Biomedical subjects

U Till

Publications and source records attributed to U Till.

At least 19 recordsLinked to original sources

PubMed2001

Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.

Aging↗

PubMed2000

Flow cytometric determination of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1 in formaldehyde-fixed endothelial cell monolayers.

BACKGROUND: Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS: TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS: HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS: The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.

Cell Membrane Permeability↗

PubMed1999

L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells.

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.

Antioxidants↗

PubMed1999

Nitric oxide inhibits proliferation of human endothelial cells via a mechanism independent of cGMP.

Endothelial cell-derived nitric oxide (NO) has been suggested to inhibit smooth muscle cell proliferation, resulting in the reduction of intimal hyperplasia during atherogenesis. The present study investigates the role of NO from exogenous and endogenous sources on the proliferation of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (CAEC). Three different NO-generating compounds [sodium nitroprusside (SNP), S-nitroso-glutathione (GSNO) and S-nitroso-acetylpenicillamine (SNAP)] were found to inhibit endothelial cell proliferation measured with three independent methods (cell counting, [3H]thymidine incorporation, DNA histograms) with significant inhibition occurring at concentrations > or = 100 microM. Growth-inhibiting effects were observed after long-term treatment (18-96 h) as well as after short stimulation with NO donors (10 min with a subsequent NO donor-free culture period of 18 h) and were comparable in culture medium (20% serum, growth factor supplementation) and serum-deficient medium (1% serum). The NO donor effects were mediated by the release of NO as they were prevented by NO scavenging. Superoxide dismutase (SOD) was found not to interfere with these effects suggesting that peroxynitrite formation was unlikely to be involved. 1H-[l,2,4]Oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ), a specific inhibitor of the soluble guanylate cyclase, was observed not to alter the antiproliferative effects of NO donors although it completely prevented NO-mediated increase of cyclic guanosine 3',5'-monophosphate (cGMP), suggesting that the NO-induced growth inhibition was not mediated by cGMP. Furthermore, inhibition of endogenous NO production by N-nitro-L-arginine methylester (L-NAME) did not affect endothelial cell growth regardless of using serum plus growth factor supplement, growth factor supplement alone, or thrombin to stimulate proliferation. We suggest that constitutively synthesized NO may not regulate endothelial cell proliferation whereas the growth-inhibiting NO effects may occur when an inducible NO synthase associated with a persistently high NO production is expressed in the atherosclerotic vessel wall.

Analysis of Variance↗

PubMed1999

Net mass transfer of plasma cholesteryl esters and lipid transfer proteins in normolipidemic patients with peripheral vascular disease.

The role of plasma cholesteryl ester transfer and lipid transfer proteins in atherosclerosis is unclear. Recent data suggest both antiatherogenic and atherogenic properties for cholesteryl ester transfer protein (CETP). The overall effect of CETP on atherosclerosis may thus vary depending on individual lipid metabolism. To test whether lipid transfer parameters are of importance even in patients without major lipid risk factors for atherosclerosis, CETP mass and activity, net mass transfer of cholesteryl esters between endogenous lipoproteins (CET), and phospholipid transfer protein (PLTP) activity were determined in plasma from 18 normolipidemic male patients with peripheral vascular disease and 21 controls. Furthermore, lecithin: cholesterol acyltransferase (LCAT) activity was tested. The results show that CETP mass, CETP activity, and LCAT activity are not different between patients and controls. However, specific CETP activity (CETP activity/CETP mass) is lower in the patients (P < .02). On the contrary, higher CET is observed in patients' plasma (P < .001). Increased plasma PLTP activity (P = .052) is demonstrable in the patients. If the data of all subjects are combined, CET correlates positively with triglycerides ([TG], r = .45, P < .001) and with PLTP activity (r = .32, P < .05) but negatively with specific CETP activity (r = -.37 P < .05). CET and specific CETP activity remain significantly different in TG-matched patients and controls and are more strongly interrelated (r = -.71, P < .001), suggesting a higher and selective influence of lipid transfer inhibitor(s) on CET and CETP activity in the patients. CET allows the best discrimination between patients and controls in univariate and multivariate analysis. Eighty-eight percent of the subjects are correctly classified by CET as a single parameter. The results suggest that increased CET in the patients may reflect atherogenic alterations in TG metabolism and/or in lipid transfer protein activities despite normal fasting lipoprotein levels.

Aged↗

PubMed1995

Activation of endothelial guanylate cyclase inhibits cellular reactivity.

The study shows that endothelial cells from human umbilical veins have a soluble guanylate cyclase which can be activated by sodium nitroprusside (SNP), SIN-1 (3-morpholinosydnonimine) and S35b (4-methyl-3-phenylsulfonylfuroxan). Cells which were pretreated with these compounds showed an inhibition of thrombin-induced arachidonic acid release, PGI2 formation, PAF synthesis and PMNL adhesion. Endothelial guanylate cyclase can also be activated by nitric oxide (NO) which is generated in endothelial cells upon stimulation with thrombin or ionomycin. It is suggested that endogenously produced NO might control cell activation and endothelial function through a cGMP-dependent mechanism.

Arachidonic Acid↗

PubMed1993

S35b, a new phenylsulfonylfuroxan compound, inhibits thrombin-induced synthesis of platelet-activating factor and prostacyclin in human endothelial cells.

Endothelial cells (EC) produce platelet activating factor (PAF) and prostacyclin (PGI2) in response to inflammatory agents such as thrombin. Upon cell stimulation a calcium-dependent phospholipase A2 (PLA2) is activated which hydrolyzes a membrane phospholipid to yield 1-0-alkyl-2-lyso-sn-glycero-3-phospho-choline (lyso-PAF) and free arachidonic acid. Lyso-PAF is in turn converted into PAF by a specific acetyltransferase and arachidonic acid is metabolized via cyclic endoperoxides to PGI2. In the present study we report that S35b (4-methyl-3-phenylsulfonylfuroxan), a new phenyl-sulfonylfuroxan compound with potent antiaggregatory effect, inhibits thrombin-induced PAF synthesis and acetyltransferase activation as well as PGI2 production in human umbilical vein endothelial cells (HUVEC) in a concentration-dependent way. Additionally, we show that S35b stimulates the production of cyclic GMP (cGMP) in HUVEC in a concentration- and time-dependent manner. At high concentration, S35b potentiates the cAMP increase induced by iloprost or forskolin without having a significant influence on cAMP level itself. Potentiation of cAMP increase during agonist-induced EC stimulation seems not to be important for the effect of S35b on cellular function as the compound is active in inhibiting PAF production when endothelial cells are pretreated with indomethacin to block PGI2 synthesis. The increase of cGMP evoked by S35b may account for the effect on endothelial cell function.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.

Acetates↗

PubMed1992

Nitrovasodilators inhibit thrombin-induced platelet-activating factor synthesis in human endothelial cells.

In response to inflammatory agents such as thrombin, cultured endothelial cells produce platelet-activating factor (PAF), which has been linked with most inflammatory and immune processes, and is a potent coronary constrictor. Sodium nitroprusside (SNP) and SIN-1 (3-morpholinosydnonimine), which spontaneously release the free radical nitric oxide (NO), cause direct relaxation of blood vessels and inhibition of platelet aggregation by activating soluble guanylate cyclase. In the present study we report that in human umbilical vein endothelial cells (HUVEC) these compounds stimulate the production of cGMP and inhibit thrombin-induced PAF synthesis in a concentration-dependent manner. 8-bromo-cGMP, a permeant non-hydrolysable analogue of cGMP, mimics the inhibitory effect of NO-generating vasodilators. PAF synthesis requires phospholipase A2-mediated hydrolysis of membrane precursors to lyso-PAF, which is in turn converted into PAF by an acetyltransferase. The thrombin-elicited activation of both enzymes is inhibited in a dose-dependent way in HUVEC pretreated with SNP and SIN-1. The inhibitory effect of SNP and SIN-1 on the thrombin-mediated PAF synthesis suggests a new mechanism of action whereby the endogenous NO can affect vascular tone and endothelium-dependent intercellular adhesion. Moreover, PAF production in endothelial cells appears to be an important target for the pharmacological action of nitrovasodilators.

Acetyltransferases↗

PubMed1992

Increase in plasma total and lipoprotein cholesterol during incubation of whole blood samples at 37 degrees C--influence of LCAT inhibitors.

Incubation of whole blood samples at 37 degrees C caused a time-dependent increase in plasma cholesterol concentrations. In samples from 40 fasting healthy males, plasma cholesterol rose by 13.6 +/- 3% during 24 h (P less than 0.001). Changes in cholesterol concentrations were found in both the HDL fraction and the VLDL/LDL fraction. The increase in lipoprotein cholesterol concentrations correlated positively with the initial levels of HDL cholesterol and apo A-I; and with the original levels of VLDL/LDL cholesterol, apo B and triglycerides. The increase in plasma total cholesterol was not related to the HDL cholesterol and apo A-I concentrations. It was more pronounced in samples with elevated plasma concentrations of total cholesterol, VLDL/LDL cholesterol, apo B and triglycerides. The elevation in plasma total cholesterol resulted from an increase in cholesteryl esters, whereas free cholesterol decreased. After LCAT inhibition no changes in total, free and esterified cholesterol were observed. Therefore, increase in plasma cholesterol seems to represent a LCAT-dependent cholesterol transport out of blood cells.

Adult↗

PubMed1992

Functional behaviour of mononuclear blood cells from patients with hypercholesterolemia.

Mononuclear cells were prepared from venous blood obtained from 20 patients with a newly diagnosed hypercholesterolemia and without clinical signs of vascular disease, and from 19 age and sex matched controls. Adhesiveness to plastic surface, phagocytic activity measured as ingestion of zymosan particles, and spontaneous motility of mononuclear cells from patients were significantly higher by 57%, 19% and 50%, respectively, when compared to controls. In controls chemotaxis induced by the chemotactic peptide FMLP was slightly higher than spontaneous motility measured in absence of FMLP, whereas in patients FMLP significantly inhibited cell motility by about 47%. With the exception of FMLP-induced chemotaxis the results indicate that mononuclear cells are hyperreactive in hypercholesterolemia.

Adult↗

PubMed1992

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. I. Human platelets.

The effect of halothane on the regulation of blood platelet free cytosolic calcium was investigated in Quin-2-loaded cells from patients susceptible to Malignant Hyperthermia (MH) and healthy controls, respectively. The resting level of free cytosolic calcium was slightly, but statistically significantly, enhanced in platelets from patients (90 +/- 10 nM vs 110 +/- 35 nM). Halothane induced a dose-dependent, rapid Ca2+ release from intracellular stores both in normal and in MH derived cells, but the resulting increase in cytosolic calcium was significantly higher in the latter (2 mM halothane: [Ca2+]i = 117 +/- 12 nM vs 218 +/- 117 nM; 4 mM halothane: 225 +/- 35 nM vs. 417 +/- 201 nM). Whereas in platelets from healthy donors a complete reversibility of the halothane effect could be observed within 30-45 min, the cytosolic Ca2+ transients in platelets from patients were different from those in normals either in a higher initial peak or in a diminished decline velocity or in both. The basal Ca2+ permeability of the platelet plasma membrane was very low. Generally, halothane caused a dose-dependent increase in Ca2+ permeability. However, the influx of external calcium was significantly higher in platelets from patients than in controls (2 mM halothane: delta [Ca2+]i = 69 +/- 12 nM vs 135 +/- 63 nM; 4 mM halothane: 127 +/- 33 nM vs. 258 +/- 111 nM). Combining the results, the suggestion can be made that susceptibility to MH is characterized by a generalized membrane defect.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1992

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. II. Pig platelets.

Since 1966 the domestic pig has served as the animal model in Malignant Hyperthermia (MH) research [1]. The use of genetically well-defined pigs rendered it possible to test the method for diagnosing MH-susceptibility of patients presented in the preceding paper. Thus, the effect of halothane on intracellular calcium movements was studied in Quin-2- and chlorotetracycline-loaded pig platelets. In 'Ca(2+)-free' suspensions the resting level of free cytosolic Ca2+ was about 60 nM. In contrast to the results with human platelets there were no significant differences between pig genotypes either in the absence or in the presence of external calcium. After addition of halothane, a mobilization of intracellular membrane-bound calcium can be observed. However, the calcium mobilization is not accompanied by a marked increase in fluorescence intensity of Quin-2-loaded platelets. Thus, in the absence of external calcium, halothane produces only a slight increase in free cytosolic Ca2+. Nevertheless, the calcium rises measured in platelets from affected animals were statistically significantly higher than those from normal subjects. However, in the presence of 1 mM external calcium, a rapid increase in free cytosolic calcium can be detected after halothane addition. This suggests that halothane causes a marked, dose-dependent increase in Ca2+ permeability of the plasma membrane. Compared to the control group, significantly enhanced calcium permeability was found, not only in homozygous positive pigs, but also in heterozygous animals.

Animals↗

PubMed1992

Ex vivo investigation of blood monocyte and platelet behaviour in pigs maintained on an atherogenic diet.

Domestic pigs aged 4 months were fed for 16 weeks an atherogenic diet rich in cholesterol and saturated fatty acid. The increase of plasma cholesterol and triacylglycerol levels was found to be accompanied by a significant increase in the number of blood monocytes and platelets when compared to control animals. Furthermore, the atherogenic diet produced a small but significant reduction in the blood monocyte phagocytic capacity and adhesion to plastic surface. No significant differences between both groups were found when spontaneous platelet aggregation in whole blood was studied. However, platelets from pigs fed the atherogenic diet had a smaller mean cell volume compared to controls. The results indicate than an atherogenic diet may affect blood monocytes and platelets in pigs.

Animals↗

PubMed1991

Stimulation of platelet-activating factor synthesis in human endothelial cells by activation of the de novo pathway. Phorbol 12-myristate 13-acetate activates 1-alkyl-2-lyso-sn-glycero-3-phosphate:acetyl-CoA acetyltransferase and dithiothreitol-insensitive 1-alkyl-2-acetyl-sn-glycerol:CDP-choline cholinephosphotransferase.

Human umbilical vein endothelial cells (HUVEC) produce platelet-activating factor (PAF) by a remodeling pathway involving a phospholipase A2 followed by an acetyl-CoA-dependent acetyltransferase which acetylates a lyso-PAF intermediate to form PAF and is stimulated by a variety of agents that generate inflammatory and allergic responses. A second route for PAF synthesis in mammalian tissues is a de novo pathway, which requires the participation of three enzymes: 1-alkyl-2-lyso-sn-glycero-3-phosphate (alkyllyso-GP): acetyl-CoA acetyltransferase, 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase, and dithiothreitol (DDT)-insensitive 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-cholinecholinephosphotransferase. In the present study we show that protein kinase C activation by phorbol 12-myristate 13-acetate (PMA) induces PAF production in HUVEC by an increase of both alkyllyso-GP:acetyl-CoA acetyltransferase and DTT-insensitive alkylacetyl-G:CDP-choline choline-phosphotransferase. PAF synthesis, labeled precursors [( 3H]acetate and [methyl-3H]choline) incorporation, and both enzyme activities of the de novo pathway increase concomitantly in response to different doses of PMA. PMA does not activate the enzymes of the remodeling pathway. We conclude that both remodeling and the de novo pathway for PAF synthesis are present in HUVEC and might be alternatively activated depending on the conditions of cell stimulation.

Acetates↗

PubMed1991

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells.

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.

Acetyltransferases↗

PubMed1991

Inhibition of PAF-induced human platelet responses by newly synthesized ether phospholipids.

A series of 10 analogues of platelet-activating factor (PAF) was evaluated for proaggregatory and inhibitory behaviour on human blood platelets in vitro. Most of the compounds did not activate platelets but inhibited the PAF-induced aggregation. The inhibition was concentration-dependent and selective for PAF. Platelet responses to ADP and collagen were not suppressed. Schild analysis of the aggregation data was consistent with a simple competitive antagonism. The pA2 of the most effective antagonist (1-O-hexadecyl-2-O-ethylglycero-3-phosphoric acid-6'-(1-chinuclidinium)-hexylester) was 5.96. There is also evidence for its ability to inhibit the high affinity binding of [3H]PAF to the platelet receptors.

Adenosine Diphosphate↗

PubMed1991

Measurements of the G-/F-actin equilibrium in ADP-stimulated human platelets.

The amounts of the different forms of actin (G-actin, F-actin) can be measured biochemically following lysis of the cells by the DNase I inhibition assay. The existing methodology for studying G-actin in unstimulated platelets was found to be inappropriate for studies during ADP-induced platelet activation. However, this problem was overcome by a simple modification of the procedure in which formaldehyde was added to the buffer used to lyse the activated platelets. Using this modification the G-actin values obtained immediately after lysis did not change during storage of the lysates on ice for more than 30 minutes. The results show rapid conversion of G-actin to F-actin in association with the shape change during ADP-stimulated activation. Examination of unstimulated platelets using the modified procedure enabled identification of a pool of actin that is rapidly dissociated to G-actin in the absence of formaldehyde, the existence of which had not previously been recognised.

Actins↗