HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Williams, S. D. Articles by Ford, D. A. Search for Related Content PubMed PubMed Citation Articles by Williams, S. D. Articles by Ford, D. A. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 41, 1585-1595, October 2000
Copyright © 2000 by Lipid Research, Inc.

Original Article

Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium

Correspondence to: David A. Ford

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The role of nuclear membrane phospholipids as targets of phospholipases resulting in the generation of nuclear signaling messengers has received attention. In the present study, we have exploited the utility of electrospray ionization mass spectrometry to determine the phospholipid content of nuclei isolated from perfused hearts. Rat heart nuclei contained choline glycerophospholipids composed of palmitoyl and stearoyl residues at the sn-1 position with oleoyl, linoleoyl, and arachidonoyl residues at the sn-2 position. Diacyl molecular species were the predominant molecular subclass in the choline glycerophospholipids, with the balance of the molecular species being plasmalogens. In the ethanolamine glycerophospholipid pool from rat heart nuclei approximately 50% of the molecular species were plasmalogens, which were enriched with arachidonic acid at the sn-2 position. A 50% loss of myocytic nuclear choline and ethanolamine glycerophospholipids was observed in hearts rendered globally ischemic for 15 min followed by 90 min of reperfusion in comparisons with the content of these phospholipids in control perfused hearts. The loss of nuclear choline and ethanolamine glycerophospholipids during reperfusion of ischemic myocardium was partially reversed by the calcium-independent phospholipase A₂ (iPLA₂) inhibitor bromoenol lactone (BEL), suggesting that the loss of nuclear phospholipids during ischemia/reperfusion is mediated, in part, by iPLA₂. Western blot analyses of isolated nuclei from ischemic hearts demonstrated that iPLA₂ is translocated to the nucleus after myocardial ischemia.

Taken together, these studies have demonstrated that nuclear phospholipid mass decreases after myocardial ischemia by a mechanism that involves, at least in part, phospholipolysis mediated by iPLA₂. — Williams, S. D., F-F. Hsu, and D. A. Ford. Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium. J. Lipid Res. 2000. 41: 1585;–1595.

Supplementary key words: phospholipids, plasmalogen, phospholipase A₂, myocardial ischemia

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the past two decades the role of plasma membrane phospholipids as precursors of second messengers such as arachidonic acid, lysophospholipids, eicosanoids, diacylglycerols and inositol phosphates has received considerable attention (1) (2) (3) (4) (5) (6). Cell regulatory mechanisms that involve phospholipolysis catalyzed by a plethora of phospholipases that target nuclear membrane phospholipids have been proposed that could potentially mediate nuclear function and thus gene regulation and programmed cell death (7) (8) (9). A role for phospholipases in nuclear signaling has been suggested by the observation that diacylglycerols derived from choline glycerophospholipid pools accumulate in the nuclear envelope of thrombin-stimulated fibroblasts by a biochemical mechanism that includes rhoA-dependent translocation of phospholipase D (PLD) to the nuclear envelope (8). In addition, cytosolic phospholipase A₂ (cPLA₂) is found in the nuclear membrane of histamine-stimulated endothelial cells (9). It is also likely that phospholipase A₂ plays a major role in myocardial nuclear phospholipid signaling because transmission electron microscopic autoradiography has demonstrated rapid arachidonic acid turnover in the nuclear membranes of cardiac myocytes (10). However, a thorough evaluation of the myocardial nuclear membrane pool phospholipids has not been forthcoming because of limitations in the detection levels of methods to analyze minute amounts of phospholipid.

Phospholipase A₂ activity has been identified in the heart and implicated as a mediator of the pathophysiological sequelae of myocardial ischemia (11) (12) (13) (14). Calcium-independent phospholipase A₂ (iPLA₂) is a predominant phospholipase A₂ activity present in myocardium (14) (15). In isolated perfused hearts rendered globally ischemic (i.e., zero-flow ischemia to the heart), iPLA₂ is translocated to myocardial membranes within 2 min of the onset of ischemia (14). However, the role of iPLA₂ in the ischemic heart remains to be fully elucidated because concomitant changes in cardiac myocyte phospholipid content have not been observed concomitant with ischemia-elicited increases in iPLA₂ activity.

Because membrane-associated iPLA₂ activity increases in a crude membrane fraction isolated from ischemic hearts (14) (15), the possibility that iPLA₂ is translocated to the nucleus during myocardial ischemia was considered. Accordingly, in the current study, the unparalleled sensitivity of electrospray ionization mass spectrometry (ESI-MS) was exploited to characterize nuclear choline and ethanolamine glycerophospholipid molecular species and to determine the possible involvement of iPLA₂ in nuclear phospholipid degradation. Herein, we report that rat myocardial nuclei contain a diverse array of both diacyl and plasmalogen molecular species and that the mass of these phospholipid pools is decreased after myocardial ischemia and reperfusion. Furthermore, the loss of nuclear choline and ethanolamine glycerophospholipids after ischemia and reperfusion is, at least in part, mediated by iPLA₂, which is translocated to the nucleus during myocardial ischemia.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials
Anti-iPLA₂ was a generous gift from Genetics Institute (Cambridge, MA). Anti-cPLA₂ (N-216), anti-secretory PLA₂ (sPLA₂), and anti-lamin B₁ were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), Cayman Chemical (Ann Arbor, MI), and Zymed (South San Francisco, CA), respectively. Secondary antibodies including goat anti-rabbit horseradish peroxidase and goat anti-mouse horseradish peroxidase were purchased from Sigma (St. Louis, MO) and Bio-Rad (Hercules, CA), respectively. Electrophoresis-grade reagents for polyacrylamide and agarose gel electrophoresis were purchased from ICN Pharmaceuticals (Costa Mesa, CA), Pharmacia (Piscataway, NJ), Bio-Rad, and Fisher (Pittsburgh, PA). Bromoenol lactone (BEL) was synthesized and prepared as previously described (16). Phospholipid internal standards, 1,2-ditetradecanoic-sn-glycero-3-phosphocholine (1,2-ditetradecanoic-sn-GPC), 1,2-ditetradecanoic-sn-glycero-3-phosphoethanolamine (1,2-ditetradecanoic-sn-GPE), and 1,2-dieicosanoic-sn-glycero-3-phosphocholine (1,2-dieicosanoic-sn-GPC), were purchased from Avanti Polar Lipids (Alabaster, AL). All other reagents were of the highest grade available and were purchased from Sigma, Fisher, or VWR Scientific (San Francisco, CA).

Preparation of myocardial nuclei from Langendorff perfused rat hearts subjected to myocardial ischemia and reperfusion
Male Sprague-Dawley rats were utilized for the preparation of Langendorff perfused hearts as described previously (14) (17). In brief, male Sprague-Dawley rats (200;–250 g body weight) were injected with heparin (200 U, intraperitoneal) 30 min before being anesthetized with pentobarbital sodium (25 mg, intraperitoneal). Hearts were rapidly removed and placed immediately in ice-cold saline solution before being perfused. Rat hearts were perfused retrograde through the aorta with a modified Krebs-Henseleit buffer (137 mM NaCl, 4.7 mM KCl, 3 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 0.5 mM Na-EDTA, 15 mM NaHCO₃, and 11 mM glucose, equilibrated with 95% O₂, 5% CO₂, pH 7.4) at 37°C for 15 min at a pressure of 60 mm Hg, followed by either control perfusions for 105 min, global zero-flow ischemia for 15 min, or global zero-flow ischemia for 15 min followed by 90 min of reperfusion. In selected experiments, 10 µM BEL was included in the perfusion buffer for 15 min after the initial 15-min equilibration period prior to ischemia and reperfusion protocols. Rat heart nuclei were prepared from isolated perfused hearts as previously described (18) (19). In brief, ventricles from perfused hearts were immediately minced in 10 volumes of homogenization buffer [10 mM Tris-HCl, 250 mM sucrose, 3 mM MgCl₂, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4] and then homogenized with a Polytron (40% setting for 10 sec). The homogenate was centrifuged at 1,000 g_max for 10 min and the pellet was subsequently resuspended in 10 volumes of homogenization buffer and rehomogenized with six strokes of a Potter-Elvehjem homogenizer at a setting of 40%. The resulting homogenate was filtered through nylon sieve mesh (100 µm) and the filtrate was centrifuged at 1,000 g_max for 10 min. The pellet was resuspended in 10 volumes of homogenization buffer. Again, the resuspended pellet was centrifuged at 1,000 g_max for 10 min and subsequently resuspended in 20 volumes of homogenization buffer adjusted to 2.2 M sucrose. The resuspended homogenate was underlaid with 5 ml of homogenization buffer containing 2.2 M sucrose and then subjected to ultracentrifugation in a swinging bucket rotor (SW-28) at 113,000 g_max for 1 h. The nuclear pellet was washed in 10 mM Tris buffer (pH 7.4) containing 3 mM MgCl₂ and centrifuged at 1,500 g_max for 20 min. The nuclear pellet was then resuspended in a small volume of the same buffer and microcentrifuged at 2,000 g_max for 15 min. The final nuclear pellet was resuspended in Tris buffer (10 mM, pH 7.4) containing 3 mM MgCl₂ and subsequently was assayed either for iPLA₂ activity or immediately frozen in liquid nitrogen prior to Western blot analysis. The purity of the nuclear preparations was verified by immunoblotting with anti-lamin B₁ as well as by comparisons with other subcellular markers (18).

Western blot analysis of iPLA₂, cPLA₂, and sPLA₂ in isolated nuclei
Nucleus-associated proteins were quantitated by the Lowry protein assay (20) and subsequently adjusted to equal protein concentrations prior to being subjected to Western blot analysis. For selected experiments, immunoblots were also performed on cytosolic proteins that were prepared from isolated perfused hearts as previously described (17). Primary antibodies were used at the indicated concentrations along with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit HRP, 1:7,000 dilution or goat anti-mouse HRP, 1:7,000 dilution). Immunoreactive bands were visualized by chemiluminescence detection on X-ray film (X-OMAT AR; Eastman Kodak, Rochester, NY), utilizing the ECL chemiluminescence system (Amersham, Arlington Heights, IL). Multiple exposures of film to the blots were developed and the exposures that had grain development within a linear range were used for quantitation of band intensity, utilizing NIH Image software after scanning and conversion of autoradiographic data to TIFF file formats.

Calcium-independent phospholipase A₂ assay and electrospray ionization mass spectrometry of nuclear membrane phospholipids
Calcium-independent phospholipase A₂ activity in nuclei prepared from isolated, perfused rat hearts was measured as described previously by measuring the release of [¹⁴C]arachidonic acid from 1-hexadecanoyl-2-[1-¹⁴C]eicosatetra-5',8',11',14'-enoyl-sn-GPE substrate (21). Lipids from isolated nuclei were extracted by the method of Bligh and Dyer (22) in the presence of internal standards. Chloroform extracts from 200 µg of nuclear protein were used for analysis of individual phospholipid molecular species, utilizing a Finnigan-MAT TSQ-7000 triple stage quadrupole mass spectrometer as described previously (23). Individual molecular species were quantitated by comparisons of the individual ion peak intensities with that of either 1,2-ditetradecanoyl-sn-GPC (14:0;–14:0-GPC) or 1,2-ditetradecanoyl-sn-GPE (14:0;–14:0-GPE) after correction for ¹³C isotope effects. Choline glycerophospholipids in the extract were either directly quantitated as their sodium adducts (M + Na⁺) or as their protonated adducts (M⁺) after the addition of dilute acetic acid. Ethanolamine glycerophospholipids in the nuclear extract were directly quantitated after the addition of 5 µmol of NaOH per milliliter. Electrospray ionization tandem mass spectrometry was utilized to substantiate individual molecular species. All values are presented as the mean ± standard deviation. Statistical evaluation between two groups was by Student's unpaired t-test. The values were considered significant when P was less than 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ischemia/reperfusion-induced alterations in the mass of nuclear choline glycerophospholipid molecular species mediated by iPLA₂
Analyses of nuclei isolated from control perfused rat myocardium by ESI-MS in the positive-ion mode demonstrated the preponderance of phosphatidylcholine molecular species predominantly containing either palmitic or stearic acids at the sn-1 position and either oleic, linoleic, or arachidonic acids at the sn-2 position [peaks at m/z 734 (16:0;–16:0 phosphatidylcholine), 758 (16:0;–18:2 phosphatidylcholine), 760 (16:0;–18:1 phosphatidylcholine), 782 (16:0;–20:4 phosphatidylcholine), 784 (18:1;–18:2 phosphatidylcholine), 786 (18:0;–18:2 phosphatidylcholine), 788 (18:0;–18:1 phosphatidylcholine), 806 (16:0;–22:6 phosphatidylcholine), 808 (18:1;–20:4 phosphatidylcholine), 810 (18:0;–20:4 phosphatidylcholine), and 834 (18:0;–22:6 phosphatidylcholine)] ( Fig 1A and Table 1). Phosphatidylcholine molecular species constituted more than 90% of the total choline glycerophospholipid mass in nuclei isolated from control perfused rat myocardium. The balance of choline glycerophospholipid molecular species was plasmenylcholine molecular subspecies [peaks at m/z 744 (16:0;–18:1 plasmenylcholine), 766 (16:0;–20:4 plasmenylcholine), 768 (18:1;–18:2 plasmenylcholine), and 792 (18:1;–20:4 plasmenylcholine)] (Fig 1A and Table 1).

View larger version (30K): [in this window] [in a new window]   Figure 1. Positive-ion electrospray ionization mass spectra of choline glycerophospholipids in myocardial nuclei prepared from isolated perfused adult rat heart. (A) Nuclear membrane phospholipids from control perfused rat myocardium were extracted, diluted with 1:2 chloroform-methanol, and infused directly into the ESI chamber with a syringe pump at a flow rate of 1.5 µl/min for positive-ion ESI mass spectroscopy as described in Materials and Methods. (B) Corresponding positive-ion ESI mass spectrum of nuclear membrane phospholipids from rat hearts subjected to 15 min of global ischemia was acquired under identical conditions. (C) Corresponding positive-ion ESI mass spectrum of nuclear membrane phospholipids from rat hearts subjected to 15 min of global ischemia followed by 90 min of reperfusion was acquired under identical conditions. (D) Corresponding positive-ion ESI mass spectrum of nuclear membrane lipids from rat hearts treated with 10 µM BEL followed by 15 min of global ischemia and 90 min of reperfusion was acquired under identical conditions. Individual choline glycerophospholipid molecular species for each experimental condition are listed in Table 1. The internal standard is 14:0;–14:0 GPC.

Comparisons of ESI mass spectra in the positive-ion mode of extracts of phospholipids from nuclei isolated from control perfused rat myocardium and phospholipids from nuclei isolated from rat hearts that were rendered globally ischemic for 15 min demonstrated that global ischemia did not significantly alter any of the choline glycerophospholipid molecular species (Fig 1B and Table 1). Furthermore, 15 min of global ischemia did not result in a measurable change in total choline glycerophospholipid mass ( Fig 2). In contrast, comparisons of ESI mass spectra in the positive-ion mode of extracts of phospholipids from nuclei isolated from control perfused rat myocardium and phospholipids from nuclei isolated from rat hearts that were rendered globally ischemic for 15 min followed by 90 min of reperfusion demonstrated that reperfusion of ischemic myocardium induced the hydrolysis of approximately 50% of the total choline glycerophospholipid molecular species (Fig 2). Statistically significant reductions were observed in all choline glycerophospholipid molecular species identified by ESI-MS with the exception of 18:1;–20:4 phosphatidylcholine (Fig 1C and Table 1). It should also be appreciated that the ratio of the mass of nuclear protein to the mass of nuclear DNA was constant under the four conditions that were compared and thus the same relative alterations of phospholipid mass were also observed when data were normalized to micrograms of nuclear DNA instead of micrograms of nuclear protein.

View larger version (36K): [in this window] [in a new window]   Figure 2. Total choline and ethanolamine glycerophospholipid mass in rat myocardial nuclei as determined by ESI mass spectrometry. Values represent the means + standard deviations from three individual independent experiments. * P < 0.05 for comparison between control perfused hearts and ischemic/reperfused hearts. P < 0.05 for comparison between ischemic/reperfused hearts and BEL-pretreated hearts.

Additional experiments were performed with the iPLA₂-specific inhibitor, BEL, in perfusions of isolated adult rat hearts subsequently rendered globally ischemic for 15 min followed by 90 min of reperfusion. Comparisons of ESI mass spectra in the positive-ion mode of extracts of phospholipids from nuclei isolated from control perfused rat myocardium and phospholipids from nuclei isolated from rat hearts that were pretreated with BEL followed by ischemia and reperfusion demonstrated a reduction in the extent of hydrolysis of phosphatidylcholine molecular species (Fig 2). Statistically significant reductions in phospholipid hydrolysis were observed in all choline glycerophospholipid molecular species identified by ESI-MS (Fig 1D and Table 1). The assignment of each molecular species to each ion was confirmed by collision-activated dissociation (CAD) tandem mass spectrometry of each precursor ion (see below).

Ischemia/reperfusion-induced alterations in the mass of nuclear ethanolamine glycerophospholipid molecular species
Examination of nuclei isolated from control perfused rat myocardium by ESI-MS in the negative-ion mode demonstrated a preponderance of phosphatidylethanolamine molecular species predominantly containing either palmitic or stearic acids at the sn-1 position and arachidonic acid at the sn-2 position [peaks at m/z 738 (16:0;–20:4 phosphatidylethanolamine), 742 (18:0;–18:2 phosphatidylethanolamine), 762 (16:0;–22:6 phosphatidylethanolamine), 762 (16:0;–22:6 phosphatidylethanolamine), 764 (18:1;–20:4 phosphatidylethanolamine), 766 (18:0;–20:4 phosphatidylethanolamine), 790 (18:0;–22:6 phosphatidylethanolamine), and 794 (20:0;–20:4 phosphatidylethanolamine)] ( Table 2). Phosphatidylethanolamine molecular species constituted 67% of the total ethanolamine glycerophospholipid mass in nuclei isolated from control perfused rat myocardium. The balance of ethanolamine glycerophospholipid molecular species consisted of plasmenylethanolamine molecular subspecies enriched with arachidonyl residues at the sn-2 position [peaks at m/z 722 (16:0;–20:4 plasmenylethanolamine), 748 (18:1;–20:4 plasmenylethanolamine), and 750 (18:0;–20:4 plasmenylethanolamine)] (Table 2).

Comparisons of ESI mass spectra in the negative-ion mode of extracts of phospholipids from nuclei isolated from control perfused rat myocardium and phospholipids from nuclei isolated from rat hearts that were rendered globally ischemic for 15 min again demonstrated that global ischemia did not significantly alter any of the ethanolamine glycerophospholipid molecular species (Table 2). Furthermore, 15 min of global ischemia did not result in a measurable change in total ethanolamine glycerophospholipid mass (Fig 2). In contrast, comparisons of ESI mass spectra in the negative-ion mode of extracts of phospholipids from nuclei isolated from control perfused rat myocardium and phospholipids from nuclei isolated from rat hearts that were rendered globally ischemic for 15 min followed by 90 min of reperfusion demonstrated that reperfusion of ischemic myocardium induced the hydrolysis of approximately 50% of the total ethanolamine glycerophospholipid molecular species (Fig 2). Statistically significant reductions were observed in all ethanolamine glycerophospholipid molecular species identified by ESI-MS with the exception of 18:1;–20:4 plasmenylethanolamine (m/z 748) (Table 2). Comparisons of ESI mass spectra in the negative-ion mode of extracts of phospholipids from nuclei isolated from control perfused rat myocardium and phospholipids from nuclei isolated from rat hearts that were pretreated with BEL followed by 15 min of ischemia and 90 min of reperfusion demonstrated a significant reduction in the extent of hydrolysis of only three of the ethanolamine glycerophospholipid molecular species (16:0;–22:6, 18:0;–20:4, and 18:0;–22:6 phosphatidylethanolamine) (Table 2). The reduction of total ethanolamine glycerophospholipid hydrolysis was relatively small and not significant ( Fig 3). The assignment of each molecular species to each ion was confirmed by CAD tandem mass spectrometry of each precursor ion (see below).

View larger version (13K): [in this window] [in a new window]   Figure 3. CAD tandem mass spectra displaying phospholipid molecular species containing arachidonyl residues. (A) Positive-ion spectrum (precursor ion scanning of m/z 489) of rat myocardial nuclear phospholipids displaying sodiated (M + 23) choline glycerophospholipid precursor ions containing arachidonic acid. (B) Negative-ion spectrum (precursor ion scanning of m/z 303) of rat myocardial nuclear phospholipids displaying ethanolamine glycerophospholipid precursor ions containing arachidonic acid.

Confirmation of individual rat myocardial nuclear phospholipid molecular species, using ESI tandem mass spectrometry
The identification of individual molecular species of rat myocardial nuclear phospholipids was achieved by CAD tandem mass spectrometry (24). Selected examples of ESI tandem mass spectra of rat myocardial nuclei are shown in Fig 3A and Fig B. For these analyses, sodiated ions (M + 23), rather than the protonated species shown in Fig 1, were analyzed after the addition of 1 mM NaOH to the lipid extracts. Positive-ion tandem mass spectroscopy of sodiated phospholipid molecular species containing arachidonic acid (precursor ion scanning of m/z 489) confirmed the presence of arachidonic acid on precursor ions at m/z 788, 804, and 832 (Fig 3A). Negative-ion tandem mass spectroscopy of deprotonated phospholipid molecular species containing arachidonic acid (precursor ion scanning of m/z 303) also confirmed the presence of arachidonic acid in precursor ions at m/z 722, 738, 748, 750, 764, 766, 778, and 794 (Fig 3B).

In addition, positive-ion tandem mass spectra (precursor ion scan of m/z 465) of nuclear phospholipids from rat myocardium containing linoleic acid confirmed the presence of linoleoyl residues on precursor sodiated ions at m/z 780, 806, and 808 ( Fig 4a). Positive-ion ESI tandem mass spectrometry of the sodiated ion at m/z 806 displayed the loss of trimethylamine (loss of 59) at m/z 747 as well as the loss of the phosphocholine (loss of 183) at m/z 623 (Fig 4b). Product ions at m/z 465 and 467 correspond to the neutral loss of the sn-1 oleic acid and the sn-2 linoleic acid, respectively, after the loss of trimethylamine. Positive-ion ESI tandem mass spectrometry of the sodiated ion at m/z 808 also displayed the loss of trimethylamine (m/z 749), loss of the phosphocholine (m/z 625), and ions at m/z 465 and 469 representing the neutral loss of the sn-1 stearic acid and sn-2 linoleic acid after the loss of trimethylamine (Fig 4c).

View larger version (52K): [in this window] [in a new window]   Figure 4. Positive-ion ESI mass spectra of selected choline glycerophospholipids containing linoleic acid. (A) Positive-ion spectrum of rat myocardial nuclear phospholipids (precursor ion scanning of m/z 465) displaying sodiated (M + 23) choline glycerophospholipid precursor ions containing linoleic acid. Precursor sodiated (M + 23) ions at m/z 806 (B) and m/z 808 (C) were mass selected and collisionally dissociated and the resultant product ions were analyzed. Collisional dissociation of the precursor ion at m/z 806 (B) demonstrated product ions at m/z 465 and 467, corresponding to the neutral loss of the sn-1 oleic acid and the sn-2 linoleic acid, respectively, identifying its structure as 18:1;–18:2 GPC. Collisional dissociation of the precursor ion at m/z 808 (C) demonstrated a product ions at m/z 465 and 469, representing neutral loss of the sn-1 stearic acid and sn-2 linoleic acid identifying its structure as 18:0;–18:2 GPC. Ions in (A) and precursor ions in (B) and (C) are sodiated (M + Na+).

iPLA₂ translocation to the nucleus during myocardial ischemia and reperfusion
Because pretreatment of ischemic and reperfused rat hearts with the iPLA₂-specific inhibitor BEL results in a reduction in the hydrolysis of nuclear phospholipids, and iPLA₂ has been shown to be activated during myocardial ischemia (14) (15), the presence of iPLA₂ in nuclear extracts from control, ischemic, and ischemic/reperfused heart was determined. Western blot analyses of nuclear protein isolated from control perfused, ischemic, and ischemic/reperfused hearts demonstrated that iPLA₂ was present in both control and ischemia/reperfused hearts and that during ischemia iPLA₂ was translocated to the nucleus within 5 min of global ischemia, which was reversible with reperfusion ( Fig 5A and Fig C). Further increases in nuclear iPLA₂ were observed with prolonged global ischemia (Fig 5A and Fig C). Western blot analysis of lamin B₁ was utilized as a control of constant nuclear protein loading (Fig 5B). The recovery and purity of nuclei from individual hearts subjected to each experimental condition were similar as demonstrated by Western blot analyses of several nuclear and subcellular marker proteins ( Fig 6). The nuclear envelope protein lamin B₁ and the myocardial nuclear transcription factor GATA4 were both enriched in the nuclei fractions under control, ischemic, and ischemic and reperfused experimental conditions. It should be appreciated that the techniques used for the isolation of nuclei in this study have been reported to result in the isolation of nuclei derived from cardiac myocytes and not endothelial cells (25). The appearance of the myocyte (fibroblast)-specific nuclear marker, GATA-4 (26), in the nuclear preparations (Fig 6) demonstrates that the nuclei isolated in the present study are predominantly derived from cardiac myocytes. Furthermore, there was a complete absence of contaminating subcellular markers of plasma membrane, sarcoplasmic reticulum (endoplasmic reticulum), and the myofibrillar apparatus in the purified nuclear fraction (Fig 6). In contrast to iPLA₂, there was minimal or no translocation of either cPLA₂ or sPLA₂, respectively, to the nucleus during myocardial ischemia ( Fig 7A and Fig B). In addition to the observed increase in iPLA₂ enzyme mass present in nuclei during myocardial ischemia, nuclear iPLA₂ was catalytically competent with measured enzyme activities of 8.41 ± 0.13 and 12.63 ± 0.04 pmol/min · mg_{nuclear protein} in control and 15-min ischemic hearts, respectively. Nuclear phospholipase A₂ activity was calcium independent and totally ablated by the addition of the iPLA₂ inhibitor, BEL, to the assays (e.g., enzyme activity in BEL-treated hearts was 0.47 ± 0.01; 0.51 ± 0.01 pmol/min · mg_{nuclear protein} for control perfused and 15-min ischemic hearts, respectively). Concomitant with the observed translocation of iPLA₂ to the nucleus during myocardial ischemia was a corresponding decrease in the presence of cytosolic iPLA₂ ( Fig 8). Similarly, the decrease in cytosolic iPLA₂ during myocardial ischemia was reversed with reperfusion of the ischemic myocardium.

View larger version (27K): [in this window] [in a new window]   Figure 5. Translocation of iPLA2 to the nucleus during myocardial ischemia. Nuclei were prepared from ventricular tissue of isolated adult rat hearts subjected to either control perfusion, 5 min of global ischemia, 15 min of global ischemia, 30 min of global ischemia, 60 min of global ischemia, 5 min of global ischemia followed by 10 min of reperfusion, 15 min of global ischemia followed by 10 min of reperfusion, 15 min of global ischemia followed by 30 min of reperfusion, or 15 min of global ischemia followed by 90 min of reperfusion as described in Materials and Methods. Nuclear proteins were subjected to SDS-PAGE and Western blot analyses using either anti-iPLA2 [Genetics Institute; rabbit, 10 µg/ml; (A)] or anti-lamin B1 [Zymed; mouse, 5 µl/10 ml; (B)] as the primary antibody. Denatured iPLA2 (E. coli inclusion body) was used as a positive control in (A). The intensity of each band from multiple analyses with anti-iPLA2 was quantitated with NIH Image software and expressed as a percentage of the intensity of the control sample (C). Values represent the means + standard deviations from three individual independent experiments. * P < 0.05 for comparison between control perfused hearts and ischemic conditions. P < 0.05 for comparison between 15 min of ischemia and 15 min of ischemia followed by reperfusion protocols.

View larger version (42K): [in this window] [in a new window]   Figure 6. Subcellular marker analysis of nuclei prepared from isolated and perfused rat hearts. Cytosol, particulate, and nuclei were prepared from isolated perfused adult rat hearts that were either control perfused (C), subjected to 15 min of global ischemia (I), or subjected to 15 min of global ischemia followed by 90 min of reperfusion (I/R) as described in Materials and Methods. Cytosolic, particulate, and nuclear proteins were subjected to SDS-PAGE and Western blot analysis using either anti-lamin B1 (Zymed; mouse, 5 µl/10 ml), anti-Na+/K+ ATPase ß (Upstate Biotechnology; mouse, 9 µl/10 ml), anti-SERCA2 ATPase (Affinity Bioreagents; mouse, 5 µl/10 ml), anti-TnI (Chemicon; mouse, 5 µl/20 ml), or anti-GATA4 (Santa Cruz; mouse, 27 µl/10 ml) as the primary antibodies as described in Materials and Methods.

View larger version (25K): [in this window] [in a new window]   Figure 7. Western blot analyses of cPLA2 and sPLA2 in nuclei from isolated perfused hearts. Nuclei were prepared from ventricular tissue of isolated adult rat hearts subjected to either control perfusion, 5 min of global ischemia, 15 min of global ischemia, 30 min of global ischemia, 60 min of global ischemia, 15 min of global ischemia followed by 30 min of reperfusion, or 15 min of global ischemia followed by 90 min of reperfusion as described in Materials and Methods. Nuclear proteins were subjected to SDS-PAGE and Western blot analysis using either anti-cPLA2 [Santa Cruz; rabbit, 20 µl/10 ml; (A)] or anti-sPLA2 [Cayman Chemical; mouse, 20 µl/10 ml; (B)] as the primary antibodies as described in Materials and Methods. Denatured human synovial sPLA2 was used as a positive control in (B).

View larger version (18K): [in this window] [in a new window]   Figure 8. Reduction of iPLA2 mass in the cytosolic fraction of isolated perfused rat hearts. Heart cytosol was prepared from ventricular tissue of isolated adult rat hearts subjected to either control perfusion, 5 min of global ischemia, 10 min of global ischemia, 20 min of global ischemia, 30 min of global ischemia, or 20 min of global ischemia followed by 90 min of reperfusion as described in Materials and Methods. Cytosolic proteins were subjected to SDS-PAGE and Western blot analysis using anti-iPLA2 (Cayman Chemical; rabbit, 10 µg/ml) as the primary antibody. The intensity of each band from multiple analyses with anti-iPLA2 was quantitated with NIH Image software and expressed as a percentage of the intensity of the control sample. Values represent the means + standard deviations from three individual independent experiments. * P < 0.05 for comparison between control perfused hearts and ischemic conditions. P < 0.05 for comparison between 20 min of global ischemia and 20 min of global ischemia followed by 90 min of ischemia followed by reperfusion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present results have characterized through electrospray ionization mass spectroscopic measurements the phospholipid molecular species present in the nuclei of cardiac cells in isolated perfused rat hearts. Mass-mass analysis of individual ions in these electrospray ionization mass spectroscopic analyses confirmed the assignment of molecular species to each ion measured. In the choline glycerophospholipid pools the primary sn-1 aliphatic chains were composed of palmitate and stearate residues while at the sn-2 position oleic and linoleic acid prevailed. In the ethanolamine glycerophospholipid pool the primary sn-1 aliphatic chains again were composed of palmitate and stearate residues while at the sn-2 position arachidonic acid was the major aliphatic group. An unusual molecular species (1-eicosanoyl-2-eicosatetra-5',8',11',14'-enoyl-sn-glycero-3-phosphoethanolamine) was observed that accounted for 11% of the total ethanolamine glycerophospholipid pool and again was confirmed by mass-mass analyses.

Although membrane-associated iPLA₂ activity is increased in ischemic myocardium, the translocation of this phospholipase to, as well as accelerated phospholipolysis within, specific subcellular membrane pools of the heart during myocardial ischemia have not been forthcoming (14) (15). The present study now demonstrates that catalytically competent iPLA₂ is translocated to the nucleus during myocardial ischemia. During ischemia and the beginning of reperfusion, significant levels of iPLA₂ are present in the nuclei. Furthermore, iPLA₂ remains detectable in the nucleus after 90 min of reperfusion. The localization of iPLA₂ to the nucleus is accompanied by accelerated nuclear phospholipid catabolism during reperfusion of ischemic myocardium, which is partially mediated by iPLA₂ based on the inhibition of phospholipolysis by the iPLA₂ inhibitor BEL. It is likely that the remaining iPLA₂ in the nucleus during reperfusion mediates the BEL-sensitive component of phospholipid degradation. In addition, it should be noted that iPLA₂ is activated by ATP (27) (28) and it is likely that the recovery of cellular ATP levels to near normal levels during reperfusion (29) contributes significantly to the hydrolysis of phospholipid during reperfusion mediated by iPLA₂. In contrast, it is possible that the absence of nuclear phospholipid degradation during ischemia despite the appearance of iPLA₂ in the nucleus is due to depressed levels of ATP during ischemia (29). Another component of the decrease in nuclear phospholipid content after ischemia/reperfusion may also result from a decrease in phospholipid biosynthesis or lysophospholipid reacylation under these conditions as well as the activity of other phospholipases (e.g., cPLA₂).

Similar to measurements of whole rat heart phospholipid composition (30) (31) (32), rat heart nuclear choline glycerophospholipid pools were not enriched with plasmalogen molecular species. However, the rat heart nuclear ethanolamine glycerophospholipid pool contained substantial amounts of plasmalogen molecular species. After reperfusion of ischemic hearts, there is a BEL-sensitive component to both diacyl and plasmalogen molecular species loss in both the ethanolamine and choline glycerophospholipid pools. The lack of specificity for phospholipid molecular species degradation suggests that, in intact myocardium, iPLA₂ is promiscuous as compared with that described in in vitro assay systems where iPLA₂ has been shown to prefer plasmalogen and diacyl species containing arachidonic acid (33) (34). It should be noted, however, that these previously reported studies (33) (34) did report iPLA₂ activity with other molecular species and other studies have demonstrated that brain iPLA₂ utilizes phospholipid molecular species containing linoleic, palmitic, and oleic acid residues at the sn-2 position (35). Taken together, as suggested by several in vitro assay systems of iPLA₂ activity, the present study utilizing intact tissue suggests that iPLA₂ is promiscuous in the ischemic/reperfused heart. Furthermore, although BEL only statistically altered the loss of diacylethanolamine glycerophospholipid molecular species and not plasmalogen loss, it should be appreciated that this enzyme can readily hydrolyze arachidonylated diacyl molecular species.

Although PLD and cPLA₂ have previously been shown to be translocated to the nucleus of stimulated cells (8) (9), the present studies are the first to demonstrate that iPLA₂ is translocated to the nucleus in response to a pathophysiological stimulus (i.e., myocardial ischemia). In addition, the present results demonstrate that cPLA₂ is present in the myocytic nuclei but does not translocate to the nucleus with the same magnitude as iPLA₂ during myocardial ischemia. Although iPLA₂ possesses no nuclear localization sequence, it is possible that its translocation is mediated via its cotranslocation with protein complexes possessing proteins with nuclear localization sequences that occurs during myocardial ischemia. It is possible that the nuclear translocation of iPLA₂ during myocardial ischemia is mediated through its association with phosphofructokinase (PFK) because iPLA₂ has been shown to exist in a tight complex with a specific 85-kDa isoform of PFK and PFK has been shown to translocate to the membrane fraction during myocardial ischemia (36) (37). In addition, a human isoform of iPLA₂ has been shown to have a consensus motif that is shared with the proline-rich middle linker domains of the Smad protein DAF-3 (38) from Caenorhabditis elegans and the mammalian protein Smad4 (39). The Smad proteins are products of tumor suppressor genes, which form hetero-oligomers with signaling proteins through the proline-rich middle linker domain (39). One intriguing possibility for iPLA₂ translocation to the nucleus during ischemia would involve its association with proteins that may bind to the Smad domain. Alternatively during ischemia, iPLA₂ may undergo a conformational change that results in surface amino acids providing a contact nuclear localization sequence site.

It is likely that nuclear membrane phospholipids are targets of phospholipases resulting in the generation of nuclear signaling messengers. In the present study, we have exploited the utility of electrospray ionization mass spectrometry to determine the phospholipid content of nuclei isolated from the cardiac myocytes of isolated perfused hearts and have revealed a 50% loss of myocytic nuclear choline and ethanolamine glycerophospholipids in hearts subjected to ischemia followed by reperfusion. The loss of nuclear choline and ethanolamine glycerophospholipids during reperfusion of ischemic myocardium is mediated, at least in part, by iPLA₂. The role of accelerated nuclear membrane phospholipid hydrolysis in the ischemic/reperfused heart remains to be resolved. However, these changes in myocytic nuclear membrane phospholipid content during ischemia and reperfusion likely lead to the production of lipidic second messengers that may have profound effects on nuclear signaling that potentially lead to changes in the genetic programming of the postischemic heart.

	ACKNOWLEDGMENTS

This research was supported jointly by HL 42665 (D.A.F.), a Research Career Development Award, and by HL 03316 (D.A.F.) and RR00954 (Washington University Mass Spectrometry Resource) from the NIH. This work was performed during the tenure of a predoctoral fellowship award (S.D.W.) from the American Heart Association, Heartland Affiliate, Inc.

Manuscript received January 11, 2000; and in revised form June 6, 2000

Abbreviations: 14:0;–14:0 (sn-1-sn-2, respectively), ditetradecanoyl-; 16:0;–16:0, dihexadecanoyl-; 16:0;–18:1, 1-hexadecanoyl-2-octadec-9'-enoyl-; 16:0;–18:2, 1-hexadecanoyl-2-octadec-9',12'-enoyl-; 16:0;–20:4, 1-hexadecanoyl-2-eicosatetra-5',8',11',14'-enoyl-; 16:0;–22:6, 1-hexadecanoyl-2-docosahex-4',7',10',13',16',19'-enoyl-; 18:1;–18:2, 1-octadec-9'-enoyl-2-octadec-9',12'-enoyl; 18:1;–20:4, 1-octadec-9'-enoyl-2-eicosatetra-5',8',11',14'-enoyl-; 18:0;–18:2, 1-octadecanoyl-2-octadec-9',12'-enoyl-; 18:0;–20:4, 1-octadecanoyl-2-eicosatetra-5',8', 11',14'-enoyl; 18:0;–22:6, 1-octadecanoyl-2-docosahex-4',7',10', 13',16',19'-enoyl-; 20:0;–20:4, 1-eicosanoyl-2-eicosatetra-5',8',11', 14'-enoyl-; BEL, bromoenol lactone; CAD, collision-activated dissociation; GPC, sn-glycero-3-phosphocholine; GPE, sn-glycero-3-phosphoethanolamine; ESI-MS, electrospray ionization mass spectrometry; HRP, horseradish peroxidase; PFK, phosphofructokinase; iPLA₂, calcium-independent phospholipase A₂; cPLA₂, cytosolic phospholipase A₂; sPLA₂, secretory phospholipase A₂; PLD, phospholipase D; PMSF, phenylmethylsulfonyl fluoride

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Nakamura, S., Nishizuka, Y. 1994. Lipid mediators and protein kinase C activation for the intracellular signaling network. J. Biochem. 115:1029-1034[Abstract/Free Full Text].

2. Yang, H. C., Farooqui, A. A., Horrocks, L. A. 1996. Plasmalogen-selective phospholipase A₂ and its role in signal transduction. J. Lipid Mediat. Cell Sign. 14:9-13[Medline].

3. Khan, W. A., Blobe, G. C., Hannun, Y. A. 1995. Arachidonic acid and free fatty acids as second messengers and the role of protein kinase C. Cell. Sign. 7:171-184[Medline].

4. Berridge, M. J., Irvine, R. F. 1984. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 312:315-321[Medline].

5. Nishizuka, Y. 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 258:607-614[Abstract/Free Full Text].

6. Van Veldhoven, P. P., Matthews, T. J., Bolognesi, D. P., Bell, R. M. 1992. Changes in bioactive lipids, alkylacylglycerol and ceramide, occur in HIV-infected cells. Biochem. Biophys. Res. Commun. 187:209-216[Medline].

7. Jarpe, M. B., Leach, K. L., Raben, D. M. 1994. Alpha-thrombin-induced nuclear sn-1,2-diacylglycerols are derived from phosphatidylcholine hydrolysis in cultured fibroblasts. Biochemistry. 33:526-534[Medline].

8. Baldassare, J. J., Jarpe, M. B., Alferes, L., Raben, D. M. 1997. Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction. J. Biol. Chem. 272:4911-4914[Abstract/Free Full Text].

9. Sierra-Honigmann, M. R., Bradley, J. R., Pober, J. S. 1996. "Cytosolic" phospholipase A₂ is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation. Lab. Invest. 74:684-695[Medline].

10. Miyazaki, Y., Gross, R. W., Sobel, B. E., Saffitz, J. E. 1990. Selective turnover of sarcolemmal phospholipids with lethal cardiac myocyte injury. Am. J. Physiol. 259:C325-C331[Abstract/Free Full Text].

11. Katz, A. M., Messineo, F. C. 1981. Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ. Res. 48:1-16[Free Full Text].

12. Corr, P. B., Gross, R. W., Sobel, B. E. 1984. Amphipathic metabolites and membrane dysfunction in ischemic myocardium. Circ. Res. 55:135-154[Free Full Text].

13. Prasad, M. R., Popescu, L. M., Moraru, I. I., Liu, X. K., Maity, S., Engelman, R. M., Das, D. K. 1991. Role of phospholipases A₂ and C in myocardial ischemic reperfusion injury. Am. J. Physiol. 260:H877-H883[Abstract/Free Full Text].

14. Ford, D. A., Hazen, S. L., Saffitz, J. E., Gross, R. W. 1991. The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A₂ during myocardial ischemia. J. Clin. Invest. 88:331-335.

15. Hazen, S. L., Ford, D. A., Gross, R. W. 1991. Activation of a membrane-associated phospholipase A₂ during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J. Biol. Chem. 266:5629-5633[Abstract/Free Full Text].

16. Hazen, S. L., Zupan, L. A., Weiss, R. H., Getman, D. P., Gross, R. W. 1991. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A₂. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A₂. J. Biol. Chem. 266:7227-7232[Abstract/Free Full Text].

17. Albert, C. J., Ford, D. A. 1999. Protein kinase C translocation and PKC-dependent protein phosphorylation during myocardial ischemia. Am. J. Physiol. 45:H642-H650.

18. Albert, C. J., Ford, D. A. 1998. Identification of specific nuclear protein kinase C isozymes and accelerated protein kinase C-dependent nuclear protein phosphorylation during myocardial ischemia. FEBS Lett. 438:32-36[Medline].

19. Jackowski, G., Liew, C. C. 1980. Fractionation of rat ventricular nuclei. Biochem. J. 188:363-373[Medline].

20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275[Free Full Text].

21. Ma, Z., Wang, X., Nowatzke, W., Ramanadham, S., Turk, J. 1999. Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A₂ (iPLA₂) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA₂ gene on chromosome 22q13. 1. J. Biol. Chem. 274:9607-9616[Abstract/Free Full Text].

22. Bligh, E. G., Dyer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.

23. Han, X., Gubitosi-Klug, R. A., Collins, B. J., Gross, R. W. 1996. Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis. Biochemistry. 35:5822-5832[Medline].

24. Hsu, F. F., Bohrer, A., Turk, J. 1998. Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 9:516-526[Medline].

25. Liew, C. C., Jackowski, G., Ma, T., Sole, M. J. 1983. Nonenzymatic separation of myocardial cell nuclei from whole heart tissue. Am. J. Physiol. 244:C3-C10[Abstract/Free Full Text].

26. Molkentin, J. D., Kalvakolanu, D. V., Markham, B. E. 1994. Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene. Mol. Cell. Biol. 14:4947-4957[Abstract/Free Full Text].

27. Hazen, S. L., Gross, R. W. 1991. Human myocardial cytosolic Ca(2+)-independent phospholipase A₂ is modulated by ATP. Concordant ATP-induced alterations in enzyme kinetics and mechanism-based inhibition. Biochem. J. 280:581-587.

28. Ramanadham, S., Wolf, M. J., Jett, P. A., Gross, R. W., Turk, J. 1994. Characterization of an ATP-stimulatable Ca(2+)-independent phospholipase A₂ from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor. Biochemistry. 33:7442-7452[Medline].

29. Reibel, D. K., Rovetto, M. J. 1979. Myocardial adenosine salvage rates and restoration of ATP content following ischemia. Am. J. Physiol. 237:H247-H252.

30. Mrnka, L., Novakova, O., Pelouch, V., Novak, F. 1996. Phospholipid composition in the rat heart exposed to pressure overload from birth. Physiol. Res. 45:83-85[Medline].

31. Spanner, S. 1966. Variations in the plasmalogen content of mammalian heart muscle. Nature. 210:637[Medline].

32. Post, J. A., Verkleij, A. J., Roelofsen, B., Op de Kamp, J. A. 1988. Plasmalogen content and distribution in the sarcolemma of cultured neonatal rat myocytes. FEBS Lett. 240:78-82[Medline].

33. Hazen, S. L., Stuppy, R. J., Gross, R. W. 1990. Purification and characterization of canine myocardial cytosolic phospholipase A₂. A calcium-independent phospholipase with absolute sn-2 regiospecificity for diradyl glycerophospholipids. J. Biol. Chem. 265:10622-10630[Abstract/Free Full Text].

34. Gross, R. W., Ramanadham, S., Kruszka, K. K., Han, X., Turk, J. 1993. Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A₂ activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells. Biochemistry 32:327-336[Medline].

35. Yang, H. C., Mosior, M., Ni, B., Dennis, E. A. 1999. Regional distribution, ontogeny, purification, and characterization of the Ca2+-independent phospholipase A₂ from rat brain. J. Neurochem. 73:1278-1287[Medline].

36. Hazen, S. L., Gross, R. W. 1993. The specific association of a phosphofructokinase isoform with myocardial calcium-independent phospholipase A₂. Implications for the coordinated regulation of phospholipolysis and glycolysis. J. Biol. Chem. 268:9892-9900[Abstract/Free Full Text].

37. Hazen, S. L., Wolf, M. J., Ford, D. A., Gross, R. W. 1994. The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia. FEBS Lett. 339:213-216[Medline].

38. Patterson, G. I., Koweek, A., Wong, A., Liu, Y., Ruvkun, G. 1997. The DAF-3 Smad protein antagonizes TGF-beta-related receptor signaling in the. Caenorhabditis elegans dauer(pathway. Genes Dev. 11):2679-2690.

39. de Caestecker, M. P., Hemmati, P., Larisch-Bloch, S., Ajmera, R., Roberts, A. B., Lechleider, R. J. 1997. Characterization of functional domains within Smad4/DPC4. J. Biol. Chem. 272:13690-13696[Abstract/Free Full Text].

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. Minahk, K.-W. Kim, R. Nelson, B. Trigatti, R. Lehner, and D. E. Vance Conversion of Low Density Lipoprotein-associated Phosphatidylcholine to Triacylglycerol by Primary Hepatocytes J. Biol. Chem., March 7, 2008; 283(10): 6449 - 6458. [Abstract] [Full Text] [PDF]

				K. A. Poulsen, S. F. Pedersen, M. Kolko, and I. H. Lambert Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1605 - C1615. [Abstract] [Full Text] [PDF]

				S. Bao, Y. Li, X. Lei, M. Wohltmann, W. Jin, A. Bohrer, C. F. Semenkovich, S. Ramanadham, I. Tabas, and J. Turk Attenuated Free Cholesterol Loading-induced Apoptosis but Preserved Phospholipid Composition of Peritoneal Macrophages from Mice That Do Not Express Group VIA Phospholipase A2 J. Biol. Chem., September 14, 2007; 282(37): 27100 - 27114. [Abstract] [Full Text] [PDF]

				F. Gruber, O. Oskolkova, A. Leitner, M. Mildner, V. Mlitz, B. Lengauer, A. Kadl, P. Mrass, G. Kronke, B. R. Binder, et al. Photooxidation Generates Biologically Active Phospholipids That Induce Heme Oxygenase-1 in Skin Cells J. Biol. Chem., June 8, 2007; 282(23): 16934 - 16941. [Abstract] [Full Text] [PDF]

				G. R. Kinsey, J. McHowat, C. S. Beckett, and R. G. Schnellmann Identification of calcium-independent phospholipase A2{gamma} in mitochondria and its role in mitochondrial oxidative stress Am J Physiol Renal Physiol, February 1, 2007; 292(2): F853 - F860. [Abstract] [Full Text] [PDF]

				W. Yan, C. M. Jenkins, X. Han, D. J. Mancuso, H. F. Sims, K. Yang, and R. W. Gross The Highly Selective Production of 2-Arachidonoyl Lysophosphatidylcholine Catalyzed by Purified Calcium-independent Phospholipase A2{gamma}: IDENTIFICATION OF A NOVEL ENZYMATIC MEDIATOR FOR THE GENERATION OF A KEY BRANCH POINT INTERMEDIATE IN EICOSANOID SIGNALING J. Biol. Chem., July 22, 2005; 280(29): 26669 - 26679. [Abstract] [Full Text] [PDF]

				B. S. Cummings, A. K. Gelasco, G. R. Kinsey, J. Mchowat, and R. G. Schnellmann Inactivation of Endoplasmic Reticulum Bound Ca2+-Independent Phospholipase A2 in Renal Cells during Oxidative Stress J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1441 - 1451. [Abstract] [Full Text] [PDF]

				K. Shinzawa and Y. Tsujimoto PLA2 activity is required for nuclear shrinkage in caspase-independent cell death J. Cell Biol., December 22, 2003; 163(6): 1219 - 1230. [Abstract] [Full Text] [PDF]

				R. V. Stahelin, J. D. Rafter, S. Das, and W. Cho The Molecular Basis of Differential Subcellular Localization of C2 Domains of Protein Kinase C-alpha and Group IVa Cytosolic Phospholipase A2 J. Biol. Chem., March 28, 2003; 278(14): 12452 - 12460. [Abstract] [Full Text] [PDF]