Biomedical subjects

H Chap

Publications and source records attributed to H Chap.

At least 199 records · Page 11Linked to original sources

PubMed1985

Carrier-mediated choline uptake by Krebs II ascites cells.

Krebs II ascites cells have a low affinity uptake system for choline (Km = 36 microM, Vm = 76 nmol/min per 2 X 10(8). Choline entered the cells and was rapidly phosphorylated (95% of total intracellular soluble label). Trans acceleration of labeled choline from cells preloaded with radiolabeled choline and postincubated in the presence of unlabeled choline indicates that choline transport in Krebs II ascites cell is carrier mediated. Ethanolamine competed for the choline carrier. The uptake was reduced by hemicholinium-3, iodoacetamide and ouabain. The mechanism of choline transport in Krebs II ascites cells is in agreement with a linear transport model.

2,4-Dinitrophenol↗

PubMed1985

Turnover of phosphocholine and phosphoethanolamine in ether-phospholipids of Krebs II ascite cells.

Krebs II ascite cells suspended in Eagle medium were incubated at 37 C for up to 6 hr in the presence of [3H] glycerol or [32P] orthophosphate. After extraction, their lipids were treated with guinea pig phospholipase A1 under conditions where all diacyl-phospholipids (diacyl-PL) became hydrolyzed with 55% recovery of lyso-PL. Using a bidimensional thin layer chromatography (TLC) involving exposure to HCl fumes between the two runs, it then became possible to determine at once the specific radioactivity of the three subclasses (diacyl-, alkylacyl- and alkenylacyl-) present in choline glycerophospholipids (CGP) and ethanolamineglycerophospholipids (EGP). Compared to diacyl-PL, a lower de novo synthesis of ether subclasses was evidenced in both CGP and EGP by [3H] glycerol incorporation. Although the same profile was obtained for CGP with [32P] orthophosphate, the three EGP subclasses displayed in this case the same specific radioactivity. These data indicate a higher turnover rate of the polar head group of ether-EGP compared to either-CGP. The simple methodology used in the present study might thus prove helpful in developing enzymatic studies dealing with the mechanism of this accelerated renewal.

Animals↗

PubMed1985

Lipoprotein and phospholipid distribution in human follicular fluids.

Human preovulatory follicular fluids, obtained in the course of stimulated cycles, were analyzed for their lipid and protein compositions. By combining different methods, a single class of lipoprotein, high-density lipoprotein, was detected in all cases. The phospholipid distribution revealed an accumulation of lysophosphatidylcholine. These data are discussed in view of the possible roles of the follicular fluid during fertilization and in relation to the high levels of estradiol and progesterone measured in those fluids.

Body Fluids↗

PubMed1985

[Cell membrane and phospholipids].

After a short review of the structure of phospholipids, the authors describe the organisation of these compounds in the cell membrane and the metabolic pathways responsible for their degradation. The latter involve the phospholipases C and A2. The first enzyme is the key to practically all cellular activation because the specific hydrolysis of phosphatidylinositol--4,5-biphosphate generates two intracellular messengers, diglycerides and inositol--1,4,5-triphosphate which activate, respectively, a specific kinase and mobilise the calcium. This mobilisation of calcium is essential for the activation of the phospholipase A2 which regulates the liberation of arachidonic acid and lysophosphatidylcholine, precursors of different lipid mediators (prostaglandins, thromboxanes, leukotrienes, platelet activating factor or PAF-acether...). The production of these derivatives represents a system of amplification and recruitment outside the cell, explaining the possible involvement of these metabolic pathways in the physiological and pharmacological regulation of the different cells of the cardiovascular system (platelets, endothelial cells, smooth muscle cells...).

Arachidonic Acid↗

PubMed1985

Relationship between phospholipid metabolism and intracellular calcium mobilization during platelet activation.

This paper reviews some of our most recent studies concerning the relationship between phospholipid metabolism and calcium mobilization in thrombin-stimulated platelets. Evidence is provided that phospholipase C activation does not require any increase in cytoplasmic calcium but is subsequent to a yet unknown membrane modification rendering the substrate accessible to the enzyme upon thrombin-receptor binding. In contrast, the much higher calcium dependence of phospholipase A2 led us to conclude that cytoplasmic free calcium might increase to in excess of 1.6 microM even in the absence of external calcium. This is supported by our estimation that calcium is present in dense tubular system in rather large excess, as measured on purified vesicles derived from this organelle. We thus suggest that the level of inositol-1,4,5-tris-phosphate is rate limiting in the process of calcium mobilization which implies that calcium influx through the plasma membrane might become a critical event with weak agonist poorly activating phospholipase C.

Aminoquinolines↗

PubMed1984

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells.

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A2 and Staphylococcus aureus sphingomyelinase C and on phospholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A1 and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).

Animals↗

PubMed1984

Different susceptibility of alkylacyl--versus diacyl--and alkenylacyl--phosphatidylcholine subclasses to stimulation of biosynthesis by phospholipase C.

Krebs II ascites cells were incubated with [3H] or [14C] choline in the presence or in the absence of Clostridium welchii phospholipase C (PLC). At enzyme concentrations where cell lysis remained limited, PLC specifically enhanced phosphatidylcholine (PC) biosynthesis, as shown by comparison with [14C] ethanolamine. Further analysis revealed that the stimulating effect of PLC remained limited to 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and 1-alkenyl-2-acyl-GPC, whereas the biosynthesis of 1-alkyl-2-acyl-GPC, the putative precursor of platelet activating factor (PAF-acether) remained unchanged. These differences reflect different localizations of the three PC subclasses in the plasma membrane and are discussed in relation to the regulation mechanism of PC biosynthesis.

Animals↗

PubMed1984

Studies on enzymes related to diacylglycerol production in activated platelets. II. Subcellular distribution, enzymatic properties and positional specificity of diacylglycerol- and monoacylglycerol-lipases.

The subcellular distribution of diacylglycerol- and monoacylglycerol-lipases has been studied in human platelets. Using a fractionation procedure on Percoll gradient (Perret, B., Chap, H. and Douste-Blazy, L. (1979) Biochim. Biophys. Acta 556, 434-446), the enzyme activity displayed the same profile as that of [3H]concanavalin A, a plasma membrane marker. This result was confirmed with highly purified platelet plasma membranes prepared by adsorption onto polyethylenimine-bonded polyacrylamide beads (Kinoshita, T., Nachman, R.L. and Minick, R. (1979) J. Cell Biol. 82, 688-696). Studies with isolated membranes or crude homogenate revealed that the enzyme requires calcium or magnesium and displays an optimal pH of 6.2, showing that it is able to hydrolyse diacylglycerol under conditions where phosphatidylinositol-specific phospholipase C is fully active. Using diacylglycerol labelled in the 1- or 2-position, it was found that the two fatty acids are released at the same rate, which is supported by the lack of monoacylglycerol accumulation and by the observation that monoacylglycerol is hydrolysed at a 20-fold faster rate than diacylglycerol. Increasing concentrations of Mg-ATP promote the conversion of diacylglycerol into phosphatidic acid by diacylglycerol kinase, but only high concentrations become inhibitory for diacylglycerol lipase. These results are discussed in the light of our former hypothesis that arachidonic acid release from platelet phospholipids might occur through the sequential action of a phosphatidylinositol-specific phospholipase C coupled to a diacylglycerol lipase (Mauco, G., Chap, H., Simon, M.F. and Douste-Blazy, L. (1978) Biochimie 60, 553-561). The possible role of this enzyme in the regulation of the activity of protein kinase C is also emphasized.

Blood Platelets↗

PubMed1984

Evidence for a two-step process in prostaglandin secretion. Intracellular accumulation of prostacyclin precedes its release from human endothelial cells in culture.

Cultured endothelial cells (EC) from human umbilical vein were incubated with [U-14C]arachidonic acid (AA) followed by a challenge with thrombin (2 units/ml) or calcium ionophore A23187 (5 microM) for 0.5-10 min at 37 degrees C. In both cases, AA was rapidly liberated from phospholipids and converted into prostaglandin I2 (PGI2), as determined by the radioactivity of the stable derivative 6-keto-PGF1 alpha. Maximal liberation of AA and synthesis of PGI2 were achieved within 2 min, but the two compounds first accumulated in EC prior to their release into supernatants. This finding, which was never reported before, raises the question of the mechanism of AA and PG release through the cell membranes and offers a convenient model to investigate this still obscure process.

6-Ketoprostaglandin F1 alpha↗

PubMed1984

Simultaneous isolation of two platelet membrane fractions: biochemical, immunological and functional characterization.

Simultaneous isolation of two platelet membrane subfractions was achieved by centrifugation on 40% sucrose from a 100.000 g crude membrane fraction. Characterization of both types of membranes was carried out by different biochemical and immunological markers. Using a surface label, 3H Concanavalin A (3HCon A), a marker enzyme, phosphodiesterase, and lipid analysis, one of the fraction has been identified as external or plasma membranes, the other consists of intracellular membranes. Further two specific antibodies directed against external membrane antigens (LeKa and IgG L) react almost exclusively with the external membranes. Finally both kinds of membranes were able to uptake calcium but the affinity for this cation was higher for the internal than for the external membranes. This suggests that both membranes are implicated in the regulation of the cytoplasmic calcium concentration and that the internal membranes (dense tubular system) play the major part in this regulation.

Antigens↗

PubMed1984

Activation of phospholipase C in thrombin-stimulated platelets does not depend on cytoplasmic free calcium concentration.

Human platelets loaded with the fluorescent Ca2+ indicator quin2 and with different radioactive compounds including [3H]serotonin, [14C]arachidonic acid (AA) and [32P]orthophosphate were stimulated by thrombin under conditions producing secretion. In the absence of external Ca2+ (Ca2+e), cytoplasmic free [Ca2+], [Ca2+]i, increased to 340 nM, against 1685 nM at 1 mM [Ca2+]e. In both cases, diglyceride and phosphatidic acid production proceeded at the same rate, whereas AA release was inhibited at low [Ca2+]i. It is concluded that, at variance with phospholipase A2, phospholipase C activation does not depend on [Ca2+]i. These results give further support to the hypothesis of a Ca2+-independent pathway of cell activation involving phospholipase C and protein kinase C.

Arachidonic Acid↗

PubMed1984

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells.

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.

Animals↗

PubMed1984

Studies on ether phospholipids. II. Comparative composition of various tissues from human, rat and guinea pig.

The ether phospholipid composition of various tissues (brain, heart, lung, liver, kidney, testis, erythrocytes and plasma) has been investigated in human, rat and guinea pig, using a new method of determination (El Tamer, A., Record, M., Fauvel, J., Chap, H. and Douste-Blazy, L. (1984) Biochim. Biophys. Acta 793, 213-220). This is based on the selective removal of diacyl phospholipid species by phospholipase A1 degradation followed by acidolysis of the plasmalogens. Our results fit rather well with other literature data available for human and rat tissues, illustrating the good reliability of the method. Among various differences noted between the three mammalian species, guinea pig is characterized by a relatively higher content of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and of ethanolamine plasmalogens in blood plasma. Alkylacyl-GPC, a putative precursor of platelet activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC), is also more abundant in guinea pig lung and in human kidney. This study also revealed a striking parallelism between the tissue content of alkylacyl-GPC and alkylacyl-GPE (1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine). This new observation is discussed in relation to a possible metabolic link between these two phospholipids.

Animals↗

PubMed1984

Phosphatidylcholine is the major phospholipid providing arachidonic acid for prostacyclin synthesis in thrombin-stimulated human endothelial cells.

Upon incubation for 24 hours with [3H]arachidonic acid (AA, 1 mu Ci/ml), cultured endothelial cells from human umbilical vein incorporated one half of the added radioactivity, mostly into phospholipids (83% of the total cell radioactivity). Distribution of the label between the various phospholipid classes was found to reflect the distribution of endogenous AA. Stimulation with human thrombin (2 U/ml) promoted a rapid release of radioactive material into supernatants, which contained essentially 6-keto-prostaglandin F1 alpha and non-converted AA. This process levelled off at 10 min, at which time phosphatidylcholine displayed a decrease accounting for 3.7% of the total cell radioactivity. Phosphatidylinositol also appeared significantly diminished, but this decrease was almost 2.5 fold less than that observed in phosphatidylcholine. It is concluded that AA availability for prostacyclin biosynthesis is mostly regulated by a phospholipase A2.

Arachidonic Acids↗

PubMed1984

Platelet activating factor (PAF-acether) is released into rat pulmonary alveolar fluid as a consequence of hypoxia.

Hypoxia provokes pulmonary constriction and because PAF-acether is a very strong pulmonary constrictor, we looked for PAF-acether in lung alveolar lavage (LAL) with a biological method based on the measurement of rabbit platelet aggregation. We first demonstrated a PAF-acether secretion during bronchoalveolar lavage with sterile isotonic NaCl (pH 7.2). PAF-acether secretion was completely suppressed with isotonic NaCl containing 5 mM EDTA but lyso-PAF-acether was still present (1.9 +/- 0.55 nmoles). Upon hypobaric hypoxia, PAF-acether was detected in LAL (1.05 +/- 0.25 10(-2)nmoles). The amount of lyso-PAF-acether increased by 6 times (12.1 +/- 4.1 nmoles). These results are given for 10(4) nmoles phospholipids of LAL. They indicate that alveolar macrophages might be activated by hypobaric hypoxia, so they produce PAF-acether in the alveole. Such a process could be involved in the well-known bronchoconstriction accompanying hypoxia.

Animals↗

PubMed1984

Studies on enzymes related to diacylglycerol production in activated platelets. I. Phosphatidylinositol-specific phospholipase C: further characterization using a simple method for determination of activity.

A simple method of determination of phosphatidylinositol-specific phospholipase C activity in soluble platelet extracts has been devised. It is based on the use of a total lipid extract from rat liver microsomes incubated with [3H]inositol in the presence of MnCl2. Phosphatidylinositol hydrolysis can thus be detected by determining hydrosoluble radioactivity formed upon incubation with enzyme fractions. Owing to the presence of other phospholipids in the assay system, phospholipase C was inhibited. However, activity was restored by sodium deoxycholate (0.1%, w/v). Optimal conditions also included calcium (1-10 mM) and a pH between 5 and 7, allowing the detection of phospholipase C without the need for purifying the substrate. Using this simplified procedure, platelet phospholipase C was submitted to preparative electrofocusing and to gel filtration chromatography on Sephacryl S-200. Phospholipase C focused in one single peak at pH 6.1. An Mr of 86 000 was found upon gel chromatography of a crude extract, against 68 000 when phospholipase C had been previously purified by electrofocusing. These data indicate that phospholipase C might be associated with lipids or with an Mr 20 000 protein, the significance of which is discussed.

Blood Platelets↗

PubMed1984

Platelet aggregating activity of lysophosphatidic acids is not related to their calcium ionophore properties.

The calcium ionophore properties of A23187 and of two lysophosphatidic acid (LPA) analogs (1-palmitoyl- and 1-hexadecyl-sn-glycero-3-phosphate or P-GPA and H-GPA, respectively) were compared using platelet membrane vesicles loaded with 45Ca. Half maximal effect (HME) was obtained at 5 microM and 10 microM for H-GPA and P-GPA, respectively, against 0.7 microM for A23187, which released 2 times more Ca. The three compounds also induced platelet aggregation with a HME at 0.5 microM, 0.3 microM and 0.01 microM for A23187, P-GPA and H-GPA, respectively. The clear dissociation between the two effects appearing for both LPA raises some doubt about the general idea that (lyso) PA participate in cell activation through their calcium ionophore properties.

Calcimycin↗

PubMed1984

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. I. Studies on neutral glycerides.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9-10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein greater than 1(3)-monoolein greater than tributyrin = triacetin greater than or equal to triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[3H]palmitoyl-2-[14C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[14C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.

Animals↗