Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Effects of fatty acids in isolated mitochondria: implications for ischemic injury and cardioprotection

Fatty acids accumulate during myocardial ischemia and are implicated in ischemia-reperfusion injury and mitochondrial dysfunction. Because functional recovery after ischemia-reperfusion ultimately depends on the ability of the mitochondria to recover membrane potential (DeltaPsim), we studied the effects of fatty acids on DeltaPsim regulation, cytochrome c release, and Ca2+ handling in isolated mitochondria under conditions that mimicked aspects of ischemia-reperfusion. Long-chain but not short-chain free fatty acids caused a progressive and reversible (with BSA) increase in inner membrane leakiness (proton leak), which limited mitochondrial ability to support DeltaPsim. In comparison, long-chain activated fatty acids promoted 1). a slower depolarization that was not reversible with BSA, 2). cytochrome c loss that was unrelated to permeability transition pore opening, and 3). inhibition of the adenine nucleotide translocator. Together, these results impaired both mitochondrial ATP production and Ca2+ handling. Diazoxide, a selective opener of mitochondrial ATP-dependent potassium (KATP) channels, partially protected against these effects. These findings indicate that long-chain fatty acid accumulation during ischemia-reperfusion may predispose mitochondria to cytochrome c loss and irreversible injury and identify a novel cardioprotective action of diazoxide.     

Stress-activated protein kinase phosphorylation during cardioprotection in the ischemic myocardium

Stress-activated protein kinases may be essential to cardioprotection. We assessed the role of p38 in an in vivo rat model of ischemia-reperfusion. Ischemic preconditioning (IPC) and the delta(1)-opioid receptor agonist 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha-octahydroquinolino [2,3,3-g]isoquinoline (TAN-67) significantly reduced infarct size (IS), expressed as a percentage of the area at risk (AAR), versus animals subjected only to 30 min of ischemia and 2 h of reperfusion (7.1 +/- 1.5 and 29.6 +/- 3.3 vs. 59.7 +/- 1.6%). The p38 antagonist SB-203580 attenuated IPC when it was administered before (34.0 +/- 6.9%) or after (25.0 +/- 3.8%) the IPC stimulus; however, it did not significantly attenuate TAN-67-induced cardioprotection (39.6 +/- 3.2). We also assessed the phosphorylation of p38 and c-jun NH(2)-terminal kinase (JNK) throughout ischemia-reperfusion in nuclear and cytosolic fractions. After either intervention, no increase was detected in the phosphorylation state of either enzyme in the nuclear fraction or for p38 in the cytosolic fraction versus control hearts. However, there was a robust increase in JNK activity in the cytosolic fraction immediately on reperfusion that was more pronounced in animals subjected to IPC or administered TAN-67. These data suggest that SB-203580 likely attenuates IPC via the inhibition of kinases other than p38, which may include JNK. The data also suggest that activation of JNK during early reperfusion may be an important component of cardioprotection.     

Transferrin modifications and lipid peroxidation: implications in diabetes mellitus

Free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation. One of the most important mechanisms of antioxidant defense is thus the sequestration of iron in a redox-inactive form by transferrin. In diabetes mellitus, increased oxidative stress and lipid peroxidation contribute to chronic complications but it is not known if this is related to abnormalities in transferrin function. In this study we investigated the role of transferrin concentration and glycation. The antioxidant capacity of apotransferrin to inhibit lipid peroxidation by iron-binding decreased in a concentration-dependent manner from 89% at > or = 2 mg/ml to 42% at 0.5 mg/ml. Pre-incubation of apotransferrin with glucose for 14 days resulted in a concentration-dependent increase of glycation: 1, 5 and 13 micromol fructosamine/g transferrin at 0, 5.6 and 33.3 mmol/l glucose respectively, p < 0.001. This was accompanied by a decrease in the iron-binding antioxidant capacity of apotransferrin. In contrast, transferrin glycation by up to 33.3 mmol/l glucose did not affect chemiluminescence-quenching antioxidant capacity, which is iron-independent. Colorimetric evaluation of total iron binding capacity in the presence of an excess of iron (iron/transferrin molar ratio = 2.4) also decreased from 0.726 to 0.696 and 0.585mg/g transferrin after 0, 5.6 and 33.3 mmol/l glucose, respectively, p < 0.01. In conclusion, these results suggest that lower transferrin concentration and its glycation can, by enhancing the pro-oxidant effects of iron, contribute to the increased lipid peroxidation observed in diabetes.     

Involvement of the mitochondrial calcium uniporter in cardioprotection by ischemic preconditioning

The objective of the present study was to determine whether the mitochondrial calcium uniporter plays a role in the cardioprotection induced by ischemic preconditioning (IPC). Isolated rat hearts were subjected to 30 min of regional ischemia by ligation of the left anterior descending artery followed by 120 min of reperfusion. IPC was achieved by two 5-min periods of global ischemia separated by 5 min of reperfusion. IPC reduced the infarct size and lactate dehydrogenase release in coronary effluent, which was associated with improved recovery of left ventricular contractility. Treatment with ruthenium red (RR, 5 μM), an inhibitor of the uniporter, or with Ru360 (10 μM), a highly specific uniporter inhibitor, provided cardioprotective effects like those of IPC. The cardioprotection induced by IPC was abolished by spermine (20 μM), an activator of the uniporter. Cyclosporin A (CsA, 0.2 μM), an inhibitor of the mitochondrial permeability transition pore, reversed the effects caused by spermine. In mitochondria isolated from untreated hearts, both Ru360 (10 μM) and RR (1 μM) decreased pore opening, while spermine (20 μM) increased pore opening which was blocked by CsA (0.2 μM). In mitochondria from preconditioned hearts, the opening of the pore was inhibited, but this inhibition did not occur in the mitochondria from hearts treated with IPC plus spermine. These results indicate that the mitochondrial calcium uniporter is involved in the cardioprotection conferred by ischemic preconditioning.     

Topogenesis of membrane proteins: determinants and dynamics

For targeting and integration of proteins into the mammalian endoplasmic reticulum, two types of signals can be distinguished: those that translocate their C-terminal sequence (cleavable signals and signal-anchors) and those that translocate their N-terminus (reverse signal-anchors). In addition to the well established effect of flanking charges, also the length and hydrophobicity of the apolar core of the signal as well as protein folding and glycosylation contribute to orienting the signal in the translocon. In multi-spanning membrane proteins, topogenic determinants are distributed throughout the sequence and may even compete with each other. During topogenesis, segments of up to 60 residues may move back and forth through the translocon, emphasizing unexpected dynamic aspects of topogenesis.     

Death, autoantigen modifications, and tolerance

Autoantibodies present in the serum of patients with a variety of inflammatory diseases have proven useful as diagnostic markers and as probes with which to elucidate biochemical and signaling pathways. The mechanisms governing the generation of autoantibodies remain elusive, constituting a critical missing link in our understanding of rheumatologic illnesses. Several lines of experimentation in recent years have strongly implicated events surrounding cell death in this process. This review will address the potential role played by death-specific modifications of autoantigens in bypassing tolerance to highly conserved autoantigens, including nucleic acids, lipids, and proteins.     

High-speed atomic force microscopy: cooperative adhesion and dynamic equilibrium of junctional microdomain membrane proteins

Junctional microdomains, paradigm for membrane protein segregation in functional assemblies, in eye lens fiber cell membranes are constituted of lens-specific aquaporin-0 tetramers (AQP0(4)) and connexin (Cx) hexamers, termed connexons. Both proteins have double function to assure nutrition and mediate adhesion of lens cells. Here we use high-speed atomic force microscopy to examine microdomain protein dynamics at the single-molecule level. We found that the adhesion function of head-to-head associated AQP0(4) and Cx is cooperative. This finding provides first experimental evidence for the mechanistic importance for junctional microdomain formation. From the observation of lateral association-dissociation events of AQP0(4), we determine that the enthalpic energy gain of a single AQP0(4)-AQP0(4) interaction in the membrane plane is -2.7 k(B)T, sufficient to drive formation of microdomains. Connexon association is stronger as dynamics are rarely observed, explaining their rim localization in junctional microdomains.     

The coxsackie B virus and adenovirus receptor resides in a distinct membrane microdomain

The coxsackie B virus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily. In addition to activity as a viral receptor, it may play a role in cellular adhesion. We asked what determines the cell membrane microdomain of CAR. We found that CAR is localized to a novel lipid-rich microdomain similar to that of the low-density lipoprotein receptor (LDLR) but distinct from that of a CAR variant that exhibited traditional lipid raft localization via fusion to a glycosylphosphatidylinositol (GPI) tail. The cytoplasmic tail determines its membrane localization, since deletion of this domain resulted in mislocalization. Results indicate that CAR, CAR-LDLR, and LDLR reside in a novel lipid raft that is distinct from caveolin-1-containing caveolae and GPI-linked proteins. Residence in a lipid-rich domain provides a mechanism that allows CAR to interact with other cell adhesion proteins and yet function as an adenovirus receptor.     

Hypoxemic and ischemic tolerance in emperor penguins

The presence of 7-O-acyl okadaic acid (OA) esters was studied by LC–MS in the digestive glands of blue mussel (Mytilus galloprovincialis) and common cockle (Cerastoderma edule) from Albufeira lagoon, located 20 km south of Lisbon. The profile of free and total fatty acids (FA) was analysed using a similar LC separation with a reversed phase C8 column and mass spectrometry detection. In mussel the free FA profile was reflected in the FA esterified to OA, being palmitic acid for instance the most abundant in both cases. In cockle, 7-O-acyl esters with palmitic acid were almost absent and esters with a C16:0 isomer were dominant, followed by esters with C15:1 and C15:0. The cockle free FA profile was similar to mussel, and in accordance with literature findings in bivalves. After hydrolysis, a major difference in the FA profile occurred in both species, presenting a high percentage of a C16:0 isomer. The isomer found in general lipids and bound to OA seemed to be related, presenting similar relative retention times (RRT) to C16:0, differing from expected RRT of monomethyl-branched isomers (iso- or anteiso-). A tentative identification was made with the multimethyl-branched isoprenoid, 4,8,12-trimethyltridecanoic acid (TMTD). TMTD is a product of phytol degradation. This was also suspected when the proportion of this compound in relation to palmitic acid was reduced in vivo in mussels fed a chlorophyll-free diet. Extensive esterification of OA by, among others, phytol-degrading bacteria is discussed as a plausible hypothesis in cockle, but not in mussel, due to the relatively high specific proportion of odd-numbered and branched FA.     

TNFalpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3

Membrane microdomains (lipid rafts) are now recognized as criticalfor proper compartmentalization of insulin signaling, but theirrole in the pathogenesis of insulin resistance has not beeninvestigated. Detergent-resistant membrane microdomains (DRMs),isolated in the low-density fractions, are highly enriched incholesterol, glycosphingolipids and various signaling molecules.Tumor necrosis factor alpha (TNF) induces insulin resistancein type 2 diabetes, but its mechanism of action is not fullyunderstood. In other studies we have found a selective increasein the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytestreated with TNF, suggesting a specific function for GM3. Inthe DRMs from TNF-treated 3T3-L1 adipocytes, GM3 levels weredoubled compared with results in normal adipocytes. Additionally,insulin receptor (IR) accumulations in the DRMs were diminished,whereas caveolin and flotillin levels were unchanged. Furthermore,insulin-dependent IR internalization and intracellular movementof the IR substrate 1(IRS-1) were both greatly suppressed inthe treated cells, leading to an uncoupling of IR–IRS-1signaling. GM3 depletion was able to counteract the TNF-inducedinhibitions of IR internalization and accumulation into DRMs.Together, these findings provide compelling evidence that ininsulin resistance the insulin metabolic signaling defect canbe attributed to a loss of IRs in the microdomains due to anaccumulation of GM3.     

Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization

Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gq alpha, Ras, integrin beta 1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-beta-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-beta-cyclodextrin-treated eggs, sperm-induced Ca(2+) transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.     

Pyruvate provides cardioprotection in the experimental model of myocardial ischemic reperfusion injury

The present study was designed to evaluate the cardioprotective potential of pyruvate and to characterize the mechanism underlying the protection. Wistar albino rats were randomly divided into three groups. Two groups were administered saline orally (sham, ischemia-reperfusion (I-R) control group) and animals of third group received pyruvate (500 mg/kg) for 4 weeks. On the 29th day, animals of the I-R control and pyruvate treated groups underwent 45 min of occlusion of the left anterior descending (LAD) coronary artery and were thereafter reperfused for 60 min. In the I-R control group, a significant cardiac necrosis, depressed mean arterial pressure (MAP) and heart rate (HR), decline in myocardial antioxidant status and elevation in lipid peroxidation were observed as compared to sham control. Pyruvate treatment restored the myocardial antioxidant status and favorably modulated the altered MAP as compared to I-R control. Furthermore, I/R-induced lipid peroxidation was significantly inhibited by pyruvate treatment. These beneficial cardioprotective effects translated into significant improvement in MAP. Histopathological examination and restored specific myocardial injury marker CK-MB isoenzyme activity further confirmed protective effects of pyruvate. In conclusion, our study has demonstrated that the beneficial effect of pyruvate likely results from improved MAP and suppression of oxidative stress.     

Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane in living yeast

We report sphingolipid-related reorganization of gel-like microdomains in the plasma membrane of living Saccharomyces cerevisiae using trans-Parinaric acid (t-PnA) and 1,6-diphenyl-1,3,5-hexatriene (DPH). Compared to control, the gel-like domains were significantly reduced in the membrane of a sphingolipid-deficient lcb1-100 mutant. The same reduction resulted from sphingolipid depletion by myriocin. The phenotype could be reverted when a myriocin-induced block in sphingolipid biosynthesis was bypassed by exogenous dihydrosphingosine. Lipid order of less-ordered membrane regions decreased with sphingolipid depletion as well, as documented by DPH fluorescence anisotropy. The data indicate that organization of lateral microdomains is an essential physiological role of these structural lipids.