F.B.St.C. Palmer

Differences in the metabolism of inositol and phosphoinositides by cultured cells of neuronal and glial origin

Phosphoinositide and inositol metabolism was compared in glioma (C6), neuroblastoma (N1E-115) and neuroblastoma X glioma hybrid (NG 108-15) cells. All cell lines had similar proportions of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Neuroblastoma and hybrid cells had almost identical phospholipid and phosphoinositide compositions and similar activities for the enzymes metabolizing polyphosphoinositides (PI kinase, PIP phosphatase, PIP kinase, PIP2 phosphatase, PIP2 phosphodiesterase). Glioma cells differed by having greater proportions of ethanolamine plasmalogen and sphingomyelin, lower PIP kinase, 3-5-fold higher PIP phosphatase activity and 10-15-fold greater PIP2 phosphodiesterase activity. Higher PIP phosphatase and PIP2 diesterase activities appear to be characteristic of cells of glial origin, since similar activities were found in primary cultures of astroglia. Glioma cells also metabolize inositol differently. In pulse and pulse-chase experiments, glioma cells transported inositol into a much larger water-soluble intracellular pool and maintained a concentration gradient 30-times greater than neuroblastoma cells. Label in intracellular inositol was less than in phosphoinositides in neuroblastoma and exchanged rapidly with extracellular inositol. In glioma, labeling of intracellular inositol greatly exceeded that of phosphoinositides. As a consequence, radioactivity in prelabeled phosphoinositides could not be effectively chased from glioma cells by excess unlabeled inositol. Such differences between cells of neuronal and glial origin suggest different and possibly supportive roles for these two cell types in maintaining functions regulated through phosphoinositide-linked signalling systems in the central nervous system.

PHOSPHOINOSITIDE KINASES IN CHICK BRAIN AND SCIATIC NERVE, A DEVELOPMENTAL STUDY

Phosphatidylinositol kinase and diphosphoinositide kinase activities were measured in homogenates of brain and sciatic nerve of developing chick embryos and chicks. Characteristics of the chick nervous system enzymes were similar to those reported for rat brain. Diphosphoinositide kinase was inhibited by high concentrations of ATP and by low concentrations of triphosphoinositide. Both activities were greatly enhanced by the non‐ionic detergent, Cutscum, and the ratio of detergent to protein in the reaction mixture was important. Optimum phosphatidylinositol kinase activity required a ratio of 7 : 1 for both tissues. The optimum ratio for diphosphoinositide kinase was 3:1 for nerve homogenates and 0.6:1 for brain. Cutscum increased the concentration of diphosphoinositide that is required for maximum diphosphoinositide kinase activity.

Phosphatidylcholine biosynthesis in cultured glioma cells: evidence for channeling of intermediates

The major pathway of choline (Cho) incorporation into phosphatidylcholine (PtdCho) in mammalian cells is sequential conversion of Cho to phosphocholine (PCho), cytidinediphosphate choline (CDP-Cho) and PtdCho. In intact cells, this sequence is usually demonstrated using radiolabeled Cho since PCho and CDP-Cho do not enter the cell intact. We have studied the incorporation of radiolabeled Cho, PCho and CDP-Cho into rat glioma (C6) cells following electropermeabilization. C6 cells were permeable as judged by [U-14C]sucrose and Erythrosin B uptake and more rapid incorporation of [1,2,3-3H]glycerol into cell lipids, and viable as assessed by uptake and incorporation of [methyl-3H]Cho, [1-14C]oleate and [1,2,3-3H]glycerol into complex lipids. Despite rapid incorporation of [methyl-3H]Cho into PtdCho in permeabilized cells, there was no incorporation of [methyl-14C]PCho or CDP-[methyl-14C]Cho into PtdCho. PCho (300 microM) and CDP-Cho (300 microM) failed to significantly reduce incorporation of 28 microM [methyl-3H]Cho into PtdCho. Radioactivity in PtdCho of cells prelabeled with [methyl-3H]Cho prior to permeabilization could be chased with 4 mM Cho but not with 4 mM PCho or 4 mM CDP-Cho. The water-soluble products of Cho metabolism--PCho, CDP-Cho and glycerophosphocholine--were retained at 37 degrees C in permeabilized cells compared with controls while there was uniform leakage from permeabilized cells at 4 degrees C. Hemicholinium-3, an inhibitor of high-affinity Cho transport, decreased [methyl-3H]Cho incorporation into PtdCho in permeabilized cells, as in controls, suggesting that even in permeabilized cells, Cho incorporation into PtdCho is linked to the transport system. We propose that individual steps of the cytidine pathway of PtdCho biosynthesis are functionally linked and that reaction intermediates are not freely diffusible within the cell but are channeled to PtdCho biosynthesis.

DHPOSITION OF LIPIDS IN THE DEVELOPING CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF THE CHICKEN

Abstract— The lipid composition of chick brain and sciatic nerve was determined during development. It was confirmed that the addition of CaCl2 to solvents during the extraction of lipids from brain results in much higher yields of diphosphoinositides particularly from unmyelinated embryo brain. Unlike the earlier report for rat brain, the recovery of triphosphoinositides was also Substantially increased. The amount of CaCl2, required to achieve optimal recoveries decreased with increasing age and addition of more than this optimal amount depressed the yields of polyphosphoinositides, particularly triphosphoinositides. CaCl2, addition did not improve the yield of diphosphoinositides from sciatic nerve of any age but drastically reduced recovery of triphosphoinositidcs. Differenccs in the effect of CaCl2 were not the result of variation in the tissue concentrations of calcium or magnesium.

The extraction of acidic phospholipids in organic solvent mixtures containing water

Abstract The observation that phosphatidyl inositol is incompletely extracted from liver tissue by chloroform-methanol mixtures containing a large proportion of water was confirmed. However, different metabolic turnover rates were not observed for the extractable and unextractable phosphatidyl inositol. It was then shown that acidic phospholipids are completely solubilized when the tissue is homogenized in chloroform-methanol but are readsorbed onto the insoluble tissue proteins if sufficient water is added to result in a two-phase system. This effect was observed in a synthetic system with both acidic and basic proteins and was found to be suppressed by low concentrations of divalent cations. Higher cation concentrations were required to suppress the readsorption of phosphatidyl inositol than the other acidic phospholipids. When tissue was extracted in this way the concentration of divalent cations originating from the tissue was adequate to suppress the readsorption of all acidic phospholipids except phosphatidyl inositol.

Fatty acid composition of human plasma lipoprotein phosphatidylinositols

Phosphatidylinositol (PI) was the only anionic phospholipid found consistently in human plasma lipoproteins. High density lipoproteins (HDL) contained a greater proportion of PI (2.6% of the phospholipids) than did either the low density (LDL) or the very low density (VLDL) lipoproteins (1.6 and 1.7%, respectively). Lipoprotein PI was enriched in stearic and arachidonic acids when compared to phosphatidylcholines from the same fractions. The fatty acid composition of the lipoprotein PI, although the same in all lipoprotein fractions, had less arachidonic acid than platelet PI and less palmitic acid than erythrocyte PI. The data suggest that significant exchange of PI between lipoproteins and cell membranes is not likely, whereas PI exchange among the different classes of lipoproteins is possible.

Lipids of Crithidia fasciculata: The occurrence and turnover of phosphoinositides

Abstract The lipids of Crithidia fasciculata have been analysed. In addition to phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine both diphosphoinositide and triphosphoinositide were identified. Plasmalogens, lysophosphatides, phosphatidylserine and phosphonolipids were absent. Experiments with 32 P i as precursor indicated that only the phosphoinositides were formed at a rate appreciably greater than that necessary to sustain new growth. The turnover of the polyphosphoinositides was very rapid and they were quantitatively decreased in cells at the end of the logarithmic phase of growth.

Measurement of 32P following colorimetric phosphorus analysis

Abstract A procedure for the determination of both 32P and phosphate contents on the same sample has been described. Following phosphate estimation by the method of Bartlett (5), the solution containing the blue reduced phosphomolybdate complex is decolorized at high pH. 32P is measured in this transparent aqueous solution with a liquid scintillation spectrometer, without the addition of scintillators, by utilizing the Cerenkov radiation.

Clofibrate and other peroxisomal proliferating agents relatively specifically inhibit synthesis of ethanolamine phosphoglycerides in cultured human fibroblasts.

Effects of several classes of peroxisomal proliferators on peroxisomal functions, hepatomegaly, hepatocarcinogenesis and lipid metabolism have been extensively investigated in rodents. Less is known about influences of these agents, some used as hypolipidemic drugs, on various metabolic parameters in humans. We examined effects of clofibrate, di(2-ethyl-hexyl)phthalate (DEHP) and pirinixic acid (WY-14,643) on phospholipid metabolism in human fibroblasts in culture. Clofibrate inhibited incorporation of [1-14C]hexadecanol and [1-14C]linolenic acid into ethanolamine phosphoglycerides in a time- and concentration-dependent manner; labeling of plasmalogens and non-plasmalogen ethanolamine phosphoglycerides was reduced by 40-80% compared to a generalized 10-30% inhibition of labeling of other phospholipids, including phosphatidylcholine. In pulse and pulse-chase experiments, selective inhibition of incorporation of [1,2-14C]ethanolamine, compared to [methyl-3H]choline, confirmed relative specificity of inhibition of ethanolamine phosphoglycerides. Similar concentration dependence and specificity for inhibition of phospholipid turnover was observed for DEHP and WY-14,643, in both control and mutant (Zellweger and adrenoleukodystrophy) fibroblasts, in the absence of major effects on peroxisomal markers. These observations that peroxisomal proliferators specifically inhibit ethanolamine phosphoglyceride turnover in human fibroblasts should be considered when assessing the efficacy and safety of such agents as hypolipidemic drugs or when evaluating mechanisms of proliferator action at the cellular level.

Biosynthesis of phosphatidylinositol in Crithidia fasciculata.

Microsomal preparations from the protozoan (Crithidia fasciculata were shown to incorporate myo-[2-3H]inositol into phosphatidylinositol by both the CDPdiacylglycerol:myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg2+ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg2+, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 micro M) both stimulated the activity 3-fold and reduced the Mg2+ required for saturation to 15-20 mM. EGTA also increased the apparent Km for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca2+ (50 micro M). This and the EGTA effect suggest a possible role for Ca2+ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn2+, was insensitive to Ca2+ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50 degrees C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme.