Specificity of phospholipases in methylcholanthrene-transformed mouse fibroblasts activated by bradykinin, thrombin, serum, and ionophore A23187.

Methylcholanthrene-transformed mouse fibroblasts synthesize prostaglandins in response to bradykinin, thrombin, serum, and the ionophore A23187. These agents activate phospholipases, thereby releasing fatty acids from phospholipids. To examine the phospholipid specificity of the phospholipases activated by bradykinin, thrombin, serum, and A23187, cells were labeled with [14C]arachidonic acid and stimulated with these agents in the presence of delipidated bovine serum albumin. Phospholipid classes were resolved by two-dimensional chromatography on silica gel-coated paper. Only phosphatidylinositol and phosphatidylcholine lost radioactivity upon stimulation. To characterize the fatty acid specificity of the phospholipases, cells were incubated with 14C-labeled stearic, oleic, linoleic, eicosatrienoic, or arachidonic acid and then exposed to the stimuli. Bradykinin, thrombin, and serum caused specific release of radioactivity into the medium only from cells labeled with arachidonic acid or eicosatrienoic acid, whereas A23187 caused release from cells labeled with any one of the five fatty acids. We conclude that bradykinin, thrombin, and serum activate phospholipases that specifically hydrolyze arachidonyl and eicosatrienoyl phosphatidylinositol and phosphatidylcholine, whereas A23187 is less specific activator of phospholipases.     

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells.

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 x 10(-10)M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30--32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.     

Acylation of lysophospholipids by rabbit alveolar macrophages. Specificities of CoA-dependent and CoA-independent reactions

Intact alveolar macrophages were found to acylate alkyl- and acyllysophospholipids with a high selectivity for arachidonate. A specific mechanism appears responsible for the incorporation of arachidonate into lysophospholipids in intact cells since the kinetic pattern for the formation of the 20:4 species was different from all other species. This specificity was investigated in more detail by examining the enzymatic acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by macrophage membranes; in the absence of CoA, ATP, and Mg2+, this lysophospholipid was acylated with a high preference for arachidonate that was independent of added free fatty acids. The addition of CoA alone increased the rate of acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine, mainly due to an increase in the formation of species other than those containing arachidonate. When CoA, ATP, and Mg2+ were present, the macrophage membranes catalyzed the acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine without preference for arachidonate. A different apparent Km and Vmax was observed for reactions involving each cofactor condition. We conclude that the acylation of alkyl- and acyllysophospholipids by rabbit alveolar macrophages occurs by three separate mechanisms: a CoA-independent transacylation, a CoA-dependent transacylation (reverse reaction catalyzed by acyl-CoA acyltransferase), and an acyl-CoA-dependent acylation. The CoA-independent transacylation reaction is unique in that it is specific for arachidonate and accounts for the selective acylation of alkyl- and acyllysophospholipids by arachidonate in membrane preparations of alveolar macrophages. This reaction appears to be extremely important in the remodeling of phospholipid molecular species and the mobilization of arachidonate into ether-linked lipids. The transfer of arachidonate to 1-alkyl-2-lyso-sn-glycero-3-phosphocholine also is of importance in the final inactivation step for platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), whereby 1-alkyl-2-arachidonoyl-sn-glycerol-3-phosphocholine (a stored precursor of both platelet activating factor and arachidonic acid metabolites) is formed.     

Density, distribution and mobility of surface anions on a normal/transformed cell pair

The distribution and mobility of cell surface anions was investigated on low passage cultures of secondary BALB/c embryonic fibroblasts and their SV40-transformed counterparts (VLM cells) using polycationized ferritin (PCF) as a label. While the absolute number of anions/nm² of membrane was equivalent on the two cell types, the topographical distribution and mobility of these anions was strikingly different. After pulse-labeling with PCF at 37 °C, anions on BALB/c fibroblasts occurred in large piled-up clusters separated by extensive areas of membrane ( = 0.47 μm) free of negative charges. Labeling at 4 °C reduced the degree of “piling up” within the clusters, but the intercluster spacing was maintained indicating that the anions have short-range mobility in the membrane and can be cross-linked into a tight lattice at 37 °C. These anions do not, however, demonstrate any long-range mobility during a 20 min post-label incubation in PBS. In contrast, anions on VLM cells are inherently present in a random configuration of microclusters and single anions with relatively small ( = 0.09 μm) intervening areas of low charge density. Short-range mobility of surface anions is not displayed, presumably as a result of the inter-site distance, but long-range mobility is indicated by the formation of large site clusters following a 20 min incubation in PBS after pulse-labeling. Very mild proteolysis of BALB/c fibroblasts induces a change in the topography of surface anions toward the random configuration typical of VLM cells. These data are discussed in relation to altered social interactions between tumor cells which may be influenced by cell-cell adhesion characteristics.     

The participation of hydroperoxides and oxygen radicals in the control of prostaglandin synthesis

Our results demonstrate that the organic hydroperoxide t-butyl-hydroperoxide (TBHP) influences the synthesis of prostaglandins in the human embryo lung fibroblast. TBHP inhibits or stimulates prostaglandin synthesis as a function of its concentration. Regardless of the concentration employed in these experiments however, TBHP stimulated the release of arachidonate from lipid stores. When the arachidonate release step in prostaglandin (PG) synthesis was bypassed by the addition of free arachidonate to the cell cultures, t-butyl hydroperoxide further stimulated PG synthesis, indicating that the hydroperoxide activates arachidonate conversion to prostaglandins. 4-Hydroxy-2,2,6,6-tetramethylpiperidinoxy radical, a scavenger of oxygen radicals, when added to cell cultures alone had no measurable effect on either arachidonate release or prostaglandin synthesis. When 4-hydroxy-2,2,6,6-tetramethylpiperidinoxy radical was administered to cell cultures in combination with t-butyl hydroperoxide, it increased prostaglandin synthesis while inhibiting arachidonate release by the hydroperoxide. The participation of hydroperoxides and trappers of oxygen radicals in the regulation of PG synthesis is not unique to lung fibroblasts. Endothelial cells from the vasculature as well as fibroblasts from the cornea also appear to be affected by these compounds with respect to prostaglandin synthesis.     

Eicosadiynoic acid: A non-toxic inhibitor of multiple enzymatic steps in the production of icosanoids from arachidonic acid

We have studied the acetylenic fatty acid 20:2Δ8a, 11a (eicosadiynoic acid, EDYA). It was found that this compound acts as an inhibitor of several steps in the production of icosanoids from arachidonic acid. First, the compound was shown to inhibit arachidonate uptake by platelets. Second, using a detergent solubilized preparation from calf brain, EDYA was found to inhibit both the arachidonoyl and the non-specific long chain acyl-CoA synthetase, which convert arachidonate to its CoA ester. Third, the compound decreased the conversion of dihomo gamma linolenic acid to arachidonate in the mouse fibrosarcoma HSDM₁C₁ cell line, acting as an apparent Δ5 desaturase inhibitor. Finally, EDYA (50 uM) inhibited cyclooxygenase activity. The compound was not toxic to cultured cells. Cells were grown for months in tissue culture medium at concentrations as high as 50 uM, with no morphologic changes by light microscopy and no prolongation of the doubling time over untreated cells. Our findings with this compound indicate that it limits icosanoid production by inhibiting cyclooxygenase and also by limiting arachidonate uptake, activation, and production from precursor fatty acids.     

Depletion of arachidonic acid from GH3 cells. Effects on inositol phospholipid turnover and cellular activation.

We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n - 9) and stearate/eicosatrienoate (n - 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n - 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.     

Ultrastructural evidence of maintenance of concanavalin A binding sites in enucleated mammalian cells

Mouse L cells were enucleated by centrifugation in cytochalasin B. Following enucleation, the enucleated cells were incubated in fresh medium for 30 min, 4, 20, or 24 h before being fixed for electron microscopy. After fixation the cells were incubated in concanavalin A and then horseradish peroxidase was bound to the ConA. Electron microscopy of these enucleates revealed that the concanavalin A-binding sites (CABS) are present on the cell surface of the enucleates even at 24 h after enucleation. Although the method does not detail the number of sites present, the inherent distribution of sites appears similar in normal and enucleated cells. Furthermore, the sites are still functional in that the live enucleated cells are agglutinated by ConA to the same extent as are normal L cells—about 80% agglutination in each instance. The results of this study indicate that surface CABS are maintained in the absence of a nucleus and they are still present even after the Golgi apparatus is morphologically disrupted. Turnover of these sites and their relationship to nuclear function are discussed.     

Studies on the uptake of fatty acids by Escherichia coli

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.     

The effects of fatty acyl CoA esters on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). We have previously shown that palmitoyl-CoA (PA-CoA) shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla at a low concentration of AA (5 microM, close to the physiological concentration of substrate). In the present study, we investigated the effects of stearoyl (SA)-, oleoyl (OA)- and linoleoyl (LA)- CoAs on the formation of PG and AA-CoA from 5microM AA in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 5microM [(14)C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2)and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. SA- and OA-CoAs increased AA-CoA formation with a reduction of PG formation, as well as PA-CoA. On the other hand, LA-CoA decreased formation of both PG and AA-CoA. These results suggest that fatty acyl CoA esters can be regulators of PG and AA-CoA formation in kidney medulla under physiological conditions.     

The effect of dietary sterol on the activity of fatty acid desaturases isolated from Tetrahymena setosa

Tetrahymena setosa has a nutritional requirement for micro amounts of sterol, a requirement which is also satisfied by relatively large amounts of either intact phospholipids or a mixture of unsaturated fatty acids normally found in these ciliates. Three microsomal fatty acyl-CoA desaturases have been isolated from T. setosa and partially characterized. These enzymes which can account for the formation of the majority of the ciliate's unsaturated fatty acids, include: a delta 9, a delta 12 and a delta 6 desaturase which catalyze the transformation of stearoyl-CoA to oleic acid, of oleoyl-CoA to linoleic acid and of linoleoyl-CoA to gamma-linolenic acid, respectively. The stearoyl CoA desaturase required NAD (or NADP), ATP and free CoA; the delta 6 and delta 12 desaturases required NADP, but not ATP or CoA. Cellular levels of the three desaturases were highest in mid-logarithmic phase cells and lowest in stationary phase cells. In order to determine if there was a relationship between the sterol requirement and the ability of the organism to desaturate, T. setosa was grown in a synthetic medium supplemented with either cholesterol or a phospholipid which permits growth in the absence of cholesterol, or with both phospholipid and cholesterol. Cells grown with phospholipid alone had only half as much stearoyl-CoA and oleoyl-CoA desaturase activity as cells of identical culture age grown either on cholesterol alone or on cholesterol plus phospholipid.     

Functional domains of the fatty acid transport proteins: studies using protein chimeras

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.     

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 × 10⁻¹⁰M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30–32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.     

Studies of energy linked reactions: a cofactor function for unsaturated fatty acids in oxidative phosphorylation; studies with a yeast auxotroph.

Mitochondria from the yeast unsaturated fatty acid auxotroph KD115 when grown with a high unsaturated fatty acid supplement catalyse normal oxidative phosphorylation and dihydrolipate-dependent ATP synthesis in the absence of added cofactors. Mitochondria from unsaturated fatty acid depleted KD115 cells have half the unsaturated fatty acid content of supplemented cells and do not catalyse oxidative phosphorylation and dihydrolipoate dependent ATP synthesis. Dihydrolipoate-dependent ATP synthesis can be restored specifically by addition of cofactor amounts of oleic acid and oleoyl CoA. The results provide further evidence for a cofactor role for an unsaturated fatty acid in oxidative phosphorylation.     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [3H]arachidonate-labeled phospholipids of HSDM1C1 cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE2. HSDM1C1 cells labeled with [3H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids )16%). Bradykinin treatment stimulated a rapid hydrolysis of [3H]arachidonate from the cellular lipids and conversion of the released acid to PGE2, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM1C1 cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE2 and to a lesser degree inhibited the release of [3H]arachidonate from the cellular lipids into the medium.     

Lipolytic enzymes of Trichophyton rubrum.

Trichophyton rubrum cells contain lipase, phospholipases A and B and acyl CoA lysolecithin acyl transferase activities. This dermatophyte excretes lipase and phospholipase A into the growth medium when cultivated in Sabouraud's broth. Extracellular lipase has optimum activity at pH 8.0 whereas the intracellular lipase is maximally active at pH 8.0 whereas the intracellular lipase is maximally active at pH 7.0. The optimum pH of phospholipase A and B activities which are localized in 15000 g sedimentable cell fragments are 7.0 and 6.0 respectively. Supernatant obtained after removal of 1,005,000 g sedimentable fragments from cell extract contains acyl CoA lysolecithin acyl transferase which requires ATP, CoA, Mg2+ and an unsaturated fatty acid for its activity.     

Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells

The intracellular distribution of specific protease, plasminogen activator (PA), has been examined in Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEF). Cellular homogenates were fractionated by differential centrifugation followed by sucrose gradient centrifugation. The activities and the percent distribution of a series of marker enzymes, specific for different subcellular organelles, were compared to those of PA. Normal CEF have been similarly fractionated and the relatively low amount of PA activity present in these cells has been analyzed in terms of its subcellular distribution. A membrane fraction was isolated from the RSV-CEF that contained the bulk of the PA activity and less than 8% of the total cellular protein. The specific activity of the PA in this fraction is 40-fold higher than that of a comparable fraction isolated from companion cultures of normal cells. This fraction contains little or no nuclear and cytoplasmic material and is contaminated only to a relatively small degree with mitochondria, lysosomes, endoplasmic reticulum. Significant amounts of a putative Golgi membrane marker are present in this fraction. The relatively high specific activities of Na+,K+-ATPase, 5'-nucleotidase, and [3H]fucose indicate that the fraction is enriched in surface membrane. Further purification of the fraction by equilibrium centrifugation on shallow sucrose gradients reduces further the contaminating activities and results in a PA distribution that closely parallels the distribution of the membrane enzyme, 5'-nucleotidase. PA was not released from its membrane association by hypotonic and hypertonic extraction and ultrasonication, while granule-bound enzymes were released by these treatments. The PA activity from hamster SV40 cells fractionated the same way as that of RSV-CEF. These results suggest that a protease that is dramatically enhanced upon malignant transformation is associated with "plasma membrane-like" elements of the cell and may serve as an intrinsic modifier of cell surface proteins after malignant transformation.     

Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase

Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-epsilon (DGKepsilon) or diacylglycerol kinase-alpha (DGKalpha) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP n ) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKepsilon knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKepsilon knockout and wild-type cells, suggesting that DGKepsilon catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and lowest for DAG. These findings indicate that DGKepsilon plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKalpha knockout mice. However, the cells from the DGKalpha knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKalpha, in contrast with DGKepsilon that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.