Anthony D. Smith

Phytohaemagglutinin stimulation of human lymphocytes Effect of fatty acids on uridine uptake and phosphogylceride fatty acid profile

1. When added to cultures of human peripheral lymphocytes, saturated (palmitate, stearate, heptadecanoate) and unsaturated (oleate, linoleate, arachidonate) fatty acids bound to albumin at an acid-albumin ratio of 2:1, inhibited the phytohaemaegglutinin-stimulated uptake of [14C]-uridine. Uridine uptake in unstimulated cells was not affected by any of these fatty acids. 2. When saturated and unsaturated acids were present simultaneously in the incubation mixture the inhibit but relieved the inhibitory effects of both saturated and unsaturated fatty acids. 4. Stimulated and unstimulated cells incorporated exogenous fatty acids into membrane phosphoglycerides. Details of the fatty acid profiles are given. 5. Evidence is presented that the inhibition results, at least in part, from modification of phosphoglyceride fatty acid profile.

Haemolysis of intact human erythrocytes by purified cobra venom phospholipase A2 in the presence of albumin and Ca2

Abstract 1. 1. When a purified phospholipase A2 from Naja naja venom is incubated with washed intact human red blood cells, phosphoglyceride hydrolysis, but no haemolysis occurs. 2. 2. The addition of bovine albumin to the incubation mixture brings about haemolysis, which increases with increasing albumin concentration up to 40 mg/ml. 3. 3. Ca2+ is essential for phospholipase action, with maximal activity of 10 mM. Complete chelation of Ca2+ with EDTA is required after enzyme action if maximal haemolysis is to be achieved. 4. 4. At 0.125% albumin, 75% of the cleaved fatty acid was removed from the membrane without appreciable haemolysis. It is suggested that one function of the albumin in causing haemolysis is to remove lysophosphatides from the membrane.

Haemolysis of washed human red cells by the combined action of Naja naja phospholipase A2 and albumin

Abstract 1. 1. Cobra venom phospholipase A 2 was found to hydrolyse phospholipids in washed human erythrocytes without causing significant haemolysis in an isotonic medium. 2. 2. This indicates that phospholipids of the cell membrane are accessible to the enzyme action in isotonic buffer. 3. 3. Addition of 2% albumin to the incubation medium either prior to or subsequent to the enzyme action causes haemolysis without any further increase in phospholipid splitting.

Desaggregation of PAF-acether-aggregated platelets by verapamil and TMB-8 with reversal of phosphorylation of 40K and 20K proteins

Verapamil at a concentration of 10(-4) M inhibited aggregation and release of [3H]5HT induced by platelet activating factor (PAF-acether, PAF) in rabbit platelet-rich plasma and washed labelled platelets. When added to platelets previously aggregated by PAF-acether verapamil caused them to desaggregate at doses as low as 2 X 10(-6) M. The desaggregated platelets were refractory to further additions of similar doses of PAF-acether but could further be aggregated by A23187. Simultaneous to full aggregation PAF-acether caused phosphorylation of 40K and 20K proteins in particular. Addition of verapamil at the concentration of 2 X 10(-6) M to platelets already aggregated by PAF-acether resulted in dephosphorylation of 40K protein and reduction of phosphorylation of 20K protein to the level of control parallel to desaggregation. TMB-8 (10(-3) M) also caused desaggregation and reversal of phosphorylation of 40K and 20K proteins. When A23187 was added to verapamil desaggregated platelets, 40K and 20K proteins were rephosphorylated. The extracellular calcium antagonists EGTA or La3+, when added to PAF-acether aggregated platelets, did not abolish the phosphorylation of 40K and 20K proteins. The experiments suggest that inhibition of intracellular calcium-dependent reactions is involved in the desaggregatory action of verapamil.

The effect of fatty acids and of albumin on the action of a purified phospholipase A2 from cobra venom on synthetic lecithins

Abstract 1. 1. A study was made of the inhibition of a purified preparation of Naja naja phospholipase A 2 by fatty acids, and of the effect of albumin on the enzyme in the presence and absence of fatty acids. 2. 2. It was confirmed that long chain fatty acids (palmitate, oleate, linoleate) cause inhibition at low concentrations (less than 0.1 mM). The degree of inhibition was dependent on the concentration of substrate. A relationship was found between the percentage inhibition and the inhibitor/substrate ratio. It was concluded that inhibition resulted, at least in part, from interaction of the fatty acid with substrate micelles. 3. 3. The enzyme hydrolysed dipalmitoyllecithin faster than dioleoyllecithin or dilinoleoyllecithin. Palmitate, oleate and linoleate inhibited in an order of effectiveness which was dependent on the substrate used. 4. 4. Bovine plasma albumin activated the enzyme in the absence of added fatty acid. At high concentrations it prevented inhibition by low concentrations of fatty acid. In the absence of added fatty acid it activated at concentrations so low (5 μg/ml) that it is unlikely that the activation resulted solely from the binding of fatty acid released by the enzyme action.

Influence of Dietary Lipids on the Effect of Chlorpromazine on Membrane Properties of Rabbit Red Cells

The effect of diets containing different types of common natural oils on physical properties of red cells was investigated by using rabbits. The rabbits were fed for 18 months on a standard diet in which 8% of its energy content was provided by safflower oil and 32% energy by either more safflower oil or fish oil, linseed oil, olive oil or palm oil. Erythrocyte deformability was significantly decreased by the fish oil diet compared with each of the other diets. Osmotic fragility was significantly less (66 mM) for red cells from rabbits fed on the linseed oil diet, and significantly greater (71 mM) for red cells from rabbits on the fish oil diet, than for red cells from rabbits on the other three diets which did not differ significantly from each other (68 mM). With rabbits on the standard diet, the resistance of their erythrocytes to osmotic haemolysis was increased by chlorpromazine at concentrations below and decreased by concentrations above 30 μM. The dietary oils caused significant changes in the effects of chlorpromazine on osmotic fragility. The concentration at which the effect of chlorpromazine reversed from antihaemolytic to prohaemolytic was decreased by the safflower and linseed oil diets and increased by the fish oil diet, compared with the olive and palm oil diets. Analysis of the fatty acid compositions of the dietary oils on the one hand and of the red cell phospholipids on the other established, specifically, that in the presence of 30 μM chlorpromazine the percentage haemolysis was directly proportional to the linoleate content of the red cell phospholipids.

Two species of phospholipase C isolated from lymphocytes produce specific ratios of inositol phosphate products

Chromatography of the soluble porcine lymphocyte phospholipase C on cellulose phosphate resolves the activity into two species. An HPLC method is described for separating the enzyme products. Ins 1,2 > P and Ins 1P. Use of these methods reveals that the two iso‐enzymes liberate, with a high degree of reproducibility, characteristic ratios of the two products. This suggests that the amount of each product produced is an inherent property of the enzyme mechanism.

Unsaturated fatty acids stimulate the formation of lipoxygenase and cyclooxygenase products in rat spleen lymphocytes

Rat spleen lymphocytes prelabelled with [14C] arachidonate were suspended in fresh medium in the presence or absence of exogenous non-radioactive fatty acids added in ethanol. It was found that fatty acids stimulate thromboxane B2, PGE2, HHT and HETE formation. The effect is specific for unsaturated fatty acids. It occurs at 50 micron concentrations and is apparent after 20 minutes incubation. Unsaturated fatty acids may serve an important role in the regulation of prostaglandin and hydroxy fatty acid metabolism in vivo. This may indicate a mechanism for the action of fatty acids on the immune response.

Accumulation of diacylglycerol induced in platelets by exogenous fatty acids

Abstract Free fatty acids added in ethanol to human platelets prelabelled with [ 14 C ]arachidonate induce an accumulation of radioactive diacylglycerol. Unsaturated fatty acids are ten times more potent than palmitate. Ethanol alone does not alter the distribution of radioactivity. Increasing the concentration of arachidonate leads to increased diacylglycerol formation. The fatty acid effect is independent of thrombin, which itself causes a relatively small change in diacylglycerol levels. Neither the labelled triacylglycerol nor the labelled free fatty acid appears to be the source of the diacylglycerol formed which may arise from the activation of phosphatidylinositol phosphodiesterase.

Formation of diacylglycerol and degradation of phosphat1dylinositol induced in rat lymphocytes by non-esterified oleic or linoleic acid

Rat spleen lymphocytes were incubated for 3 h with [14C]arachidonic acid in foetal calf serum. It was found that arachidonic acid distributed into phospholipids in the order phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol. After labelling with arachidonic acid the lymphocytes were washed, and incubated for up to 2 h with non-radioactive palmitic, oleic or linoleic acid dissolved in ethanol. The presence of ethanol or palmitic acid during a 2 h post-incubation had little effect on the amount of radioactivity found in different lipid fractions. Both oleic acid and linoleic acid, however, brought about an accumulation (up to 8-fold) of radioactivity in the diacylglycerol fraction. These fatty acids also brought about a change of radioactivity in several phospholipids, notably in phosphatidylinositol, which lost more than 50% of its counts during the 2 h incubation. Although maximum effects were seen at 2 h, diacylglycerol radioactivity was increased by 100% within 5 min after adding the fatty acids. The minimum concentration of fatty acids used (50 microM) gave an almost maximum response. The results indicate that unsaturated fatty acids may activate phosphatidylinositol phosphodiesterase in lymphocytes, as they do in brain. The possibility that a phospholipase A is activated is discussed. Possible implications for any experiments in which cells are incubated with fatty acids are pointed out.