Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells.

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.

Endogenous inhibitors of human choline acetyltransferase present in Alzheimer's brain: preliminary observation.

We have previously demonstrated the presence in Alzheimer's disease brain of an endogenous inhibitor of choline acetyltransferase activity. Selected properties of these compounds were investigated. There appear to be two distinct classes of inhibitor present, both phosphomonoesters and nonphosphorylated substances. They are not proteins, pass through 500 mm dialyses membranes and are not lipoidal. There are both different sensitivities of individual control cytosotic activity to inhibition and differences in intrinsic inhibitory activity present in individual Alzheimer's disease brain samples. There is a competitive type of inhibition with respect to acetyl CoA as substrate and a noncompetitive type with respect to choline as substrate.

Developmental changes and regional distribution of phospholipase D and base exchange enzyme activities in rat brain.

Phospholipase D (PL-D) activity per mg protein of whole homogenate increased 5.1 fold between Embryonic (E) day 17 and Postpartum (P) day 14 and slightly decreased by P 30 days. This was due to the decrease of PL-D activity of the P2 fraction. The PL-D activity of P2 and P3 fractions increased 11.2 and 6.1 fold respectively between E 17 and P 14. The 3 base exchange enzyme (BEE) activities per mg protein of whole homogenate increased up to P 14 or P 21 and then decreased. This decrease was greater in the P2 fraction and the P3 fraction increased after P 14. Brains from 1 day to 25 month old rats were dissected into 7 separate regions and both PL-D and BEE activities were measured. In adult rats, the hippocampus and hypothalamus had the highest PL-D activities while medulla + pons and cerebellum had the lowest PL-D activities. The developmental patterns of 5 regions except for hippocampus and hypothalamus were similar. PL-D activity in the hippocampus was maximum at P 7 followed by a steep decrease till P 30 suggesting that the PL-D activity of the hypothalamus develops later and that of the hippocampus develops earlier than any other region. The distributions of BEE activities were quite different from those of PL-D activities. In adult rats, the cerebellum had the highest activity while the striatum and medulla + pons had the lowest. The BEE activities of cerebellum were lowest at P 1 and showed steep increase during the next 2 weeks.

Phosphatase and phospholipase activity in Alzheimer brain tissues.

There is a reduction in the ability of Alzheimer brain tissue homogenates to release choline from both lecithin and phosphorylcholine. This suggests that dietary treatment with choline or lecithin supplements will not be useful.

Reduced phosphorylcholine hydrolysis by homogenates of temporal regions of Alzheimer's brain.

The hydrolysis of p-nitrophenylphosphorylcholine, p-nitrophenylphosphate, and phosphorylcholine was quantitated in homogenates prepared from the occipital and temporal regions of control and Alzheimer's Disease patient brain. There were no significant differences detected in activities of enzymes cleaving the first 2 compounds. A 78% reduction in the capacity for hydrolyzing phosphorylcholine by temporal homogenates from Alzheimer's patients compared to controls was demonstrated. The hydrolysis of this substrate by occipital homogenates was identical to control values. These results may explain the lack of cognitive improvement of Alzheimer's patients receiving dietary supplements of either choline or lecithin.

The role of endogenous phosphatidylcholine and ceramide in the biosynthesis of sphingomyelin in mouse fibroblasts.

The intracellular location of sphingomyelin formation via the cholinephosphotransferase reaction from both endogenous an exogenous phosphatidylcholine and ceramide substrates has been studied in the subcellular membrane fractions prepared from mouse fibroblasts. The enzyme was found to be located in both the plasma membrane and the Golgi fractions. Activity in the Golgi fraction was stimulated to a greater extent by the addition of exogenous ceramide than was the activity in the plasma membrane fraction. It is concluded that endogenous phosphatidylcholine is available to the cholinephosphotransferase at saturating concentration and, therefore, is not rate-limiting. In contrast, the very low concentration of endogenous ceramide seems to limit the reaction rate, necessitating supplementation with exogenous material Both endogenous substrates are shown to be utilized in an intramembranous rather than an intermembranous reaction. The capacity of the plasma membrane fraction to synthesize sphingomyelin from endogenous phosphatidylcholine and ceramide was found to be sufficiently high to account for the rate of net synthesis of plasma membrane-bound sphingomyelin observed in the logarithmically multiplying cell culture. In contrast, the Golgi fraction displayed only 26% of the expected capacity, but it was stimulated 6-fold by the addition of exogenous ceramide. These results demonstrate that the total cellular sphingomyelin of the mouse fibroblasts can be provided via the cholinephosphotransferase pathways and that the plasma membrane and the Golgi fraction are most probably the intracellular sites of sphingomyelin biosynthesis.