Biomedical subjects

F B Palmer

Publications and source records attributed to F B Palmer.

At least 37 records · Page 2Linked to original sources

PubMed1992

Cultured fibroblasts from patients with Niemann-Pick disease type C and type D exhibit distinct defects in cholesterol esterification.

The Niemann-Pick group of diseases can be broadly classified into two types based on clinical and biochemical characteristics. Type I is characterized by a primary deficiency of lysosomal sphingomyelinase while Type II may have a defect in the regulation of intracellular cholesterol metabolism. We have studied cholesterol esterification in cultured fibroblasts from patients with two phenotypes of Type II disease: an Acadian population of southwestern Nova Scotia (Canada) with a form of the disease known as Niemann-Pick type D (NPD) and a group of panethnic origin with Niemann-Pick type C (NPC). Addition of whole serum to normal fibroblasts grown initially in lipoprotein-deficient serum caused a rapid (within 6 h) increase in cholesterol esterification, reaching maximum values at around 24 h, while NPC fibroblasts showed little increase (less than 10% of normal). In contrast, cholesterol esterification in NPD fibroblasts increased slowly during the first 6-12 h and reached 50% of normal values by 24 h. 25-Hydroxycholesterol, a non-lipoprotein stimulator of cholesterol esterification, caused a similar stimulation of cholesterol esterification in NPC, NPD and normal cells. This was inhibited by addition of serum in mutant but not in normal cells. Within 24 h of serum addition, free cholesterol accumulated in all cell types with NPC greater than NPD greater than normal. These observations indicate that (a) regulation of cholesterol esterification in response to serum lipoproteins (but not 25-hydroxycholesterol) is abnormal in both NPC and NPD fibroblasts, and (b) the biochemical phenotypes of fibroblasts from NPC and NPD patients are distinct.

Cells, Cultured↗

PubMed1992

Niemann-Pick type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis.

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (greater than 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [3H]oleic acid into cholesterol-[3H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimulated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquine abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.

Cell Line↗

PubMed1991

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis by cytochalasin B in cultured glioma cells: potential regulation of biosynthesis by Ca(2+)-dependent mechanisms.

The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.

Animals↗

PubMed1991

Channeling of intermediates in the CDP-choline pathway of phosphatidylcholine biosynthesis in cultured glioma cells is dependent on intracellular Ca2+.

The major route of phosphatidylcholine (Ptd-choline) biosynthesis in mammalian cells is the CDP-choline pathway which involves stepwise conversion of choline to phosphocholine (P-choline), cytidine diphosphate choline (CDP-choline), and Ptd-choline. Our previous studies with electropermeabilized (EP) rat glioma (C6) cells have indicated that the intermediates of this pathway are not freely diffusible in the cell but are channeled toward synthesis of Ptd-choline (George, T.P., Morash, S.C., Cook, H.W., Byers, D.M., Palmer, F. B. St.C., and Spence, M.W. (1989) Biochim. Biophys. Acta 1004, 283-291). In this study, Ca(2+)-[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers were used to investigate the role of intracellular free Ca2+ levels in functional organization of this pathway in EP glioma cells. In EP cells reduction of free Ca2+ in the medium from 1.8 mM to less than 200 nM resulted in 2-3-fold stimulation of exogenous [3H]choline and [14C]P-choline incorporation into Ptd-choline whereas incorporation of exogenous CDP-[14C]choline was augmented 100-fold; there was no uptake or incorporation of labeled P-choline or CDP-choline in intact cells. In EP cells incubated at 1.8 mM Ca2+ the water-soluble products of choline metabolism (choline, P-choline, CDP-choline, and glycerophosphocholine) were retained at 37 degrees C; in contrast, in the presence of 100 nM Ca2+ there was uniform leakage of these metabolites. Experiments with hemicholinium-3, an inhibitor of choline transport, and EP cells at 100 nM Ca2+ show that linkage of choline transport and Ptd-choline biosynthesis is also dependent on Ca2+. These results suggest that channeling of intermediates in the CDP-choline pathway of Ptd-choline biosynthesis in glioma cells is mediated by intracellular Ca2+ levels that may coordinately regulate the steps involved in conversion of choline to Ptd-choline.

Animals↗

PubMed1991

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells.

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.

Animals↗

PubMed1991

Alternate pathways in the desaturation and chain elongation of linolenic acid, 18:3(n-3), in cultured glioma cells.

Cultured C6 glioma cells rapidly incorporate and metabolize the essential fatty acids, 18:2(n-6) and 18:3(n-3), to 20- and 22-carbon polyunsaturated fatty acids. Using several deuterated fatty acid substrates we have obtained data that suggest alternate pathways, one possibly involving delta 8-desaturation, may exist in glioma cells for formation of 20:5(n-3) and 22:6(n-3) from 18:3(n-3). With 18:3(n-3)-6,6,7,7-d4 practically no 18:4(n-3)-6,7-d2 or 20:4(n-3)-8,9-d2 was detected whereas 20:3(n-3)-8,8,9,9-d4 accounted for 3.4% and delta 5,11,14,17-20:4-8,8,9,9-d4 for 21.1% of the total deuterated fatty acids recovered in phospholipids after a 16 h incubation; 20:5(n-3)-8,9-d2, 22:5(n-3)-10,11-d2, and 22:6(n-3)-10,11-d2 accounted for 42.4%, 13.2%, and 2.8% of deuterated acyl chains, respectively. When added exogneously, 20:3-8,8,9,9,-d4 was extensively converted to delta 5,11,14,17-20:4(n-3)-8,8,9,9-d4 (45%) and 20:5(n-3)-8,9-d2 (24%); a small amount (4%) of 18:3(n-3)-d4 also was detected. Both 20:4(n-3)-8,9-d2 and 18:4(n-3)-12,13,15,16-d4 were also converted to 20:5(n-3) and 22:6(n-3) with 8 and 0% of the respective original deuterated substrate remaining after 16 h. A possible pathway for 18:3(n-3) metabolism in glioma cells is described whereby an initial chain elongation step is followed by successive delta 5 and delta 8 desaturation reactions resulting in 20:5(n-3) formation and accounting for the ordered removal of deuterium atoms. Alternatively, extremely effective retroconversion may occur to chain shorten 20:3(n-3)-d4 to 18:3(n-3)-d4 followed by rapid conversion through the classical desaturation and chain elongation sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

The role of sphingomyelin in phosphatidylcholine metabolism in cultured human fibroblasts from control and sphingomyelin lipidosis patients and in Chinese hamster ovary cells.

Human fibroblasts in culture take up exogenous [choline-Me-3H,32P]sphingomyelin (SM) from the medium and incorporate it into cellular SM and phosphatidylcholine [Spence, Clarke & Cook (1983) J. Biol. Chem. 258, 8595-8600]. The ratio of [3H]choline/[32P]Pi is similar in SM and phosphatidylcholine, indicating that the phosphocholine (P-Cho) moiety is transferred intact. Similar results are obtained with Niemann-Pick (NP) cells which are deficient in lysosomal sphingomyelinase activity, suggesting that the P-Cho transfer may not be mediated by the lysosomal sphingomyelinase and that alternative pathways of sphingomyelin catabolism are present in cultured cells. In this study we have shown that: (1) the P-Cho pool in control and NP cells incubated with exogenous labelled SM has a specific radioactivity intermediate between that of SM and PtdCho; (2) expansion of the intracellular P-Cho pool by incubation with exogenous choline reduces the incorporation of [3H]choline from SM into PtdCho; and (3) incorporation of P-Cho from SM into PtdCho is decreased at the non-permissive temperature in Chinese hamster ovary cells with a temperature-sensitive mutation in the cytidylyltransferase reaction. These results suggest that incorporation of P-Cho from SM into PtdCho involves a reaction sequence in which P-Cho is hydrolysed from SM by a sphingomyelinase, followed by incorporation of P-Cho into PtdCho via the cytidine pathway of biosynthesis (SM----P-Cho----CDP-Cho----PtdCho). The appreciable incorporation of P-Cho from SM into PtdCho in sphingomyelinase-deficient NP cells suggests a more substantial or effective lysosomal sphingomyelinase activity in intact cells than is measured in vitro, and/or a significant contribution by other sphingomyelinase activities in these cells.

Animals↗

PubMed1990

Incorporation of polyunsaturated fatty acids into plasmalogens, compared to other phospholipids of cultured glioma cells, is more dependent on chain length than on selectivity between (n - 3) and (n - 6) families.

In several tissues and cells, polyunsaturated fatty acids (PUFA) are esterified to plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3 phosphoethanolamine). Some studies have implicated selectivity for (n - 3) fatty acids, particularly of 20- and 22-carbons, over the (n - 6) family of fatty acids. We have investigated selectivity for esterification of both families of PUFA to plasmalogens in cultured C6 glioma cells. By 24 h, approx. 40% of cell-associated label from [1-14C]18:3(n - 3) was incorporated into plasmalogens and that label consisted almost exclusively of desaturation and chain elongation products [80% 20:5(n - 3) and 15% 22:5(n - 3)]. Relative incorporation of label from PUFA into plasmalogens was 20:5(n - 3) greater than 20:4(n - 6) greater than 18:3(n - 3) much greater than 18:2(n - 6); incorporation of unaltered 18-carbon chains was highly restricted. Cells incubated with [1-14C]18:3(n - 3) and 20-150 microM competing unlabeled fatty acids showed 20:5(n - 3) greater than 20:4(n - 6) greater than or equal to 22:4(n - 6) greater than 18:3(n - 3) as inhibitors of plasmalogen labeling. Chase experiments in cells prelabeled with [1-14C]18:3(n - 3) for 2 h showed limited reduction of label in plasmalogen. Reduction of plasmalogen label did occur when (n - 3) or (n - 6) fatty acids were added to cells prelabeled for 48 h, accounting for losses of 20-35% compared to controls. Accordingly, little selectivity occurs in esterification of plasmalogens from mixtures of (n - 3) and (n - 6) fatty acyl chains. Subsequent remodeling of (n - 3) acyl chains occurs, but is more dependent on acyl chain length than on selectivity between (n - 3) and (n - 6) families. Our data are consistent with a stable plasmalogen pool enriched in PUFA, but not specifically with (n - 3) fatty acids.

Binding, Competitive↗

PubMed1990

Phosphoinositide metabolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane.

The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentially recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but significant proportion of PtdInsP2-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP2 (diacylglycerol kinase and PtdInsP kinase) also were present. CTP:phosphatidate cytidylyltransferase and CMP-phosphatidate:inositol phosphatidyltransferase were located exclusively in a microsomal fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol from agonist-stimulated cleavage of PtdInsP2, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP2 completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP2 phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PtdInsP2 in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP2 in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.

Animals↗

PubMed1990

Enzymes that degrade phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate have different developmental profiles in chick brain.

The activities and subcellular distributions of the hydrolases that degrade polyphosphoinositides were compared in the developing chick central nervous system. Specific activities increased 2- 3-fold and total activities increased 13- to 16-fold. Phosphatidylinositol 4-phosphate phosphatase is localized in membranes (78%), but is preferentially associated with nonmyelin membranes, since the increase in specific activity preceded myelination and proportions of membrane and soluble activities were constant during accumulation of myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphatase is largely soluble in embryonic (57%) and myelinated brain (50%). Although specific activity increased coincident with myelination, approximately equal increases in soluble and membrane activity indicate no preferential association with myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphodiesterase activity increased only in the early stages of myelination, but showed some preferential association with myelin membranes, since the proportion of soluble diesterase declined from 40 to 25%.

Animals↗

PubMed1990

Detection of young children in need of reading help. Evaluation of specific reading disability formulas.

To determine whether formula-based definitions of specific reading disability (SRD) were related to the actual classroom performance of children aged 7 1/2, the authors applied five definitions of SRD to a cohort of 243 children. The children were followed from birth and not selected on the basis of cognitive, reading, or academic performance criteria. The authors determined the concurrent validity of each of the formulas by comparing the prediction of each definition to whether the child received additional reading help in the classroom. Although the years behind method was significantly better than other approaches, no method identified more than 48 percent of the children who were given additional reading help. This study questions the ability of formula-based definitions adequately to identify young children with reading dysfunction.

Child↗

PubMed1990

Precursors of reading delay: neurodevelopmental milestones.

The relationship between current reading ability and the achievement of early language and motor developmental milestones was evaluated in 240 children, aged 7 1/2 years, whose language and motor achievement had been charted at each well baby visit during the first 2 years of life. Those children whose composite reading score was 6 months behind their chronologic age on the Woodcock-Johnson Psychoeducational Battery were classified as having reading delay. Relationships to reading outcome were assessed for individual infant milestones, for critical screening values, and by statistical techniques that characterized the developmental process rather than single milestones. Significant differences (P less than .05) were noted between children with and without reading delays for the following milestones: 4 to 6 words, 7 to 20 words, 50 words, 2-word sentences, and 5 and 8 body parts. The positive predictive value of slower milestone achievement ranged from 0% to 50%. Techniques that focused on the developmental process during the first 2 years (either rate of achievement of neurodevelopmental milestones or order of milestone acquisition) were better able to classify children with reading delay (sensitivity = .73, specificity = .78). Although the language milestone measures did not classify children sufficiently well to be diagnostic, the data served to determine whether a child would be at high risk based on performance rather than historical factors.

Child↗

PubMed1990

Infant stimulation curriculum for infants with cerebral palsy: effects on infant temperament, parent-infant interaction, and home environment.

To assess the effects of intervention in cerebral palsy, 48 infants 12 to 19 months of age, with mild to severe spastic diplegia, were randomly assigned to receive either 6 months of infant stimulation followed by 6 months of physical therapy (test group) or 12 months of neurodevelopmental physical therapy (contrast group). The infant stimulation protocol consisted of cognitive, motor, sensory, and language activities. Outcome was assessed after 12 months by using Carey Infant Temperament Questionnaire subscores (activity, rhythmicity, adaptability, approach, threshold, intensity, mood, distractibility, and persistence); Roth Mother-Child Relationship Evaluation subscores (acceptance, overprotection, overindulgence, rejection); and Home Observation for Measurement of the Environment subscores (maternal responsiveness, avoidance of restriction and punishment, organization of environment, play materials, maternal involvement, and variety of daily stimulation). Motor and cognitive outcomes suggesting advantage for the test group have been reported previously. After 12 months of intervention, mothers with infants in the contrast group showed a greater improvement in emotional and verbal responsiveness as measured by the Home Observation for Measurement of the Environment (mean score change in control group = 1.2, test group = 0.3 P less than .04). None of the 19 other measures differed significantly between treatment groups in change from baseline. This study demonstrates no short-term systematic effect on temperament, maternal-infant interaction, or home environment attributable to the inclusion of an infant stimulation curriculum in an intervention program for infants with spastic diplegia. It suggests that motor and cognitive advantages associated with infant stimulation are not mediated by measurable changes in the psychosocial variables studied.

Cerebral Palsy↗

PubMed1989

Defective activity of acyl-CoA:cholesterol O-acyltransferase in Niemann-Pick type C and type D fibroblasts.

The activity of acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) was measured in fibroblast homogenates from Niemann-Pick Type C (NPC) and Type D (NPD) patients to determine whether these cells exhibit similar defects in the regulation of cholesterol esterification. ACAT activity in normal cells cultured in the absence of serum lipoproteins responded rapidly (within 6 h) to the addition of serum and reached peak levels at 12-24 h, whereas little stimulation of activity in NPC cells was observed. In contrast, ACAT activity in NPD fibroblasts (cell lines from four different patients) began to increase between 6 and 12 h after serum addition, reaching levels up to 50% of normal values at 24 h. ACAT activity in NPC and NPD cell extracts could not be stimulated by preincubation with normal cell homogenates, nor was complementation between NPC and NPD homogenates observed. Addition of 25-hydroxycholesterol to fibroblasts cultured in delipidated serum increased ACAT activity for all three cell types, although stimulation in NPD cells was less than that observed in NPC cells. ACAT activity of deoxycholate-solubilized homogenates reconstituted into phosphatidylcholine vesicles was independent of the presence of serum lipoproteins during culture and dependent on cholesterol present in the vesicles for all cell types. However, ACAT activities of mutant fibroblasts in vesicles plus cholesterol were significantly (about 40%) lower than control levels. These results suggest that the metabolic lesions in NPC and NPD cells are biochemically distinct and that both may involve factors in addition to the availability of cholesterol substrate for the ACAT enzyme.

Cells, Cultured↗

PubMed1989

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.

Animals↗

PubMed1989

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.

1-Phosphatidylinositol 4-Kinase↗

PubMed1989

A new Zn2+-stimulated sphingomyelinase in fetal bovine serum.

Fetal bovine serum contains a Zn2+-dependent sphingomyelinase with optimal activity at pH 5.5 in vitro. Activity could be demonstrated with a liposomal sphingomyelin substrate suspension but was stimulated up to 15-fold by Triton X-100. Under a variety of conditions tested, phosphatidylcholine, lysophosphatidylcholine, glycerophosphocholine, and p-nitrophenyl phosphate were not substrates for this activity. Several inhibitors of serum alkaline and acid phosphatases had no effect on the activity. The enzyme resembles the acid lysosomal sphingomyelinase in pH optimum and inhibition by AMP but differs in inhibition by EDTA, stimulation by Zn2+, and heat lability at 55 degrees C. It resembles the neutral, Mg2+-stimulated enzyme in inhibition by EDTA and heat lability but differs in metal ion requirement and pH optima. Of the sera tested, activity was highest in fetal bovine serum, with fetal bovine greater than newborn bovine greater than horse greater than human; more than 95% of the activity is in the lipoprotein-free infranatant of serum (d greater than 1.21). This activity appears to be a hitherto undescribed sphingomyelinase. Its biological functions are not known but may subserve a special role in sphingomyelin catabolism in the circulation, in blood vessel walls, or in the tissue(s) of origin.

Animals↗

PubMed1989

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells.

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.

Animals↗