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Biomedical subjects

G Mauco

Publications and source records attributed to G Mauco.

At least 55 records · Page 3Linked to original sources
PubMed1987

Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors.

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.

Blood Platelets↗
PubMed1985

Measurement of arachidonic acid liberation in thrombin-stimulated human platelets. Use of agents that inhibit both the cyclooxygenase and lipoxygenase enzymes.

The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
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

Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets.

Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.

Blood Platelets↗
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

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↗
PubMed1983

Platelet activating factor (PAF-acether) promotes an early degradation of phosphatidylinositol-4,5-biphosphate in rabbit platelets.

32P-Labelled washed rabbit platelets were incubated with 0.6 nM platelet activating factor (PAF-acether), giving a full aggregation and release response within 30-60 s. The major phospholipid changes observed under these conditions were: (1) An increased labelling of phosphatidic acid (PA) within 10 s and of phosphatidylinositol (MPI) at 30 s, reflecting the activation of the MPI cycle via the cytosolic phospholipase C; (2) an enhancement of phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI) labelling at later incubation times; (3) an early degradation of TPI with a counterbalancing formation of DPI. The latter changes suggest a receptor-mediated stimulation of TPI-phosphomonoesterase, the role of which in the mechanism of platelet activation is discussed.

Animals↗
PubMed1982

Platelet arachidonic acid metabolism in severe cerebrovascular disease.

The ability of platelets to synthetise thromboxane B2 and hydroxylated fatty acids from arachidonic acid was studied simultaneously with arachidonic acid-induced aggregation in 42 patients suffering from severe cerebral atherosclerosis and also in 34 healthy controls. Additionally, phospholipase-A2-induced aggregation was performed as a probe for arachidonic acid located at the platelet surface. All the assays were performed with washed platelets, eliminating a possible influence of plasma. Platelets from patients were found responsive to significantly lower concentrations of arachidonic acid whereas thromboxane and hydroxylated fatty acid biosynthesis did not differ from controls. In the experimental conditions used, 75% of the control platelets underwent aggregation with phospholipase A2 plus sphingomyelinase C, in comparison to only 50% for the patients, indicating the necessity for further analysis of the platelet membrane lipids in atherosclerosis.

Adult↗
PubMed1982

Organization and role of platelet membrane phospholipids as studied with phospholipases A2 from various venoms and phospholipases C from bacterial origin.

Phospholipases A2 from various snake or bee venoms and phospholipases C secreted as exotoxins by several bacteria have been used to study the transverse distribution of phospholipids in the platelet plasma membrane and their role in platelet activation. An asymmetric distribution was described for phospholipids, characterized by a preferential localization of sphingomyelin and phosphatidylcholine in plasma membrane outer leaflet, whereas the inner half contains almost all of the anionic procoagulant phosphatidylserine and phosphatidylinositol. Such a distribution might explain the latency of procoagulant activity in resting platelets and implies an intracellular localization of arachidonic acid, the precursor of prostaglandins and thromboxanes. The external arachidonic acid is involved in phospholipase A2-induced aggregation, whereas phospholipase C from Clostridium welchii stimulates platelets through a thromboxane-independent pathway. The latter one is directly linked to the formation of phosphatidic and lysophosphatidic acids, which are able to activate cells through calcium mobilization. So, phospholipase C represents an interesting tool for studying the biochemical processes accompanying stimulation, since it is shown that it mimics the effects of an intracellular phospholipase C, the role of which in platelet activation is discussed.

Animals↗
PubMed1981

Studies on topological distribution of arachidonic acid replacement in platelet phospholipids and on enzymes involved in the phospholipid effect accompanying platelet activation.

In this short review recent results obtained on platelet phospholipid metabolism are summarized. The first part reports a topological study of arachidonic acid (AA) replacement in platelet phospholipids. It is shown that incubation of platelets with radioactive free arachidonic acid leads to a labelling of the phospholipids present inside the platelet, whereas the exchange of intact phosphatidylcholine (PC) molecules with the plasma lipoproteins occurs on the platelet outer surface. This should allow a selective labelling of the small external pool of AA in order to follow its behaviour during platelet activation. In the second part, some enzymes involved in the metabolism of phosphatidylinositol (PI) have been further characterized. The first one is a diglyceride-lipase, which is located in the plasma membrane and releases the two fatty acids esterifying the diglycerides formed from PI by the action of the platelet phospholipase C. Such an enzyme is probably responsible for the release of AA from PI occurring upon platelet activation. On the other hand, cytosolic phospholipid exchange proteins able to catalyse the transfer of PI between membranes have been identified. The possible role of the enzymes involved in the acceleration of PI turnover occurring during platelet activation is discussed.

Arachidonic Acid↗
PubMed1978

Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase.

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.

Animals↗