Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.     

The solution structure of the recombinant hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 in its hemichrome state

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin displays bis-histidine coordination of the iron ion. In addition, this protein is capable of covalently attaching a reactive histidine to the heme 2-vinyl group. The structure of the protein in the low-spin ferric state with intact vinyl substituents was solved by NMR methods. It was found that the structure differs from that of known truncated hemoglobins primarily in the orientation of the E helix, which carries His46 (E10) as the distal ligand to the iron; the length and orientation of the F helix, which carries His70 (F8) as the proximal ligand to the iron; and the H-helix, which carries His117 (H16), the reactive histidine. Regions of enhanced flexibility include the short A helix, the loop connecting the E and F helices, and the last seven residues at the carboxy end. The structural data allowed for the rationalization of physical properties of the cyanobacterial protein, such as fast on-rate for small ligand binding, unstable apoprotein fold, and cross-linking ability. Comparison to the truncated hemoglobin from the green alga Chlamydomonas eugametos also suggested how the endogenous hexacoordination affected the structure.     

Mechanisms of cardioprotection by lysophospholipids

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphosphatidic acid (LPA) reduce mortality in hypoxic cardiac myocytes. S1P is also cardioprotective in both mouse and rat models of cardiac ischemia/reperfusion (I/R) injury. Although these results are consistent with prior work in other cell types, it is not known what signaling events are critical to cardioprotection, particularly with respect to ceramide and the preservation of mitochondrial function, which is essential for cardiac cell survival. Neither receptor regulation nor signaling has been studied during I/R in the heart with or without the application of S1P or LPA. The role of sphingosine kinase in I/R and in ischemic preconditioning (IPC) has not been defined, nor has the fate or function of S1P generated by this enzyme, particularly during preconditioning or I/R, been elucidated. Whether S1P infused systemically in animal models of myocardial infarction in which survival is an end-point will be hemodynamically tolerated has not been determined. If not, the substitution of agents such as the monosialoganglioside GM-1, which activates sphingosine kinase, or the development of alternative ligands for S1P receptors will be necessary.     

Stem cell regulation by lysophospholipids

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) regulate a diverse range of mammalian cell processes, largely through engaging multiple G protein-coupled receptors specific for these lysophospholipids. LPA and S1P have been clearly identified to have widespread physiological and pathophysiological actions, controlling events within the reproductive, gastrointestinal, vascular, nervous and immune systems, and also having a prominent role in cancer. Here we review the recent literature showing the additional emerging role for LPA and S1P in the regulation of stem cells and their progenitors. We discuss the role of these lysophospholipids in regulating the proliferation, survival, differentiation and migration of a range of adult and embryonic stem cells and progenitors, and thus are likely to play a substantial role in the maintenance, generation, mobilisation and homing of stem cell and progenitor populations in the body.     

Reversible and irreversible hemichrome generation by the oxygenation of nitrosylmyoglobin

The repeated oxygenation/reduction/nitrosylation of nitrosylmyoglobin produces low-spin ferric heme hemichromes which have been characterized by electron spin resonance spectroscopy. The predominant myoglobin hemichrome is a chemically reversible dihistidyl complex identified by the g values 1.53, 2.21, and 2.97. Also present is a low-spin ferric hydroxide derivative which is represented by the g values 1.83, 2.18, and 2.59. The formation of these species goes undetected by UV-vis spectroscopy, but the oxygenation of myoglobin to metmyoglobin is correlated with complete conversion of nitric oxide to nitrate which is released following a clear induction period. These results are interpreted in terms of the intermediates generated during the MbNO oxygenation reaction.     

Hemoglobin conversion to hemichrome under the influence of fatty acids

The effect of free fatty acids on the process of hemoglobin conversion and lipid peroxidation has been studied in model systems and erythrocytes. It has been found that methemoglobin and oxyhemoglobin are converted to the low spin oxidized form, namely, reversible hemichrome under the action of fatty acids. In the case of oxyhemoglobin, an increase in the level of active oxygen forms is observed in the system which initiates the formation of primary and secondary lipid peroxidation products. Incubation of erythrocytes with free fatty acids causes the formation of Heinz bodies and is accompanied by an increase of the lipid peroxidation level.     

Activation of peritoneal macrophages by orally administered ether analogues of lysophospholipids.

Oral administration of dodecylglycerol, inflammatory product of cancerous tissues, and the alkyl lysophospholipid derivative, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3-choline), greatly activated mouse peritoneal macrophages. The activation was dose related and was assessed as increased Fc-mediated ingestion of red blood cells, superoxide production, chemiluminescence activity, and incorporation of radioactive thymidine and leucine. Furthermore, the data show that dodecylglycerol or ET-18-OCH3-choline was capable of inducing equally high levels of macrophage activation and cytotoxic action on tumor cells, just as occurs with intraperitoneal administration. Dodecylglycerol appeared to activate the macrophages at a relatively lower dose (5 micrograms/mouse) than ET-18-OCH3-choline (15 micrograms/mouse). The optimal oral doses required to activate macrophages for ingestion and cytotoxic activities were relatively higher than previously observed when these agents were administered intraperitoneally. Thus, the dose difference provided crucial information for correlating oral dosages with in vivo concentration of these agents as bioassayed by macrophage activation. These observations have extended and further support our earlier findings that these agents are effective immunopotentiators and thus could therapeutically be used to activate macrophages for cytotoxic effects on tumor cells via the oral route.     

Damage to hemoglobin by radiation-generated serum albumin radicals.

We have studied the effects of the interaction of radiation generated human serum albumin radicals (HSA*) with human hemoglobin molecules (Hb). Diluted Hb aqueous solutions were irradiated under N2O or argon without HSA and in the presence of HSA. Analysis of Hb absorbance spectra in the visible range, cross-linking of HSA* radicals with Hb molecules and functional properties of Hb were investigated. The degree of Hb destruction estimated on the basis of changes in the absorption spectra indicated that the effectiveness of HSA* radicals generated under N2O for Hb destruction was approximately equal to that of *OH radicals. In this case mainly *OH radicals formed the secondary HSA* radicals. However, during the irradiation Hb + HSA under argon the presence of equivalent amounts of oxidizing and reducing products of water radiolysis lowers the degree of Hb destruction. Some reactions of HSA* radicals with Hb molecules lead to the formation of covalent bonds between the molecules of both proteins. The following types of hybrids could be distinguished: Hb monomer-HSA, Hb dimer-HSA and higher aggregates. Structural changes of Hb by HSA* radicals were reflected by alterations in the oxygen affinity (increase) and cooperativity (decrease) of Hb. The results obtained indicate that in the experimental systems studied, the HSA* radical reactions with Hb molecules are favoured over recombination reactions of HSA* radicals. On this basis one can suggest that in the studied systems Hb plays the role of an acceptor of radical energy located on HSA.     

Activation of macrophages by ether analogues of lysophospholipids

Summary Inflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L--lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkylglycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkylglycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.     

Sensing of lysophospholipids by TRPC5 calcium channel

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.     

Capacitation of bovine spermatozoa by lysophospholipids and trypsin

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.     

Regulation of protein kinase C by lysophospholipids. Potential role in signal transduction

Certain lysophospholipids, lysophosphatidylcholine (lyso-PC) in particular, stimulated protein kinase C at low concentrations (less than 20 microM) but, conversely, inhibited it at high concentrations (greater than 30 microM). Protein kinase C stimulation by lyso-PC required the presence of phosphatidylserine (PS) and Ca2+ and was associated with a decreased Ka for PS and increased Ka for Ca2+ of the enzyme. Cardiolipin and phosphatidic acid could partially substitute for PS in supporting the stimulatory effect of lyso-PC. Lyso-PC also biphasically regulated protein kinase C activated by diolein. Of several synthetic lyso-PC preparations tested, the oleoyl, myristoyl and palmitoyl derivatives were most active. Data from the Triton X-100 mixed micellar assay indicated that 1.4 and 14.0 mol of lyso-PC/micelle produced a maximal stimulation and a complete abolishment of the stimulation of protein kinase C, respectively. Protein kinase C stimulation by lyso-PC, with a pH optimum of about 7.5, was observed for phosphorylation of histone H1, myelin basic protein, and the 35- and 47-kDa proteins from the rat brain, but not for that of other histone subfractions and protamine. Lyso-PC acted synergistically with diacylglycerol in stimulating protein kinase C, whereas the stimulation by lyso-PC was additive to that by oleic acid. Protein kinase C inhibitors (alkyllysophospholipid, sphingosine, tamoxifen, and polymyxin B) inhibited more potently the protein kinase C activity stimulated by PS/Ca2+/lyso-PC than that stimulated by PS/Ca2+. The stimulatory and inhibitory effects of lyso-PC were not observed for myosin light chain kinase and cAMP-dependent protein kinase, indicating a specificity of its actions. The present findings suggested that lyso-PC, likely derived from membrane PC by the action of phospholipase A2, might play a role in signal transduction via a dual regulation of protein kinase C, and that it could further modulate the enzyme and hence the cellular activity by interplaying with diacylglycerol and unsaturated fatty acid, the two other classes of cellular mediators also shown to be activators of protein kinase C.     

Reversible skeletal neuromuscular paralysis induced by different lysophospholipids

Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion-pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine>lysophosphatidylethanolamine>lysophosphatidic acid>lysophosphatidylserine>lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis.     

Binding of spin-labeled fatty acids and lysophospholipids to hydrophobic region of calmodulin.

In the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phospholipids, described previously [Suzuki, T., Katoh, H., & Uchida, M.K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.     

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.     

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes.

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.     

Metabolic relationship between arachidonate activation and its transfer to lysophospholipids by brain microsomes

Evidence is presented to indicate a metabolic relationship between arachidonic acid activation and its transfer to lysophospholipds by brain microsomes. Thus, in the presence of 1-acylglycerophosphocholines or 1-acyl-glycerophosphoinositols, the activation of labeled arachidonate to its acyl-CoA was enhanced, and the acyl-CoA formed was, in turn, transferred to the lysophospholipids to form the respective diacyl-glycerophospholipids. The coupling effect seems to pertain mainly to the lysophospholipids which are good substrates of the acyltransferase. Other lyso-compounds were either not effective or inhibitory to the arachidonate activation process. The activation-transfer activity mediated by the fatty acid ligase and acyltransferase could be dissociated by Triton X-100, which apparently stimulated the acyl-CoA ligase activity but inhibited the acyltransferase. These results suggest that fatty acid ligase and acyltransferase are located in close proximity within the membrane domain. The existence of a close metabolic relationship between these two enzymic reactions is important for maintaining a dynamic equilibrium between the free fatty acids and the membrane phospholipids. The mechanism is also useful in regulating the cellular acyl-CoA and lysophospholipid metabolism, because both compounds have membrane perturbing properties when present in excessive quantity.