 X g for 15 min (17). The material at the interface, containing myelin, and the pellet, shown to contain synaptosomes by electron microscopy, were collected. In some cases, the postnuclear supernatant was centrifuged at 20,000 x g for 20 min to obtain the P2 pellet (containing synaptosomes, mitochondria, and myelin). The post-P2 supernatant was centrifuged at 100,000 X g for 60 min to obtain a microsomal pellet.PtdEtnMeTase Assay. PtdEtnMeTase activity was assayed by a modification of the method described previously (4). The standard assay medium was changed to 50 mM Tris HCl, pH 7.5/5 mM MgCl2/0.2 mM EDTA/0. 1 mM ATP and tissue preparation (0.2-0.4 mg of protein) in a final volume of 120 1.l. The effect on phospholipid [3Himethyl incorporation of omitting various components of this medium was examined (see Table 1). Norepinephrine-HCI (Sigma), dopamineHCl (Sigma), haloperidol lactate (5 mg/ml, containing 1.8 mg of methylparaben and 0.2 mg of propylparaben; a gift from McNeil, Fort Washington, PA), were added to some tubes before incubation. Reactions were started by addition of 2.5 jCi of S-adenosyl[methyl-3H]methionine (New England Nuclear; 13-15 Ci/mmol; 1 Ci = 37 GBq) to a final AdoMet concentration of 1.2-1.6 ,uM. Samples were incubated for 30 min at 37TC. Reactions were stopped by addition of 3 ml of chloroform/methanol/HCI, 100:50:1 (vol/vol). Water-soluble radioactive. materials were extracted by washing the incubation mixture with two 2-ml portions of 0....

Brain serotonin (5HT) metabolism in rats at weaning was examined in relation to their maternal dietary protein (PC) and tryptophan (TRP) concentration and to their protein feeding behavior. Pup brain TRP, 5HT, and 5-hydroxyindoleaceticacid (5HIAA) concentration was unaffected by maternal diet PC (10, 20, or 40%) but was increased by TRP additions (0.66 or 1.86%) to 40% casein diets. Turnover of brain 5HT, measured at weaning after probenecid injection, was inversely related to maternal diet PC (r = .54, P less than 0.05) but was not affected by TRP supplementation. Pups allowed to select from 10 and 60% casein diets selected during the first 3 days post-weaning, dietary PC which reflected the PC (r = 0.65, P less than 0.01), but not RRP content of their maternal diet, and also reflected brain 5HT turnover (r = .50, P less than 0.05), but not concentration, of their littermates. Thus, the relationship dietary PC to protein selection by the offspring is associated with maternal diet-maternal diet-induced changes in their brain 5HT turnover, at least as measured after probenecid administration.

Weanling rats fed a choice of two diets varying only in protein concentration (10 and 60% casein) select a lower proportion of dietary energy as protein when the dietary minerals are supplied by a mineral mixture high in Na, K, and Cl but low in P and Cu (Hi Cl) than when the diets contain the Bernhart-Tomarelli (BT) mineral mixture. The addition of Na and/or K as acetate salts to the BT mineral mixture, to levels in the Hi Cl mixture, had no effect on regulation of protein intake. However, the addition of Na and/or K as chloride salts to the BT mineral mixture resulted in the rats selecting a protein concentration equivalent to the Hi Cl fed group. Weight gains and energy consumption were similar for all groups. It was therefore concluded that dietary chloride affects protein feeding behavior.

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