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Biomedical subjects

O B Tysnes

Publications and source records attributed to O B Tysnes.

48 records · Page 3Linked to original sources
PubMed1988

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets.

Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the gamma-phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20-fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP gamma-phosphate. Based on constant rates of synthesis and removal, this thrombin-induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min-1/10(11) cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.

Adenosine Triphosphate↗
PubMed1987

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets.

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.

Blood Platelets↗
PubMed1987

Turnover of the phosphomonoester groups of polyphosphoinositol lipids in unstimulated human platelets.

The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by short-term labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] had the same specific 32P radioactivity as the gamma-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4-13% compared to that of the ATP-gamma-phosphate. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)P2, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)P2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)P2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the gamma-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.

Adenosine Diphosphate↗
PubMed1987

Phosphoinositide metabolism in resting and thrombin-stimulated human platelets. Evidence against metabolic heterogeneity.

The specific radioactivity of the phosphodiester and phosphomonoester moieties of the phosphoinositides was determined in resting and thrombin-stimulated platelets that were prelabelled with [32P] Pi. In the unstimulated cells, the specific radioactivity of the monoester phosphates of PIP and PIP2 was similar. Both prolonged incubation at 37 degrees C and upon stimulation with 0.5 U/ml of thrombin, the specific radioactivity of the monoester phosphates decreased 10-15% in both polyphosphoinositides. In the unstimulated cells, the specific radioactivity of the diester phosphates was similar in PI, PIP and PIP2 but amounted only to 3% of the activity of the monoester groups. Prolonged incubation of the unstimulated cells as well as stimulation with thrombin induced a similar 5-6 fold increase in specific radioactivity of the diester phosphate of PI, PIP and PIP2. The results indicate that the phosphoinositides in both resting and thrombin-stimulated platelets exist in a metabolically homogeneous pool.

Adenosine Triphosphate↗
PubMed1987

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides.

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.

Adenosine Triphosphate↗
PubMed1987

Neomycin inhibits agonist-stimulated polyphosphoinositide metabolism and responses in human platelets.

The effect of neomycin on agonist-induced changes in polyphosphoinositide metabolism was studied in human platelets. Neomycin induced no changes in the 32P-labeled phospholipids of unstimulated cells. Between 1 and 5 mM of neomycin the initial thrombin-induced decrease in [32P]phosphatidylinositol-4,5-bis-phosphate and production of [32P]phosphatidic acid were gradually inhibited. In contrast, the production of [32P]phosphatidylinositol-4-phosphate was increased. The thrombin-induced decrease in mass of phosphatidylinositol was completely inhibited at 5 mM of neomycin. Collagen- and PAF acether-induced changes in platelet phosphoinositide metabolism as well as aggregation and dense granule secretion induced by all three agonists were inhibited by neomycin. The results indicate that neomycin inhibit platelet responses by selective interference with the interconversions and hydrolysis of polyphosphoinositides upon thrombin stimulation.

Blood Platelets↗
PubMed1986

Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets.

Human platelets were pulse-labelled with [32P]Pi and extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. Immediately after pulse-labelling, the specific 32P radioactivity of phosphatidylinositol (PI) was only 3.4% of that of the gamma-phosphoryl of ATP. Upon incubation of the platelets at 37 degrees C, the specific 32P radioactivity of ATP (beta- and gamma-phosphoryls) remained constant. However, specific 32P radioactivity in PI increased continuously to 17% of specific [gamma-32P]ATP at 90 min of incubation. Stimulation with 0.5 U/ml of thrombin induced a 35% decrease in mass of PI which was unaffected by the time after the pulse-labelling. In contrast, the thrombin-induced changes in [32P]PI differed markedly at the various times after the [32P]Pi-pulse. Immediately after pulse-labelling, [32P]PI initially decreased but increased thereafter to 260% of control values after 180 s. With increasing specific 32P-radioactivity in PI before stimulation, the thrombin-induced increase in [32P]PI gradually disappeared. After 90 min of incubation, thrombin induced a continuous decrease in [32P]PI that almost parallelled mass. The data are explained by an initial breakdown of PI to diacylglycerol through the PI cycle or the polyphosphoinositide cycle, followed by resynthesis of PI through phosphatidic acid. In contrast to pre-existing PI, the resynthesized PI is in full isotopic equilibrium with ATP. This allowed us to estimate that 14% of the PI that is consumed between 30 and 180 s of stimulation, is recycles. From our data we calculate that the rate of PI resynthesis increased from 2.4 to 20 nmol/min per 10(11) cells upon thrombin stimulation of platelets.

Blood Platelets↗
PubMed1985

Thin-layer chromatography of polyphosphoinositides from platelet extracts: interference by an unknown phospholipid.

Different ratios of radioactive polyphosphoinositides in platelets pulse-labelled with 32p-orthophosphate have been reported by various laboratories. We studied whether these differences originate from differences in methodology. Extracts of 32p-Pi labelled human platelets were prepared at various times after gel-filtration and phosphatidylinositol (PI)-, mono (PIP)- and bisphosphate (PIP2) were separated by thin-layer chromatography using four different solvent systems. The 32p-levels in PIP and PIP2 remained constant during one hour after gel-filtration, whereas 32p-PI increased continuously and more than doubled within the first h. In two of the systems PIP co-chromatographed with a radioactive compound which separated well from PIP in the two other systems. This unknown compound was also labelled with 3H-glycerol, 3H-inositol and 3H-arachidonic acid, but it was metabolically and functionally different from the polyphosphoinositides. Both the co-chromatography of this unknown phospholipid and the increase in 32p-PI in gel-filtered platelets can explain the difference in 32p-labelling in phosphoinositides reported in the literature.

Blood Platelets↗
PubMedDate not supplied

Combined effect of alkyl-lysophospholipid and vincristine on proliferation, migration and invasion in human glioma cell lines in vitro.

The aim of the present study was to investigate the combined effect of the alkyl-lysophospholipid Et-18-0-CH3 (ALP) and vincristine (VCR) on the proliferative, migratory and invasive properties of human glioma cells. Both drugs have previously been shown to act upon the locomotive apparatus of the cell. In the glioma cell lines D37MG and GaMG we found that ALP and VCR inhibited proliferation in monolayer growth as well as in multicellular spheroids in a dose-dependent manner. When combined, the two drugs had a significantly enhanced effect upon proliferation in the cell line GaMG but not in D37MG. Migration was inhibited by ALP and VCR in a dose-dependent manner in both cell lines. An enhanced effect by combining the drugs was also observed. The invasion of glioma cells into the brain cell aggregates was markedly inhibited at lower concentrations than needed for the inhibition of proliferation and migration when ALP and VCR were applied alone, without any further enhancement by combining the drugs. It is concluded that the combination of ALP and VCR have additive anti-proliferative and anti-migratory properties in human glioma cell lines.

Antineoplastic Agents↗
PubMedDate not supplied

Differential effects of 12-O-tetradecanoyl-13-phorbol acetate (TPA) on growth, migration and invasion of a human glioma cell line.

To evaluate the effects of protein kinase C (PKC) activation on a human glioma cell line (GaMG), this study examined the effects of the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA) on the tumor properties proliferation, migration and invasion. GaMG monolayer growth was reduced by 30% (p < 0.05) in the presence of TPA and, concomitantly, cells accumulated in the G2M phase of the cell cycle as determined by flow-cytometric analyses. In studies of directional migration of cells from GaMG spheroids, low concentrations of TPA increased the area of migration by 60% (p < 0.005). The effects at higher concentrations of the phorbol ester were still present but less pronounced. Glioma invasion was assessed with a glioma spheroid/brain aggregate co-culture technique and revealed TPA-induced enhancement of the invasive process (p < 0.005) at low concentrations of the phorbol ester. Down-regulation of PKC by passaging the cells in a TPA containing medium significantly decreased the migratory response to TPA. Moreover, the PKC inactive phorbol ester 4-alpha-phorbol-12,13-didecanoate had no effect on the migratory response of GaMG cells. It is concluded that metabolic pathways under the control of PKC may play an important role in the control of glioma growth, migration and invasion. The effects of PKC activation are probably dependent on the growth conditions. Studies on PKC activation under different culture conditions in three-dimensional systems are required to evaluate further the role of PKC in glioma biology.

Brain Neoplasms↗
PubMedDate not supplied

Adhesion and migration of human glioma cells are differently dependent on extracellular matrix molecules.

To analyse the interactions between glioma cells and extracellular matrix (ECM) proteins, the adhesive and migratory capacity of five human glioma cell lines (D37MG, D54MG, GaMG, U118MG and U251MG) were studied. The expression of integrins was analysed and correlated to the adhesive and migratory abilities of the cells. All cell lines were able to adhere to and migrate on the extracellular matrix proteins collagen IV, fibronectin, laminin and vitronectin. Laminin was superior in propagating adhesion and migration in all five cell lines. As analysed by flow cytometry, the expression of the integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 3, beta 4, alpha v, and integrin alpha v beta 5 proved to be uniform between the cell lines. All integrins except alpha 4, alpha 6, beta 3 and beta 4 were expressed on more than 85% of the cells. Inhibition of adhesion with synthetic peptides and antibodies directed against integrins demonstrated that adhesion on laminin was independent of integrins and the 67 kD laminin receptor. On the other hand, migration was shown to be integrin-dependent on all substrates. The results indicate that the mechanism responsible for cell adhesion in human gliomas differ from those present during migration, and that integrins play an important role in regulating these two mechanisms.

Cell Adhesion↗