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Biomedical subjects

H Chap

Publications and source records attributed to H Chap.

At least 163 records · Page 9Linked to original sources
PubMed1988

Effect of PCR 4099 on ADP-induced calcium movements and phosphatidic acid production in rat platelets.

Antiplatelet activity of PCR 4099, an analogue of ticlopidine, resides in its specific effect against exogenous as well as released ADP. This study investigated in rat platelets the effects of the drug on ADP-induced shape change, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Shape change and influx of Ca2+ ions across the plasma membrane were not modified after PCR 4099 administration using aspirin-treated platelets. On the other hand, phosphatidic acid formation and calcium mobilization from internal stores were strongly inhibited. These results suggest that PCR 4099 leaves intact the machinery involved in ADP-induced platelet shape change and influx of calcium ions, but inhibits an early step in the ADP-response coupling leading to inositol phospholipid hydrolysis and aggregation.

Adenosine Diphosphate↗
PubMed1988

Characterization of plasma membranes from A431 cells, isolated by self-generating Percoll gradient: a rapid isolation procedure to obtain plasma membranes with functional epidermal growth factor receptors.

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers. Plasma membranes displayed a 4.5- and 4.4-fold enrichment in [3H]concanavalin A and 5'-nucleotidase, respectively. This proteic fraction was further characterized by its lipid composition and phospholipid analysis. The cholesterol/phospholipid molar ratio was 0.45 in plasma membranes against 0.19 in lysate. Sphingomyelin increased from 7.5% of total phospholipids in lysate to 16.2% in plasma membranes, as well as phosphatidylserine which displayed a 1.5-fold enrichment in the plasma membrane fraction. This was at the expense of phosphatidylcholine (45.2% in lysate, against 35% in plasma membranes). Electron microscopy of the isolated material showed vesicles essentially free from endoplasmic reticulum and organelles. These plasma membranes retained the ability to bind 125I-labelled epidermal growth factor (125I-EGF) with a Kd = 4.7 nM and Bmax = 63 pmol/mg protein. EGF binding resulted in a stimulation of the phosphorylation protein reaction in the presence of [gamma-32P]ATP and sodium dodecyl sulfate polyacrylamide gels of phosphorylated proteins indicated that the radioactivity of the major band of molecular weight 170,000 was clearly enhanced by EGF binding. These results indicate that the EGF receptor and its intrinsic protein kinase activity were preserved during our plasma membrane isolation procedure.

Carcinoma, Squamous Cell↗
PubMed1988

Effect of a stimulant of guanylate cyclase, sin 1, on calcium movements and phospholipase C activation in thrombin-stimulated human platelets.

The effects of sin 1, a metabolite of an antianginal agent, molsidomine, were investigated on human platelet activation induced by thrombin. This drug promoted a slight inhibition of serotonin release in a medium containing 1 mM Ca2+ or 1 mM EGTA (from 63% to 46% and from 57% to 41% of total serotonin secretion, respectively, with the highest dose used). Under these conditions, Ca2+ movements, monitored by quin 2 fluorescence, were markedly impaired. The most pronounced effect was towards Ca2+ influx, which presented a rapid inhibition with low doses. In the presence of external calcium, thrombin raised cytoplasmic free Ca2+ concentration from 100 nM to 1277 nM. This was reduced to 466 nM and 175 nM with 10(-7) M and 10(-4) M sin 1, respectively. Ca2+ mobilization from internal stores was less inhibited, since cytoplasmic free Ca2+ movements, sin 1 was tested on [32P] phosphatidic acid synthesis resulting from phospholipase C activation induced by thrombin. Phosphatidic acid labelling displayed a maximal inhibition of 43-50% with the highest doses of sin 1 (10(-4) M-10(-3) M) with or without Ca2+ in the incubation medium. However, this effect appeared much more sensitive to sin 1 in the presence of external Ca2+ (25% at 10(-7) M sin 1 with external Ca2+ against 12% at the same sin 1 concentration with EGTA). This discrepancy might be explained by the difference of cGMP level obtained when platelets were treated by sin 1 in the presence or in the absence of Ca2+ in the medium. This study shows that the major target of sin 1 via cGMP is not platelet phospholipase C as previously described, but inhibition of Ca2+ influx through plasma membrane.

Blood Platelets↗
PubMed1988

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum.

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.

5'-Nucleotidase↗
PubMed1988

Potential anticoagulant activity of lipocortins and other calcium/phospholipid binding proteins.

Thrombin generation was determined in the presence of phospholipids, coagulation factors Xm, Vm and II (prothrombin), calcium, and various calcium/phospholipid bindings proteins, including lipocortins I and II, 35 kDa calelectrin, and 32.5 kDa endonexin. All of these proteins induced a dose-dependent inhibition of thrombin generation similar to the inhibition of pig pancreas phospholipase A2. It is suggested that the ability of lipocortins and other related proteins to interact with anionic phospholipids in the presence of Ca++ is responsible for both their anticoagulant and anti-phospholipase A2 activity.

Animals↗
PubMed1988

Uptake of HDL unesterified and esterified cholesterol by human endothelial cells. Modulation by HDL phospholipolysis and cell cholesterol content.

Human HDL (1.070-1.210), doubly labelled with 3H/14C-labelled unesterified cholesterol and 3H-labelled esterified cholesterol were incubated for 1-5 h with monolayer cultures of human endothelial cells. HDL were preincubated for 60-120 min the presence of albumin and with/without purified phospholipase A2 (control HDL, phospholipase A2 HDL) before dilution in the cell culture medium. Average phosphatidylcholine (PC) degradation was 62.10% +/- 2.57% (range 45-80%). A purified lipase/phospholipase A1 from guinea pig pancreas was used in some experiments (range of PC hydrolysis: 16-70%). (1) 3H/14C-labelled unesterified cholesterol and 3H-labelled esterified cholesterol appeared in cells during 0-5 h incubations. Trypsin treatment allowed a simple adsorption of HDL onto the cell surface to be avoided, and most of the 3H-labelled esterified cholesterol transferred to cells was hydrolysed. Cell uptake of radioactive cholesterol increased as a function of HDL concentration but no saturation was achieved at the highest lipoprotein concentration used (200 micrograms cholesterol/ml). Flux of 3H/14C-labelled unesterified cholesterol was related to the cell cholesterol content, suggesting that it might partly represent an exchange process. The cell cholesterol content was slightly increased after 5 h incubation with HDL (+16%). (2) Pretreatment of HDL with purified phospholipase A2 doubled on average the amount of cell recovered 3H-labelled esterified cholesterol, while the flux of 3H/14C-labelled unesterified cholesterol was enhanced by 15-25%. Both transfer and cell hydrolysis of 3H-labelled esterified cholesterol were increased. A stimulation was also observed using purified lipase/phospholipase A1, provided that a threshold phospholipid degradation was achieved (between 27 and 45%). (3) Endothelial cells were conditioned in different media so as to modulate their charge in cholesterol. The uptake of 3H-labelled esterified cholesterol was found to be significantly higher in cholesterol-enriched cells compared to the sterol-depleted state. Finally, movements of 3H-labelled esterified cholesterol from HDL to endothelial cells were essentially unaffected by cell density or by the presence of partially purified cholesterol ester transfer protein. The possible roles of the transfer of HDL esterified cholesterol to endothelial cells and its modulation by phospholipases are discussed.

Biological Transport↗
PubMed1988

Glycerophosphocholine in seminal plasma of fertile and infertile men.

Glycerophosphocholine (GPC) was measured in seminal plasma from 65 fertile men, 276 infertile men and 10 men before and after vasectomy, using a new enzymatic method. Extra-epididymal excretion of GPC accounted for 30% of the total seminal levels of GPC. From a diagnostic point of view, GPC determination did not appear to be a specific tool which could discriminate between secretory and excretory azoospermia. Although the seminal content of GPC was related positively to the total sperm count in both fertile and infertile men, there was an inverse relationship between the level of GPC and sperm motility when considering classes displaying the same total sperm count. This was observed in all classes from infertile men as well as in fertile men with a total sperm count lower than 200 x 10(6) sperm/ejaculate. These results suggest a possible role of GPC in the regulation of human sperm motility, which warrants further investigation.

Glycerylphosphorylcholine↗
PubMed1988

[Effects of pentoxifylline and propentofylline on the turnover of polyphosphoinositides of the erythrocyte membrane and on the metabolism of arachidonic acid in platelets].

[gamma-32p] ATP incorporation into polyphosphoinositides of the erythrocyte membrane has been studied in the presence of increasing concentrations of pentoxifylline and propentofylline. Our results show an inhibitory effect on the labelling of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, higher with propentofylline. We have then demonstrated that these two drugs block the metabolism of arachidonic acid in platelets stimulated by thrombin. The inhibition is located at the first biochemical steps of platelet activation, probably on phospholipase C. These results are discussed in relation with the modifications of platelet cyclic AMP content.

Blood Platelets↗
PubMed1987

Broad spectrum anti-platelet activity of ticlopidine and PCR 4099 involves the suppression of the effects of released ADP.

Aggregation and serotonin secretion were studied in washed rat platelets after oral administration of ticlopidine or its more potent analog PCR 4099. Besides a complete suppression of the ADP-induced aggregation, the two drugs significantly inhibited aggregation and secretion induced by three protein kinase C activators (1-oleoyl-2-acetyl-sn-glycerol, OAG; 12-0-tetradecanoyl phorbol-13-acetate, TPA; phospholipase C), by the calcium ionophore A 23187 and by thrombin. The highest inhibition was observed at low stimuli concentrations but could be partly or almost completely overcome by increasing their concentrations. The combination of aspirin (ASA) with the ADP scavenging system, creatine phosphate/creatine phosphokinase (CP/CPK) in vitro resulted in an inhibition similar to that observed ex vivo after ticlopidine or PCR 4099 treatment. Moreover, these in vitro and ex vivo treatments were not additive. As identical results were obtained with CP/CPK alone but not with ASA, it is concluded that ticlopidine and PCR 4099 do not interfere with protein kinase C or calcium movements but specifically inhibit the effects of released ADP, which might explain the broad spectrum anti-platelet activity of these drugs.

Adenosine Diphosphate↗
PubMed1987

Isolation of two 67 kDa calcium-binding proteins from pig lung differing in affinity for phospholipids and in anti-phospholipase A2 activity.

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholipase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.

Amino Acids↗
PubMed1987

Inhibition of the metabolism of platelet activating factor (PAF-acether) by three specific antagonists from Ginkgo biloba.

Washed rabbit platelet suspensions were incubated in the presence of 1-[3H]O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H] PAF-acether), which was metabolized into 1-[3H]O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) through the sequential action of cytosolic acetylhydrolase and membrane transacylase. Within 60 min at 37 degrees, percentage of [3H] PAF-acether metabolized was 50.3 +/- 5.2% (9 experiments). This conversion was inhibited in a dose-dependent manner by various concentrations of ginkgolides A, B and C (BN 52020, 52021, 52022) known as specific antagonists of PAF-acether. The three compounds displayed the following order of potency: BN 52021 (IC50 = 3.6 X 10(-6) M) greater than BN 52020 (IC50 = 9.7 X 10(-6) M) greater than BN 52022 (IC50 = 37.6 X 10(-6) M). As this order is the same as that previously defined for inhibition of platelet aggregation to PAF-acether or for inhibition of PAF-acether binding to platelets, our data bring further support to the view that PAF-acether metabolism in platelets involves in some way its binding to its membrane receptor.

Animals↗
PubMed1987

Phospholipase C from human sperm specific for phosphoinositides.

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.

Humans↗
PubMed1987

Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187.

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.

Acrosome↗
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↗
PubMed1987

Lipocortin-like anti-phospholipase A2 activity of endonexin.

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X-100/calcium; chromatography on DEAF-TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide-immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [3H]oleic acid-labelled Escherichia coli membranes as substrate, endonexin inhibited phospholipase A2 from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium-dependent manner.

Amino Acid Sequence↗
PubMed1987

Pathways of arachidonic acid liberation in thrombin and calcium ionophore A23187-stimulated human endothelial cells: respective roles of phospholipids and triacylglycerol and evidence for diacylglycerol generation from phosphatidylcholine.

Cultured endothelial cells from human umbilical vein were incubated for 20 h at 37 degrees C in the presence of [U-14C]arachidonic acid. Around 60-70% of the radioactive fatty acid was incorporated into cell lipids and was predominantly found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerol (39%, 33%, 13% and 6.5% of total incorporated radioactivity, respectively). Stimulation of the cells with human thrombin (2 U/ml) or calcium ionophore A23187 (5 microM) promoted the release into supernatants of arachidonic acid, 6-ketoprostaglandin F1 alpha, prostaglandins E2 and F2 alpha, in decreasing order of importance. The amount of secreted material was 4-fold higher with A23187, compared to thrombin. Parallel to the liberation process, phosphatidylcholine underwent a rapid decrease of radioactivity with both agonists, suggesting the involvement of a Ca2+-dependent phospholipase A2. Phosphatidylethanolamine displayed a minor decrease with A23187, whereas some reacylation was observed at 10 min with thrombin. Phosphatidylinositol was non-significantly affected in thrombin-stimulated cells, whereas A23187 promoted an early but minor decrease, followed by resynthesis. In contrast to A23187, thrombin was also able to promote a significant hydrolysis of triacylglycerol, which might thus be implicated in the process of arachidonate liberation. Finally, radioactive phosphatidic acid and diacylglycerol appeared in endothelial cells, in response to the two agonists. However, diacylglycerol formation did not parallel that of phosphatidic acid, especially with A23187. Determination of the 14C/3H ratio of the different lipids upon cell labelling with both [14C]arachidonic acid and [3H]palmitic acid revealed that diacylglycerol and phosphatidic acid are hardly derived from inositol-phospholipid breakdown by phospholipase C. Other possible pathways involving for instance phospholipase C splitting of phosphatidylcholine are discussed.

Arachidonic Acid↗
PubMed1987

Effect of BN 52021, a specific antagonist of platelet activating factor (PAF-acether), on calcium movements and phosphatidic acid production induced by PAF-acether in human platelets.

32P-labelled human platelets loaded with quin 2 and pretreated with aspirin were stimulated with 1-100 nM platelet activating factor (PAF-acether or 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in a medium containing the ADP-scavenging system creatine phosphate/creatine phosphokinase. Under these conditions, PAF-acether evoked a characteristic fluorescence change allowing to quantify elevations in cytoplasmic free Ca2+ from internal stores (Ca2+ mobilization) or from external medium (Ca2+ influx), as well as an increased production of phosphatidic acid, reflecting phospholipase C activation. These effects, which can be attributed to PAF-acether only and not to released products such as ADP or thromboxane A2, were strongly inhibited in a dose-dependent manner by BN 52021, a specific antagonist of PAF-acether isolated from Ginkgo biloba. As the drug remained inactive against the same effects elicited by thrombin, it is concluded that BN 52021 does not interfere directly with the mechanism of transmembrane signalling involving inositol-phospholipids or (and) some putative receptor-operated channels, but rather acts on the binding of PAF-acether to its presumed membrane receptor.

Aminoquinolines↗