COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

The role of calcium and cyclic adenosine 3′,5′-monophosphate in the regulation of glycogen metabolism in phagocytozing human polymorphonuclear leukocytes

Incubation of human polymorphonuclear leukocytes in a glucose-free Krebs-Ringer bicarbonate buffer for 2 h resulted in glycogen depletion, decreased phosphorylase activity and increased synthase-R activity. Addition of dialyzed latex particles to starved leukocytes revealed a very rapid ingestion rate (half-maximal ingestion within 30 s). This uptake is followed by glycogenolysis associated with an immediate two-fold increase in phosphorylase a activity and a synthase-R to -D conversion within 30 s. Furthermore, in rapid time-course experiments with phagocytozing cells we found that the concentration of cyclic AMP increased by 93% within 15 s and returned to baseline values at 1 min. In a medium without added calcium and with 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid, phagocytosis was blocked, cyclic AMP formation decreased by 50% and phosphorylase activation was abolished, but the conversion of synthase-R to -D was preserved. Addition of calcium ions to cells suspended in a calcium-free buffer without added latex results in phosphorylase activation and glycogenolysis, but not in cyclic AMP increase or synthase-R to -D conversion. Measurements of ⁴⁵Ca efflux during phagocytosis suggest an initial increase in cytosolic calcium obtained by a release of membrane-bound ⁴⁵Ca. Activation of phosphorylase during phagocytosis is thus presumably due to an increase in cytosol Ca²⁺ and subsequent activation of phosphorylase kinase, and is independent of the simultaneous increase in concentration of cyclic AMP. Phosphorylation of synthase R to the D form does not depend on the presence of Ca²⁺ in the extracellular phase.     

Auxin-polyamine interaction with the calcium-controlled secretion of peroxidase by sugarbeet cells

Spermidine and ornithine given to normal auxin-requiring cell suspensions of sugarbeet inhibited peroxidase secretion in the absence of Ca²⁺. Habituated (organogenic or not) cells did not respond. Both compounds counteracted the Ca²⁺ - promoted enzyme secretion by three cell lines. Auxins (2,4-D and BSAA) did not modify the extracellular level of peroxidase activity in the absence of Ca²⁺ When Ca²⁺ was added, auxins increased its effect in normal cells and had practically no effect in habituated cells. The inhibitory effect of spermidine and ornithine was somewhat reduced by auxins in normal cells and increased in habituated cells. It was hypothesized that the effect of auxins did not involve the mediation of polyamines and that both types of compounds directly interacted with Ca²⁺ at the membrane level.Abbreviations BSAA [benzo(b)selenienyl-3]acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - HNO habituated non-organogenic - HO habituated organogenic - NNO normal non-organogenic     

The influence of cellular amino acids and the Na+: K+ pump on the membrane potential of the Ehrlich ascites tumor cell

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na⁺: K⁺ pump. The membrane potential (monitored by the fluorescent dye, 3,3′-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na⁺ but not cellular ATP. Na⁺ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na⁺ and amino acids.Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na⁺ (approx. 30 mM), the Na⁺: K⁺ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na⁺ was raised to 60 mM, the activity of the pump changed the membrane potential from the range ?25 to ?30 mV to ?44 to ?63 mV. This hyperpolarization required external K⁺ and was inhibited by ouabain.     

Macular cherry-red spots and myoclonus with dementia: coexistent neuraminidase and beta-galactosidase deficiencies.

A patient was previously characterized as having a variant form of G_M1 gangliosidosis based on severe deficiencies in β-galactosidase activity in both leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-galactoside and G_M1 ganglioside. Reexamination of her cultured fibroblasts revealed a severe deficiency in neuraminidase activity using neuramin lactose, fetuin and 2-(3′-methoxyphenyl)-N-acetyl-D-neuraminic acid as substrates, but normal neuraminidase activity using G_M3 ganglioside as a substrate. The presence of normal levels of β-galactosidase activity in leukocytes from the mother of the patient indicates that the β-galactosidase deficiency is not the primary enzyme defect in this type of patient.     

DYNAMICS OF LEUKOCYTE MIGRATION INTO THE MOUSE ASCITES TUMOR

There is a marked increase in the number of peritoneal leukocytes (lymphocytes, monocytes and granulocytes) during the growth of Ehrlich ascites tumor in mice. No local proliferation (as indicated by a labeling at 1 hr following a single ³H-TdR injection) was observed in the normal peritoneal leukocytes or those in the ascites tumor, except for a very minor labeling of some tumor macrophages. Kinetics of peritoneal leukocytes was studied with a series of twelve injections of ³H-thymidine (20 μCi every 8 hr) in normal mice as well as mice injected with 10⁶ tumor cells i.p. 2 hr after the last ³H-TdR injection. Animals were sacrificed at intervals up to 6 days. Granulocyte labeling in the blood as well as peritoneal space was near 100% in both groups of animals at all the intervals. Temporal changes in the labeling of lymphocytes (from 10% at 0 day to 22% at day 6), and monocytes (from 20% at 0 day to 57% at day 6) were identical in the blood and peritoneal space of normal animals, indicating a free exchange of cells between these compartments. Higher labeling indices than those in the controls were attained in the blood of tumor-bearing hosts (viz 40% for lymphocytes and 80% for monocytes at 6 days) suggesting an increased turnover of these cells in the circulation. In addition, peritoneal mononuclear cells of tumor-bearing mice showed even a higher labeling than those in the blood (viz 65% for lymphocytes and 92% for monocytes at 6 days) indicating a selective migration and/or retention of newly formed cells within the tumor, in contrast to a random migration into the normal peritoneal cavity. Furthermore, an identical labeling of macrophages to that of monocytes within the tumor indicated a short monocyte-macrophage transition. The preferential accumulation of young mononuclear cells into the tumor may be of functional importance.     

Effect of quercetin on human polymorphonuclear leukocyte lysosomal enzyme release and phospholipid metabolism

Quercetin inhibited in a concentration-dependent manner the release of beta-glucuronidase from human polymorphonuclear leukocytes stimulated with zymosan-activated serum. ³H-arachidonic acid-prelabelled polymorphonuclear leukocytes released ³H-arachidonic acid upon stimulation with zymosan-activated serum and this was associated with a decrease of radioactivity in the phospholipid fraction as determined by thin layer chromatography. Quercetin inhibited the release of ³H-arachidonic acid. These observations suggest that the zymosan-activated serum stimulus activates phospholipase A₂ and that phospholipase A2 is inhibited by quercetin. Thus, quercetin alters polymorphonuclear leukocyte phospholipid metabolism and responses to stimulation.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Myeloperoxidase-Based Chemiluminescence of Polymorphonuclear Leukocytes and Monocytes

Luminol and lucigenin chemiluminescence (CL) responses produced by separated human blood polymorphonuclear leukocytes (pmn) and monocytes (mono) have been studied following stimulation with the surface-receptor agonist fMLP (a synthetic chemotactic peptide) and the protein kinase C activator phorbol myristate acetate (PMA). Pmn produced two- to threefold the luminol CL and superoxide anion (O₂⁻) levels of mono; lucigenin CL was similar for both cell-types. The myeloperoxidase (MPO) inhibitor salicylhydroxamic acid (SHA) abrogated luminol but not lucigenin CL in both cell types, but did not further inhibit the already grossly subnormal luminol CL responses seen with MPO-deficient cells which produced normal lucigenin CL. SHA also profoundly inhibited the luminol CL response in a cell-free MPO–H₂O₂ system. Mono lucigenin CL does not appear to specifically measure O₂⁻ production. These data show that luminol CL provides a useful measure of pmn and also mono MPO activity. However, analysis of the effects of various reactive oxygen species (ROS) scavengers, assessed on phagocyte and cell-free CL systems (both MPO–H₂O₂ and superoxide generating) suggest that the luminol CL signal is not entirely dependent on MPO activity.     

Further characterization of Ca2+-activated phospholipase A2 in cat adrenal cortex

When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved $\text{E.}$$\text{coli}$) in the presence of corticotropin, there was a Ca²⁺-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca²⁺ concentration. The addition of increasing Ca²⁺ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [¹⁴C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [¹⁴C]palmitic acid failed to release fatty acid in response to Ca²⁺, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca²⁺-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca²⁺-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca²⁺-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca²⁺-mediated activation of phospholipase A₂ participates in the control of adrenocortical activity. However, since Ca²⁺ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca²⁺ on fatty acid release.     

Effects of fatty acids on superoxide radical generation in leukocytes

Acetylated ferricytochrome c was employed for the detection of superoxide radicals (O₂^?) generated both in intact cells and in subcellular fractions of leukocytes. Certain saturated fatty acids, myristate in particular, induced the production of O₂^? in leukocytes, suggesting a correlation between the formation of O₂^? and the hydrophobic interaction of fatty acids with the leukocyte plasma membrane. As compared with O₂^? radical generation from phagocytizing leukocytes, a greater stimulation of O_2? formation was observed in cells in which myristate was added. The enhanced activity which generated O₂^? in the cell-free system was located in a particulate fraction but not in the cytosol. The rate of O₂^? generation in the particulate fraction was higher in the presence of NADPH than in the presence of NADH. The effects of reagents such as KCN, 2,4-dichlorophenol and aminotriazole on the O₂^? generation in this fraction are examined and the nature of the O₂^? generating system is discussed.     

Effect of blood serum, caffeine and heparin on in vitro phagocytosis of frozen-thawed bull sperm by neutrophils derived from the peripheral blood of cows

Although polymorphonuclear leukocytes (PMNs) are recruited into the uterine lumen to phagocytize sperm, factors controlling the phagocytotic ability of PMNs in cattle are not well documented. The objective was to determine the effects of blood serum, caffeine, and heparin on chemotaxis of PMNs for sperm and phagocytosis of sperm by PMNs in cows. Polymorphonuclear leukocytes were obtained (centrifugation) from a cow's peripheral blood. In Experiment 1, the chemotactic activity of PMNs increased (P < 0.01) when fresh serum was included in the medium (1226 cells/mm² in serum vs. 1110 cells/mm² in BSA), regardless of the presence of sperm, whereas heat-inactivated serum (1099 cells/mm²) did not affect their activity (P = 0.65). Phagocytosis of live and dead sperm by PMNs both increased (P < 0.01) in the presence of fresh serum (incidences of 54.5 and 48.0%, respectively), but stimulation was decreased (P < 0.01) by supplementation of the medium with ≥1 mM caffeine (20.6-30.3%). Serum-stimulated chemotactic activity of PMNs (1218 cells/mm²) was also decreased (P < 0.01) in the presence of caffeine (1090 cells/mm²). Furthermore, supplementation of PMNs with heparin in the presence of serum decreased (P < 0.01) both phagocytotic (from 43.8% to 21.5-31.7%) and chemotactic activities of PMNs (from 1124 to 1048-1108 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, and that both caffeine and heparin decreased serum-stimulated phagocytotic and chemotactic activities of PMNs.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E₂ production as well as a smaller increase in non-cyclooxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E₂ production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F_2α, prostacyclin and thromboxane A₂. Prostaglandins E₂ and F_2α are the principal cyclo-oxygenase products of this interaction.We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F_2α by leukocytes.     

Biological activities of lipid components ofLeptospira interrogans serovarcopenhageni avirulent strain Shibaura

The gas chromatogram showed that the polar lipids (PL) extracted fromLeptospira interrogans serovarcopenhageni avirulent strain Shibaura consisted mainly of palmitic acid, palmitoleic acid, and oleic acid, and that the free fatty acids (FFA) from the strain contained oleic acid at a concentration as high as 67.5%. The intracutaneous injection of FFA induced much stronger edematous and vascular permeability reactions in guinea pigs than did that of PL. In addition, O ₂ ^– generation of polymorphonuclear leukocytes in vitro was enhanced with FFA to much greater extent than with PL.Among the FFA, oleic acid caused the strongest biological activities such as edematous reaction, vascular permeability reaction, O ₂ ^– -generating activity, and hemolytic activity. It is, therefore, very likely that oleic acid may play an important role in toxicities of leptospiral lipids.     

Drug inhibitable ecta-ATPase in leukocytes.

In rat leukocytes, the total ATP-hydrolyzing activity was highest when determined in cells with an intact plasma membrane. The ecto-ATPase, exhibiting a Km for ATP of 0.3 mM, was inhibited 30% by sulfhydryl reagents, whereas dinitrophenol, oligomycin or Ca²⁺ had little or no effect. The ouabain inhibitable, Na, K-activated component amounted to 7% of the total ATPase activity in leukocytes. A major characteristic of the ecto-ATPase was its marked inhibition by micromolar concentrations of both phenothiazine tranquilizers and tricyclic antidepressants.     

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

 We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR⁺ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3^–, CD19^–, CD20^–, CD14^–, CD11b^–, CD16^–, CD56^–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system. Received: 20 June 1997 / Accepted: 14 August 1997     

Protease inhibitors antagonize the activation of polymorphonuclear leukocyte oxygen consumption.

We report that the burst of oxygen consumption, as well as the resultant production of O₂^?? and H₂O₂, occurring in activated human polymorphonuclear leukocytes is inhibited by various compounds which have in common the ability to antagonize the effects of proteolytic enzymes. This effect of protease inhibitors was observed with a variety of stimuli, both phagocytic and non-phagocytic, used to activate O₂^?? production in human polymorphonuclear leukocytes. Inhibition was also noted in rat polymorphonuclear leukocytes and alveolar macrophages. The results indicate that proteolysis may be involved in activating the burst of oxygen consumption following stimulation of phagocytic cells.     

Effectors of fatty acid synthesis in hepatoma tissue culture cells

An investigation was undertaken to better understand the process of fatty acid synthesis in hepatoma tissue culture (HTC) cells. By comparing the findings to the normal liver some of the differences between normal and cancer tissue were defined. Incubation of the HTC cells in a buffered salt-defatted albumin medium showed that fatty acid synthesis was dependent upon the addition of substrate. The order of stimulation was glucose + pyruvate ～- glucose + alanine ～- glucose + lactate ～- pyruvate > glucose > alanine ? no additions. Fatty acid synthesis in HTC cells was decreased by oleate. In these respects HTC cells are similar to the liver; however, in contrast to the normal liver, N⁶, O²-dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl-cAMP) did not inhibit glycolysis or fatty acid synthesis. The cytoplasmic redox potential, as reflected by the lactate to pyruvate ratio, was found to be elevated compared to normal liver but unchanged by the addition of dibutyryl cAMP. Since higher rates of fatty acid synthesis are associated with lower lactate-to-pyruvate ratios in normal liver, it was expected that by decreasing the lactate-to-pyruvate ratio in HTC cells the rate of fatty acid synthesis would increase. One way to lower the lactate to pyruvate ratio is to increase the activity of the malate-aspartate shuttle. Stimulators of the hepatic malate-aspartate shuttle in normal liver (ammonium ion, glutamine, and lysine) had mixed effects on the redox state and fatty acid synthesis in HTC cells. Both ammonium ion and glutamine decreased the redox potential and increased the rate of fatty acid synthesis. Lysine was without effect on either process. Since NH₄Cl and glutamine stimulate the movement of reducing equivalents into the mitochondria and decrease the redox potential, then the stimulation of fatty acid synthesis by NH₄Cl and glutamine may be due to an increase in the movement of reducing equivalents into the mitochondria. However, if the shuttle were rate determining for fatty acid synthesis the rate from added lactate would be the same as from glucose alone but would be lower than from pyruvate which does not require the movement of reducing equivalents. This was not the case. Lactate and pyruvate gave comparable rates which were higher than glucose alone. Other possible sites of stimulation were investigated. The possibility that NH₄⁺ and glutamine stimulated fatty acid synthesis by activating pyruvate dehydrogenase was excluded by finding that dichloroacetate, an activator of pyruvate dehydrogenase, did not stimulate fatty acid synthesis when glucose was added. Stimulation by NH₄⁺ and glutamine at steps beyond pyruvate dehydrogenase was ruled out by the observation that NH₄⁺ caused no stimulation from added pyruvate. NH₄⁺ and glutamine did not alter the pentose phosphate pathway as determined by ¹⁴CO₂ production from [1-¹⁴C]- or [6-¹⁴C]glucose. Ammonium ion and glutamine increased glucose consumption and increased lactate and pyruvate accumulation. The increased glycolysis in HTC cells appears to be the explanation for the stimulation of fatty acid synthesis by NH₄⁺ and glutamine, even though glycolysis is much more rapid than fatty acid synthesis in these cells. The following observations support this conclusion. First, the percentage increase in glycolysis caused by NH₄⁺ or glutamine is closely matched by the percentage increase in fatty acid synthesis. Second, the malate-aspartate shuttle, the pentose phosphate pathway, and the steps past pyruvate are not limiting in the absence of NH₄⁺ or glutamine.