Phosphatidylcholine formation from exogenous lysophosphatidylcholine in isolated hamster heart

40

PHOSPHATIDYLCHOLINE FORMATION FROM EXOGENOUS LYSOPHOSPHATIDYLCHOLINE IN ISOLATED HAMSTER HEART

(Received

August

I I th, 1981)

The formation of phosphatidylcholine in hamster heart by reacylation and transacylation of exogenous lysophosphatidylcholine was investigated. Isolated hamster hearts were perfused with labeled lysophosphatidylcholine in Krebs-Henseleit buffer. Uptake of total radioactivity by the heart was maximum at 30 min of perfusion and was also linear from S-20 PM of lysophosphatidylcholine in the perfusate. About 17*3% of total radioactivity taken up by the heart was recovered in phosphatidylcholine. Perfusion of the isolated heart with 1-I“C]palmitoylglycerophospho[metby/-3H]choline indicated that labeled phosphatidylcholine was formed by reacylation of lysophosphatidylcholine with acyl-CoA and not by transacylation with another molecule of lysophosphatidylcholine. From the pool size of total cardiac lysophosphatidylcholine, the amount of phosphatidylcholine formed via the reacylation process was estimated to be 6.6 nmol/min per g heart.

Introduction

phatidylcholine is a facile mechanism for the cell to obtain ‘tailor-made’ phosphatidylcholine with the required acyl groups [3,5]. Reacylation of exogenous lysophosphatidylcholine may also be an important pathway for new formation of phosphatidylcholine. Although the actual existence of this pathway in mammalian heart was not demonstrated, it was reported that labeled lysophosphatidylcholine administered intravenously into squirrel monkeys was taken up rapidly by various

Phosphatidylcholine is the major phospholipid in mammalian heart [ 11. It is important not only as a structural component of the cardiac membranes, but also as a modulator to a number of membrane-bound enzymes [2]. Although the biosynthesis of this phospholipid was studied extensively in a number of mammalian organs [3], only limited information is available for its biosynthesis in the mammalian heart. A recent report from our laboratory indicated that the majority of phosphatidylcholine is synthesized de novo via the CDPcholine pathway in the isolated hamster heart. The contribution of the progressive methylation of phosphatidylethanolamine pathway and base exchange reaction towards phosphatidylcholine formation is relatively minor [4]. An alternate pathway for phosphatidylcholine formation is via reacylation of lysophosphatidylcholine (Fig. 1). It was demonstrated that the deacylation - reacylation of intracellular phosOOOS-2760/X2/0000-Oooo/so2.75

S’ 1982 Elsevier Biomedical

v.eMBrt*NE,

Fig. I. Possible pathways dylcholine from exogenous

Press

w*fw

for the formation of phosphatilyaophosphatidylcholinc.

41

organs, including the heart, and a significant portion of the radioactivity was recovered subsequently as phosphatidylcholine [6]. However, it was not clear whether lysophosphatidylcholine was transported into the heart prior to acylation, or the lysophosphatide was acylated by an energyindependent acyltransferase in the plasma ]‘7-9] or elsewhere and, subsequently, taken up by the heart as labeled phosphatidylcholine. In this report, we demonstrated that in isolated hamster heart, lysophosphatidylcholine is transported actively into the heart, and the lysolipid is reacylated exclusively by the action of lysophosphatidylcholine: acyl CoA acyltransferase. Materials and Methods Materials. L-cY-Lysophosphatidylcholine, ~-aphosphatidylcholine, phospholipase A, (Naja naja venom), oleoyl-CoA (free acid) and bovine serum albumin (fatty acid-free) were obtained from Sigma Chemical Company, St. Louis, MO. I-[ l-‘4C]Palmitoylglycerophosphocholine was purchased from New England Nuclear. ACS (aqueous counting scintillant) was the product of Amersham Corporation. Thin-layer chromatographic plates (SILG25) were obtained from Brinkmann. All other chemicals were of reagent grade and were obtained from Fisher Chemical Company. All solutions were prepared with glass-distilled water and were adjusted to the desired pH. Experimental animals. Syrian golden hamsters, 80-120 g, were maintained on Purina Hamster Chow and tap water, ad libitum, in a light- and temperature-controlled room. Preparation of 1 -acyl-sn-glycerophospho[ meth.ylI- Acyl-sn - glycerophospho[ methyl-‘H]chofine. 3H]choline was prepared from phosphatidyl[methyl-3H]choline by the hydrolytic action of phospholipase A, [lo]. The labeled phosphatidylcholine (5 pmol of lipid phosphorus) was dissolved in 5 ml diethyl ether and the reaction was initiated by adding 50 FM of 0.02 M TrisHC1/0.05 M CaCl, buffer, pH 8.0, which contained 1 mg of phospholipase A, (210 units/mg protein). The reaction was allowed to incubate for 60 min at 20°C. After the incubation, 0.5 ml of H,O and 0.5 ml methanol were added and the reaction mixture was incubated for a further 60

min at 20°C to ensure complete hydrolysis. The reaction mixture was evaporated to complete dryness after the second incubation, and resuspended in 1 ml of CHCl,/CH,OH (2/l; v/v). 1 ml of 0.1 M KC1 was added to the suspension and the phases were allowed to separate. The upper phase was re-extracted once more with 1 ml theoretical lower phase buffer [11] and the resultant lower phase was pooled with the original lower phase. The lipids in the lower phase were separated by thin-layer chromato~aphy in a solvent system containing CHCl,/CH,OH/_H 20/ CH,COOH (70 : 30 : 4: 2, v/v). Over 90% of the phosphatidyl[methyf-3 HIcholine was hydrolyzed and 85% of the radioactivity was recovered in lysophosphatidyl[ methyb3 HIcholine. Protein and phospholipid determination. Protein concentrations were determined by the method of Lowry et al. [12]. Phosphorus contents of the extracted lipids were measured by the procedure of Parker and Peterson [ 133 after acid digestion. In some experiments, the phosphorus content of the lipids were also assayed directly by the method of Raheja et al. 1141. Radioactivity was measured by a Searle Mark III liquid scintillation counter with channel ratio calibration method. Enzyme assays. Acyl-CoA : lysophosphatidylcholine acyltransferase was assayed by a modified procedure of Van Heusden et al. [15). The assay mixture contained 5 PM 1 -[ 1 -I4 Clpalmitoylglycerophosphocholine (specific activity 1 . lo5 cpm/nmol), 0.1 M Tris-HCl, pH 7.4, 0.2 mM oleoyl-CoA and 0.2-0.8 mg protein in a final volume of 0.5 ml. The mixture was incubated at 37°C for O-15 min. Aliquots of 0.1 ml were taken from the reaction mixture and the aliquots were placed immediately in 1 ml of CHC13/CH,0H (l/2; v/v). The precipitate was removed by centrifugation and the lipids were extracted as previously reported [ 161. Labeled phosphatidylcholine formed from the reaction was separated from lysophosphatidylcholine by thin-layer chromatography in a solvent system containing CHCl,/CH,OH/H,O,‘CH,COOH (70: 30:4: 2; v/v). Carrier phosphatidylcholine and lysophosphatidylcholine were added to facilitate separaLysophosphatidylcholine : lysophosphati tion. dylcholine transacylase and lysophospholipase were assayed by the procedures of Brumley and

42

Van den Bosch [ 171. The reaction mixture contained 200 nmol of 1-[ 1-I4 Clpalmitoyl-sn-glycerophosphocholine (spec. act. 1000 dpm/nmol) in potassium phosphate buffer, pH 7.4, and 0.05-0.1 ml enzyme preparation. The final volume of the mixture was 0.2 ml. The mixture was incubated at 37°C for 15 min and 1 ml of CHCI,/CH,OH (1 : 2, v/v) was added to terminate the reaction. The precipitate was removed by centrifugation and the lipids were extracted as previously reported. Labeled phosphatidylcholine, fatty acids and lysophosphatidylcholine were separated by thinlayer chromatography in a solvent system containing CHCl,/CH,OH/H,O/CH,COOH (70 : 30 : 4 : 2, v/v). Lysophospholipase activity was estimated by the amount of radioactivity in the fatty acids and lysophosphatidylcholine : lysophosphatidylcholine transacylase activity was calculated from the radioactivity in phosphatidylcholine. Since a small amount of acyl-CoA may be present in some subcellular fractions, a small amount of labeled phosphatidylcholine may be formed from lysophosphatidylcholine: acyl-CoA acyltransferase reaction. In order to distinguish clearly the presence of lysophosphatidylcholine: lysophosphatidylcholine transacylase, 1-[ 1-I4 Clpalmitoyl -sn -glycerophospho[ methyl-3 HIcholine was also used as a substrate. Phosphatidylcholine formed from transacylation reaction will have a 3H/‘4C ratio half that of the substrate, whereas phosphatidylcholine formed from acylation with an acyl-CoA moiety will retain the same 3H/‘4C ratio as the lysophosphatidylcholine substrate. Perfusion of isolated hamster heart and uptake of 1 -[I -‘4C]lysophosphatidylcholine. Perfusion and uptake of labeled lysophosphatidylcholine was performed in the same manner as previously described [4]. Hamsters were killed by decapitation and the hearts were removed rapidly and placed in Krebs-Henseleit buffer [18] saturated with 95% C&/5% CO, at room temperature. The heart was cannulated via the aorta in the Langerdorff mode [19] and the pulmonary artery was incised to ensure adequate coronary drainage. Perfusion was performed at 37°C with a coronary flow rate of 2-3 ml/min. Electrocardiac recording of the isolated heart was obtained during perfusion. One electrode was attached to the aortic cannula and the other was placed in the solution bathing the

heart. The signals were amplified and recorded by a Gould Brush 2400 paper recorder. 1-[ l-‘4C]Palmitoyllysophosphatidylcholine (0.2 pCi/ml) was placed in a vial, dried under a stream of nitrogen and mixed thoroughly with KrebsHenseleit buffer by vortex. In some experiments, bovine serum albumin was also added to the buffer. Doubly labeled lysophosphatidylcholine was added to the buffer in the same manner. Subsequent to perfusion for the prescribed interval, the heart was reperfused with 5 ml buffer to remove any labeled compounds in the vascular space. Finally, 5 ml of air was forced into the cannula to remove the buffer. The heart was cut open, blotted dry and the wet weight of the organ was determined. The heart was cut into small pieces and homogenized in 20 ml CHCl, /CH,OH (2 : 1, v/v) and an aliquot was taken to determine total radioactivity incorporation into the heart. The uptake of labeled lysophosphatidylcholine was calculated from the specific radioactivity of the lysophosphatidylcholine in the perfusate. Phospholipids in the heart homogenate were separated by thin-layer chromatography. Standard phospholipids were used for positive identification. The amount of radioactivity incorporated into each phospholipid and fatty acid was removed, and the radioactivity was determined by scintillation counting. Preparation of hamster heart subcellular fractions. Hamsters were killed by decapitation and the hearts were removed rapidly and placed in 20 vol. of ice-cold 0.25 M sucrose solution. The hearts were weighed and homogenized with a polytron homogenizer for 25 s. The homogenate was filtered through four layers of cheese-cloth, and centrifuged at 600 X g for 2 min. The supernatant was centrifuged at 1000 X g for 5 min and the nuclei pellet was resuspended in 0.25M sucrose. The mitochondrial, microsomal and cytosolic fractions were obtained by differential centrifugation as previously described [4]. The subcellular fractions were used immediately for enzyme assays. Measurement of lysophosphatidylcholine in hamster plasma and subcellular fractions. Blood was obtained from the hamster by cardiac puncture with a heparinized syringe. Plasma was separated from other blood components by centrifugation. A small amount of 1 - [ 1 -I4 C]palmitoylglycero -

43

phosphocholine was added to 8 ml plasma followed by the addition of 1 ml 0.145 M NaCl and 3.75 ml CH,OH/CHCl, (2: 1, v/v). The mixture was centrifuged at 1000 X g for 10 min. The supernatant was removed and the pellet was washed twice with 2.4 ml CH,OH/CHCl, /Hz0 (2 : 1: 0.8, v/v) [16]. The extracts (supernatant and subsequent washes) were pooled and 2.5 ml each of CHCl, and H,O were added. The solution was mixed and centrifuged. The lower phase was removed and upper phase was re-extracted with 2 ml Theoretical Lower Phase [ll]. The pooled lower phase was evaporated to dryness. Lysophosphatidylcholine in lower phase was separated by thinlayer chromatography using CHCl, /CH,OH/ H,O,‘CH,COOH (70 : 30 : 4 : 2, v/v). Lysophosphatidylcholine was visualized by iodine vapor, and subsequently eluted from the silica gel by CHCl,/CH,OH (2: 1, v/v). The concentration of lysophosphatidylcholine in hamster plasma was determined by the amount of inorganic phosphate present after acid digestion of the lipid [13]. Results Uptake choline

of

0

10

20

30

40

50

60

TIME (min) Fig. 2. Total lysophosphatidylcholine uptake and incorporation of label into phosphatidylcholine in isolated hamster hearts. Isolated hamster hearts were cannulated via the aorta and perfused with Krebs-Henseleit buffer containing 5 pM I[ I-‘4C]palmitoylglycerophosphocholine (0.2 pCi/ml) for 5-60 min. After perfusion, the hearts were homogenized in chloroform/methanol (2: I, v/v) and total radioactivities in the homogenates were determined. The phospholipids in the homogenates were separated by thin-layer chromatography and the amount of label incorporated into phosphatidylcholine was determined. l , Total radioactivity uptake by the isolated heart. A. Amount of radioactivity incorporated into phosphatidylcholine. Each point represents the mean of two separate experiments.

I-[ 1 -14C] palmitoylglycerophospho-

by the isolated hamster

heart. Our initial approach was to demonstrate that exogenous lysophosphatidylcholine, and not phosphatidylcholine was actually taken up by the isolated perfused heart. Isolated hamster hearts were perfused in Krebs-Henseleit buffer containing l-[ las described I4 Clpalmitoylg 1y cerophosphocholine in Materials and Methods. As shown in Fig.2, total uptake of radioactivity reached maximum at 30 min of perfusion with 5 PM of 1 -[l -I4 Clpalmitoylglycerophosphocholine (0.2 pCi/ml). Linearity of uptake was observed with 5-20 PM of the lysophosphatide (Fig. 3). However, higher concentrations of lysophosphatidylcholine in the perfusate (over 25 PM) inevitably caused cardiac arrhythmia and occasionally, cessation of mechanical pumping activity. Addition of bovine serum albumin (0.5 mg/ml) in the perfusate appeared to protect the heart from arrhythmia at higher concentrations of lysophosphatidylcholine, but did not affect the amount of radioactivity taken up by the heart. In the presence of albumin, uptake of radioactivity

was linear from lo-75 PM of lysophosphatidylcholine (Fig. 3). The unspecific binding of labeled lysophosphatidylcholine to the membrane surface of the heart cells was also investigated by a pulse-chase experiment. The heart was perfused 1 -[ 1 -I4 Clpalmitoylglycerophosphocholine with (5pM) (0.2 pCi/ml) for 20 min and chased with the same concentration of unlabeled palmitoylglycerophosphocholine for 10 min. Less than 2% of the labeled material in the heart was recovered in the effluent during the entire chase period. Our results demonstrate that labeled lysophosphatidylcholine was not bound unspecifically to the membrane surface, but was actually taken up by the heart cells. From the double reciprocal plot of lysophosphatidylcholine uptake versus lysophosphatidylcholine concentrations in the perfusate, the apparent substrate concentration for half saturation velocity (K,) of lysophosphatidylcholine taken up by the isolated heart was determined. The K, of lysophosphatidylcholine

PC LUMI Fig. 3. Total lysophosphatidylchohne (LPC) uptake in isolated hamster hearts with different concentrations of lysophosphatidylcholine in the perfusate. (A) Hearts were perfused with IO-300 PM I -[I -I4 C]palmitoylglycerophosphocholine (0.2 pCi/ml) with the addition of 0.5 mg/ml bovine strum albumin. At concentration of 300 PM I-[l-‘4C]palmitoylglycerophosphochohne, I .O mg/ml bovine serum albumin was added. Total lysophosphatidylcholine uptake was calculated from specific radioactivity of lysophosphatidylcholine in the perfusate. Each point represents the mean of three separate experiments, the vertical bars indicate standard deviations. (B) Hearts were perfused with 5-20 pM, I -[I -I4 Clpalmitoylglycerophosphocholine (0.2 aCi/ml), without the addition of bovine serum albumin. Each point represents the mean of two separate experiments.

uptake by the heart in the presence and absence of bovine serum albumin was estimated to be 250 and 220 PM, respectively. In order to investigate whether acylation of lysophosphatidylcholine occured prior to uptake, the hamster heart was perfused with phosphatidyl[methyl-3H]choline. At 30 min of perfusion, only 12% of the radioactivity in the perfusate was taken up by the heart. Under identical experimental conditions, more than 70% of radioactivity was taken up when the heart was perfused with 1 -[ 1 -I4 Clpalmitoylglycerolphos phocholine. Our results clearly indicate that lysophosphatidylcholine was not acylated prior to uptake. Lysophosphatidylcholine

concentrations

in ham-

ster plasma and hamster heart subcellular fractions.

The

lysophosphatidylcholine

concentrations

in

hamster plasma and subcellular fractions before and after perfusion were determined. The lipids from the hamster heart were extracted into the organic phase and, subsequently, lysophosphatidylcholine was separated from other lipids by thin-layer chromatography. Total lysophosphatidylcholine from unperfused hearts, hearts perfused with Krebs-Henseleit buffer in 0.5 mg/ml bovine serum albumin, or perfused with buffer and 5- 100 PM of lysophosphatidylcholine in 0.5 mg/ml bovine serum albumin were determined. A high concentration of lysophosphatidylcholine was found in the hamster plasma (291 c 32 PM). Lysophosphatidylcholine concentration in hamster hearts did not change significantly before or after perfusion with up to 10 PM of lysophosphatidylcholine in the perfusate. A 2-fold increase in total cardiac lysophosphatidylcholine was observed when the heart was perfused with 100 FM lysophosphatidylcholine (Table I). The majority of the exogenous lysophosphatidylcholine taken up by the heart during perfusion appears to be located in the cytosolic and microsomal fractions. Phosphatidylcholine lysophosphatidylcholine

formation in isolated

from

exogenous

hearts.

Subsequent to perfusion of the hamster hearts with of 1 - [ 1 -I4 Clpalmitoylglycero 5 - 300 PM phosphocholine (0.2 pCi/ml) for 30 min, the labeled metabolites in the heart homogenates were analyzed. About l-2% of the total radioactivity taken up by the heart was recovered in the aqueous phase, and the remainder of the radioactivity in the homogenate was recovered in the organic phase. Analysis of the organic phase by thin-layer chromatography indicated that over 95% of the radioactivity was located in lysophosphatidylcholine, phosphatidylcholine and free fatty acid. Less than 1% of total radioactivity was detected in phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and phosphatidylinositol or sphingomyelin. The amount of radioactivity in phosphatidylcholine increased linearly from 5-30 min of perfusion (Fig. 2, insert). A linear relationship was also observed between total uptake of lysophosphatidylcholine and phosphatidylcholine formation up to 10 PM of lysophosphatidylcholine in the perfusate (Table II). Hence, at low concentrations of lysophosphatidylcholine in the perfusate, the amount of radioactivity incorporated

45

TABLE

I

LYSOPHOSPHATIDYLCHOLINE AND AFTER PERFUSION

CONCENTRATIONS

IN HAMSTER

HEART

SUBCELLULAR

Subcellular fractions were prepared from hamster hearts without perfusion, perfused with Krebs-Henseleit PM lysophosphatidylcholine in Krebs-Henseleit buffer for 30 min. Values are mean*S.D. The figures number of experiments. Lysophosphatidylcholine

Homogenate Nuclei Mitochondria Microsomes Cytosol

concentration

(nmol/g

No perfusion

Krebs-Henseleit

buffer

13Ot-27 IS-’ 8 192 6 40’- I4 45-‘ll

1472 I8 (3) 18i 5 (3) 21’ 4(3)

(6) (3) (3) (4) (4)

TABLE

BEFORE

buffer or perfused with 100 in parentheses represent the

heart) 100 PM lysophosphatidylcholine in Krebs-Henseleit buffer 271*31 (3) 24* 9 (3) 3O”ll (3) 84i-21 (3) 167*24 (3)

385 I I (3) 46* 7 (3)

into phosphatidylcholine appears to correlate closely with the total radioactivity uptake by the heart. At higher concentrations of lysophosphatidylcholine in the perfusate (over 10 PM) the amount of radioactivity incorporated into phosphatidylcholine was no longer proportional to the total amount of radioactivity uptake. However, total uptake was still linear up to 75 PM of lysophosphatidylcholine in the perfusate. The amount of labeled lysophosphatidylcholine and free fatty acid in the organic phase after perfusion were analyzed also (Table 11). At low concentrations of lysophosphatidylcholine in the perfusate, a substantial amount of radioactivity taken up by the heart was converted into free fatty

FRACTIONS

acid, whereas at higher concentrations of lysophosphatidylcholine, the majority of the radioactivity remained as lysophosphatidylcholine (Table II). Since lysophospholipase activity was detected in the homogenate and subcellular fractions of the heart (Table III), the presence of labeled free fatty acid may result from the hydrolytic action of lysophospholipase on lysophosphatidylcholine. Pathways for reacylation of Iysophosphatidylcholine. Although lysophosphatidylcholine was reacylated to phosphatidylcholine in the hamster heart, it was not clear whether phosphatidylcholine was reacylated by the action of lysophosphatidylcholine: acyl-CoA acyltransferase or by transacylation with another lysophosphatidylcho-

II

RADIOACTIVITY INCORPORATION INTO PHOSPHATIDYLCHOLINE, LYSOPHOSPHATIDYLCHOLINE FATTY ACIDS IN HAMSTER HEARTS PERFUSED WITH I-[I-‘4C]PALMITOYLGLYCEROPHOSPHOCHOLINE

AND

FREE

Isolated hamster hearts were perfused with 5-100 PM of I-[l-‘4C]palmitoylglycerophosphocholine (0.2 pCi/ml) for 30 min. The amount of radioactivity incorporated into phosphatidylcholine. lysophosphatidylcholine and free fatty acids were analyzed by thin-layer chromatography. Each value represents three separate experiments. Values are cpm. IO -“/g heart (mean*S.D.). Lysophosphatidylcholine in perfusate 5 IO 25 75 100

Phosphatidylcholine

Lysophosphatidylcholine

Free fatty acid

o.g2*0.19 o.g4*0.17 0.76*0.02 0.58-tO.02 0.42*0.04

0.57-co.35 0.61 ‘0.31 1.06*0.38 l.16kO.44 1.21 to.21

0.94kO.33 0.91 kO.49 0.72*0.16 0.65 -CO.67 0.56 * 0.44

(PM)

46

TABLE

III

TABLE V

LYSOPHOSPHOLIPASES ACTIVITIES IN GENATE AND SUBCELLULAR FRACTIONS HAMSTER HEART

HOMOOF THE

Enzyme activities were assayed in 0. I M phosphate buffer, 7.4, for 15 min with 0.2 mg of protein from homogenate subcellular fractions. Each value represents three separate periments (mean*S.D.). Subcellular

fraction

pH or ex-

Lysophospholipase (nmol/min per mg protein)

Homogenate Microsomes Cytosol

0.12*0.07 0.71 iO.24 1.6X*0.41

line catalyzed by lysophosphatidylcholine : lysophosphatidylcholine transacylase. Hence, the isolated hamster heart was perfused with l-[ 1-‘4C]palmitoylglycerophospho[ merfryl-3 HIcholine in Krebs-Henseleit buffer. The 3H/‘4C ratio in phosphatidylcholine should remain the same as in lysophosphatidylcholine if reacylation was catalyzed by lysophosphatidy’lcholine : acyl- CoA acyltransferase whereas reacylation catalyzed by lysophosphatidylcholine : lysophosphatidylcholine

TABLE

IV

LABELED PHOSPHATIDYLCHOLINE FORMATION IN HAMSTER HEART PERFUSED WITH I -[I -I4 ClPALMITOYLGLYCEROPHOSPHO[mer~r+~H]CHOLINE Isolated hamster hearts were perfused with doubly labeled lysophosphatidylcholine (200 PM) and bovine serum albumin (I .O mg/ml) in Krebs-Henseleit buffer for I5 min. The amount of 14C and 3H in the perfusate (per ml) and lipids from the perfused heart (per g wet weight) were determined. The results shown are the mean of two separate experiments. Values are dpm.lO-‘. ‘H Lysophosphatidylcholine in perfusate Lysophosphatidyl choline in hamster heart after perfusion Phosphatidylcholine in hamster heart after perfusion

‘4c

‘H/14C

475

x2

5.8

7 202

I 200

6.0

524

76

6.9

LYSOPHOSPHATIDYLCHOLINE : ACYL-CoA ACYLTRANSFERASE ACTIVITIES IN HOMOGENATE AND SUBCELLULAR FRACTIONS OF THE HAMSTER HEART Enzyme activities were assayed in 0. I M Tris-HCI buffer, pH 7.4, for 3 min with 4 mg protein from homogenate and 0.2 mg protein from each subcellular fractions. The value for homogenate is the mean of two experiments; other values are mean-+ SD. of the number of experiments, shown in parentheses. Subcellular fraction

Lysophosphatidylcholine: acyl-CoA acyltransferase (nmol/min per mg protein)

Homogenate Mitochondria Microsomes

0.1 I 3.43’1.90 (3) 7.92) 1.36 (4)

transacylase would decrease the 3H/‘4C ratio in phosphatidylcholine. By perfusing the isolated hamster heart with 1 - [ 1 -I4 Clpalmitoylglycerophospho[ methyl -3 HIcholine, the phosphatidylcholine formed had a 3H/‘4C ratio similar to that of lysophosphatidylcholine (Table IV). Our results indicate that reacylation of lysophosphatidylcholine in hamster was catalyzed exclusively by lysophosphatidylcholine: acyl-CoA acyltransferase. Enzyme studies. In order to confirm the exclusive presence of lysophosphatidylcholine: acyl-CoA acyltransferase but not lysophosphatidylcholine : lysophosphatidylcholine transacylase in hamster heart, the activities of these two enzymes were assayed in both the heart homogenate and subcellular fractions. Since it was reported that product formation by lysophosphatidylcholine: acyl-CoA acyltransferase was linear only during the first few minutes of incubation [20], the linearity of product formation by this enzyme in hamster heart subcellular fractions was also investigated. We are able to confirm that product formation is linear only up to a maximum of 3 min of incubation, and majority of the enzyme activity was located in the microsomal fraction (TableV). No lysophosphatidylcholine : lysophosphatidylcholine transacylase activity was detected.

Discussion In this study we demonstrated that phosphatidylcholine was formed in the isolated hamster heart by reacylation of exogenous lysophosphatidylcholine. Since the hamster heart was isolated and perfused in Krebs-Henseleit buffer, we eliminated the possibility that the lysophosphatide was reacylated in the plasma prior to uptake. In addition, this mode of perfusion provided us with a facile mechanism to monitor the electrocardiac activity of the beating heart throughout the perfusion period, especially when the hamster heart was perfused with a cytolytic agent [21]. Development of cardiac arrhythmia was observed when the isolated heart was perfused with concentrations of lysophosphatidylcholine greater than 25 PM. However, the arrhythmogenic effect of the lysolipid was removed by the addition of bovine serum albumin or hamster serum in the perfusate (Man, R. and Choy, P., unpublished data). In the presence of 0.5 mg/ml bovine serum albumin, no change in electrocardiac function was observed when the hearts were perfused with lo-100 PM of lysophosphatidylcholine. A higher concentration of bovine serum albumin (1 .O mg/ml) was required to protect the hamster heart from arrhythmia when 300 PM of lysophosphatidylcholine was added to the perfusate. The ability of hamster heart to reacylate exogenous lysophosphatidylcholine is demonstrated clearly. About 14-20s of the total radioactivity taken up by the heart was reacylated to phosphatidylcholine at all concentrations and periods of perfusion. It can be argued that lysophosphatidylcholine may be reacylated on the outside of the sarcolemma and the lipid is transported into the cell as phosphatidylcholine and the labeled lysophosphatidylcholine found in the heart is formed from subsequent deacylation of the labeled phosphatidylcholine (Fig. 1). This does not seem to be the case since the rate of phosphatidylcholine uptake by the heart was much lower than the uptake of lysophosphatidylcholine. Furthermore, the amount of labeled lysophosphatidylcholine and free fatty acid found in the heart at different concentrations of lysophosphatidylcholine in the perfusate was not directly proportional to the amount of labeled phosphatidylcholine (Table II).

Hence, we conclude that lysophosphatidylcholine must be transported into the cell prior to reacylation. It is clear from this study that phosphatidylcholine in the hamster heart is not formed from transacylation of two lysophosphatidylcholine molecules but by a reacylation process. Although the acyl group may come from a variety of sources, the detection of lysophosphatidylcholine: acyl-CoA acyltransferase activity in the subcellular fractions enabled us to postulate that the reacylation of lysophosphatidylcholine and acyl-CoA represents a major, or possibly an exclusive, pathway for the resynthesis of phosphatidylcholine. This is in agreement with the results obtained from the gallbladder, where phosphatidylcholine is reacylated exclusively by lysophosphatidylcholine : acyl-CoA acyltransferase [22]. Also, the bifunctional property of hamster heart lysophospholipase was investigated. It was reported that the rat lung lysophospholipase is a bifunctional enzyme which exhibits transacylase activity [15,17]. We have demonstrated the presence of Iysophospholipase in the perfused heart as well as in the subcellular fractions. However, we were unable to detect any lysophosphatidylcholine : lysophosphatidylcholine transacylase activity in both in vivo experiments and in vitro assays. In this aspect, hamster heart lysophospholipase is similar to that in bovine pancreas and liver, which was shown to be nonbifunctional [ 171. One intriguing result pertaining to this study is the apparent discrepancy of lysophosphatidylcholine concentrations in non-perfused hearts and hearts perfused with 100 PM of lysophosphatidylcholine, where a 2-fold increase was observed (Table I). Since the non-perfused hearts were circulated in blood which contained 291 PM of Iysophosphatidylcholine, we expected that its cardiac lysophosphatidylcholine content should be equal or higher than those obtained after perfusion with 100 PM lysophosphatidylcholine. However, the total cardiac lysophosphatidylcholine level in the non-perfused hearts were similar to those obtained when the hearts were perfused with O-10 PM of the lysophospholipid in the perfusate. One possible explanation is that some lysophosphatidylcholine in hamster plasma is in tightly bound form, e.g. to serum lipoproteins [23], and thus is

48

not available for uptake. If this is the case, it appears that only up to 10 FM (3-4%) of total plasma lysophosphatidylcholine is present in such forms that are available for uptake by the heart. Although we are not clear about the different forms of lysophosphatidylcholine in the hamster plasma, it was reported that the critical micelle concentration of 1 - palmitoylglycerophosphocholine is 2-7 PM [21]. Hence, it is conceivable that the form of lysophosphatidylcholine in hamster plasma available for uptake by the heart is the micelle form. The amount of phosphatidylcholine formed by the reacylation pathway at 10 PM of lysophosphatidyicholine in the perfusate was estimated to be 6.6 nmol/min per g heart. This calculation is based on the total pool size of cardiac lysophosphatidylcholine and the amount of radioactivity in lysophosphatidylcholine and phosphatidyicholine in the heart after 30 min of perfusion (Tables I and II), It was shown in our earlier report [4] that 40 nmol/min per g heart of phosphatidylcholine was synthesized via the CDPcholine pathway and from methylation of phosHence, we estimate that phatidylethanolamine. 14% of the total phosphatidylcholine in the heart may be formed via the reacylation of lysophosphatidylcholine. Acknowledgements This study was supported Medical Research Council of Canadian Heart Foundation Pauline Robinson and Tracy technical assistance.

by a grant from the Canada. P.C.C. is a Scholar. We thank Slater for excellent

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