Biosynthesis of plasmalogens in mammalian cells and their accelerated catabolism during cellular activation

BIOSYNTHESIS OF PLASMALOGENS IN MAMMALIAN CELLS AND THEIR ACCELERATED CATABOLISM DURING CELLULAR ACTIVATION

David A. Ford and Richard W. Gross

ABSTRACT I. INTRODUCTION II. PHOSPHOLIPID STRUCTURE ill. PLASMALOGEN BIOSYNTHESIS A. DeTVovo Biosynthesis of Alkyl Ether Glycerophospholipids B. De Novo Synthesis of the Vinyl Ether Bond of Plasmenylcholine C. PlasmenylchoHne Biosynthesis D. Plasmalogen Catabolism During Cellular Perturbation E. Plasmalogen Catabolism in Myocardium . F. Plasmalogen Catabolism in Smooth Muscle Cells G. Plasmalogen Catabolism in Neutrophils IV. CONCLUSION ACKNOWLEDGMENT REFERENCES

Advances in Lipobiology Volume 1, pages 163-191. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 163

164 164 165 167 167 169 169 179 179 183 185 187 188 188

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DAVID A. FORD and RICHARD W. GROSS

ABSTRACT Plasmalogens are specialized phospholipids which have a unique conformation, possess distinct molecular dynamics, and serve as the major endogenous phospholipid storage depot for arachidonic acid in many mammalian cells. Recently, several novel intracellular phospholipases A2 have been identified which selectively hydrolyze plasmalogen substrate. Quantitative analysis of phospholipid molecular species in several cell types demonstrate that plasmalogen molecular species containing arachi­ donic acid are selectively hydrolyzed during cell stimulation and serve as the major source of arachidonic acid mass released during cellular activation. Collectively, these results underscore the importance of plasmalogen hydrolysis as a major mechanism for the release of eicosanoid metabolites during agonist stimulation. Accordingly, this chapter will review recent insights on the de novo synthesis of plasmalogens, address the molecular mechanisms responsible for the enrichment of arachidonic acid in plasmalogen molecular species and,finally,will focus on the mechanisms which mediate accelerated plasmalogen catabolism and the release of arachidonic acid during cellular activation.

I. INTRODUCTION Biological membranes are critical cellular constituents w^hich serve a multiplicity of distinct functional roles in cellular metabolism and physiology. First and fore­ most, biological membranes constitute the permeability barrier that provides the appropriate physical interface for the sequestration of critical enzymes and meta­ bolites v^hich allows each organism to propagate and reproduce. Without the barrier function of the membrane, life itself would be impossible. Second, biological membranes are the storage depot for many of the chemical precursors utilized in the generation of lipid second messengers (e.g., eicosanoids, diglycerides) which are synthesized during cellular stimulation through the activation of intracellular phospholipases. Third, biological membranes are dynamic, interactive matrices that facilitate the appropriate interactions of multiple transmembrane proteins which, in turn regulate cellular function and adaptive responses. To accomplish these multiple functions, biological membranes have undergone an enormous amount of chemical specialization yielding an astonishing diversity of the individual molecu­ lar constituents present in specific membrane compartments. The functional diver­ sity and scope of adaptive membrane responses results, in part, from the presence of thousands of distinct chemical moieties in biological membranes including polar lipids, nonpolar lipids, and proteins interdigitated in a complex dynamic matrix which allows each cell to fulfill its biological destiny. Through the appropriate juxtaposition of unique combinations of these distinct chemical entities within a membrane bilayer, nature has created a mechanism where critical stereoelectronic relationships, membrane physical properties, and membrane dynamics can be

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precisely tailored to facilitate each cell's adaptation to its metabolic history, current external milieu and genetically pre-destined function. The purpose of this chapter is to examine the biology, chemistry, and physiologic significance of one specialized phospholipid subclass in mammalian cell mem­ branes, the plasmalogens. Plasmalogens are phospholipid membrane constituents which possess a distinct covalent structure, adopt a unique molecular conformation, possess specific molecular dynamics, represent the major phospholipid storage depot of arachidonic acid in many cell types and are the target of certain phospholipases activated during cellular stimulation.

11. PHOSPHOLIPID STRUCTURE Mammalian membranes are comprised of a diverse array of polar lipids which (for the most part) are glycerol-based and typically contain two aliphatic chains covalently linked to the sn-l and sn-2 positions of the glycerol backbone. The sn-3 position contains a polar head group covalently attached to the glycerol backbone through a phosphodiester linkage. Variations in the chemical nature of the polar head group (e.g., choline, ethanolamine, inositol, serine) give rise to the structural diversity known as phospholipid classes (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, etc.). Variations in the polar head group produce substantial changes in the surface charge characteristics of the membrane and in critical stereoelectronic relationships within the membrane bilayer. Each phospholipid class can contain (in theory) three subclasses representing variations in the nature of the covalent attachment of the sn-\ aliphatic linkage to the glycerol backbone. Phospholipid subclasses include diacyl phospholipids (ester linkages at both the sn-\ and sn-2 positions), alkyl ether phospholipids (an alkyl ether linkage at the sn-\ position and an ester linkage at the sn-2 position) and plasmalogen molecular species (a vinyl ether linkage at the sn-1 carbon and an ester linkage at the sn-2 position) as shown in Figure 1. In most mammalian membranes, diacyl phospholipids are far more abundant than either alkyl ether- or vinyl ether-containing phospholipids. Furthermore, phospholipids with either alkyl ether or vinyl ether constituents occur predominantly within the choline and ethano­ lamine glycerophospholipid classes. Each cell type and subcellular membrane compartment has a distinct and highly regulated complement of specific phos­ pholipid classes and subclasses. For example, myocardial cells have a high content of plasmenylcholine (plasmalogens with a choline polar head group) while hepatocytes contain only diminutive amounts of plasmenylcholine. This diversity in mammalian phospholipids is further amplified by a vast array of different aliphatic constituents linked to the sn-\ and sn-2 carbon of the glycerol backbone which vary in their chain length and the degree and position of their olefinic carbons. Common aliphatic constituents typically include saturated fatty acids, predominantly found at the sn-\ position (palmitic and stearic acids) and

166

DAVID A. FORD and RICHARD W. GROSS Rj

Phospholipid Subclass

H H rO-Ri

-C=C-R '

Plasmalogen

O II

I-O-C-R2 O

H H

-c-C-Ri' I I H H

Alkyl Ether

i-O-p-o-X O

II -C-Ri'

Diacyl

Figure / . Phospholipid subclasses. Phospholipid subclasses are distinguished by the nature of covalent bond connecting the sn-^ aliphatic constituent to the glycerol backbone. The aliphatic constituent of plasmalogen, alkyl ether and diacyl subclasses of phospholipids contains a vinyl ether, alkyl ether, or ester bond at the sn-^ position, respectively. The abbreviations used are R2, long chain aliphatic constituent (e.g., C19H31), X, polar head group (e.g., choline) Ri', aliphatic chain.

unsaturated fatty acids, predominantly present at the sn-2 carbon (oleic, linoleic and arachidonic acids). The combined diversity present in the multiplicity of phospholipid classes (~ 10 different polar head groups), subclasses (three different covalent linkages at the sn-1 carbon), and individual molecular species (20 different fatty acid combinations at the sn-l and sn-2 carbons) gives rise to hundreds of specific chemical entities which collectively comprise the phospholipids of bio­ logical membranes. The complexity inherent in the membrane is further demon­ strated by the presence of specific nonpolar lipids in individual membrane compartments (e.g., cholesterol in the plasma membrane) and individual protein constituents, which are each present in specific subcellular membrane compart­ ments. The evolutionary design of these multiple constituents to facilitate cellular adaptation to diverse perturbations underscores the importance of these specialized features in the organization, surface charge characteristics, molecular dynamics, conformation, and physical properties of the membrane bilayer. Although the dramatic differences precipitated by changes in the physical char­ acteristics of the membrane bilayer elicited by alterations in phospholipid class composition (i.e., changes in the polar head group) or changes in individual molecular species (i.e., alterations in the sn-l and. ^«-2 constituents) are well characterized, the significance of subclass specialization in biologic membranes has just begun to be appreciated. The structural difference between plasmalogen and diacyl phospholipids (i.e., the replacement of an ester linkage with a vinyl ether linkage), however subtle, results in a substantially different molecular geometry of plasmalogen molecular species in comparison to their diacyl phospholipid coun-

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167

terparts (a consequence of the presence of two sp^ carbon atoms in the proximal portion of the sn-l aliphatic chain) and resultant changes in lipid packing and molecular dynamics in membrane bilayers comprised of plasmalogens (Han and Gross, 1991).

III. PLASMALOGEN BIOSYNTHESIS Although the de novo biosynthetic pathways responsible for alkyl ether glycerophospholipid synthesis, as well as for the synthesis of the ethanolamine plasmalo­ gens (plasmenylethanolamine) have been known for many years, the biosynthetic pathway for plasmenylcholine remained enigmatic until recently. The oxidation of plasmanylethanolamine to plasmenylethanolamine is catalyzed by a mixed-function oxidase and represents the only known mechanism for the introduction of a vinyl ether linkage into mammalian phospholipids. Thus, while it was generally believed that the direct metabolic precursor of plasmenylcholine is plasmenylethanolamine, the biochemical mechanisms responsible for this conversion were unknown. The transformation of plasmenylethanolamine to plasmenylcholine could result from either polar head group remodeling or from the sequential iV-methylation of the ethanolamine head group of plasmenylethanolamine. This section will first review the biosynthesis of plasmenylethanolamine and subsequently focus on recent insights into the de novo pathways of plasmenylcholine biosynthesis. A.

De Novo Biosynthesis of Alkyl Ether Glycerophospholipids

The biosynthetic mechanism responsible for the generation of the ether bond in ether-containing glycerophospholipids was described independently by Hajra and Snyder et al. over two decades ago (Hajra, 1970; Snyder et al., 1971). The critical reaction in the introduction of the ether bond was demonstrated to result from an exchange between a fatty alcohol (e.g., palmitoyl alcohol) and the acyl moiety of monoacyl dihydroxyacetone phosphate, resulting in the synthesis of monoalkyl dihydroxyacetone phosphate (Hajra, 1970; Lumb et al., 1971) (Figure 2). Both substrates in this reaction are present in substantial quantities in eukaryotic cells. Monoacyl dihydroxyacetone phosphate is produced by the acylation of the gly­ colytic intermediate, dihydroxyacetone, and fatty alcohol is synthesized by reduc­ tion of fatty acid (Rizzo et al., 1987). The next step in alkyl ether glycerophospholipid de novo biosynthesis is the reduction of the alkyl dihy­ droxyacetone phosphate by an NADPH-dependent alkyl dihydroxyacetone phos­ phate reductase (Figure 2). The product of this reaction, l-O-slkyl-snglycero-3-phosphate, is then sequentially acylated at the sn-2 carbon and sub­ sequently dephosphorylated at the sn-3 carbon. Dephosphorylation is mediated by a microsomal phosphatase that requires magnesium ion. The resultant alkyl ether diglyceride, l-(9-alkyl-2-acyl-5A2-glycerol, represents a branch point intermediate in the biosynthesis of polar and nonpolar ether lipids since the alkyl-acyl glycerol

DAVID A. FORD and RICHARD W. GROSS

168

HjC-O-C-R, 0=C

O

I

II

HjC-0-P-OH

r R, CH2CH2OH

A

O II

R,C-OH ^

H2C-O-CH2CH2R,

o=i

o

H,C-0-P-OH

NADP

'NADPH

J

HjC-O-CHjCHjR, HO-CH

O

H,C

0-P-OH

RjC-SCoA-^ CoASH

O

^

H2C-O-CH2CH2R,.

RjC - 0 - C H 0 I II HjC-0-P-OH H2O ->^| O® O e II 0-P-OH ^ I OH

;:i

O

H,C-0-CH2CH2R,

RjC - 0 - C H HjC - O H CDP-Ch^

CDP-Etn ^ *

O

H2C-O-CH2CH2RV

O 11

RjC-O-CH

O

H,C - 0 - P - 0 - C H 2 C H 2 N ( C H 3 ) 3

l^RiCSCoA

')_ CMP^

CMPV^

H^C-O-CHjCHjR, ' 1

R2C - 0 - C H

?

?•

1

O

H^C-O-P-O-CHjCHj^Hj

HjC-0-CH2CH2R, R2C - O - C H O I II H,C-0-C-R,

O^

Figure 2. The de novo biosynthetic pathway for plasmanylethanolamine. Monoacyl dihydroxyacetone phosphate and fatty alcohol are the precursors of the alkyl ether phospholipids. Fatty alcohol exchange with the fatty acyl moiety of monoacyl dihydroxyacetone phosphate is catalyzed by alkyl dihydroxyacetone phosphate syn­ thase and is the first committed step in the biosynthesis of ether containing lipids. Through the sequential reduction and acylation at the sn-2 carbon, alkyl ether phosphatidic acid is synthesized. Dephosphorylation of alkyl ether phosphatidic acid generates 1-0-alkyl-2-acyl-sn-glycerol which can be utilized by amino alcohol phosphotransferases or diradyl glycerol acyl transferase for the synthesis of either choline and ethanolamine alkyl-ether glycerophospholipids or alkyl-ether triradyl glycerols, respectively.

Plasma Iogen Metabolism

169

can either be subsequently acylated (by a diglyceride acyl transferase) to generate a triglyceride, phosphorylated to generate phosphatidic acid, or utilized as a cosubstrate for condensation with CDP-choline or CDP-ethanolamine to generate plasmanylcholine or plasmanylethanolamine (Figure 2). B. De Novo Synthesis of the Vinyl Ether Bond of Plasmenylcholine

The only known enzymic mechanism for the introduction of a vinyl ether linkage into phospholipids utilizes a pyridine nucleotide-dependent mixed-function oxi­ dase which has an absolute specificity for desaturation of l-O-alkyl-2-acyl-GPE (plasmanylethanolamine) to generate the corresponding l-O-alk-r-enyl-2-acylGPE (plasmenylethanolamine) (Schmid et al, 1972; Wykle et al., 1972). This mixed-function oxidase is inhibited by cyanide, but not by carbon monoxide, and requires molecular oxygen, reduced pyridine nucleotide (NADH or NADPH), and cytochrome b5. These characteristics are similar to those of the fatty acid desaturase that catalyzes the synthesis of monoenoic fatty acids. Multiple l-O-alkyl-2-acylGPE molecular species can be utilized by the mixed-function oxidase (Blank et al, 1986). However, neither 3-0-alkyl-2-acyl-5«-glycero-l-phosphorylethanolamine, 1 -0-alkyl-GPE, 1 -0-alkyl-2-acyl-5«-glycero-3-phosphoryl(A^-dimethyl)ethanolamine, nor, most importantly, plasmanylcholine is utilized by this mixed-function oxidase (Schmid et al., 1972; Wykle et al., 1972; Paltauf and Holasek, 1973; Blank et al., 1986). No alkyl ether desaturase activities have been demonstrated which can catalyze the desaturation of plasmanylcholine to plasmenylcholine despite numerous attempts to demonstrate this activity in broken cell preparations, intact cells or intact tissue. Accordingly, the only pathway for introduction of the vinyl ether linkage into cellular lipids is by oxidation of plasmanylethanolamine to plasmenylethanolamine. Thus, it is generally accepted that plasmenylcholine must be generated from plasmenylethanolamine. C. Plasmenylcholine Biosynthesis

Three pathways which could potentially generate plasmenylcholine from plas­ menylethanolamine have been deduced predominantly from broken cell experi­ ments as well as from a limited number of studies with intact cells and intact tissue. Pathway I involves the condensation of CDP-choline with l-O-alk-r-enyl-2-acyl5«-glycerol and was initially suggested by Kiyasu and Kennedy (1960). The key intermediate in this pathway, 1 -0-alk-1 '-enyl-2-acyl-5«-glycerol, was subsequently identified as an endogenous constituent of myocardium (Ford and Gross, 1988; Ford et al., 1992). The strategy employed by Pathway I is the exploitation of polar head group remodeling (initiated by either phospholipase C directly or by the sequential actions of phospholipase D coupled with phosphatidate phosphohydrolase) to facilitate the introduction of the vinyl ether linkage into plasmenylcholine. The subsequent condensation of l-0-alk-r-enyl-2-acyl-5«-glycerol with CDPcholine catalyzed by choline phosphotransferase results in the synthesis of plas-

CMP Ethanolamine Phosphotransferase

CDP - Ethanolamine CDP - Choline

Phosphorylethanolamine Phosphorylcholine

Choline Phosphotransferase

CMP

II

O "2-0-0-^

O

j - O - C=C-R, C O 0

II

R,-C-0

L o - PP-O-CH2CH2NH3

"

■ - O - P - G - CCH2CH2N(CH3)3 h A

J

CDP-Choline^ O

|-0-C=C-R,

•00--C C=C-Ri

II

R,-C-0

L-0-P-0-CH2CH2NH3

{

OH O e >■ O-P-OH I

♦ ■C=C-R, O LO-P-OH

O

.-T'

—7^

OH

r-0-C=C-Ri

II

R2-C-O

O

L-O-P-OH

R2CSC0A

III

3 S-adenosyl methionine

-C=C-R, O

3 S-adenosyt homocysteine

k0-C-R2

I-O-C-R2

N - methyl transferase

o II

pO-C=C-R, O ■>

II

II

®

'-0-P-0-CH2CH2N(CH3), 0^

«

l-O-P-O-CHjCHjNHa O^

Figure 3. Proposed pathways for de novo plasmenylcholine biosynthesis. Three pathways have been proposed for the de novo biosynthesis of plasmenylcholine which each utilize plasmenylethanolamine as the precursor of the vinyl ether aliphatic group of plasmenylcholine. Through Pathway I, plasmenylcholine is synthesized by polar head group remodeling of plasmenylethanolamine which is initiated by phospholipase C (or a phospholipase C equivalent) catalyzed hydrolysis of plasmenylethano­ lamine. Pathway II is initiated by phospholipase A2 catalyzed hydrolysis of plasmenylethanolamine and through remodeling of the sn-2 and sn-3 constituents generates l-0-alk-V-enyl-2-acyl-SA7-glycerol which condenses with CDP choline resulting in the biosynthesis of plasmenylcholine. Pathway III is mediated by the sequential N-methylationoftheethanolamine head group of plasmenylethanolamine. 170

Plasmalogen Metabolism

171

menylcholine. Pathway II was initially proposed by Wykle and co-workers (Wykle and Schremmer, 1974; Wykle and Snyder, 1976) and is initiated by phospholipase A2-mediated hydrolysis of plasmenylethanolamine, resulting in the generation of lysoplasmenylethanolamine. The subsequent hydrolysis of the ethanolamine head group catalyzed by lysophospholipase D can generate plasmalogenic lysophosphatidic acid. Plasmalogenic lysophosphatidic acid can then be sequentially acylated and dephosphorylated resulting in l-0-alk-r-enyl-2-acyl-5«-glycerol production. Plasmenylcholine can be subsequently synthesized by choline phosphotransferasemediated condensation of l-0-alk-r-enyl-2-acyl-5«-glycerol with CDP choline. Thus, Pathway I and Pathway II share common final steps in the generation of plasmenylcholine (i.e., phosphatidate phosphohydrolase and choline phos­ photransferase) and utilize the same key intermediate (l-0-alk-r-enyl-2-acyl-5«glycerol) which is generated by different enzymatic pathways. Pathway III utilizes the sequential 7V-methylation of the ethanolamine moiety of plasmenylethano­ lamine mediated by one or more A^-methyl transferases to introduce the vinyl ether linkage into plasmenylcholine (Mogelson and Sobel, 1981). Through sequential A^-methylations utilizing S-adenosylmethionine as the methyl donor, monomethyl plasmenylethanolamine, dimethyl plasmenylethanolamine and,finally,plasmenyl­ choline are produced. The evidence supporting each of these pathways will be briefly summarized and their quantitative importance, mechanisms of regulation, and some of the directions of future research in this area will be discussed on the following pages. The discovery in 1985 of a neutral-active phospholipase C in myocardium that utilizes choline and ethanolamine glycerophospholipids (including plasmalogen molecular species) (Wolf and Gross, 1985) led to the hypothesis that plasmenyl­ choline biosynthesis in myocardium is initiated by phospholipase C-mediated hydrolysis of plasmenylethanolamine. The direct generation of the key intermedi­ ate, l-0-alk-r-enyl-2-acyl-^«-glycerol, from plasmenylethanolamine in conjunc­ tion with the known ability of choline phosphotransferase to utilize l-0-alk-r-enyl-2-acyl-5«-glycerol to generate plasmenylcholine would provide a direct route to the synthesis of plasmenylcholine molecular species through polar head-group remodeling (e.g., Pathway I, Figure 3). Initial experimental support for this pathway included the demonstration of l-0-alk-r-enyl-2-acyl-5«-glycerol as an endogenous lipid in rabbit myocardium and the demonstration that endogenous microsomal l-0-alk-r-enyl-2-acyl-5«-glycerol is selectively utilized by myocar­ dial microsomal choline phosphotransferase (Ford and Gross, 1988). The selectiv­ ity of myocardial choline phosphotransferase for the synthesis of plasmenylcholine was demonstrated in studies in which the water soluble metabolite, CDP-choline, was added to rabbit myocardial microsomes and the utilization of each endogenous microsomal l,2-diradyl-5«-glycerol by microsomal choline phosphotransferase was quantified. These experiments demonstrated that plasmenylcholine and phos­ phatidylcholine molecular species were synthesized at similar rates (corresponding to their tissue distribution in rabbit myocardial microsomal lipids), despite the fact

172

DAVID A. FORD and RICHARD W. GROSS

that over a 20-fold molar excess of l,2-diacyl-5«-glycerol as compared to 1-0-alk1 '-enyl-2-acyl-5«-glycerol was present in myocardial microsomes (Ford and Gross, 1988). Furthermore, these studies also documented the metabolic significance of this pathway in maintaining myocardial plasmenylcholine levels, since the rate of plasmenylcholine synthesis by choline phosphotransferase utilizing endogenous l-0-alk-r-enyl-2-acyl-5«-glycerol and physiologic concentrations of CDPcholine was comparable to the rate of plasmenylcholine catabolism in intact tissue (Ford and Gross, 1988). Taken together, these studies demonstrated the presence of the key intermediate of Pathway I (e.g., l-0-alk-r-enyl-2-acyl-^«-glycerol), the selectivity of the choline phosphotransferase for alk-r-enyl-2-acyl diradyl glyc­ erol, and the metabolic capacity of myocardial choline phosphotransferase to catalyze the major portion of plasmenylcholine biosynthesis in intact tissues utilizing endogenous concentrations of l-0-alk-r-enyl-2-acyl-5«-glycerol and CDP-choline. To further identify the salient kinetic characteristics resulting in the molecular species distribution of choline and ethanolamine plasmalogens in mammalian cells, the substrate selectivity and kinetic profile of myocardial ethanolamine phos­ photransferase were characterized. Quantification of the initial rates of CDPethanolamine condensation with l-(9-alk-r-enyl-2-acyl-5«-glycerol (AAG) compared to l,2-diacyl-^«-glycerol (DAG) catalyzed by ethanolamine phos­ photransferase demonstrated a 16-fold selectivity for utilization of vinyl ether diradyl glycerol in comparison to diacyl diradyl glycerol (Ford et al., 1992). Furthermore, direct competition experiments employing mixed micelles comprised of equimolar amounts of AAG and DAG (containing palmitic acid at the sn-l position and arachidonic acid at the sn-2 position) demonstrated a 20:1 ratio of plasmenylethanolamine production compared to phosphatidylethanolamine pro­ duction. Additionally, incubation of rabbit myocardial microsomes with CDPethanolamine utilizing endogenously produced diradyl glycerols as substrate resulted in the robust production of 16:0-20:4 plasmenylethanolamine, 18:1—20:4 plasmenylethanolamine, and 18:0-20:4 plasmenylethanolamine with only diminu­ tive amounts of phosphatidylethanolamine synthesized. Thus, these results demon­ strate the substantial substrate selectivity of mammalian ethanolamine phospho­ transferase for plasmenylethanolamine synthesis in direct comparison to utilization of DAG substrate for phosphatidylethanolamine synthesis, and underscore the importance of the ethanolamine phosphotransferase reaction in determining the subclass distribution of endogenous ethanolamine glycerophospholipids in mam­ malian cells. Comparisons of the molecular species selectivity of rabbit myocardial microso­ mal ethanolamine phosphotransferase demonstrated that its reaction velocity was predominantly determined by the subclass distribution of diradyl glycerols and not the molecular species distribution since utilization of l-0-alk-l-enyl-2-acyl-5«glycerol containing either oleic or arachidonic acid by the ethanolamine phos­ photransferase proceeded with similar velocities. Similarly, all molecular species

Plasmalogen Metabolism

1 73

of l-0-alkyl-2-acyl-5«-glycerols were utilized with similar reaction velocities. Additional experiments demonstrated a modest selectivity for utilization of diacyl glycerophospholipid molecular species containing arachidonic compared to oleic acid at the sn-2 position. However, this selectivity was dwarfed by the fact that the reaction velocity utilizing any molecular species of AAG was over 20-fold greater than that manifest by the best diacyl glycerol substrate. Collectively, these results demonstrated that the primary determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity is the covalent nature of the sn-l aliphatic group of diradyl glycerol acceptors. Based upon these observations, several biochemical correlates concerning the subclass and molecular species distribution of plasmalogen and diacyl phos­ pholipids in mammalian cells can be made. First, the dramatic selectivity of myocardial ethanolamine phosphotransferase for the synthesis of plasmenylethanolamines (in comparison to phosphatidylethanolamines) identifies one biochemical mechanism responsible for the abundance of the plasmalogen subclass in ethanolamine glycerophospholipids. Furthermore, identification of the substan­ tial enrichment of arachidonic acid in l-0-alk-r-enyl-2-acyl-5«-glycerol molecu­ lar species in conjunction with the selectivity of ethanolamine phosphotransferase for l-0-alk-r-enyl-2-acyl-5«-glycerol demonstrates one mechanism contributing to the high content of arachidonic acid in plasmenylethanolamine molecular species. Thus, the subclass distribution of plasmalogens in ethanolamine glycero­ phospholipid pools results, in large part, from the preferred utilization of 1-0-alkr-enyl-2-acyl-5'«-glycerol moieties by ethanolamine phosphotransferase and the enrichment of arachidonic acid in vinyl ether containing diradyl glycerols. The selective synthesis of arachidonylated l-0-alk-r-enyl-2-acyl-5«-glycerol could occur either through selective phospholipase C- or D-mediated cleavage of plas­ malogens containing arachidonic acid at the sn-2 position. Based on these studies, the biochemical mechanisms responsible for plasmenylcholine biosynthesis have been refined as depicted in Scheme I. The essential features inherent in this scheme include: (a) the initial generation of the vinyl ether linkage in plasmenylethanolamine by de novo synthesis catalyzed by alkyl ether desaturase; (b) the subsequent shuttling of vinyl ether equivalents carried by l-6)-alk-r-enyl-2-acyl-5«-glycerols; and (c) the de novo synthesis of plasmenylcholine or the regeneration of plasmenylethanolamine by their respective amino alcohol phosphotransferases each utilizing a common l-0-alk-r-enyl-2-acyl-5«glycerol intermediate. There are several important physiologic correlates which are implicit in this scheme. First, the content of vinyl ethers present in choline and ethanolamine glycerophospholipid classes is determined, at least in part, by the substrate selectivity of each amino alcohol phosphotransferase for 1 -0-alk-1 '-enyl2-acyl-5n-glycerol versus l,2-diacyl-5«-glycerol. This explains why the fractional percentage of phospholipids containing vinyl ethers in the ethanolamine glycero­ phospholipid pool is substantially higher than that in the choline glycerophos­ pholipid pool (i.e., the selectivity of the ethanolamine phosphotransferase greatly

174

DAVID A. FORD and RICHARD W. GROSS iPLASMANYLETHANOLAMirJEl

IPLASMENYLETHANOLAMINEI

Scheme 1. The plasmenylethanolamine: 1-0-alk-r-enyl-2-acyi-sA7-glycerol cycle: Regulation of de novo biosynthesis of plasmenylcholine by 1-0-alk-1'-enyl-2-acylsn-glycerol. The abbreviations used are: EPT, ethanolamine phosphotransferase; Etn, ethanolamine; PA, phosphatidic acid; PAP, phosphatidic phosphohydrolase; PEtn, phosphorylethanolamine; PLC, phospholipase C; PLD, phospholipase D.

exceeds that of the choline phosphotransferase). Secondly, we point out that the enrichment of endogenous l-0-alk-r-enyl-2-acyl-5«-glycerol molecular species in arachidonic acid contributes to the enrichment of both the plasmenylethano­ lamine and plasmenylcholine pools in arachidonic acid, since neither choline phosphotransferase nor ethanolamine phosphotransferase possesses an inherent ability, at least in vitro, to discriminate between diradyl molecular species contain­ ing oleic acid or arachidonic acid at the sn-2 position. Since the molecular species of l-(9-alk-r-enyl-2-acyl-5«-glycerol are similar to the molecular species distribu­ tion of plasmenylethanolamine in rabbit membranes, these results suggest that plasmenylethanolamine is the predominant substrate for the generation of 1 -O-alkr-enyl-2-acyl-5w-glycerol in this system by phospholipase C or phospholipase D. The selective utilization of plasmenylethanolamine by myocardial phospholipase C or phospholipase D would thereby facilitate the flux of vinyl ether linkages into the choline glycerophospholipid pool and the generation of plasmenylcholine enriched in arachidonic acid. Recently, pulse-chase radiolabeling techniques have been utilized with precur­ sors of the sn-l, sn-2 and sn-3 functionalities of plasmenylethanolamine and plasmenylcholine to quantitatively compare the rates of de novo synthesis to the rates of turnover of the sn-2 aliphatic chain and the sn-3 polar head group in intact contracting myocardium. Through this approach, the relative magnitudes of de novo synthesis versus remodeling can be ascertained. Since utilization of fatty alcohol is an obligatory reaction in the de novo synthetic pathway, de novo

Plasmalogen Metabolism

175

plasmalogen biosynthesis was assessed by pulse-chase radiolabeling of Langendorff perfused rabbit hearts with l-[-^H]-hexadecanoL Pulse-chase radiolabeling of perfused rabbit hearts with I -[^H]-hexadecanol resuhed in the rapid and progressive incorporation of radiolabel into plasmenylethanolamine molecular species (e.g., after 0.5 hours of radiolabeling, 10% of l-[-'H]-hexadecanol incorporated into ethanolamine glycerophospholipid was in plasmenylethanolamine, and after 1.5 hours, 21% was in plasmenylethanolamine), with no detectable radiolabeling of plasmenylcholine until three hours after the pulse (Figure 4). The temporal course of the appearance of plasmanylethanolamine, plasmenylethanolamine, and plas-

Plasmanyicholine

Plasmanylethanolamine

10 15 PERFUSION INTERVAL (Hours) BEGINNING OF CHASE INTERVAL BEGINNING OF PULSE LABELING

Figure4, Thetemporalcourseof1-[^H]-pa(mitoyl alcohol {1-[^H]-hexadecanol) into myocardial choline and ethanolamine glycerophospholipid pools. Rabbit hearts were perfused in a recirculating Langendorff mode with modified Krebs-Henseleit buffer containing 1-[^H]-palmitoyl alcohol for selected intervals. After a 1.5 hour radiola­ beling interval the hearts were perfused with unlabeled palmitoyi alcohol (20 \xN\). At the indicated times, perfusions were terminated by freeze-clamping. Choline and ethanolamine glycerophospholipids were purified by straight phase HPLC and incor­ poration of radiolabel into ether lipids was determined by the generation of fatty aldehyde after exposure to acid fumes as well as by reverse phase HPLC separation of the molecular species oi each glycerophospholipid.

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DAVID A. FORD and RICHARD W. GROSS

menylcholine is consistent with precursor-product relationships between both plasmanylethanolamine and plasmenylethanolamine as well as between plasmenylethanolamine and plasmenylcholine in intact tissue. Analysis of the molecular species of plasmenylcholine and plasmenylethano­ lamine formed in the l-[^H]-hexadecanol pulse-chase radiolabeling experiments demonstrated that the predominant plasmenylethanolamine molecular species syn­ thesized were 16:0-20:4 and 16:0-18:1, whereas the predominant plasmenyl­ choline molecular species synthesized was 16:0-20:4. Thus, the pulse-chase labeling profiles of individual molecular species suggest that arachidonoylated molecular species of plasmenylethanolamine are the preferred precursors for shut­ tling into plasmenylcholine by the selective phospholipase C- or phospholipase D-mediated hydrolysis of plasmenylethanolamine molecular species. These results are entirely consistent with the aforementioned in vitro studies demonstrating the selective synthesis of arachidonoylated plasmenylethanolamine molecular species by ethanolamine phosphotransferase and identifying the predominant endogenous molecular species of l-0-alk-r-enyl-2-acyl-5«-glycerol in myocardium as 1-0hexadec-r-enyl-2-eicosatetra-5',8',ir,14'-enoyl-5«-glycerol (Ford et al., 1992). Thus, plasmenylethanolamine molecular species enriched in arachidonic acid are both synthesized and degraded more rapidly than their monounsaturated counter­ parts. To substantiate the importance of polar head group remodeling and to further exclude the participation of an alkyl ether desaturase which could utilize plasmanylcholine, additional studies utilizing l-(9-[^H]-alkyl-GPC (^H-lysoPAF) were per­ formed. Although ^H-lysoPAF was rapidly incorporated into plasmanylcholine through acylation, no detectable plasmenylcholine or plasmenylethanolamine was synthesized even after extended perftision intervals. Thus, even in intact tissue, plasmanylcholine is not a direct precursor of plasmenylcholine. To assess the relative rates of polar head group remodeling, sn-2 remodeling of plasmalogens, and de novo vinyl ether biosynthesis, comparisons of the flux of either choline, ethanolamine (polar head group remodeling), arachidonic acid (sn-2 remodeling), or hexadecanol into plasmenylcholine and plasmenylethanolamine were performed. Remarkably, the rate of polar head group remodeling of plasmalo­ gens was over 300-fold more rapid than the rate ofde novo plasmalogen synthesis in intact, ftinctioning myocardium (Table 1). Furthermore, sn-2 remodeling of plasmalogens occurs at rates over 100-fold greater than that ofde novo plasmalogen biosynthesis (Table 1). Additionally, sn-2 group remodeling of plasmalogen mo­ lecular species likely contributes to their enrichment in arachidonic acid, since the rate of incorporation of arachidonic acid into plasmenylcholine and plas­ menylethanolamine was threefold greater than the rate of incorporation of oleic acid. In contrast, rates of incorporation of oleic acid were more rapid into phospha­ tidylcholine and phosphatidylethanolamine in comparison to their plasmalogen counterparts.

Plasmalogen Metabolism

1 77

Table 1. Incorporation Rates of Polar Head Group and Aliphatic Precursors of Diacyl and Plasmalogen Glycerophospholipids in Isolated Perfused Rabbit Hearts Precursors (nmol/g^^,h) Product Plasmenylcholine Phosphatidylcholine Plasmenylethanolamine Phosphatidylethanolamine Note:

Hexadecanol

Choline

<0.01

21 131 — —

0.09

Ethanolamine — — 31 139

Arachidonic acid Oleic acid 35 113 12 16

11 93 4 19

Rabbit hearts were perfused in a recirculating Langendorff mode with modified Krebs-Henseleit buffer containing either l-pH]-hexadecanol, pH] choline, or [-^H] ethanolamine for 1.5 hours or alternatively with modified Krebs-Henseleit buffer containing either [^H] oleic acid or [^H] arachidonic acid for 20 minutes. The flux of precursors into each lipid pool was calculated from the specific activity of radiolabel in each lipid pool and the incorporation of radiolabel into the designated products.

Thus, independent results utilizing a variety of methodologies employing both broken cells and intact tissue have elucidated the pathways for plasmenylcholine biosynthesis and the biochemical mechanisms responsible for the enrichment of plasmalogen molecular species in arachidonic acid. These studies underscore the quantitative importance of remodeling in comparison to de novo synthesis and the inherent selectivity of the salient enzymic machinery for remodeling, which is critical in facilitating the rapid hydrolysis and resynthesis of specific phospholipid constituents in mammalian cells. Conceptually, Pathway I and Pathway II are similar since they both (ultimately) require plasmenylethanolamine polar head group remodeling and employ the common intermediate, l-(9-alk-r-enyl-2-acyl-5«-glycerol, for plasmenylcholine biosynthesis. However, Pathway II involves a multi-step process for the remodeling of the sn-2 and sn-?> constituents of plasmenylethanolamine which is initiated by a plasmalogen-selectivephospholipase A2 (Figure 3). Pathway II was first envisioned nearly 20 years ago, based on the discovery of a lysophospholipase D activity which demonstrated selectivity for ether-linked lipids (Wykle and Schremmer, 1974; Wykle and Snyder, 1976). Pathway II is initiated by phospholipase A2-mediated hydrolysis of plasmenylethanolamine followed by hydrolysis of the sn-2> polar head group of the resultant lysoplasmenylethanolamine by lysophospholipase D to generate lysophosphatidic acid containing a vinyl ether linkage at the sn-1 position. After acylation and dephosphorylation, the resultant l-(9-alk-r-enyl-2-acyl-5«glycerol, the intermediate which represents the convergence point of Pathways I and II, could be utilized by choline phosphotransferase resulting in the synthesis of plasmenylcholine. Thus, in this scheme the shuttling of vinyl ether moieties from plasmenylethanolamine into the choline glycerophospholipid pool is initiated by

178

DAVID A. FORD and RICHARD W. GROSS

phospholipase A2 and requires multiple sequential reactions, while Pathway I generates the key intermediate directly. Although Pathway II was based on broken cell studies measuring lysophospholipase D activity (Wykle and Schremmer, 1974; Wykle and Synder, 1976), recent studies demonstrating the existence of plasmalogen-selective phospholipase A2 and the rapid disappearance of lysoplasmalogen have supported the presence of Path­ way II. Further support for Pathway II as an enzymatic mechanism responsible for plasmenylcholine biosynthesis has been gained through studies utilizing [1-^H]-1O-alk-l'-enyl-GPE to label plasmalogen pools in intact HL-60 cells (Blank et al., 1993). In these studies, [l-^H]-l-0-alk-r-enyl-GPE was initially incorporated into plasmenylethanolamine molecular species which predominantly contain 20:3 fatty acid at the sn-2 carbon. Subsequently, radiolabel appeared in plasmenylcholine molecular species predominantly containing 16:0 at the sn-2 carbon. The disparity of radiolabeled individual molecular species of plasmenylcholine and plas­ menylethanolamine was interpreted to support the role of phospholipase A2 in Pathway 11. Additionally, since both plasmenylethanolamine and plasmenylcholine contained molecular species with 16:0 aliphatic chains at the sn-2 carbon, it was concluded that at least some plasmenylcholine biosynthesis is initiated by phos­ pholipase C (or phospholipase D, or their equivalents) (e.g.. Pathway I) or by direct base exchange (e.g.. Pathway III). The third pathway for plasmenylcholine biosynthesis (Figure 3) involves the A^-methylation of plasmenylethanolamine. Support for this pathway was obtained through demonstration that myocardium contains enzymes which catalyze the 7V-methylation of plasmenylethanolamine to plasmenylcholine (Mogelson and Sobel, 1981). It is not known if more than one enzyme is required for the complete A^-methylation of plasmenylethanolamine as in the case of phosphatidylethanolamine (Higgins, 1981) or if the enzyme(s) catalyzing the A^-methylation of plas­ menylethanolamine are identical to those previously characterized for phosphatidylethanolamine. However, it seems unlikely that this pathway contrib­ utes substantively to plasmenylcholine biosynthesis since: (a) the measured biosynthetic capacity of this pathway is relatively low (Mogelson and Sobel, 1981) as compared to the metabolic capacity of enzymes responsible for plasmenylcholine catabolism; (b) myocardial phospholipase D activity is diminutive (Schmid et al., 1983); and (c) at each concentration of S-adenosyl methionine studied phospha­ tidylethanolamine was A^-methylated more rapidly than plasmenylethanolamine (Mogelson and Sobel, 1981) and A^-methylation of phosphatidylethanolamine is a quantitatively minor component of phosphatidylcholine synthesis. However, A^methylation of plasmenylethanolamine may be of importance under some patho­ physiologic conditions in which accelerated A^-methylation has been demonstrated (e.g., Horrocks et al., 1986a,b).

Plasmalogen Metabolism D.

179

Plasmalogen Catabolism During Cellular Perturbation

The identification of the unique conformational motif of plasmalogen molecular species (Han and Gross, 1990), their enrichment in arachidonic acid (Chilton et al., 1984; Gross, 1984; Ford and Gross, 1989a), their accelerated turnover during cellular stimulation (Ford and Gross, 1989a; Tessner et al., 1990), and the identifi­ cation of plasmalogen-selective phospholipases A2 each serve to underscore the importance of plasmalogen molecular species in cellular activation. This portion of the chapter will focus on salient aspects of plasmalogen catabolism including the turnover of plasmalogen molecular species in activated cells, recognition of plasmalogens by intracellular phospholipases, and the importance of plasmalogens as storage depots for the arachidonic acid mass released during cellular activation in several representative cell types including cardiac myocytes, smooth muscle cells, and neutrophils. E. Plasmalogen Catabolism in Myocardium

The demonstration that plasmalogens are localized in specific subcellular loci (i.e., sarcolemma and sarcoplasmic reticulum. Gross, 1984; Gross, 1985) suggested their importance as critical components essential for the appropriate physiologic function of myocardium. It has long been appreciated that ischemia is accompanied by accelerated phospholipid metabolism (Boime et al., 1970) which results in the accumulation of amphiphilic metabolites (e.g., lysophospholipids and arachidonic acid) as well as the release of oxygenated arachidonate metabolites from ischemic myocardium subjected to reperfusion (Hsueh et al., 1977). Since sarcolemmal plasmalogens are enriched in arachidonic acid (Gross, 1984), and since myocar­ dium contains a plasmalogen-selective phospholipase A2, sarcolemmal plasmalo­ gens have been implicated as a source of the arachidonic acid released during myocardial ischemia. Recently, evidence demonstrating the importance of acceler­ ated sarcolemmal plasmalogen catabolism in ischemic myocardium has been accrued utilizing quantitative electron microscopic autoradiography of isolated myocytes subjected to metabolic deprivation. Sarcolemmal phospholipids were the predominant target of accelerated catabolism during metabolic deprivation as assessed by the selective incorporation of arachidonic acid into sarcolemmal phospholipids resulting from deacylation-reacylation cycling in reversibly injured cardiac myocytes (Miyazaki et al., 1990). These observations in tissue culture systems have been substantiated in intact tissue studies which demonstrated the activation of microsomal calcium-independent plasmalogen-selective phospholi­ pase A2 during brief (i.e., reversible) intervals of myocardial ischemia. Activation of this phospholipase A2 is reversible upon reperfusion (Ford et al., 1991; Hazen et al., 1991a) and the temporal course of alterations in phospholipase A2 activation precisely parallels the temporal course of alterations in glycolytic flux. The physi­ ological significance of accelerated plasmalogen catabolism during myocardial

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DAVID A. FORD and RICHARD W. GROSS

ischemia is underscored by the enrichment of arachidonoylated molecular species of plasmalogens in the sarcolemma of cardiac muscle cells and the fact that arachidonic acid has profound effects on ion channel function. Accordingly, the accelerated hydrolysis of sarcolemmal phospholipids results in alterations in the physicochemical properties of the membrane due to accumulation of amphiphilic constituents which are tightly coupled with the function of critical transmembrane proteins such as ion channels (Lenaz, 1987; Kim and Clapham, 1989; Alvermann et al., 1992). Thus, the selective hydrolysis of sarcolemmal plasmalogens and the consequent release of arachidonic acid during myocardial ischemia results in a sarcolemmal microenvironment enriched in arachidonic acid and lysophospholipids which would have profound effects on K"^ channel function, ligand-receptor coupling, and ion pump function. Accordingly, the attenuation of accelerated plasmalogen turnover and arachidonic acid release mediated by the activation of calcium-independent phospholipase A2 in ischemic myocardium represents an important target for pharmacologic intervention in the treatment of electrophysi­ ologic dysfunction and myocytic cellular necrosis during ischemia. Although numerous studies suggested the importance of accelerated phos­ pholipid metabolism during ischemic injury, previous attempts to document acti­ vation of myocardial phospholipase A2 during ischemia have been unsuccessful utilizing diacyl phospholipid substrates (Das et al., 1986; Bentham et al., 1987). Recently, the synthesis of plasmalogen substrates have been instrumental in the identification of the activation of phospholipase A2 This ischemia-activated phos­ pholipase A2 is membrane-associated, calcium-independent and preferentially cleaves plasmalogen substrate (Ford et al., 1991; Hazen et al., 1991a). Remarkably, membrane-associated, calcium-independent, plasmalogen-selective phospholipase A2 activity was demonstrated to increase over fivefold during two minutes of global ischemia, was nearly maximally activated (>eightfold) after only five minutes of ischemia, and remained activated throughout a one hour ischemic interval (Figure 5). Activation of plasmalogen-selective phospholipase A2 was shown to be rapidly reversible after reperfiision of ischemic tissue (Figure 5). Furthermore, the activa­ tion of plasmalogen-selective phospholipase A2 occurred concordantly with acti­ vation of anaerobic metabolism (as assessed by myocardial lactate levels). Thus, activation of calcium-independent phospholipase A2 is one of the earliest measur­ able biochemical manifestations of acute myocardial ischemia, occurs prior to irreversible myocardial ischemic injury (as determined by the absence of electron microscopic evidence of cellular damage at these early time points), and is entirely reversible with reperfiision. Furthermore, the membrane-associated calcium-inde­ pendent phospholipase A2 is specifically inhibited by the mechanism-based inhibi­ tor, (£)-6-(bromomethylene)tetrahydro-3-( 1 -naphthalenyl)- 2H-pyran-2-one. Accelerated plasmalogen catabolism during myocardial ischemia can also occur by the acfivation of myocardial phospholipases C or D. The possibility that l-(9-alk-r-enyl-2-acyl-5«-glycerol accumulation during myocardial ischemia oc­ curs through phospholipase C activation is suggested by the demonstration that

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Metabolism 300

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Experimental Interval (min) Figure 5. The concordant activation of membrane-associated phospholipase A2 activity and glycolysis during myocardial ischemia and their reversibility during reperfusion. Ischemic and reperfused rabbit hearts were initially perfused for a 10 minute equilibration interval before a 0-60 minute experimental period. After the initial 10 minute pre-equilibration interval, hearts were rendered ischemic for 2, 5, or 60 minutes (—), or rendered ischemic for five minutes and subsequently reperfused for an additional 10 minutes (i.e., 15 minute data points), or reperfused for an additional 55 minutes (i.e., 60 minute data points) ( ). Phospholipase A2 activity was assessed by incubating microsomes (8 ^g) from either control, ischemic, or reperfused rabbit hearts with 16:0, [ H] 20:4 plasmenylcholine (•) in the presence of 4 m M EGTA as previously described (Hazen et al. 1991). Tissue lactic acid content (n) was quantitated spectrophotometrically. In control hearts phospholipase A2 activity as well as lactate production were not elevated.

myocardium contains a neutral active phospholipase C that catalyzes the hydrolysis of plasmalogen molecular species whose activity is regulated by an endogenous inhibitor (Wolf and Gross, 1985). The demonstration that l-O-alk-l'-enyl-l-acylsn-g\ycQTo\ accumulates during brief myocardial ischemia (Ford and Gross, 1989b) (two and fivefold following 20 and 60 minutes of global ischemia, respectively) illustrates the likelihood of accelerated plasmalogen polar head group turnover during the ischemic interval (Figure 6). In sharp contrast, diacyl glycerol content decreases during myocardial ischemia (Ford and Gross, 1989b). The discordant changes in l-0-alk-r-enyl-2-acyl-5«-glycerol and diacyl glycerol content in is­ chemic myocardium likely reflects the disparate rates of metabolic clearance of each diradyl glycerol molecular subclass. Since 1 -(9-alk-1 '-enyl-2-acyl-5«-glycerol is a poor substrate for both diglyceride kinase and diglyceride lipase which function to remove diacyl glycerol, the potential for accumulation of r-O-alk-enyl-2-acylglycerol is substantially greater than that of diacyl glycerol (Ford and Gross, 1990a).

DAVID A. FORD and RICHARD W. GROSS

182

J 20

o

E

U

/^ J-

Globaliy Ischemic Hearts \

o o

T

^^^^ J1

'

Control Perfused Hearts

/ y ^

■

'

20 40 Experimental Interval (min)

'

60

Figure 6. The accumulation of 1-0-alk-r-enyI-2-acyl-sn-gIycerol in perfused and ischemic rabbit myocardium. Rabbit hearts were either perfused normally or rendered ischemic for the indicated times. Rabbit myocardial 1-0-alk-1'-enyl-2-acyl-sn-glycerol was quantitated after purification as previously described (Ford and Gross, 1988). Values represent the mean ± SEM for six determinations.

It should be recognized that l-0-alk-r-enyl-2-acyl-5«-glycerol content was ob­ served to increase prior to arachidonic acid accumulation (i.e., arachidonic acid accumulation was not detected until 60 minutes of global ischemia) (Ford and Gross, 1989b). The only l-0-alk-r-enyl-2-acyl-5«-glycerol molecular species that was detected in both control and ischemic rabbit hearts was l-O-hexadec-T-enyl2-acyl-5«-glycerol. Since this molecular species is enriched in plasmenylcholine, but only present in moderate amounts in plasmenylethanolamine, these findings suggested that l-0-alk-r-enyl-2-acyl-5«-glycerol production was mediated by phospholipase C-catalyzed hydrolysis of plasmenylcholine. Alternatively, it should be recognized that another potential source of l-O-alk-T-enyl-l-acyl-^w-glycerol is via the selective hydrolysis of specific molecular species of plasmenylethano­ lamine containing a 16:0 vinyl ether at the sn-\ carbon by phospholipase C or its equivalent (i.e., sequential phospholipase D and phosphatidate phosphohydrolase activities). The temporal course of 1 -O-alk-1 '-enyl-2-acyl-5n-glycerol accumulation is simi­ lar to that of the increase in intracellular free calcium during myocardial ischemia. Accordingly, 1 -0-alk-1 '-enyl-2-acyl-5'«-glycerol and calcium could synergistically

Plasmalogen Metabolism

183

activate specific myocardial protein kinase C isozymes during the ischemic event. It is of interest that l-(9-alk-r-enyl-2-acyl-5«-glycerol may induce the specific phosphorylation of critical myocardial proteins since it is a potent activator of myocardial protein kinase C which possesses an obligatory requirement for physi­ ologic increments in free calcium concentration (Ford and Gross, 1990b). In contrast, myocardial protein kinase C activity activated by diacyl glycerol is partially calcium-independent (Ford and Gross, 1990b). The calcium requirement for the activation of individual protein kinase C isozymes by l-O-alk-r-enyl-2acyl-5«-glycerol was further elucidated after the demonstration that the purified P isozyme of protein kinase C is activated by diacyl glycerol in the absence of free calcium while l-0-alk-r-enyl-2-acyl-5«-glycerol-mediated activation of the P isozyme has an absolute requirement for physiologic increments in free calcium (Ford et al., 1989). In sharp contrast, activation of the purified a isozyme of protein kinase C by both l-0-alk-r-enyl-2-acyl-5«-glycerol and diacyl glycerol requires physiologic increments in calcium ion. Taken together, these results demonstrate that accelerated plasmalogen polar head group hydrolysis results in the generation of a plasmalogen diradyl glycerol, l-0-alk-r-enyl-2-acyl-5«-glycerol, that poten­ tially mediates alterations in myocardial performance through the activation of distinct isozymes of myocardial protein kinase C which, in turn, phosphorylates specific myocardial proteins. F. Plasmalogen Catabolism in Smooth Muscle Cells

The regulation of phospholipid catabolism in smooth muscle cells has been the focus of intense investigation since phospholipid-derived second messengers in­ cluding eicosanoids, diglycerides, and platelet activating factor have profound effects on vascular smooth muscle contractility. In fact, the production of specific eicosanoids in the vascular bed represents a major mechanism responsible for the regulation of vascular tone and the appropriate distribution of blood flow to specific organs to fulfill each tissue's hemodynamic requirement. Accordingly, the mecha­ nisms responsible for the release of arachidonic acid from vascular smooth muscle cells, as well as other cells in the vascular bed (e.g., endothelial cells), have been extensively investigated. The goal of many of these studies is to identify the source of released arachidonic acid, the phospholipase(s) mediating release with each agonist (i.e., the subcellular compartment targeted for hydrolysis and the individual molecular species which are hydrolyzed) and to identify the physiologic importance of the released metabolites in the vasculature. Much of the work in the field of arachidonic acid release in activated smooth muscle cells has focused on inositol phospholipids which are rapidly hydrolyzed after stimulation with many agonists (e.g., vasopressin, angiotensin). However, the majority of arachidonic acid mass released in stimulated smooth muscle cells likely results from phospholipase A2-mediated cleavage of choline and ethanolamine glycerophospholipids. Several lines of evidence support this conclusion. First,

184

DAVID A. FORD and RICHARD W. GROSS

plasmenylethanolamine is the major endogenous phospholipid storage depot of arachidonic acid in rabbit aortic intimal smooth muscle. In fact, 80% of the arachidonic acid present in aortic smooth muscle cell phospholipid is sequestered in plasmenylethanolamine molecular species (Ford and Gross, 1989a). Second, vasopressin stimulation of cells results in major losses in arachidonic acid content in choline and ethanolamine glycerophospholipids. Utilizing aortic rings prelabeled with [^H] arachidonic acid the specific activities of the choline and inositol glycerophospholipid pools were similar while ethanolamine glycerophospholipid had a specific activity of only 20% of that present in the choline and inositol glycerophospholipid pools. Despite the marked disparity in the specific activities of these three phospholipid classes after the prelabeling interval employed, angiotensin II stimulation resulted in nearly identical fractional losses (35-41%) of [^H] arachidonic acid from aortic smooth muscle cell choline, ethanolamine, and inositol glycerophospholipid classes demonstrating that plasmenylethanolamine, due to its high arachidonic acid content and low specific activity, was the major source of released arachidonic acid mass in this system. Reverse phase HPLC confirmed that over 60% of the arachidonic acid released from ethanolamine glycerophospholipids during angiotensin II stimulation originated from plas­ menylethanolamine molecular species (Ford and Gross, 1989a). The demonstration that arachidonic acid was selectively released from vascular smooth muscle cell plasmenylethanolamine pools suggested that vascular smooth muscle contained plasmalogen-selective phospholipase(s) A2 which facilitated the release of arachidonic acid from specific pools. To clarify the salient kinetic characteristics of the phospholipase(s) A2 in smooth muscle cells, in vitro analyses of their calcium requirements, substrate selectivities, and kinetic properties were performed. Three separate and distinct phospholipase A2 activities are present in vascular smooth muscle including: (a) a cytosolic calcium-independent phospholi­ pase A2 that is activated by nucleotide di- and triphosphates; (b) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (c) a microsomal calcium-independent phos­ pholipase A2 which is highly selective for plasmenylcholine substrate (Miyake and Gross, 1992). Traditionally, the importance of a given enzyme or protein responsible for a specific physiologic or pathophysiologic effect after cellular perturbation has been clarified through utilization of inhibitors which possess substantial specificity for the reactions or processes under consideration. In the case of phospholipases, the assignment of the role of individual polypeptides in the release of arachidonic acid after agonist stimulation represents a critical issue both for the understanding of smooth muscle cell biochemistry as well as identification of the mechanisms of potential therapeutic effects. Our ability to dissect critical biochemical events leading to the release of arachidonic acid after smooth muscle cell activation has been hampered by the relatively poor selectivity of existing pharmacologic agents for the inhibition of specific types of phospholipase A2. In many cases, utilization

Plasmalogen Metabolism

185

of nonspecific inhibitors has produced ambiguous results which has led to substan­ tial confusion regarding the importance of a given type of phospholipase in mediating arachidonic acid release during a specific agonist-mediated event. Ac­ cordingly, the identification of inhibitors which possess selectivity for each specific type of intracellular phospholipase would be of great utility in identifying the contributions of each of the individual phospholipases A2 in the release of arachi­ donic acid. Recognizing that one suicide inhibitor previously developed by Katzenellenbogen and co-workers (Daniels et al., 1983; Daniels and Katzenellenbogen, 1986), contained a vinyl ether linkage (a chemical moiety which was specifically recognized by calcium-independent phospholipases) and an oxyester linkage which was potentially susceptible to hydrolysis by calcium-independent phospholipase A2, Hazen et al. (1991b) identified (F)-6-(bromomethylene)tetrahydro-3-(l-naphthalenyl)-2H -pyran-2-one (HELSS) as a specific mechanism-based inhibitor which possessed over a 1,000-fold selectivity for inhibition of calciumindependent phospholipase A2 in comparison to calcium-dependent phospholipase A2. Since HELSS did not contain a charged functionality, it was reasonable to expect that it could freely diffuse through cellular membranes. The thoracic aortic cell line, A10 smooth muscle cells, expresses vasopressin receptors of the VI subtype, whose stimulation results in the selective release of arachidonic acid. Accordingly, we exploited the specificity inherent in mechanism-based inhibition to identify the intracellular phospholipase(s) responsible for arachidonic acid release during vasopressin stimulation of aortic smooth muscle cells. Prelabeling of smooth muscle A10 cells with [^H]-arachidonic acid followed by treatment with 1 )LiM arginine vasopressin for five minutes resulted in the release of approximately 4% of total [^H] arachidonic acid in cellular phospholipids (Lehman et al., 1993). The only metabolite released by these cells was [^H]-arachidonic acid without demonstrable lipoxygenase or cyclooxygenase metabolites observed (i.e., these smooth muscle cells do not contain substantial quantities of eicosanoid oxidative enzymes). Remarkably, treatment of smooth muscle A10 cells with only 1 JLIM HELSS resulted in the inhibition of over one-half of pH] arachidonic acid release, and treatment with 5 JLIM HELSS resulted in the inhibition of over two-thirds of [^H]-arachidonic acid release. Thus, the majority of arachidonic acid release by arginine vasopressin was attributed to calcium-independent phospholipase A2. Since arachidonic acid attenuates the rate of myosin light chain dephosphorylation and is associated with increased smooth muscle cell contractility, it seems likely that calcium-independent phospholipase A2 is an important modulator of the increase in contractile force in smooth muscle mediated by arginine vasopressin stimulation. G. Plasmalogen Catabolism in Neutrophils Accelerated plasmalogen catabolism in activated neutrophils plays a major role in the production of eicosanoids as well as platelet acfivating factor during the

186

DAVID A. FORD and RICHARD W. GROSS

inflammatory response. The importance of accelerated ether lipid catabolism, arachidonic acid release, and platelet activating factor biosynthesis in neutrophils can easily be demonstrated simply by recognizing that 66% of the arachidonic acid in the choline glycerophospholipid pool is present in alkyl ether choline glycerophospholipids (i.e., the precursor of platelet activating factor) and 71% of arachi­ donic acid in the ethanolamine glycerophospholipid pool is present in the plasmalogen subclass (Chilton and Connell, 1988). Initial investigations predomi­ nantly focused on the concomitant production of arachidonic acid and platelet activating factor utilizing calcium ionophore- or zymosan-stimulated neutrophils (Chilton et al., 1984). Since platelet activating factor production is initiated by the hydrolysis of arachidonic acid from alkyl-acyl-glycerophosphoryl choline, it was envisaged that a single enzymatic reaction (i.e., phospholipase A2) could result in the initiation of two classes of lipid mediators (i.e., eicosanoids and platelet activating factor). The concept that platelet activating factor and eicosanoid pro­ duction are initiated through phospholipase A2-mediated hydrolysis of alkyl-acylGPC was further supported by experiments demonstrating similar specific activities in alkyl-acyl-GPC, leukotriene B4, and 20-hydroxy-leukotriene B4 in stimulated neutrophils that were prelabeled with [^H]-arachidonic acid (Chilton, 1989). Since phospholipase A2 likely mediates the release of arachidonic acid and the initiation of platelet activating factor production in stimulated neutrophils, substan­ tial effort has been directed toward identifying the mechanism of phospholipase A2 activation in neutrophils. One hypothesis that has been given considerable attention is that the activation of phospholipase A2 in stimulated neutrophils is mediated by the activation of protein kinase C through diradyl glycerols produced after agonist stimulation. This hypothesis was first supported by the demonstration that neutro­ phils stimulated with the chemotactic peptide,flVILP,accumulate l-(9-alkyl-2-acyl^«-glycerol and l,2-diacyl-5«-glycerol (Rider et al., 1988). The production of 1-0-alkyl-2-acyl-5«-glycerol in stimulated neutrophils is mediated by phospholi­ pase D-catalyzed hydrolysis of alkyl-acyl-GPC followed by phosphatidate phosphohydrolase-mediated hydrolysis of the resultant phosphatidic acid (Chabot et al., 1992; Strum et al, 1993). Diradyl glycerol-mediated regulation of neutrophil phospholipase A2 has been supported by the demonstration that 1,2-diacyl-5«-glycerol and l-(9-alkyl-2-acyl-5/7-glycerol not only prime neutrophil respiratory bursts for stimulation by fMLP, but l,2-diacyl-5«-glycerol and l-(9-alkyl-2-acyl also prime neutrophils for arachidonic acid release during fMLP stimulation (Bauldry et al, 1988). Of further interest is the observation that only l,2-diacyl-5'«-glycerol and not l-0-alkyl-2-acyl-5«-glycerol primes neutrophil production of LTB4 (Bauldry et al., 1991). Taken together, it is likely that diradyl glycerols modulate neutrophil phospholipase A2 and possibly lipoxygenase activities. Several recent studies have elucidated the role of plasmalogen catabolism in neutrophil arachidonic acid release and platelet activating factor production. The

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observation that plasmenylethanolamine catabolism resulted in lysoplasmenylethanolamine accumulation in stimulated neutrophils (Tessner et al., 1990) led to the discovery of a novel enzymatic pathway for platelet activating factor biosynthesis in neutrophils which is initiated by phospholipase A2-mediated hy­ drolysis of plasmenylethanolamine (Nieto et al, 1991; Uemura et al., 1991). Via this pathway, the subsequent transacylation of lysoplasmenylethanolamine with arachidonic acid from l-(9-alkyl-2-arachidonoyl-GPC generates lyso-platelet acti­ vating factor. Lyso-platelet activating factor is then readily acetylated by a reaction mediated by acetyl CoA transferase or a CoA-independent transacetylation path­ way (Lee et al., 1986) resulting in the synthesis of platelet activating factor. Accelerated plasmalogen catabolism in activated neutrophils also results in the accumulation of an acetylated plasmenylethanolamine species (Tessner and Wykle, 1987). Although this plasmalogenic platelet activating factor analog is chemically similar to platelet activating factor, it does not share the biological activities of platelet activating factor and it has not yet been assigned a biological activity. The biosynthetic pathway for plasmalogenic platelet activating factor resembles that of platelet activating factor, since lysoplasmenylethanolamine can be converted to its acetylated platelet activating factor analog by either a CoA-independent transacetylase activity utilizing the acetate of platelet activating factor as the donor or via an acetyl transferase pathway (Lee et al., 1992). Collectively, multiple studies have underscored the importance of the hydrolysis of arachidonoylated plasmenylethanolamine in activated neutrophils catalyzed by phospholipase A2 resulting in the generation not only of arachidonic acid and its bioactive oxygenated products, but also the initiation of the synthesis of other bioactive lipid second messengers. Since these lipid second messenger molecules likely are key elements in inflammatory responses, specific inhibitors would be of great utility in attenuating the accumulation of these biomolecules. Furthermore, since the hydrolysis of plasmenylethanolamine is an initial step in the cascade in the synthesis of these molecules, the identification of inhibitors that selectively inhibit plasmenylethanolamine hydrolysis by phospholipase A2 represent a particu­ larly attractive pharmaceutical target.

IV. CONCLUSION The last decade has witnessed remarkable advances in our understanding of the structure and function of ether phospholipids. The observation that plasmalogens represent the predominant phospholipid constituents of the sarcolemmal membrane of myocardium has underscored the importance of plasmalogen constituents in membrane function. The enrichment of arachidonic acid in plasmalogen molecular species in conjunction with the demonstration of the activation of plasmalogen-selective phospholipases A2 has now provided unambiguous evidence delineating the

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role of the plasmalogen subclass as a reservoir of eicosanoids released during cellular activation. Concomitant with the rapid expansion of our knowledge on plasmalogen conformation, catabolism and the role of plasmalogens in cellular function, the current concepts of rapid polar head group turnover in the de novo synthesis of plasmenylcholine have evolved. It is important to realize that these insights on plasmalogen structure, function, and metabolism do not circumscribe the entire plasmalogen dynamic but, rather, provide the initial insights into the molecular mechanism and covalent structures which underlie nature's own special­ ized system integrating form and function.

ACKNOWLEDGMENT This research was supported by NIH grant HL42665.

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