Determination of platelet-activating factor by a chemiluminescence method and its application to stimulated guinea pig neutrophils

A simple, rapid and sensitive chemiluminescence method has been developed to measure platelet-activating factor (PAF). Hydrogen peroxide generated from PAF, upon phospholipase D cleavage, by choline oxidase is determined as chemiluminescence by a luminol-microperoxidase system. The detection limit of PAF by this method is 5 pmol/tube. The method is reproducible with a 5.5% coefficient of variation at 10 pmol of PAF (n=5). Lipids were extracted from guinea pig neutrophils after stimulation with cytochalasin B andN-formyl-methionyl-leucylphenylalanine, and PAF was isolated by high-performance liquid chromatography and determined by chemiluminescence measurements. The amount of PAF detected was 96.1±39.7 (mean ± SD, n=7) pmol/10⁸ cells. This highly sensitive method could be useful for the determination of PAF generated under pathophysiological conditions. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Regulation of the biosynthesis of platelet-activating factor in alveolar macrophages

Activities of enzymes which metabolize lysoplatelet-activating factor (lysoPAF) and platelet-activating factor (PAF) were studied in rabbit alveolar macrophage lysates. Substantial acetyltransferase activity was noted in the presence of 100 μM acetyl-coenzyme A (CoA), and this activity was increased in A23187-stimulated cell lysate. On the other hand, in the absence of exogenous acetyl-CoA, lysoPAF was mainly acylated through a transacylation pathway rather than by acetyltransferase in both control and A23187-stimulated cell lysates. We confirmed that the intracellular concentration of acetyl-CoA is relatively low. The observations suggest that the transacylation system may play an equally important role in the regulation of the availability of lysoPAF in intact cells. Intracellular lysoPAF was also maintained at relatively low levels. Interestingly, large amounts of PAF were produced even in unstimulated cells upon addition of an excess of exogenous lysoPAF, suggesting that generation of an adequate amount of lysoPAF within cells may be sufficient to trigger PAF synthesis in this type of cells. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-[1-¹⁴C]arachidonoyl PC or 1-acyl-2-[1-¹⁴C]linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-[1-¹⁴C]linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label being transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-[1-¹⁴C]arachidonoyl PC released¹⁴C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca²⁺-ionophore A₂₃₁₈₇. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.     

The response of peritoneal macrophages to dapsone covalently attached on the surface of carbon nanotubes

Dapsone is an anti-microbial and anti-inflammatory drug. Water-dispersible dapsone-modified multi-wall carbon nanotubes (dap-MWCNTs) were prepared by chemical modification of the carboxyl groups introduced on the surface of the nanotubes using O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (N-HATU) and N,N-diisopropylethylamine (DIEA). The modification was confirmed by Fourier-transform infrared spectroscopy, transmission election microscopy and thermogravimetric analysis. The biological effect of dap-MWCNTs was tested using rat peritoneal macrophages (PMØ). By confocal laser microscopy and flow cytometry, it was shown that the dap-MWCNTs were rapidly ingested by PMØ as were the control, oxidized o-MWCNTs. Neither dap-MWCNTs at lower concentrations (up to 50 μg/ml), nor o-MWCNTs, at equivalent concentrations, respectively affected the viability of PMØ, while higher concentrations triggered apoptosis. Apoptosis of PMØ induced by the control, o-MWCNTs, was higher than that induced by dap-MWCNTs and it correlated with the induction of oxidative stress. In contrast, dap-MWCNTs did not trigger oxidative stress but caused apoptosis of PMØ predominantly after prolonged cultivation (3 days). Although equivalent concentrations of soluble dapsone induced oxidative stress, they were anti-apoptotic. Cumulatively, the obtained results show the complexity of dap-MWCNT/PMØ interactions and suggest that this complex could be investigated for the treatment of dapsone-sensitive intracellular microorganisms or inflammatory diseases responding to dapsone therapy.     

Cardiovascular effects of platelet-activating factor

Sudden release of platelet-activating factor (PAF) into the circulation can cause hypotension, tachycardia, and circulatory collapse. To further examine this response, we performed detailed studies of cardiovascular function after PAF administration to young domestic pigs and newborn piglets. Our results indicate that circulatory dysfunction after PAF reflects severe constriction of pulmonary resistance vessels and consequent acute right ventricular failure. Although PAF-induced coronary artery constriction and contractile depression may be complicating problems, left ventricular underperfusion and dysfunction after PAF are mainly the result of systemic arterial hypotension and diminished left ventricular filling. The adverse hemodynamic effects of PAF are accompanied by substantial release of thromboxane A₂ (TxA₂). These effects are mimicked by the TxA₂ agonist U-46619 and partially blocked by specific and nonspecific inhibitors of TxA₂ synthesis (OKY-046 and indomethacin). Even more potent blockade of PAF action is exerted by the TxA₂ receptor blocker, SQ 29,548. Taken together, these findings indicate that severe pulmonary vascular constriction and hemodynamic collapse soon after intravenous PAF are at least partially mediated by PAF-induced TxA₂ release. Tachyphylaxis to PAF influence has been observed in studies of leukocyte and platelet function. We hypothesized that tachyphylaxis to PAF might also occur in our studies of constrictor responses in pulmonary vessels. Recently, we have examined the capacity of PAF to produce sustained pulmonary vasoconstriction in openchested, anesthetized newborn piglets. Infusions sufficient to produce 100% increase in mean pulmonary artery pressure after 3 min showed no loss of efficacy when sustained for 30 min. The same was true for infusions of U-46619. Thus, the pulmonary vasoconstrictor influence of PAF or U-46619 is not readily diminished by tachyphylaxis. These findings favor the viewpoint that PAF or TxA₂ release during inflammatory processes could have prolonged adverse actions on pulmonary and systemic circulations. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989. The opinions or assertions contained here are those of the authors. They do not reflect the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported here were conducted according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, U.S. Department of Health and Human Services (NIH Publ. 85-23, 1985).     

Significance of platelet-activating factor in mesenteric ischemia-reperfusion

Reperfusion of the ischemic mesenterium is frequently followed by acute circulatory collapse. This review focuses on the possible role of platelet-activating factor (PAF) in ischemia-induced damage. It provides evidence that (i) PAF concentrations are elevated in the mesenteric circulation following temporary ischemia; (ii) administration of exogenous PAF into the superior mesenteric vein mimics many events observed during reperfusion; and (iii) pretreatment of the experimental animals with specific PAF receptor antagonists prevent the circulatory collapse. These findings suggest that PAF may play an important role in the development of circulatory collapse caused by mesenteric ischemia-reperfusion. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Guinea pig bone marrow cells treated with platelet-activating factor generate factor(s) which affects their DNA synthesis and microbicidal activity

We have previously reported that platelet-activating factor (PAF) induces proliferation and microbicidal activity of guinea pig bone marrow cells. In the present study, we have found that the conditioned medium of PAF- or nonmetabolizable PAF agonist-treated guinea pig bone marrow cells augmented DNA synthesis and induced microbicial activity of bone marrow cells. A PAF specific antagonist, CV-6209, inhibited generation of the active conditioned medium by PAF. Addition of the PAF antagonist only partially suppressed the augmentative effect of the active conditioned medium on DNA synthesis; this is consistent with the fact that, because of the rapid breakdown, no appreciable amount of PAF remained in the conditioned medium of PAF-treated cells. Although mouse bone marrow cells did not respond to PAF unlike guinea pig cells, their DNA synthesis was significantly enhanced by the conditioned medium of PAF-treated guinea pig bone marrow cells. Thus, some newly generated factor(s) distinct from the originally inoculated PAF seemed to modulate the bioactions of PAF on bone marrow cells. An appreciable amount of PAF was produced by calcium ionophore-treated guinea pig bone marrow cells. These findings indicate that PAF synthesized in guinea pig bone marrow cells induces generation in the cells of some factor(s) which affects proliferation or microbicidal activity. Presented at The Third International Conference on Platelet-Activating Factor and Structurally Related Ether Lipids, Tokyo, Japan, May 1989.     

The contribution of platelet-activating factor to allergen-induced eosinophil infiltration and bronchial hyperresponsiveness

Platelet-activating factor (PAF) is an ether-linked phospholipid having a range of biological properties of relevance to our understanding of the pathogenesis of bronchial asthma and related allergic diseases. In particular the ability of PAF to induce eosinophil activation and recruitment has received considerable attention. Since in experimental animals PAF and allergen-induced eosinophil infiltration are dependent upon platelet activation, it is suggested that platelet activation may be an important component of the allergic process. The ability of PAF antagonists to inhibit various aspects of the allergic response has been demonstrated in a number of animal models but not all of them have been extended into man. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Involvement of platelet-activating factor in zymosan-induced rat pleurisy

The role of platelet-activating factor (PAF) in inflammatory reactions was studied in zymosan-induced rat pleurisy. Pleurisy was induced by injection of a 2% zymosan suspension into the pleural cavity of rats. The time course of pleural exudate accumulation, the exudation rate, and exudate leukocyte numbers were followed then for 96 hr. Peak pleural exudate accumulation was about 3 mL at 24 hr, whereas the exudation rate increased biphasically with peaks at 0.5 hr and 5 hr. The migration of leukocytes into the pleural cavity increased with time up to 48 hr. The polymorphonuclear leukocytes were the dominant white cells in the exudate between 5 and 16 hr, but mononuclear leukocytes started to outnumber them around 24 hr. Pretreatment with cyproheptadine (5 mg/kg), an inhibitor of both histamine and seotonin, significantly suppressed pleural fluid accumulation and the exudation rate at 0.5 hr. The PAF antagonist CV-6209 (1 mg/kg) significantly suppressed pleural fluid accumulation and the exudation rate at both 0.5 and 5 hr. At either time point, the parameters were not suppressed by indomethacin. We detected PAF activity in the high-performance liquid chromatography (HPLC) fraction (with a retention time corresponding to that of authentic PAF) of the exudates at 0.5 hr, 5 hr, and 16 hr using an aggregation bioassay with washed rabbit platelets. The results suggest that in zymosan-induced rat pleurisy, histamine and/or serotonin are the main mediators of exudation at 0.5 hr and that PAF may be partly responsible for exudation at 0.5 hr and later at 5 hr to 16 hr. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Presence of platelet-activating factor in pyuria in humans

The relationship between the occurrence of platelet-activating factor (PAF) and neutrophils in urine from patients with urinary tract infection was examined. PAF was detected in human pyuria, when leukocyte levels reached at least 300 cells/μL (n=45), but not in normal urine (n=12). The amount of PAF found in pyuria, measured by platelet aggregation assay, was 0.01 to 13.3 pmol/mL. A close correlation was seen between the amount of PAF present and the number of urinary leukocytes (p<0.01, r=0.70). The leukocytes in pyuria consisted almost entirely of neutrophils (96±4%, mean ±S.D.). Our findings suggest that the occurrence of PAF is associated with the accumulation of neutrophils in urine. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Phospholipid composition of guinea pig lung lavage

Phospholipids from guinea pig lung lavage were analyzed. The total lavage phospholipid content was 2.65+0.67 mg, per gram of lung, which accounted for 85% of the total lipids in lung wash. Phosphatidylcholine (PC) accounted for over 60% of the total phospholipids. The other phospholipids factions, in order of pedominance, were phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SPH), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and lysophosphatidyl-choline (LPC). Disaturated phosphatidylcholine (DSPC) comprised 80% of the total PC, and it contained ostly palmitic acid. The DSPC content of the lung lavage fluid per square meter of alveolar surface area was 5.76±0.42 mg.     

Hepatic phospholipid molecular species in the guinea pig adaptations to pregnancy

Incorporation of polyunsaturated fatty acids (PUFA), particularly 22∶6n−3, into fetal brain at specific gestational ages is critical for development of normal brain function. We have studied adaptations to maternal liver phospholipid molecular species compositions that may be related to the supply of PUFA to fetal brain. The increment of 22∶6n−3 in brain phosphatidylethanolamine (PE) was maximal at day 25 to day 35 of gestation, consistent with early prenatal development of guinea pig brain. At the same gestational ages, there was a transient increase in maternal liver concentration of 16∶0/22∶6 phosphatidylcholine (PC), which preceded the progressive increase in total PC concentration toward term (day 68). This effect was specific for thesn-1 16∶0 species, as, there was no significant increase in 18∶0/22∶6 PC concentration. These results are consistent with a specific role for 16∶0/22∶6 PC in the directed supply of 22∶6n−3 from maternal liver to the fetus. Concentrations of all PE species in maternal liver decreased at day 25 and day 35 of gestation. The gradual accumulation of 22∶6n−3 in fetal liver throughout gestation did not correlate with the pattern of acquisition of 22∶6n−3 into fetal brain PE. Maternal plasma PC and cholesterol concentrations decreased dramatically by day 25 of gestation, and remained low until term. This hypolipidemia of pregnancy in the guinea pig may be due to increased lipase-mediated turnover of plasma lipoproteins and contrasts strongly with the well-characterized hyperlipidemia in human and rat gestation.     

Biphasic action of platelet-activating factor on isolated guinea-pig ileum

1-0-Hexadecyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) at 10⁻¹⁰-10⁻⁹ M induced slow contraction of isolated guinea-pig ilcal muscles and the contraction persisted for a long time. At a higher concentration of 10⁻⁷ M, this phospholipid induced more rapid, but not greater, contraction. At higher concentrations (10⁻⁶-10⁻⁵ M), this phospholipid induced a biphasic response: rapid contraction followed by relaxation. At high concentrations, this compound inhibited acetylcholine-induced contractions. The stimulatory effect of this phospholipid was ca. 300 times that of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine, while its inhibitory potency on induced contraction was similar to those of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine and its lyso derivative. It was suggested that the differences in effects on contraction of different concentrations of 1-0-hexadecyl- and 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine were due to the dual effects of these compounds on the ileum: a strong stimulatory effect and a moderate inhibitory effect on contraction.     

Metabolism of platelet-activating factor by blood platelets and plasma

High performance liquid chromatography in combination with a radioactivity detector was used to study the metabolism of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by washed platelets, platelet-free plasma and platelet-rich plasma obtained from rabbits and humans. Degradation of platelet-activating factor in plasma was completely inhibited by diisopropylfluorophosphate and was partially inhibited by ethylenediamine tetraacetic acid. Washed platelets metabolized platelet-activating factor not only to the 2-lyso compound but also, by reacylation of this lyso intermediate, to an analogue of platelet-activating factor probably containing a long-chain acyl group at thesn-2 position. These transformations occurred, but to a lesser extent, in platelet-rich plasma.     

Characterization of the coronary vascular responses to platelet-activating factor in the isolated perfused heart

Platelet-activating factor (PAF) is a potent phospholipid mediator with diversein vivo andin vitro coronary vascular effects. In the present study, the coronary vascular responses to bolus injections of PAF were compared in rat hearts perfused under constant flow and under constant pressure. Low levels of PAF (1 pmol) produced vasodilatation only, while higher PAF concentrations (100 pmol) produced initial vasodilatation which was followed by a vasoconstriction under both experimental conditions. To determine species differences in PAF action, the effect of PAF was tested on perfused guinea pig hearts. Unlike in perfused rat hearts, only a dosedependent vasoconstrictor response was observed in perfused guinea pig hearts following a bolus injection of 1 fmol to 10 pmol of PAF. The results from repeated injections of PAf indicated that depletion of vasoactive mediators induced by PAF or receptor desensitization may explain a failure of a second injection of PAF to initiate a vasoconstrictor response. After PAF injection, the coronary vascular response to leukotriene was not altered, indicating that the reduced vasoconstrictor effect of a second injection of PAF cannot be due to a reduced ability of the smooth muscle to constrict. The study demon-strates that similar coronary vascular responses to PAF are observed in perfused rat hearts under either constant flow rate or constant pressure and that some of the variable coronary vascular responses reported may be due to the difference between animal species.     

Bioactions of 5-hydroxyicosatetraenoate and its interaction with platelet-activating factor

In a variety of stimulated cells, platelet-activating factor (PAF) and numerous arachidonate derivatives are coproducts that form as a consequence of receptor-mediated phospholipid mobilization. These lipid co-products produce a plethora of biological effects in a wide variety of cell systems. Furthermore, they often have a fascinating, although less widely appreciated, interaction. 5-HETE, at submicromolar concentrations, exerts relatively few direct bioactions. It does, however, potently (16–160 nM) raise cytosolic free calcium [Ca²⁺]_i and augment PAF-induced responses in human polymorphonuclear neutrophils (PMN) by as much as 100- to 1000-fold. 5-HETE acts on PMN by a structurally specific, stereospecific and pertussis toxin-inhibitable mechanism. In addition, PMN exposed to 5-HETE exhibit homologous but not heterologous desensitization. These findings suggest that 5-HETE, like PAF, may bind to its own specific plasmalemmal receptors to exert its unique set of bioactions. However, further investigation is required to demonstrate any putative 5-HETE receptors. Other potential mechanisms of 5-HETE-induced bioactions together with the possible effects of 5-HETE on PAF transduction mechanisms are also discussed. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Production of platelet-activating factor by human normodense and hypodense eosinophils

Normodense eosinophils and neutrophils from normal donors produced considerable amounts of plateletactivating factor (PAF) when stimulated with ionophore A23187. PAF produced by eosinophils appeared to be degraded more rapidly than PAF formed by neutrophils, suggesting a higher activity of PAF-degrading enzyme in eosinophils. Substantial proportions of PAF newly formed by both eosinophils and neutrophils were shown to be cell-associated. By comparison, hypodense eosinophils obtained from a patient with idiopathic hypereosinophilic syndrome produced an extremely large amount of PAF and released much of it into the incubation medium. The accelerated formation of PAF in hypodense eosinophils may be related to various cardiovascular complications associated with hypereosinophilic syndrome. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Effect of platelet-activating factor on lipoprotein lipase and blood lipids

We investigated the effect of platelet-activating factor (PAF) and of the PAF specific antagonist CV-6209 on plasma lipid metabolism, and particularly on post-heparin plasma lipolytic activity in male Wistar rats. Lipoprotein lipase (LPL) activity was enhanced by intravenous injection of PAF before intravenous injection of heparin when the PAF dose was low (0.2 μg/kg). PAF activated hepatic triacylglycerol lipase (HTGL) activity dose-dependently. Plasma triacylglycerols (TG) significantly decreased with the activation of LPL and/or HTGL. Plasma total cholesterol (TC) and phospholipid (PL) levels decreased at a low dose of PAF (0.2 μg/kg), but increased when higher doses were used. The PAF antagonist CV-6209 partially reversed the PAF induced effects on HTGL, TC and PL. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

The hormonal regulation of platelet-activating factor acetylhydrolase activity in plasma

We have previously reported that certain fetal tissues including the lung and kidney have an increased platelet-activating factor (PAF) content and enzymatic mechanism for its elevated biosynthesis during the latter stages of pregnancy. In contrast, in the maternal plasma compartment of both the rabbit and human, a decreased capacity to inactivate PAF has been demonstrated. The PAF acetylhydrolase in the fetal plasma is also suppressed. The present study was undertaken to determine the mechanism(s) involved in the regulation of PAF acetylhydrolase. The 17α-ethynylestradiol was administered (intraperitoneal [i.p.] 2.5 mg/kg body wt 5 days) to female and male rats. The plasma PAF acetylhydrolase activity decreased 5-fold. A decrease was observed when a concentration of the estrogen as low as 50 μg/kg was employed. The injection of dexamethasone (i.p., 1.3 mg/kg body wt, 5 days) to male and female rats resulted in a 3-fold increase in the plasma PAF acetylhydrolase activity. The activity returned to the values prior to hormone treatment 4 days after cessation of treatment. Testosterone and progesterone were without effect on plasma acetylhydrolase activity. The change in PAF acetylhydrolase activity caused by estrogen and the glucocorticoid was reflected by a change in the activity in the HDL fraction and not due to the presence of an inhibitor or activator in the plasma of the hormone-treated animals. Human serum obtained from a group of women, in which the 17β-estradiol concentration was elevated in preparation for anin vitro fertilization procedure, showed an inverse relationship between the plasma estrogen concentration and the PAF acetylhydrolase activity. It is suggested that estrogen is responsible for the regulation of PAF acetylhydrolase and the decrease in the plasma PAF acetylhydrolase during the latter stages of pregnancy in both the maternal and fetal plasma caused by the hyperestrogenic state that occurs during this period. The observed increase in PAF acetylhydrolase by dexamethasone may account for, in part, the known anti-inflammatory properties of this steroid by decreasing the concentration of this potent autacoid. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

The degradation of platelet-activating factor and related lipids: Susceptibility to phospholipases C and D

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) is an ether-linked lipid that exhibits selective cytotoxicity toward several types of tumor cells and is relatively inactive toward normal cells under the same conditions of treatment. The mechanism of this selective cytotoxicity is unknown. We conducted studies to determine whether this compound is metabolized by phospholipases C and D and, if so, whether sensitive and resistant cells differ in their ability to degrade ET-18-OCH₃ by these enzymes. We have examined the metabolism of the L-isomer of ET-18-OCH₃, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH₃), by lysophospholipase D of rat liver microsomes and by a phospholipase D from the marine bacteriumVibrio damsela. The metabolism of L-ET-18-OCH₃ was also examined in cell culture using Madin-Darby canine kidney cells, human promyelocytic leukemia cells and human myelocytic leukemia cells. In these studies, L-ET-18-OCH₃ and related 1-O-alkyl-linked phosphocholine analogs radiolabeled with³H in the 1-O-alkyl chain were used. L-ET-18-OCH₃ was not hydrolyzed by lysophospholipase D from rat liver microsomes under conditions where cleavage of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine was observed. However, phospholipase D from the marine bacteriumV. damsela readily hydrolyzed L-ET-18-OCH₃ to 1-O-[³H]octadecyl-2-O-methyl-sn-glycero-3-phosphate, demonstrating that L-ET-18-OCH₃ can be degraded by a phospholipase D. Platelet-activating factor (PAF) and lyso-PAF were also substrates for the bacterial phospholipase D. When intact cells were incubated with radiolabeled L-ET-18-OCH₃ a product was formed that was identified as 1-O-[³H]octadecyl-2-O-methyl-sn-glycerol. There are two mechanisms that could account for the appearance of this product. The first involves cleavage of the compound by a phospholipase C, resulting in direct release of the diglyceride. The second possible mechanism involves cleavage by a phospholipase D to form the phosphatidic acid analog with subsequent hydrolysis to the diglyceride by a phosphohydrolase. Preliminary data support the phospholipase C-type mechanism. Regardless of which mechanism operates in intact cells, the metabolic degradation of L-ET-18-OCH₃ does not appear to be a significant factor in the selective cytotoxicity of this antitumor agent.