Arachidonic Acid and Other Unsaturated Fatty Acids Alter Membrane Potential in PC12 and Bovine Adrenal Chromaffin Cells

Abstract: The action of arachidonic acid and other fatty acids on membrane potential in PC 12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1–40 μ M ) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K⁺-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC 12 and bovine chromaffin cells by modulating K⁺ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca²⁺ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca²⁺ influx and secretion. We propose a model where internally generated fatty acids act as a feed-back to desensitize the stimulated cell via inhibition of receptor-dependent Ca²⁺ influx and induction of membrane hyperpolarization.     

Arachidonic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils.

Arachidonic acid (20:4) and other cis-unsaturated fatty acids exert direct effects on a variety of cells, effects that do not depend on the metabolism of fatty acids via cyclooxygenase or lipoxygenase pathways. In these studies arachidonic acid and other cis-unsaturated fatty acids (but not trans-unsaturated or saturated fatty acids) increased the specific binding of the nonhydrolyzable analog of GTP, [35S]GTP gamma S, to purified neutrophil membrane preparations and elicited superoxide anion generation from intact neutrophils. There was a positive correlation (r = 0.70) between the capacity of fatty acids to increase nucleotide binding and to elicit the respiratory burst. Scatchard plot analysis of binding at equilibrium demonstrated an increase in the number of available GTP binding sites in the presence of 50 microM arachidonic acid. Nonsteroidal antiinflammatory agents interfered with the arachidonic acid effect on [35S]GTP gamma S binding. ADP-ribosylation of the pertussis toxin substrate Gi alpha within the plasmalemma-reduced specific [35S]GTP gamma S binding and blocked arachidonate-dependent enhancement of binding. Moreover, pertussis toxin treatment of intact neutrophils inhibited arachidonic acid-induced superoxide anion generation. The data indicate that arachidonic acid directly activates a GTP binding protein in the neutrophil plasma membrane and may thereby act as a second messenger in signal transduction.     

Lipid transfer between endothelial and smooth muscle cells in coculture

A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell-cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.     

Arachidonic acid inhibits phosphate transport by cultured renal cells

Because arachidonic acid and its metabolites are reported to be intracellular messengers of various exogenous stimuli, we studied whether arachidonic acid influences phosphate transport by cultured mouse renal epithelial cells. Arachidonic acid, at 10(-7)-10(-4)M, inhibited phosphate transport without influencing cyclic adenosine 3':5'-monophosphate production. Nordihydroguaiaretic acid and indomethacin, inhibitors of arachidonic acid metabolism, did not cancel the arachidonic acid-induced inhibition of phosphate transport. Furthermore, unsaturated fatty acids other than arachidonic acid also inhibited phosphate transport and their inhibitory effect increased as the number of double bond increased. These data demonstrate that arachidonic acid inhibits the phosphate transport by the cultured renal epithelial cells, probably not via conversion to its metabolites.     

Role of triglycerides in endothelial cell arachidonic acid metabolism

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Lysine 63-linked ubiquitination is important for arachidonic acid-induced cellular adhesion and migration

Arachidonic acid, a dietary cis-polyunsaturated fatty acid, stimulates adhesion and migration of human cancer cells on the extracellular matrix by activation of intracellular signaling pathways. Polyubiquitin chains bearing linkages through different lysine residues convey distinct structural and functional information that is important for signal transduction. We investigated whether ubiquitination was required for arachidonic acid-induced cellular adhesion and migration of MDA-MB-435 cells on collagen type IV. An E1 (ubiquitin-activating enzyme) inhibitor, PYR-431, completely abrogated arachidonic acid-stimulated adhesion. Additionally, expression of a lysine null mutant ubiquitin prevented activation of cellular adhesion. Cells expressing ubiquitin in which lysine 63 (K63) was mutated to arginine (K63R) were unable to adhere to collagen upon exposure to arachidonic acid. When K63 was the only lysine present, the cells retained the ability to adhere, indicating that K63-linked ubiquitin is both necessary and sufficient. Moreover, K63-linked ubiquitin was required for the induction of cell migration by arachidonic acid. The ubiquitin mutants and PYR-431 did not prevent arachidonic acid-induced phosphorylation of TGF-β activated kinase-1 (TAK1) and p38 MAPK, suggesting K63-linked ubiquitination occurs downstream of MAPK. These novel findings are the first to demonstrate a role for K63-linked ubiquitination in promoting cell adhesion and migration.     

Short-term and long-term effects of fatty acids in rat hepatoma AS-30D cells: the way to apoptosis

Arachidonic acid and, to a smaller extent, oleic acid at micromolar concentrations decreased the mitochondrial membrane potential within AS-30D rat hepatoma cells cultivated in vitro and increased cell respiration. The uncoupling effect of both fatty acids on cell respiration was partly prevented by cyclosporin A, blocker of the mitochondrial permeability transition pore. Arachidonic acid increased the rate of reactive oxygen species (ROS) production, while oleic acid decreased it. Both fatty acids induced apoptotic cell death of AS-30D cells, accompanied by the release of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and association of proapoptotic Bax protein with mitochondria; arachidonic acid being a more potent inducer than oleic acid. Trolox, a potent antioxidant, prevented ROS increase induced by arachidonic acid and protected the cells against apoptosis produced by this fatty acid. It is concluded that arachidonic and oleic acids induce apoptosis of AS-30D hepatoma cells by the mitochondrial pathway but differ in the mechanism of their action: Arachidonic acid induces apoptosis mainly by stimulating ROS production, whereas oleic acid may contribute to programmed cell death by activation of the mitochondrial permeability transition pore.     

Shear stress attenuates endothelin and endothelin-converting enzyme expression through oxidative stress

Shear stress is known to dilate blood vessels and exert an antiproliferative effect on vascular walls. These effects have partly been ascribed to shear stress-induced regulation of the secretion of endothelium-derived vasoactive substances. In this study, to elucidate the role of shear stress in endothelin production by endothelial cells, we examined the effect of physiological shear stress on the mRNA expression of endothelin-converting enzyme-1 (ECE-1) as well as endothelin-1 (ET-1) in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs), using a parallel plate-type flow chamber. ECE-1 mRNA expression was significantly down-regulated by shear stress in an intensity- and time-dependent manner within the physiological range (1.5 to 15 dyn/cm(2)). ET-1 mRNA expression decreased together with ECE-1 mRNA expression. Shear stress at 15 dyn/cm(2) for 30 min induced a significant increase in the intracellular peroxide concentration, and the down-regulation of ECE-1 and ET-1 mRNA expression by shear stress was attenuated almost completely on treatment with N-acetyl cysteine (NAC), an antioxidant (20 mM). Furthermore, when H(2)O(2) (0.5 to 2 mM) was added to BAECs in static culture, the ECE-1 as well as ET-1 mRNA expression was attenuated in proportion to the concentration of H(2)O(2). It is suggested that endothelial cells sense shear stress as oxidative stress and transduce signal for the regulation of the gene expression of ECE as well as ET to attenuate vascular tone and inhibit the proliferation of vascular smooth muscle cells.     

The role of arachidonic acid in rat heart cell metabolism

Although it is known that arachidonic acid accumulates in the ischemic myocardium and that cardiac prostaglandin formation from the precursor arachidonic acid is altered during disease states, the role of arachidonic acid in the myocyte itself is not yet clear. Using isolated Ca-tolerant adult rat heart muscle cells, we were able to study cardiac metabolism of arachidonic acid without the effects induced by endothelial or other non-muscle tissue. Myocytes rapidly incorporate arachidonic acid as well as other fatty acids into their lipid pools, the predominant acceptor being the triacylglycerols at an extracellular fatty acid concentration of 20 microM. As exogenous arachidonic acid is decreased, the distribution pattern shifts to favor phospholipid esterification. Cardiocyte prostaglandin production from arachidonic acid added to the incubation medium was limited (less than 1% conversion of added arachidonic acid) and lipoxygenase pathway activity was not detected. Oxidation rates of arachidonic acid were 3-fold lower than for palmitic acid, indicating that it is of secondary importance in energy-yielding reactions. Our results suggest that arachidonic acid serves primarily as a structural component of myocardial membranes and that its release during ischemia would permit its use as a substrate for prostaglandin production by coronary vascular tissue.     

Characterization of arachidonic acid-induced apoptosis

Tumor necrosis factor (TNF) can induce apoptosis in a number of different cell types. This response often depends on the activity of cytosolic phospholipase A₂ (cPLA₂), which catalyzes the release of arachidonic acid from the sn-2 position of membrane phospholipids. In this study, we investigate the ability of arachidonic acid itself to cause cell death. We show that in assays with 10% fetal bovine serum (FBS) arachidonic acid will not kill, nor does act synergistically with TNF. In contrast, by lowering the concentration of FBS to 2% it is possible to use arachidonic acid to induce cell death. Arachidonic acid-induced cell death was judged to be apoptotic based on morphology and the cleavage of poly (ADP) ribose polymerase. Arachidonic acid was able to kill all cell lines tested including two human melanoma-derived cell lines, and susceptibility to arachidonic acid was not influenced by adenovirus gene products that control susceptibility to TNF. Finally, we show that arachidonic acid is unique among 20 carbon fatty acids for its ability to induce apoptosis and that several other unsaturated, but not saturated fatty acids can also induce apoptosis.     

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.     

Free fatty acid release from endothelial cells

Cultured bovine aortic endothelial cells that have been previously enriched with fatty acid are able to release free fatty acid (FFA) into the extracellular fluid. No stimulus other than the presence of albumin in the medium is needed to elicit the FFA release. Intracellular triglycerides appear to be the source of most of the FFA that is released. The released FFA is composed of a mixture of fatty acids, with the fatty acid used to enrich the cells contributing about half of the total. Under certain conditions sufficient fatty acid can be released to increase the FFA concentration of the extracellular fluid. Cells enriched initially with arachidonic acid released 1.7- to 2.9-times more FFA as compared to cells enriched with corresponding amounts of oleic acid. Neither prostaglandins nor lipoxygenase products contributed appreciably to the amount of FFA released from cells enriched with arachidonic acid. Porcine pulmonary artery endothelial cells also can release net amounts of FFA. These findings indicate that endothelial cells have the capacity to release fatty acid in the form of FFA. This process could possibly play a role in the transfer of fatty acids, particularly arachidonic acid, across the endothelium.     

Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.     

Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.     

Effects of Arachidonic Acid on Glutamate and γ-Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

The effects of arachidonic acid on glutamate and gamma-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 mumol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015-0.6 mumol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 mumol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.