Masaru Takahashi

Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca2+ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca2+ ionophore, appeared to parallel the decrease in Ca2+ uptake activity. Phospholipase A2 activity, monitored by the production of [3H]lysophosphatidylcholine and [3H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca2+ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca2+ content.

Secreted Candida albicans phospholipases: purification and characterization of two forms of lysophospholipase-transacrylase

Candida albicans secreted three types of phospholipase (lysophospholipase, lysophospholipase-transacylase and phospholipase B) at different rates in culture. Clinical isolates of C. albicans showed variable activities of these phospholipases. Two forms of lysophospholipase-transacylase (LPTA) were purified to homogeneity from the culture filtrate of C. albicans 3125. The two purified enzymes, designated LPTA-I and LPTA-II, showed some differences in molecular mass (81 kDa for LPTA-I and 41 kDa for LPTA-II), amino acid composition and enzymatic properties. Antibody raised against purified C. albicans LPTA-II reacted strongly with LPTA-II, but not with LPTA-I. Furthermore, the biochemical properties of C. albicans lysophospholipase-transacylase were distinct from those of the corresponding mammalian enzyme.

Purification and characterization of lysophospholipase-transacylase of pathogenic fungus Candida albicans

A lysophospholipase-transacylase was purified to homogeneity from the culture broth of Candida albicans by ammonium sulfate precipitation and chromatographs on DEAE-cellulose, Ultrogel AcA-44, first Mono Q, hydroxyapatite, TSKgel-3000 and second Mono Q columns. The purified protein was a single band (Mr 41,000) as inferred by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a specific activity of 78 mumol/min per mg protein for fatty acid release and 320 mumol/min per mg protein for phosphatidylcholine formation. Fatty acid release obeyed Michaelis-Menten kinetics and the apparent Km was 76 microM of 1-palmitoyl-sn-glycero-3-phosphatidylcholine, but Lineweaver-Burk plots of transacylase activity was parabolic. The ratio of hydrolase to transacylase activity of the purified enzyme was varied depending upon the concentration of lysophosphatidylcholine. Transacylation was prominent at high concentration of substrate and the ratio of hydrolase to transacylase was 0.24. Low concentration of palmitoylcarnitine (50 microM) inhibited markedly phosphatidylcholine formation but stimulated fatty acid release. The degree of esterification of 1-acyllysophosphatidylcholine was altered with mixtures of different molecular species of substrate, demonstrating acyl chain selectivity in the transfer process. These results suggest that C. albicans lysophospholipase-transacylase is different from the corresponding mammalian enzymes in enzymatic properties.

Effects of dibutyryl cyclic AMP and forskolin on phospholipid biosynthesis in thrombin-stimulated human platelets.

The influence of forskolin, an adenylate cyclase activator, and of dibutyryl cyclic AMP (Bt2cAMP) on [3H]glycerol incorporation into glycerolipids was investigated in human platelets. It was found that preincubation with 2.5 mM Bt2cAMP produced a 2-4-fold increase in thrombin-induced incorporation into phospholipids compared to platelets activated by thrombin alone. Pretreatment with forskolin, which increased cellular cAMP content, also resulted in an increase in thrombin-stimulated [3H]glycerol incorporation into phospholipids. These findings demonstrate that a rise in platelet cAMP can accentuate thrombin-induced de novo synthesis of phospholipids from [3H]glycerol. Since the content of cellular cAMP was correlated with its ability to inhibit platelet activation monitored by serotonin release, it seems likely that glycerolipid, in particular phospholipid biosynthesis, is involved in controlling platelet activation by thrombin.

Phospholipid metabolism in human platelets preserved at 22 °C: Differential effects of storage on phospholipase A2- and C-mediated reactions

The effects of preservation at 22 degrees C on phospholipid metabolism were studied in human platelets. Stimulation of fresh platelets with thrombin caused a rapid and transient rise of 1,2-diacylglycerol (DG) which was derived from phosphatidylinositol (PI) by its strictly specific phospholipase C. Lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) were also accumulated as a result of the action of phospholipase A2. No significant changes in phospholipid metabolism were detected in platelets preserved at 22 degrees C up to 6 hr. However, platelets stored for more than 12 hr showed (1) an accumulation of both lysoPC and lysoPE before thrombin activation, (2) a subsequent decrease in the formation of lysoPC and lysoPE after thrombin activation when compared to fresh platelets, (3) a threefold lower rate of liberation of arachidonic acid than fresh platelets after activation, and (4) a lower rate and extent of aggregation than fresh platelets. Nevertheless, the amount of 1,2-DG produced during preservation up to 48 hr was similar to that observed in fresh platelets. The results indicate that the markedly enhanced activity of phospholipase A2, but not phospholipase C, that occurs during platelet storage leads to the deterioration of aggregation and arachidonic acid liberation in response to thrombin.

Phospholipid and fatty acid composition of erythrocyte membrane from wild Japanese serow (Capricornis crispus).

The phospholipid composition and fatty acid composition of the individual phospholipids were determined in erythrocyte membrane of wild Japanese serow, Capricornis crispus, and compared with those of Japanese cattle. Sphingomyelin (SM) contributed more than 50% to the total phospholipids, with only 3% phosphatidylcholine, 30% phosphatidylethanolamine and 11% phosphatidylserine. This phospholipid composition and ratio of phospholipid to protein in erythrocyte membrane of wild serow were quite similar to those of Japanese cattle. However, marked differences in fatty acid composition were found, especially in lignoceric acid 24:0 and nervonic acid 24:1 of sphingomyelin which were major constituents (approximately 60%) of that phospholipid.

Erythrocyte membranes in biliary obstruction: phospholipase A2-induced alterations in lipid composition and morphology.

Abstract Without significant hemolysis, phospholipase A2 (N. naja) hydrolyzed lecithin of the outer monolayer of erythrocytes from control subjects and patients with biliary obstruction. The hydrolytic treatment with this enzyme induced more prominent alterations in the morphology of red blood cells in cholestasis—crenation of red blood cells—compared to control cells. Phospholipase A2 hydrolyzed 20 and 40% of erythrocyte membrane lecithin from control subjects and patients with biliary obstruction, respectively. The hydrolysis of lecithin by this enzyme revealed that 85 to 90% of released free fatty acids were unsaturated ones whereas the lysolecithin acyl chain formed was found to be composed mostly of saturated fatty acids (85%). Freeze-fracture electron microscopy showed that the treatment of erythrocyte membranes with polyene antibiotic filipin produced more than twice as many filipin-cholesterol complexes in cholestatic cells as in control cells. The electron spin resonance studies on intact red blood cells, extracted membrane total lipids and phospholipids showed that the red blood cells in cholestasis were more fluid than those in control subjects. The increased susceptibility of erythrocyte membranes in cholestasis to phospholipase A2 is discussed on the basis of physicochemical properties of red cells.