Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.

Platelet activity and phosphoinositide turnover increase with advancing age.

PURPOSE: Blood platelet activity increases with advancing age. This study was designed to determine if changes in a key signal-transducing mechanism in the platelet, phosphoinositide turnover, are associated with the enhanced platelet activity seen in aging. PATIENTS AND METHODS: Platelets were harvested from a total of 40 healthy, non-obese, 22- to 62-year-old individuals, free of any clinical evidence of atherosclerotic vascular disease, and having normal serum laboratory lipid levels. Studies of platelet activity included measurement of in vitro platelet aggregation and plasma beta-thromboglobulin (beta-TBG), a marker of in vivo platelet secretion. Basal and thrombin-stimulated phosphoinositide turnover was measured following [32P]-orthophosphate incorporation into the various phospholipids, isolation of the phosphoinositides and phosphatidic acid by thin-layer chromatography and autoradiography, and quantification by liquid scintillation spectroscopy of these radiolabeled phospholipids. RESULTS: There was a positive correlation with age for both adenosine diphosphate (ADP)-induced aggregation (1.25 microM, r = 0.464, p less than 0.001; 2.5 microM, r = 0.386, p less than 0.05) and plasma beta-TBG (r = 0.381, p less than 0.055). There was a time-dependent increase of [32P]orthophosphate (32Pi) incorporation into platelet phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), and isotopic equilibrium was reached by 120 minutes at 37 degrees C. A positive correlation was found between age and basal 32P-PIP2 (r = 0.640, p less than 0.001) and 32P-PIP (r = 0.676, p less than 0.0005). Basal 32Pi incorporation into PIP2 correlated positively with in vitro aggregation (1.25 microM ADP, r = 0.795, p less than 0.0001; 2.5 microM ADP, r = 0.755, p less than 0.0005) as did 32Pi incorporation into PIP (1.25 microM ADP, r = 0.815, p less than 0.0001; 2.5 microM ADP, r = 0.795, p less than 0.0001). There was also a positive correlation between plasma beta-TBG levels and basal 32P-PIP2 (r = 0.768, p less than 0.005) and 32P-PIP (r = 0.505, p less than 0.066). Finally, increasing age correlated with thrombin (4 U/mL)-stimulated 32P-PIP2 hydrolysis (r = 0.694, p less than 0.01) and phosphatidic acid formation (r = 0.556, p less than 0.05).

Albumin-bound docosahexaenoic acid and collagen-induced human platelet reactivity.

An in vitro system designed to mimic the effect of various plasma nonesterified (polyunsaturated) fatty acids on platelet function and metabolism was employed. Human platelet aggregation induced by submaximal (1.8 micrograms/ml) collagen stimulation was significantly inhibited by 2 min preincubation with 20 microM albumin-bound docosahexaenoic acid (22:6n-3) (DHA), but not by the other fatty acids tested. [3H]Phosphatidic acid (PA) formation, an indicator of phospholipase C activation following platelet stimulation, was moderately inhibited by eicosapentaenoic acid (20:5n-3), 11,14,17-eicosatrienoic acid (20:3n-3), dihomo-gamma-linolenic acid (20:3n-6), as well as DHA, but not by arachidonic acid (20:4n-6); this inhibition of phospholipase C activation could not explain the differential effect of DHA on platelet aggregation. The decreased production of thromboxane A2 (TxA2), as assessed by [3H]12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) formation, may account for the inhibition of collagen-induced aggregation by 20 microM DHA. Surprisingly, preincubation with 40 microM albumin-bound DHA, even though resulting in greater inhibition of collagen-induced aggregation, had less impact on HHT formation. A small but significant increase in [3H]prostaglandin D2 (PGD2) levels following 3-min collagen stimulation may have contributed to the greater antiaggregatory effect of 40 muM DHA. It is concluded that increased plasma nonesterified DHA may contribute to the dampened platelet activation and altered metabolism following fish oil supplementation of the diet.

The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets.

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [3H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA-versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.

Fatty fish-induced changes in membrane lipid composition and viscosity of human erythrocyte suspensions.

The effect of a daily supplement of 75-100 g fatty fish, providing 3 g of omega 3 fatty acids, on the lipid composition and deformability of erythrocytes was studied in six healthy male subjects. After 4 weeks of the fish supplementation the mean degree of unsaturation, expressed as double bond index, of erythrocyte phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) had increased significantly by 10% and 4%, respectively. The mean cholesterol/phospholipid ratio (C/P ratio) in the membranes had increased by 28% (p less than 0.001). No change was seen in the distribution of the four major phospholipid classes. After withdrawal of the supplement for 4 weeks the changes in PC were reversed, but the changes in PE persisted and the mean C/P ratio still was higher than basal by 16% (p less than 0.01). The effect on the C/P ratio by the fatty fish is in contrast with the previously observed lack in effect of cod-liver oil. The viscosity of erythrocyte suspensions with a packed cell volume fraction of 0.80 and measured at shear rates less than or equal to 8.11 s-1 was decreased after the fish. We suggest that the fall in the viscosity of erythrocyte suspensions, reflecting increased cell deformability, is probably due to the change in the fatty acid composition of erythrocyte PC.

Phosphorus labeling of proteins and phospholipids in intact platelets in response to pH 5.3.

Previous studies had shown that when gel-filtered or washed human platelets were incubated at pH 5.3, the cells secreted their granule-stored materials suggesting that low pH can act as a platelet activator. We determined here whether the effects of low pH on platelet protein phosphorylation and on platelet lipid metabolism were consistent with this view. When washed human platelets were incubated for 20 min at pH 5.3 and electrophoresed on SDS-PAGE, there was a great increase in 32P-label in the 20,000 and 47,000 dalton protein bands. There was also an increase in the labeling of phosphatidic acid and a small decrease in phosphatidyl inositol. When the platelets were returned to pH 7.6, the 32P labeling of the 20,000 and 47,000 dalton bands was greatly reduced, and that of phosphatidic acid reduced to the control value, while the labeling of phosphatidyl inositol was increased above control. Incubation at pH 5.3 for 60 min gave the same pattern, but return to pH 7.6 resulted in only partial reversal of labeling of the two protein bands and little decrease in the label associated with phosphatidic acid, but the radioactivity in phosphatidyl inositol was greatly increased. The changes in the 32P-labeling of phospholipids and proteins after incubation of platelets at pH 5.3 may reflect an increase in cytoplasmic Ca++ resulting from leakage of Ca++ from intracellular storage sites, a process which becomes irreversible after longer time exposure to the low pH.(ABSTRACT TRUNCATED AT 250 WORDS)