Arachidonic acid channelling in the phospholipid fractions and subcellular compartments of cultured myocardial cells

PROSTAWANDINSLE%KOTRENES ANDEilWNW&FAmAClDS Prostaglandins 0 Longman

Leukotrienes and Essential Group UK Ltd I992

Fatty

Acids

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46, 323-32X

Arachidonic Acid Channelling in the Phospholipid Fractions and Subcellular Compartments of Cultured Myocardial Cells A. Halabi, N. Nachas and A. Pinson Laboratory for Myocardial Research, Institute of Biochemistry, Hebrew University-Hadassah Medical School, POB 1172, Jerusalem 91010, Israel (Reprint requests to AP) ABSTRACT.

Arachidonic acid (AA) channeling in cultured heart cells was studied following pulse labelling for 1 h. AA was shown to be esterified immediately and equally distributed between the neutral lipids and phospholipids. A rapid constant flow to various phospholipid classes occurred thereafter, while the AA oxidation was only between 12%. The s&cellular distribution of AA was studied by nitrogen cavitation followed by fractionation on 6.7% percoll in sucrose-EDTA. After 1 h pulse labeling and 2 h post-pulse incubation, most of the radioactivity was found in the sarcolemmal fraction with a much smaller amount in the mitochondrial fraction.

Miyazaki et al (5), who studied AA distribution in neonatal cultured heart cells from l-24 h following AA pulses. In these experiments, AA was preferentially incorporated into PLs and located in the mitochondrial and inner cytoplasmic membrane subcellular fractions. AA oxidation was also found to be negligible. During cardiac ischemia, activation of the sarcolemmal lipases leads to release of AA, which either accumulates or is metabolized by the cyclooxygenase pathway (6, 7). In addition to thrombolytic activity, some of the metabolites of this pathway have a ‘cell preservation effect’ (8). However, the concomitant lysophoshoglyceride production and free AA accumulation may have adverse effects, which have been implicated in irreversible cell damage (9, 10). Clearly, more information on AA metabolism during normal aerobic conditions and under anoxic injury might advance strategies designed to enhance cell preservation. The present study was directed toward characterizing the AA channelling pathways among the various lipid classes and subcellular compartments in cultured heart cells following relatively short AA pulses.

INTRODUCTION Arachidonic acid (AA) is one of the most abundant fatty acids (FA) in the heart both in situ and in culture, particularly in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) the two main phospholipids (PLs) in the sarcolemma. AA esterifies the Sn;! position of the glyceryl moiety (1, 2). Phosphotidylinositol (PI), which accounts for about 10% of cellular PLs, has a fixed FA composition - stearic and arachidonic acids in the Sni and Sri* positions, respectively (2). There are conflicting data regarding the AA distribution in cardiac muscle. Thus, in the perfused heart, AA is preferentially incorporated into the PLs with &fold higher levels in PC than in PE (3). In calcium-resistant cardiomyocytes, Hohl and Rosen (4) found the AA distribution to be concentration dependent: in the presence of 20 PM AA, it was preferentially located in the triacylglycerols, while at lower concentrations, AA became incorporated into the PLs.. They also detected low, but significant, levels of AA oxidation. They also suggested that AA serves primarily as a structural component in myocardial membrane. Its sarcolemmal location and release during ischemia would initiate the AA cascade and prostaglandin (PG) production in coronary vascular tissue. These findings are in conflict with those of

MATERIALS AND METHODS Cell cultures Heart cells culture from l-day-old rats (Wistarderived Hebrew University strain) were prepared as

Date received 5 September 1991 Date accepted 8 January 1992 323

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previously described (11, 12) at seeding densities of 1.8-2.1 X 18 cell per 35 mm-diameter Petri dish (Falcon 3001). The initial proportion of muscle cells @O-90%) is maintained for at least 6 days in culture (11, 12).

Arachidonate preparation Potassium arachidonate (Sigma, Israel), supplemented with 13-‘4C-arachidonate of specific activity 39 mCi/mmol (CEA, France), was added to Ham F-10 culture medium (Gibco, USA) containing 8 g/l fraction V, FA-free albumin (Sigma, Israel) to give a final AA concentration of 0.2 mM and an arachidonate/albumin ratio of about 2. The substrate was stored at -20°C.

Cell labeling Cell labeling was carried out on the fifth day in culture by incubating the Petri dishes with 1 ml of the substrate at 37°C for periods of up to 1 h. After the AA pulse, the medium was changed to Ham F-lOalbumin (w/o FA supplementation) and allowed to incubate further for various periods. The AA distributions for pulses of various durations of up to 1 h were also determined.

Lipid extraction At the end of the incubation, the cells were washed with albumin-supplemented medium at 4°C and then with cold saline. The saline was removed and the lipids were then extracted by the direct addition of 1.5 ml isopropanol (Fluka, analytical grade) to the Petri dish (13) at room temperature for 1 h. The solvent was then removed and the cells were washed with 0.5 ml isopropanol. The combined extracts were then evaporated to dryness under a stream of nitrogen and redissolved in chloroform/methanol (2/l v/v). Centrifugation was then carried out for 10 min at 3000g in order to remove the traces of plastic that had been dissolved in the ispropanol.

Lipid analysis Separation of NL and PL was conducted on minicolumns (SepPak silica cartridges, Water Associates, Mitford, MA), eluted with 5 ml chloroform and then 5 ml methanol to give NLs and PLs, respectively. The lipid classes were separated by TLC on 0.2 mm layers of silica gel 60 on aluminium sheets (Merck), eluting for NLs and PLs, respectively. The lipid spots were visualized by staining with iodine vapor, they were scraped off and were then counted for radioactivity using Lumax as a scintillation fluid and a fi-scintillation counter.

Cell fractionation Cell fractionation was carried out according to the method of Ziegler and Bach (14), modified for use with cardiomyocytes. Briefly, cardiomyocytes were pulse-labeled with 13-‘4C-arachidonate followed by 2 h post-pulse incubation in 0.8% FA-free albumin Ham F-10 medium. Cells were then removed from the Petri dishes by controlled trypsinization for 2 min, washed twice with saline, and then with sucrose/EDTA (0.25 mM/O.l mM) and disrupted by nitrogen cavitation, resuspended in the same solution and then centrifuged (in a Sorvall centrifuge) at 2000 rpm for 10 min at 4°C. The supernatant was layered onto 6.7% percoll in sucrose-EDTA, PH 6.8 on a 0.3 ml cushion of 2.5 M sucrose in a quick-seal centrifuge tube (Beckman Instrument, CA). Fractionation was carried out by 30-min centrifugation at 4°C in a Kontron I or II centrifuge in a VT1 65 vertical head at 20 000 rpm. About 20 fractions 0.25 ml in volume were collected and assayed for radioactivity, proteins (15), and for several subcellular marker enzymes (5’-nucleotidase (16), succinic dehydrogenase (17)) and P-hexosaminidase (18)). Arachidonate oxidation Arachidonate oxidation was determined by using 0.2 mM or either 13-i4C-arachidonate or 1-14Carachidonate of specific activity 58 mCi/mmol Arachidonate oxidation was (CEA, France). evaluated in a special incubation chamber at 37°C (19). The [‘VI0 2 was trapped in 1 M hyamine hydroxide (Packard), and the soluble CO2 (carbonates) were released from the medium at the end of the experiment by injecting 1 ml 1N HCI.

RESULTS The preliminary assays indicated that all the cellular radioactivity was extracted by isopropanol. Samples for TLC separation were taken in quadruplicate, each from the pooled cells from two Petri dishes. A plot of the the time-dependent incorporation of 1314C-arachidonate into TG and PLs is presented in Figure 1. During the specified time period, the radioactivity was equally distributed between TGs and PLs. Non-esterfied fatty acid (NEFA) labeling remained constant and high due to the continuous arachidonate uptake during the pulse. Figure 2 shows the distribution of AA in the main PL classes during short pulses of up to 1 h. The incorporation of 14C-AA into PC was 3-fold higher than in PE or PI. The incorporation of AA into the other PL classes was between 1% and 2% throughout.

AA Channelling in Cardiomyocytes

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0

Time

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Fig. 1

Time-dependent incorporation of 13-‘“C-arachidonate into cultured myocardial cells (2.060 dpm = 1 nmol). The diglyceride were found to be constant and low throughout, and cholesterol labeling was low but increased from 1200 to 800 dpm. TG - triglyceride W--B PL - phospholipids 0-o - nonesterified fatty acids*--*

7000 6000

Time

(min)

Fig. 2

Time-dependent incorporation of 13-“C-arachidonate into the main PL classes. The sphingomyelin. phosphatidylserine and cardiolipin levels were low throughout. PC - phosphatidylcholine l ---o PI - phosphatidylinositol o---o PE - phosphatidylethanolamine A---A

Q,1

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Time

LI

T

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n-o+ 4

5

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Fig. 3

Time-dependent decrease in triglyceride radioactivity after pulse labeling with 13-‘4C-archidonate for 1 h (time 0). There was a concomitant increase in PL labeling. TG - triglyceride A----A DG -- diglyceride a----a FA - nonesterified fatty acids o--o

The Table gives a more detailed summary of the relative distribution of AA in the various PL fractions. The increase in PI_/NL radioactivity ratio reflects a flow toward the PLs. The relative percent labeling in the various PL classes remain constant throughout. To determine the subcellular channelling of AA, following a l-h pulse, the cultured cardiac myocytes were separated into subcellular fractions and then left in AA-free medium for 2 h. As shown in Figure 4, distinct radioactive peaks, identified by markers, were found in the lysosomal, mitochondrial and sarcolemmal fractions. The largest radioactive peak was associated with the sarcolemma (fractions 6-11). There were two smaller peaks: the main one in the mitochondria, which also has some 5’nucleotidase activity (fraction 4), and the other in the supernatant (fraction 18). Oxidation, as reflected by [14C]0 2 release, only accounted for up to 2% of the incorporated arachidonate. Major differences in [‘4C]02 release with either 13-‘4C- or l-‘4C-labeled arachidonate were not detected.

DISCUSSION Arachidonate channelling after 1 h of pulse labeling was followed by replacing the medium with a FA-free medium and incubating the cells further for periods of up to 6 h. Figure 3 shows a linear decrease in TG labeling. A concomitant increase in PL labeling (not shown) occurs. Thus, a flow of arachidonate between these two classes takes place. The labeling levels of NEFA, which exhibited high labeling during the pulse (cf. Fig. l), were low throughout the post-pulse period.

This study utilized short 14C-AA pulses in cultured cardiac myocytes to probe AA channelling into various lipid classes and cellular compartments. The spontaneously synchronously beating cultured myocardial ceil model offers several advantages in such studies. Compared to the intact heart, the cultures provide a more homogeneous cell population. Since a large number of identical units (Petri dishes) with controlled environments may easily be

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Table Incorporation classes Lipid class

PI + PS SM PC PE CL PC/PE PL/NL

of 13-14C-arachidonate in various phospholipid

After t 1 h-pulse

20.8 1.2 56.4 19.3 2.3

+ f + f f

2.9 0.95

Post-pulse incubation time 1h

2h

3h

Percent 2.1 19.7 f 2.4 23.2 0.3 1.4 f 0.1 1.6 4.7 57.4 f 3.9 55.9 2.1 19.1 _+ 1.9 17.4 0.1 2.4 + 0.1 1.9 Ratio 3.0 3.2 2.9 6.5

PI - phosphatidylinositol PS - phosphatidylserine SM - sphingomyelin PC - phosphatidylcholine

Fig. 4 Distribution of 13-‘?-arachidonic acid in cardiomyocyte subcellular fractions after pulse labeling for 1 h and post-pulse incubation for 2 h (for details, see Materials and Methods). - A - A - proteins - l - l - radioactivity

Z!I2.4 22.6 + 2.5 22.7 + 2.4 + 0.3 1.2 f 0.1 1.0 f 0.3 f 4.9 61.4 f 4.8 60.6 f 4.4 + 1.9 12.5 f 1.3 14.0 + 1.7 f 0.2 2.3 f 0.4 1.7 f 0.2 4.9 6.3

4.3 7.4

PE - phosphatidylethanolamine CL - cardiolipin n= 4+SEM

o 1d, o‘o_o-~9_o.~p_~.~p-~.~.~.~ , 2 r* 6 .5 10 12 14 16 18 Fraction Number

4h

prepared, kinetic studies based on the quantitative recovery of radioactive labels may be carried out conveniently in cultures. Arachidonic acid is one of the most abundant FAs in cultured heart cells. It displays a greater affinity for the fatty-acid-binding protein than, for example, palmitic acids (ko C16:o is 0.83 and ko C2”:4 is 0.27) (21). For this reason, palmitic acid is more available for oxidation than arachidonate. In addition, transcyclase exhibits a strong preference for AA (22), which may explain the high AA levels occurring in the microsomal FA-PL fraction. Therefore, only a small amount of AA is available for P-oxidation. Most of the AA is channelled towards the ‘structural’ PLs, where it plays roles in maintaining the physical state and the enzyme and receptor activities of the membrane. In addition to its role in prostglandin synthesis (20), AA seems to play an essential role in myocardial membrane function, since even under conditions of extreme AA deficiency, whereas most tissues lose AA, cardiac and renal cells ‘paradoxically’ accumulate arachidonate in the PE fraction (but not in PI) (23). Notably in AA deficiency, renal and cardiac PG production is decreased in response to angiotensin II but it is maintained during ischemia (23). The use of short pulses for AA channelling in this study should allow more precise determination of AA flow patterns. Indeed, we followed the fate of AA by pulse-labeling the cells in a medium containing only AA, and then examined the post-pulse AA distribution. In contrast to the report of Hohl and Rosen (4) of the predominant incorporation of AA into the TG fraction, which was obtained at 20 ,uM AA, we found an equal distribution in the NLs and the PLs, and a relatively high degree of labeling in the NEFAs during the pulse (Fig. 1). Immediately after the pulse, there was a constant AA flow from

AA Channelling in Cardiomyocytes

the NLs towards the PLs (Fig. 2 & Table). Since 2 h after the pulse almost all the radioactivity is found in the PLs with only very minor changes in distribution occurring thereafter (Fig. 3) and very little AA undergoes oxidation, AA would be expected to be located in the membranous cell structures. Hohl and Rosen (4) suggest that AA is channelled towards the sarcolemma, where it serves primarily as a structural component of myocardial cell membranes. Surprisingly, Miyazaki et al (5) could not detect AA in the sarcolemma of cultured heart cells or in adult animals. Even after 24 h of incubation with 3H-AA, they found 90% of the radioactivity associated with the mitochondria and sarcoplasmic reticulum. They therefore concluded that the AA released during high energy phosphate depletion is primarily derived from these subcellular fractions. By contrast, other researchers (6, 7) found a decrease in the sarcolemmal PLs, and AA accumulation concomitant with ATP depletion. In order to shed new light on AA channelling into cellular structures, we used a different approach based on a mild cell fractionation technique, which maintains the integrity of cellular structures while providing highly purified subcellular fractions, as indicated by the specific markers for the various fractions. Thus, as Figure 4 clearly shows, after 1 h of pulse and 2 h of post-pulse labeling, the radioactivity is predominantly located in the sarcolemmal fraction. It is difficult to account for the differences in AA subcellular channelling by Miyazaki et al (5) with this report and the findings of others (4, 6, 7). Indeed, only during AA deficiency is this essential fatty acid channelled primarily into mitochondrial PE, where it plays an essential structural role in the membrane and in oxidative phosphorylation coupling (23). This was not observed in Miyazaki’s experimental systems, using either normal adult rats or cultured cells grown in a serum supplemented medium. They explain their findings by assuming that the turnover of arachidonic PLs is ‘much slower’ than that of cellular membranes. However, their assumption is somewhat difficult to reconcile with the high turnover of sarcolemmal PLs (24). Since the total AA incorporation is greater than that metabolized by the P-oxidation and PG pathways by two orders of magnitude, their explanation would imply that following 24 h incubation with AA, the mass of mitochondria and the SR would increase while that of the sarcolemma would remain unchanged. Moreover, considering the rapid turnover of the PI-IP cycle and the role it plays in the sarcolemma-modulated cardiomyocyte contractility, and since AA exclusively esterifies the Sn2 position of PI, PI channelling primarily directed toward the sarcolemma would be expected.

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Acknowledgements This research was supported by the American Heart Disease Prevention Foundation Inc. and by grants from the National Council for Research and Development, Israel, the South African Medical Research Council, Mr and Miss D. VidalMadjar (Paris), Mrs F. Berk (Brussels) in memory of her daughter Mrs Iva Mis, and Mrs R. Missistrano (Cannes) in memory of her husband Mr Henri Missistrano.

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