The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A2 during myocardial ischemia.

Recent studies have demonstrated the existence of two members of a novel family of calcium-independent plasmalogen-selective phospholipases A2 in mammalian myocardium (Wolf, R. A., and R. W. Gross. 1985. J. Biol. Chem. 260:7295-7303; and Hazen, S. L., D. A. Ford, and R. W. Gross. 1991. J. Biol. Chem. 266:5629-5633). To examine the potential role of these calcium-independent phospholipases A2 in mediating membrane dysfunction during early myocardial ischemia, the temporal course of alterations in phospholipase A2 activity during global ischemia in Langendorf perfused rabbit hearts was quantified and compared with traditionally accepted markers of myocytic ischemic injury and anaerobic metabolism. We now report that membrane-associated calcium-independent plasmalogen-selective phospholipase A2 activity increased over 400% during 2 min of global ischemia (P less than 0.01), was near maximally activated (greater than 10-fold) after only 5 min of ischemia, and remained activated throughout the entire ischemic interval examined (2-60 min). Activation of membrane-associated plasmalogen-selective phospholipase A2 after 5 min of myocardial ischemia was rapidly reversible during reperfusion of ischemic tissue. Both the activation of phospholipase A2 and its reversibility during reperfusion were temporally correlated to alterations in myocytic anaerobic metabolism. Furthermore, activation of membrane-associated phospholipase A2 was essentially complete before electron microscopic evidence of cellular damage. Collectively, these results identify dynamic alterations in calcium-independent plasmalogen-selective phospholipase A2 activity during myocardial ischemia which precede irreversible cellular injury and demonstrate that activation of plasmalogen-selective phospholipase A2 is amongst the earliest biochemical alterations in ischemic myocardium.

Isolation of a human myocardial cytosolic phospholipase A2 isoform. Fast atom bombardment mass spectroscopic and reverse-phase high pressure liquid chromatography identification of choline and ethanolamine glycerophospholipid substrates.

Recent studies have demonstrated the existence of a novel family of calcium-independent plasmalogen-selective phospholipases A2 in canine myocardium that have been implicated as enzymic mediators of ischemic membrane damage. We now report that human myocardium contains two functionally distinct isoforms of cytosolic calcium-independent phospholipase A2. The major cytosolic phospholipase A2 isoform preferentially hydrolyzes plasmalogen substrate, possesses a pH optimum of 7.0, and is chromatographically resolvable from a minor cytosolic calcium-independent phospholipase A2 isoform that hydrolyzes plasmenylcholine and phosphatidylcholine substrates at similar rates and possesses a pH optimum of 8.5. The major cytosolic calcium-independent phospholipase A2 isoform was identified as a 40-kD polypeptide after its 182,000-fold purification by sequential column chromatographies to a final specific activity of 67 mumol/mg.min. The purified 40-kD human myocardial phospholipase A2 preferentially hydrolyzes plasmalogens containing arachidonic acid at the sn-2 position. Both reverse-phase HPLC and fast atom bombardment mass spectroscopic analysis of human myocardial ethanolamine and choline glycerophospholipids demonstrated that plasmenylethanolamine and plasmenylcholine molecular species containing arachidonic acid at the sn-2 position are prominent constituents of human myocardium. Collectively, these results identify and characterize the major human myocardial cytosolic calcium-independent phospholipase A2 activity, demonstrate the presence of functionally distinct human myocardial cytosolic calcium-independent phospholipase A2 isoforms, and document the abundance of arachidonoylated plasmalogen molecular species in human myocardium that serve as substrates.

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine.

Mitochondrial thioesterase from rabbit myocardium was purified to homogeneity by sequential ion exchange, hydroxylapatite, chromatofocusing and gel filtration chromatographies. The purified protein had an apparent molecular mass of 170 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining revealed a single band (Mr 43 000) demonstrating that the protein is a tetramer. The specific activity of the purified thioesterase for palmitoyl-CoA hydrolysis was 1.8 mumol/mg per min. Thioesterase activity was maximal at pH 8 and was activated by Mg2+ but inhibited by Ca2+. Pathophysiological concentrations of L-palmitoyl carnitine (20-400 microM) competitively inhibited enzymic activity. The purified enzyme was also inhibited by high concentrations of substrate (over 20 microM palmitoyl-CoA).

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.

Alterations in membrane dynamics elicited by amphiphilic compounds are augmented in plasmenylcholine bilayers.

The dynamics of binary mixtures of choline glycerophospholipids and lysophospholipids were examined by fluorescence spectroscopy to compare and contrast the effects of each subclass of lysophospholipids on plasmenylcholine and phosphatidylcholine membrane motional characteristics. The decrease in steady-state anisotropy resulting from the introduction of lysoplasmenylcholine into plasmenylcholine bilayers was 4-6-fold greater than that manifest from the introduction of lysophosphatidylcholine into phosphatidylcholine bilayers (i.e., delta r = 0.017 vs. 0.004 or 0.011 vs. 0.002 at 5 C degrees and 10 C degrees above their phase transition temperatures, respectively). Lysoplasmenylcholine was also more potent than lysophosphatidylcholine in perturbing the dynamics of membrane bilayers comprised of phosphatidylcholine as measured by alterations in the steady-state anisotropy of the diphenylhexatriene probe. Finally, lipid matrices comprised of plasmenylcholine were uniformly more susceptible to amphiphilic perturbation (mediated by lysoplasmenylcholine, lysophosphatidylcholine or long chain acylcarnitine) than matrices comprised of phosphatidylcholine. Collectively, these results demonstrate that accumulation of plasmalogen catabolites resulting from activation of plasmalogen-selective phospholipases A2 can potentiate alterations in membrane dynamics during signal transduction in plasmalogen-enriched bilayers.

Proton nuclear magnetic resonance studies on the molecular dynamics of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol bilayers.

Physiologically relevant molecular species of plasmenylcholine and phosphatidylcholine were synthesized and their molecular dynamics and interactions with cholesterol were compared by determination of salient proton spin-lattice relaxation times and apparent activation energies for 1H-NMR observable motion. The molecular dynamics of PA PhosCho (1-hexadecanoyl-2-eicosatetra-5',8',11',14'-enoyl-sn-glycero-3-pho sphocholine) in multiple regions of the bilayer. Furthermore, the fluidity gradient of PA PhosCho was larger than that of PA PlasCho as ascertained by 1H spin-lattice relaxation time measurements. Introduction of cholesterol into each bilayer resulted in disparate effects on the dynamics of each subclass including: (1) increased motional freedom in the polar head group of PA PlasCho without substantial alterations in the dynamics of the polar head group of PA PhosCho; and (2) increased immobilization of the membrane interior in PA PlasCho in comparison to PA PhosCho. Analysis of Arrhenius plots of T1 relaxation times demonstrated that the apparent activation energies for vinyl and bisallylic methylene proton NMR observable motion in PA PhosCho were greater than that in PA PlasCho. Thus, comparisons of spin-lattice relaxation times and apparent activation energies demonstrate that vesicles comprised of PA PlasCho and PA PhosCho possess differential molecular dynamics and distinct interactions with cholesterol. Collectively, these results underscore the significance of the conjoint presence of the vinyl ether linkage and arachidonic acid as an important determinant of membrane dynamics in specialized mammalian membranes.

Dynamics of binary mixtures of plasmenylcholine/arachidonic acid and phosphatidylcholine/arachidonic acid--a study using fluorescence and NMR spectroscopy.

Arachidonic acid is selectively released during signal transduction in many cell types. To examine the effects of physiologically relevant amounts of arachidonic acid on membrane bilayers, alterations in membrane dynamics induced by arachidonic acid were investigated utilizing fluorescence and nuclear magnetic resonance spectroscopy. We demonstrate that decreases in the fluorescence steady-state anisotropy of diphenylhexatriene are induced by incorporation of physiologically relevant amounts (i.e., 5 mol%) of arachidonic acid into either phosphatidylcholine or plasmenylcholine membrane bilayers. Furthermore, examination of the motional dynamics of the bis-allylic protons in arachidonic acid by analyses of their spin-spin relaxation times demonstrated that these protons are more restrained when arachidonic acid is present as a substitutional impurity in plasmenylcholine vesicles than in phosphatidylcholine vesicles. Collectively, these results demonstrate that arachidonic acid, when present in physiologically relevant mole fractions, can modify the molecular dynamics of biological membranes and that the motional dynamics of arachidonic acid in membrane bilayers is influenced by the type of covalent linkage present in the proximal portion of the sn-1 aliphatic chain in host bilayer matrices.

Reconstitution of membrane fusion between pancreatic islet secretory granules and plasma membranes: catalysis by a protein constituent recognized by monoclonal antibodies directed against glyceraldehyde-3-phosphate dehydrogenase.

An isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolated and purified from rabbit brain cytosol has previously been demonstrated to catalyze membrane fusion (Glaser and Gross, Biochemistry 33 (1994) 5805-5812; Glaser and Gross, Biochemistry 34 (1995) 12193-12203). Herein, we provide evidence suggesting that this GAPDH isoform can reconstitute in vitro protein-catalyzed fusion between naturally occurring subcellular membrane fractions involved in insulin exocytosis. Utilizing purified rat pancreatic beta-cell plasma membranes and secretory granules, we show that a brain cytosolic factor catalyzed the rapid and efficient fusion of these two purified membrane fractions which could be inhibited by a monoclonal antibody directed against the brain isoform of GAPDH. Moreover, the brain cytosolic factor also catalyzed the fusion of reconstituted vesicles prepared from lipid extracts of islet plasma membranes and secretory granules. Although the brain cytosolic factor rapidly catalyzed membrane fusion between islet plasma membranes and secretory granules, it did not catalyze fusion between one secretory granule population with another. To identify the potential importance of brain cytosolic factor catalyzed membrane fusion in islet cells, we examined extracts of hamster insulinoma tumor cells (HIT cells) for fusion-catalyzing activity. A protein constituent was present in HIT cell cytosol which was immunologically similar to the rabbit brain GAPDH isoform. Although native HIT cell cytosol did not catalyze membrane fusion, removal of an endogenous protein inhibitor unmasked the presence of the protein which catalyzed membrane fusion activity and such fusion was ablated by a monoclonal antibody directed against the brain isoform of GAPDH. Collectively, these results suggest the possibility that an isoform of brain GAPDH, also evident in HIT cells, can catalyze fusion between the two naturally occurring subcellular membrane compartments involved in insulin secretion and suggest a novel paradigm potentially coupling glycolytic flux with insulin release.

Characterization of the sphingomyelin content of isolated pancreatic islets. Evaluation of the role of sphingomyelin hydrolysis in the action of interleukin-1 to induce islet overproduction of nitric oxide.

Inflammatory cytokines may participate in the destruction of pancreatic islets during the pathogenesis of insulin-dependent diabetes mellitus, and the cytokine interleukin-1 (IL-1) strongly inhibits insulin secretion from rat pancreatic islets by a process which involves induction of expression of the inducible isoform of nitric oxide synthase and the overproduction of nitric oxide. The signaling events between IL-1 receptor occupancy and induction of nitric oxide synthase in rat islets involve activation of the transcriptional activator NFkappa B. Because sphingomyelin hydrolysis has been implicated as a signaling process both in NFkappa B activation and in IL-1 action in some cells, we have examined the potential involvement of sphingomyelin hydrolysis in the induction of islet nitric oxide overproduction by IL-1. Rat islet sphingomyelin pools were radiolabeled with [3H]choline, and sphingomyelin was then isolated by normal phase HPLC. Electrospray ionization-mass spectrometric analysis revealed islet sphingomyelin consists of at least 4 distinct molecular species, and the most abundant of them contained sphingosine as the long chain base and a residue of palmitic acid as the fatty acid substituent. Molecular species containing residues of stearic acid and arachidic acid were also observed. Neither interleukin-1 nor tumor necrosis factor-alpha was found to induce hydrolysis of islet sphingomyelin species, and neither an exogenous, cell-permeant ceramide species (N-acetyl-D-sphingosine) nor exogenous sphingomyelinase mimicked or potentiated the effect of IL-1 to increase rat islet nitric oxide generation, as reflected by nitrite production. Similar findings were obtained with RINm5F insulinoma cells and with mouse pancreatic islets. These findings provide the first information on the molecular species of sphingomyelin in pancreatic islets and suggest that sphingomyelin hydrolysis is not involved in the signaling pathway whereby IL-1 induces the overproduction of nitric oxide by pancreatic islets.

Amplification of insulin secretion by lipid messengers.

D-glucose induces a rise in pancreatic islet beta-cell cytosolic [Ca2+] by processes requiring both glucose metabolism and Ca2+ entry from the extracellular space, and this Ca2+ signal is thought to be critical to the induction of insulin secretion. Insulin secretagogues also induce phospholipid hydrolysis and accumulation of phospholipid-derived mediators in islets, including the lipid messengers DAG, nonesterified arachidonic acid, and arachidonate 12-LO products. This study offers the following viewpoints on potential roles of these lipid messengers in insulin secretion as working hypotheses: 1) the Ca2+ signal provided to the beta-cell by D-glucose induces insulin secretion only in the context of amplifying background signals provided by the beta-cell content of messengers including DAG; 2) muscarinic receptor agonists amplify glucose-induced insulin secretion in part by altering the beta-cell content of DAG; 3) the Ca2+ signal provided by metabolism of D-glucose is amplified by the level of nonesterified arachidonic acid in beta-cell membranes, which acts to facilitate Ca2+ entry; 4) metabolism of glucose induces accumulation of nonesterified arachidonate in beta-cells via activation of a recently identified ASCI-PLA2 enzyme, which may be a component of the beta-cell fuel sensor apparatus; and 5) arachidonate 12-LO metabolites are potential candidates as adjunctive modulators of beta-cell K(+)-channel activity.

Myocardial phospholipases A(2) and their membrane substrates.

Many of the adverse sequelae of acute myocardial ischemia result from the degradation of sarcolemmal phospholipid constituents mediated by the activation of intracellular phospholipases. The consequent deleterious changes in sarcolemmal membrane properties precipitate ischemic membrane dysfunction resulting in electrophysiologic alterations and myocytic cell death. In myocardium, the overwhelming majority of phospholipase activity is catalyzed by a novel class of calcium-independent plasmalogen-selective phospholipases A(2) that is rapidly and reversibly activated within minutes of myocardial ischemia. Elucidation of the molecular mechanisms underlying the regulation of these phospholipases A(2) will define novel therapeutic targets that can potentially be pharmacologically manipulated to attenuate the deleterious effects of ischemia and reperfusion on myocardial function.

Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2.

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.

Long-term potentiation requires activation of calcium-independent phospholipase A2.

The predominant phospholipase activity present in rat hippocampus is a calcium-independent phospholipase A2 (302.9 +/- 19.8 pmol/mg.min for calcium-independent phospholipase A2 activity vs. 14.6 +/- 1.0 pmol/mg.min for calcium-dependent phospholipase A2 activity). This calcium-independent phospholipase A2 is exquisitely sensitive to inhibition by the mechanism-based inhibitor, (E)-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran -2-one (BEL). Moreover, treatment of hippocampal slices with BEL prior to tetanic stimulation prevents the induction of LTP (40.8 +/- 5.6% increase in excitatory post-synaptic potential (EPSP) slope for control slices (n = 6) vs. 5.8 +/- 8.5% increase in EPSP slope for BEL-treated slices (n = 8)). Importantly, LTP can be induced following mechanism-based inhibition of phospholipase A2 by providing the end product of the phospholipase A2 reaction, arachidonic acid, during the application of tetanic stimulation. Furthermore, the induction of LTP after treatment with BEL is dependent on the stereoelectronic configuration of the fatty acid provided since eicosa-5,8,11-trienoic acid, but not eicosa-8,11,14-trienoic acid, rescues LTP after BEL treatment (37.6 +/- 16.1% increase in EPSP slope for eicosa-5,8,11-trienoic acid vs. -3.7 +/- 5.2% increase in EPSP slope for eicosa-8,11,14-trienoic acid). Collectively, these results provide the first demonstration of the essential role of calcium-independent phospholipase A2 in synaptic plasticity.

The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia.

Myocardial calcium-independent phospholipase A2 (PLA2) activity is mediated by a 400 kDa catalytic complex comprised of a tetramer of phosphofructokinase (PFK) and a 40 kDa catalytic subunit [1,2]. During myocardial ischemia, calcium-independent PLA2 activity rapidly and reversibly translocates from the cytosol to a membrane-associated compartment where it has been implicated as a mediator of ischemic damage [3,4]. Herein we demonstrate that the majority of both PFK mass and activity is translocated from the cytosol to a membrane-associated compartment prior to the onset of irreversible myocytic injury and that translocated PFK is catalytically inactive while membrane-associated. Furthermore, reperfusion of ischemic myocardium, or treatment of membranes derived from ischemic myocardium with ATP results in the conversion of both PFK mass and activity from its membrane-associated state to a soluble, catalytically-competent form. Collectively, these studies demonstrate that the concomitant changes in glycolysis and phospholipid hydrolysis during early myocardial ischemia result, at least in part, from the translocation of a common regulatory polypeptide critical in both processes.

Structural determinants of haloenol lactone-mediated suicide inhibition of canine myocardial calcium-independent phospholipase A2.

Haloenol lactones are potent mechanism-based inhibitors of a novel class of calcium-independent phospholipases A2 which have been implicated as the enzymic mediators of membrane dysfunction during myocardial ischemia (Hazen, S. L.; et al. J. Biol. Chem. 1991, 266, 7227-7232). Herein we demonstrate that the ring size, hydrophobic group, and cryptic electrophile in the haloenol lactone moiety are important and modifiable determinants of the inhibitory potency of haloenol lactone-mediated inhibition of calcium-independent phospholipase A2. Direct comparisons between haloenol lactone-mediated inhibition of calcium-independent phospholipase A2 and the absence of inhibition with calcium-dependent phospholipase A2 further underscore the marked differences in the catalytic strategy employed by these two classes of intracellular phospholipases A2.

Sequencing of cloned DNA using bacteriophage lambda gt11 templates.

A procedure for isolating and directly sequencing recombinant bacteriophage lambda gt11 DNA templates is described. Approximately 250-300 bases of sequence can be obtained directly from the lambda gt11 template, eliminating the need for subcloning prior to dideoxynucleotide sequencing of clones.

Autoradiography of phosphatidyl choline.

Saturated choline phosphatides are extracted during conventional tissue processing for electron microscopy. To facilitate autoradiographic subcellular localization of arrhythmogenic myocardial phospholipids, we evaluated tissue processing procedures for preservation of saturated phosphatidyl choline (PC). Suspensions, of a murine plasmacytoma were incubated with negative, unilamellar liposomes containing 14C-choline-labeled PC or 14C-1-palmitate dipalmitoyl PC. Extraction of radioactivity was monitored at each processing step by liquid scintillation spectrometry. Conventional fixation with glutaraldehyde and osmium tetroxide followed by acetone dehydration and Spurr's plastic embedding led to extraction of nearly all radioactivity. However, treatment of cells with 1.5% tannic acid after glutaraldehyde but before osmium tetroxide fixation preserved 93.1 +/- .6% of 14C-choline-labeled PC. Virtually identical results were obtained with dipalmitoyl PC. Autoradiography demonstrated no significant translocation of labeled PC from plasmacytoma cells to unlabeled avian erythrocytes, mixed in equal proportions after fixation but before dehydration and embedding.

Structural determination of picomole amounts of phospholipids via electrospray ionization tandem mass spectrometry.

The remarkable sensitivity of electrospray ionization was exploited to achieve great increases in the sensitivity of tandem mass spectrometric analyses of phospholipids derived from both synthetic and biologic sources. Herein, we demonstrate that (1) product-ion spectra after electrospray ionization can be obtained easily by utilizing ≤ 5 pmol of phospholipid with a mass-selected window of less than 2 mass units, (2) the low energy inherent in the electrospray ionization method facilitates analysis of labile molecular ions that are not easily detected with the relatively high energy employed during fast-atom bombardment desorption, and (3) collision-induced dissociation of precursor ions generated from electrospray ionization often resulted in novel product-ion patterns. Collectively, these results underscore the utility of electrospray ionization tandem mass spectroscopy for the structural determination of diminutive amounts of phospholipids.

Differential molecular dynamics and transmembrane fluidity gradients in canine myocardial sarcolemma and sarcoplasmic reticulum.

The molecular dynamics of highly purified preparations of canine myocardial sarcolemma (SL) and sarcoplasmic reticulum (SR) were quantified by electron spin resonance spectroscopy (ESR). Canine myocardial SL and SR have substantially different motional regimes in their membrane interiors as demonstrated by alterations in the relative peak height ratios, peak widths and peak splittings in ESR spectra of 16-doxylstearate incorporated into SL and SR. Quantification of the apparent order parameters (S) of 16-doxylstearate in SL and SR by analyses of ESR spectra demonstrated that the interior of the SL membrane was substantially more immobilized than the interior of the SR membrane (e.g. S = 0.168 +/- 0.002 for SL and S = 0.128 +/- 0.003 for SR). In contrast, only modest differences in membrane dynamics near the hydrophobic-hydrophilic interface were present in SL and SR as ascertained by ESR spectra of the probe 5-doxylstearate incorporated into these membranes. Myocardial sarcolemma contained heretofore unsuspected amounts of cholesterol (1.4 +/- 0.1 mumol cholesterol/mg protein) while sarcoplasmic reticulum contained only small amounts of cholesterol (0.17 +/- 0.06 mumol cholesterol/mg protein). Model systems employing binary mixtures of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol demonstrated that the observed alterations in molecular dynamics were due, in large part, to the differential cholesterol content in these two subcellular membrane compartments. Taken together, these results demonstrate that these two functionally distinct myocardial subcellular membranes have markedly disparate molecular dynamics and transmembrane fluidity gradients which may facilitate their performance of specific functional roles during excitation-contraction coupling in myocardium.

The synthesis and molecular dynamics of phospholipids having hydroxylated fatty acids at the sn-2 position.

We have devised a general method for the synthesis of phospholipids containing hydroxylated fatty acids and have utilized this methodology to synthesize two naturally occurring hydroxylated lecithins (i.e. 1-palmitoyl-2-[15(S)-hydroxy-5E, 8E,11E,13Z-eicosatetraenoyl]-sn-glycero-3-phosph ocholine (1-palm-2-15HETE PC) and 1-palmitoyl-2-[5-hydroxy-6Z,8E,11E, 14E-eicosatetraenoyl]-sn-glycero-3-phosphocholine (1-palm-2-5HETE PC]. After protection of the hydroxylated fatty acid as its t-butyldimethylsilyl derivative, the anhydride of the protected fatty acid was formed utilizing dicyclohexylcarbodiimide and subsequently was condensed with a regiospecific lysolecithin utilizing pyrrolidinopyridine as catalyst to form the protected hydroxylated lecithin. Finally, hydroxylated lecithins were formed after removal of the t-butyldimethylsilyl group with acetic acid. Electron spin resonance spectroscopy was utilized to interrogate the molecular dynamics of lipid bilayers comprised of mixtures of these hydroxylated lecithins and naturally occurring lecithins. Remarkably, the molecular dynamics of spin-labeled phospholipids in liposomes comprised of cholesterol and phosphatidylcholine were altered substantially after addition of only 3.5 mol% 1-palm-2-15HETE PC as assessed by the order parameter of 16-doxylstearoyl phosphatidylcholine.