The effect of methyl lidocaine on lysophospholipid metabolism in hamster heart

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada R3E OW3

RICKY Y. K. MAN

Department of Pharmacology, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada R3E 0 W3

AND

CHRISTOPHER R. MCMASTER AND PATRICK C. CHOY'

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada R3E OW3

Received September 20, 1989

TARDI, P. G., MAN, R. Y. K., MCMASTER, C. R., and CHOY, P. C. 1990. The effect of methyl lidocaine on lyso- phospholipid metabolism in hamster heart. Biochem. Cell Biol. 68: 745-750.

An important feature in the remodelling of fatty acyl chains in cellular phospholipids is the acylation of lysophospholipids. Since Iysophospholipids are cytolytic at high concentrations, the acylation reaction may provide an alternate pathway for the removal of cellvIar lysophospholipids. However, the physiological role of the acylation process in the maintenance of lysophospholipid levels in mammalian tissues has not been clearly defined. In this study, methyl fdocaine was found to inhibit both Iysophosphatidylchohe:acyl-CoA and lysophosphatidylethano1amine:acyl-CoA acyltransferase activities in the hamster heart, but the drug had no effm on the other lysophospholipid metabolic enzymes. When the heart was perfused with 0,s mg methyl lidocaine/mL, acyltransferase activities were attenuated, but there was no change in the activities of phospholipase A or lysophospholipase. The levels of the major lysophospholipids in the heart were not altered by methyl lidocaine perfusion. When the hearts were perfused with labelled lysophospholipid In the presence of methyl lidocaine, there was a reduction in the formation of the phospholipid and an increase in the release of the free fatty acid. However, the labelling of lysophospholipid in the heart was not altered by methyl lidocaine. We postulate that the acylation reaction has no direct contribution to the maintenance of the lysophospholipid levels in the heart.

Key words: lysophosphatidylcholine, lysophosphatidylethanolarnine, acyltransferase, methyl lidocaine, hamster heart.

TARDI, P. G., MAN, R. Y. K., MCMASTER, C. R., et CHOY, P. C. 1990. The effect of methyl lidocaine on lysophospholipid metabolism in hamster heart. Biochem. Cell Biol. 68 : 745-750.

L'acyIation des lysophospholipides est un aspect important du remodelage des chaines d'acides gras dans les phospholipides cellulaires, Cornme les lysophospholipides sont cytolytiques a concentrations Clevkes, la reaction d'acylation peur fournir une auue voie pour I'enltvernent des lysophospholipides cellulaires. Cependant, le rBle physiologique du processus d'acylation dans le maintien des taux de lysophospholipides dans les tissus mammaliens n'est pas clairement defini. Dans ce travail, il est dCmontrC gue la methyl lidocai'ne inhibe l'activite des lysophosphatidylcho1ine:actyl-CoA et 1ysophosphatidyltthanolamine:acyl-CoA acyltransf~rases dans le coeur du hamster. En revanche, cette substance n'exerce aucun effet sur Ies autres enzymes du mttabolisme des lysophospholipides. Quand le coeur est perfuse avec O,5 rng methyl lidocai'ne/rnL I'activiti des acyltransf&rases est attenuee, mais l'activite de la phospholipase A ou Lysophosphdipase ne change pas. Les taux des principaux lysophospholipides dans le coeur ne sont pas alttres par la perfusion de mhhyl lidocake. Quand les cwurs sont perfuses avec des lysophospholipides marques en presence de mkthyl lidocdine, il y a reduction dans la Formation des phospholipides et augmentation dans la liberation des acides gras libres. Cependant, Ie marquage des lysophospholipides dans le coeur n'est pas affect6 par la methyl lidocaine. Nous suggCrons gue la r4action d'acylation ne contribue pas directement au maintien des taux des lysophospholipides dans Ie coeur.

Mots clPs : lysophosphatidylcholine, lysophosphatidylethanolamine, acyltransferase, mtthyl lidocaine, coeur du hamster.

[Traduit par la revue]

Introduction Phosphatidylcholine and phosphatidylethanolamine are

the major phospholipids in the mammalian heart (White 1973). The principal pathway for their catabolism is through the hydrolytic action of phospholipase A, which releases the acyl group and leads to the formation of the respective lysophospholipids (van Golde and van den Bergh 1977). The

ABBREYTATIONS: LPC. lysophosphatidylcholine; LPE, tysophosphatidylethanoIamine; ACS. aqueous counting scintillant.

I Author to whom all correspondence should be smr at the 7ollowing address: Department of Biochemjstry and Molecular Biology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man.. Canada R3E QW3. 'nnted m Canada / Impnme au Canada

lysophospholipids can be further deacylated by lysophos- pholipase or, alternatively, they can be reacylated back to the parent phospholipids by acyl-CoA acyltransferases (van Golde and van den Bergh 1977).

The acylation of lysophospholipids serves a number of important functions in the cell. It is part of the mechanism for the remodelling of the fatty acyl chains of cellular phospholipids (Lands 1960). fn addition, the acylation reac- tion may provide an alternate pathway for the removal of the cellular lysophospholipids, which are potent cytolytic agents at high concentrations (Weltzein 1979). Indeed, the accumutation of lysophospholipids in the ischemic heart has been suggested as a biochemical cause for cardiac dysfunc-

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746 BIOCHEM. CELL BIOL. VOL. 68, 1990

tions, including the development of cardiac arrhythmias (Katz and Messineo 1981; Corr et al. 1984; Man and Choy 1982). In the hamster heart, the majority of newly syn- thesized phosphatidylcholine undergoes the deacylation- reacylation process to acquire the appropriate acyl groups (Arthur and Choy 1984). Although the acyltransferases for the acylation of lysophosphatidylcholine and lysophospha- tidylethanolamine have been studied for a number of years, most of these studies were directed towards the acyl specificity of the enzymes (Choy and Arthur 1989). At present, only limited information is available on their physiological role in the maintenance of lysophospholipid levels in mammalian tissues.

A number of agents, including local anesthetics (Shier 1977; Sanjanwala et al. 1988), clofibric acid (Kawashima et al. 1986), and neuroanabolic drugs (Parthasarathy et al. 1981) were found to modulate acyl-CoA acyltransferase activities. However, the effects of these compounds on the other lysophospholipid metabolic enzymes or on the acyla- tion of lysophospholipids in vivo are not known. Methyl lidocaine is a local anesthetic with anti-arrhythmic properties (Man and Dresel 1977). In this study, methyl lidocaine was found to inhibit LPC:acyl-CoA acyltransferase and LPE:acyl-CoA acyltransferase activities in the hamster heart, but had no effect on the other lysophospholipid metabolic enzymes. Perfusion of the isolated hamster heart with methyl lidocaine did not cause any change in the levels of lysophosphatidylcholine and lysophosphatidyletha- nolamine.

Materials and methods 1 , 2 - ~ i [ l - ' ~ ~ ] ~ a l m i t o ~ l phosphatidylethanomaline, 1-[l -14c]

palmitoyl lysophosphatidylcholine, [ I - ' ~ C ] O ~ ~ O ~ ~ - C O A , and ACS were obtained from Amersham Canada Ltd. (Oakville, Ont.). Lysophosphatidylcholine (pig liver), lysophosphatidylethanolamine (pig liver), phosphatidylcholine (pig liver), and phosphatidyletha- nolamine (pig liver) were purchased from Serdary Research Lab- oratories (London, Ont.). Methyl lidocaine was a gift from Astra Pharmaceutical Products (Worcester, MA). The reagents for gas- liquid chromatography were obtained from Supelco Canada Ltd. (Oakville, Ont.). Phospholipase A, (Crotalus adamanteus venom) was obtained from Sigma Chemical Co. (St. Louis, MO). Thin- layer chromatographic plates (Sil-G25) were produced by Macherey-Nagel and purchased through Brinkmann Instrument (Rexdale, Ont.). All other chemicals were of reagent grade and were obtained through Canlab Division of Travenol Canada Inc. AII solutions were prepared with glass-distilled water and adjusted to the desired pH.

Syrian golden hamsters, 120 + 20 g, were obtained from Charles River Canada Inc. (Saint-Constant, Que.). The animals were main- tained on Purina chow and tap water, ad libitum, in a light- and temperature-controlled room.

Preparation of microsomes from hamster hearts Hamsters were sacrificed by decapitation and the hearts were

removed and placed on ice. The hearts were washed, weighed, cut into pieces, and homogenized in 0.25 M sucrose - 10 mM Tris- HCI (pH 7.4) - 1 mM EDTA at 4°C to give a 5% (w/v) crude homogenate. The crude homogenate was centrifuged at 100 x g for 5 min to remove the connective tissues and other cell debris. The supernatant was collected and used for enzymes assays. The microsomal fraction was prepared from the cell homogenate by differential centrifugation (Zelinski et a/. 1980). The microsomes were resuspended in 0.25 M sucrose - 10 mM Tris-HC1 at pH 7.4. Protein concentrations in the subcellular fractions were determined by the method of Lowry et al. (1951).

Acyl-CoA:I-ocylgIycemphosph~~holine and acyCCoA:I-awl- glycerophosphoethanolarnine ucyltransferase assays

The 1-acylglycerophosphocholine and l-acyfglycerophopho- ethanolamine acyltransferase activities were assayed in hamster heart homogenate and microsomes using labefled oleoyl-CoA (Arthur and Choy 1986; Arthur er al. 1987). Briefly, the reaction mixture contained 20 mM Tris-HCI (pH 8.5), 100 nmol l-acylgly- cerophosphocholine (pig liver) or 100 nmol l-acylglycerophos- phoethanolamine (pig liver), 86 nmol l - [ - 1 4 ~ ] o l e o y l - ~ o ~ , and the appropriate amount of enzyme protein. Methyl lidocaine was added into the reaction mixture and the reaction was initiated by the addition of labelled oleoyl-CoA to a final volume of 0.7 mL. The mixture was incubated at 25°C for 30 min and the reaction w a s terminated by the addition of 1.5 mL of chloroform-methanol (2:1, v/v). The phosphatidylcholine or phosphatidylethanolamine fraction in the lipid extract was isolated by thin-layer chromatog- raphy using a solvent containing chloroform - methanol - water - acetic acid (70:30:4:2, by volume) (Zelinski et al. 1980). Enzyme activity was calculated from the radioactivity associated with the appropriate phospholipid fraction. Assays without I -acylglycero- phosphocholine or I-acylglycerophosphoethanoIamine were used as controls.

Other enzyme assays Phospholipase A and lysophospholipase activities in the hamster

heart homogenate were determined as previously described (Tam et al. 1984; Savard and Choy 1982).

Preprotion of Iysophosphatidylethanolamine Labelled l-[l-'4~]palmitoyl glycerophosphoethanolarnine was

prepared by the hydrolysis of I,2-[-'4~]dipalmitoyl phosphatidyl- ethanolamine with Cratalus adamanteus venom phospholipase A, according to the procedure o f Hanahan et al. (1954). Briefly, the labelled phosphatidylethanolarnine (7.5 pnol) was dissolved in 6 mL of diethyl ether. The reaction was initiated by adding 150 pL of 0.1 M Tris-HCI (pH 7.4), 0.01 M CaCl,, and 100 U of phosphoIipase A2 (295 U/mg protein). The reaction mixture was incubated at 25°C for 3 h with occasional mixing. Subsequent to the reaction, labelled Iysophosphatid ylethanolamine was isolated by thin-layer chromatography (Zeiinski et ul. 1980). The yield of lysophosphatidylethanolamine was over 80%.

Perfusion of isolated hamster heart and determination of phospholipid composition

T h e isolated hamster heart was perfused in the Langendorff mode (Langendorff 1895) with Krebs-Henseleit buffer (Krebs and Henseleic 1932) saturated with 95% O2 and 5% CO,. Appropriate amounts of methyl lidocaine or labelled materials were added to the perfusate. Perfusion rook place at 37OC with a coronary flow rate of 3.0 mL/min. Subsequent to perfusion, the heart was washed with 10 mL Krebs-Henseleit buffer, cut into small pieces, and homogenized in chloroform-methanol (1: 1, v/v). The homogenate was centrifuged at 1000 x g for 10 min, and the precipitate was washed twice with chforoforrn-methanol (1 : 1 , v/v). The super- natants were pooled, and water and chloroform were added to cause phase separation. The lower phase was collected and the phospholipid classes were separated by thin-layer chromatography (Zelinski et al. 1980). In some experiments, fatty acid was separated from the other lipids by thin-layer chromatography (Skipski and Barclay 1969). Lipid phosphorus contents were determined by the procedure of Bartlett (1959).

Uptake of /ahlied compounds by the iso/c~ted hewt Hearts were perfused with ~ - [ l - ' ~ ~ ] p a l r n i t o y l glycero-

phosphocholine or ~-[l-'~CJpalrnitoyl glycerophosphoethanol- arnine. The isolated hearts were perfused in Krebs-Henseleit buffer containing labelled 1ysophosphatidylchoIine (10 pM, 20 &Ci/pmol; 1 Ci = 37 CBq) or labelled lysophosphatidylethanolamine (10 pM. 5 pCi/pmol) in bovine serum albumin (0.1 rng/mL) and 0.5 mg methyl lidocaine/mL for 30 min. Perfusion with the labelled lyso-

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TABLE 1. Effect of methyl lidocaine on LPC:acyl-CoA acyltransferase and LPE:acyl-CoA acyltransferase activities in hamster heart homogenate and microsomes

-

LPC:acyl-CoA acyltransferase activity, LPE:acyl-CoA acyltransferase activity, nmol/(h.mg protein) nmol/(h-mg protein)

Methyl lidocaine, mg/mL Homogenate Microsomes Homogenate Microsomes

Control (no addition) 72+ 14 343 + 20 7.0 + 2.4 32 + 7 0.2 50 + lo* 214 + 22* 5.4 k 1.2 25 + 6 0.5 20 + 6* 101 + 21* 4.2+ 1.1 19+5* 1 .O 13 + 3* 57 + 13* 2.5 +0.5* 12+4* 2.0 5+1* 25 + 6* 2.0 + 0.5* 9+4*

NOTE: Acyltransferases activities were assayed in hamster heart homogenate and microsomal preparation in the presence and absence of methyl lidocaine. Each value represents the mean + SD of four separate experiments (* p < 0.05).

FIG. 1. Double-reciprocal plot of microsomal LPE:acyl-CoA acyltransferase activity versus substrate concentrations. LPE:acyl- CoA acyltransferase activity was determined at 2S°C with 0.18 mg microsomal protein and expressed as nanomoles per hour per milligram protein. Enzyme activity in the upper panel was assayed with 140 pM LPE and 10-50 pM oleoyl-CoA. Enzyme activity in the lower panel was assayed with 120 pM oleoyl-COA and 50-500 pM of LPE. The assays were performed in the absence (A) and the presence of 0.5 (0) or 1.0 (a) mg methyl lidocaine/mL.

phospholipid, but without methyl lidocaine, was used as control. Subsequent to perfusion, the heart was washed with 10 mL of Krebs-Henseleit buffer, cut open, blotted dry, and weighed. The hearts were homogenized in 0.25 M sucrose - 10 mM Tris-HC1 (pH 7.4) - 1 mM EDTA and subcellular fractions were prepared as described previously (Zelinski et al. 1980).

Other analyses Radioactivity was determined by scintillation counting using

channel ratio calibration method. Student's t-test was used for statistical analysis. The level of significance was set at p < 0.05.

Results The in vitro effect of methyl lidocaine on the lysophospho-

lipid metabolic enzymes in hamster heart Since acyltransferases have been shown to be modulated

by local anesthetics, the effects of methyl lidocaine on both LPC:acyl-CoA and LPE:acyl-CoA acyltransferase activities were examined. Methyl lidocaine inhibited both enzyme activities in the tissue homogenate and also in the microsomal fraction to the same extent, but the inhibitory effect was more prominent in LPC:acyl-CoA acyltransferase than LPE:acyl-CoA acyltransferase (Table 1). Methyl lido- caine at 0.5 mg/mL caused a 71% inhibition of the microsomal LPC:acyl-CoA acyltransferase activity, whereas at the same concentration, a 41% inhibition of the microsomal LPE:acyl-CoA acyltransferase activity was obtained.

The mode of inhibition of the acyltransferases by methyl lidocaine was investigated by kinetic studies. Enzyme activ- ities were determined at various substrate concentrations in the presence and absence of methyl lidocaine. The data obtained were analyzed by double-reciprocal plots of acyltransferase activities versus oleoyl-CoA or lysophos- pholipid concentrations. As depicted in Fig. 1, the inhibi- tion of methyl lidocaine on LPE:acyl-CoA acyltransferase was essentially noncompetitive in nature. The inhibition of LPC:acyl-CoA acyltransferase by methyl lidocaine was also found to be essentially noncompetitive (data not shown).

The effects of methyl lidocaine on the other lysophospho- lipid metabolic enzymes were also studied. As depicted in Table 2, methyl lidocaine has no effect on phospholipase A activity in the hamster heart homogenate at low concentra- tions (0.2-0.5 mg/mL), but became inhibitory at higher con- centrations (1-2 mg/mL). The activity of lysophospholipase in the presence of methyl lidocaine was also investigated (Table 2). Methyl lidocaine (0.2-2.0 mg/mL) had no effect on the lysophospholipase activity in hamster heart homogenate.

The in vivo effect of methyl lidocaine on lysophospholipid metabolism and phospholipid composition in the isolated hamster heart

To study the contribution of the acylation process on the metabolism of lysophospholipids, hamster hearts were per- fused in Krebs-Hanseleit buffer containing 0.5 mg methyl lidocaine/mL for 30 min. Subsequent to perfusion, the heart was homogenized and enzyme activities for the metabolism of lysophospholipids in the homogenate were assayed. No

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TABLE 2. The effect of methyl lidocaine on phospholipase A and lysophospholipase in hamster heart homogenate

Phospholipase A Methyl lidocaine, activity,

mg/mL nmol/(h.mg protein)

Control (no addition) 4.5 -t 0.3(4) 0.2 4.2 + 0.4(4) 0.5 4.6 -t 0.3(4) 1 .O 3.8k 0.3(4)* 2.0 3.6 + 0.4(4)*

Lysophospholipase activity,

nmol/(h.mg protein)

16.3 + 1.1(4) 16.0 f 0.8(4) 15.8+0.7(4) 16.1 +0.8(4) 16.4 f 1.0(4)

NOTE: Phospholipase A and lysophospholipase activities in hamster heart homogenate were assayed in the presence and absence of methyl lidocaine. Each value represents the mean + SD (number of experiments) ( * p i 0.05).

TABLE 3. Activities of lysophospholipid metabolic enzymes in hamster heart perfused with methyl lidocaine

Enzyme activities, nmol/(h-mg protein)

Control Methyl lidocaine

Phospholipase A Homogenate 5.1 f 0.4(3) 4.8 * 0.4(3)

Lysophospholipase Homogenate 18.7+ 1.5(3) 18.0+ 1.2(3)

LPC:acyl-CoA acyltransferase Homogenate 78.8 + 11.7(3) 44.9 f 12.5(3)* Microsomes 343 f 20(8) 205 + 1 1 (3)*

LPE:acyl-CoA acyltransferase Homogenate 6.8 + 0.6(3) 4.8 f 0.4(3)* Microsomes 32.0 f 6.5(9) 22.4 + 2.6(4)*

NOTE: Hamster hearts were perfused in Krebs-Henseleit buffer in the presence and absence of 0.5 mg methyl lidocaine/mL. Subsequent to perfusion, the hearts were homogenized and microsomal fractions were prepared from the homogenates. Enzyme activities were determined in the homogenates or microsomal prep- arations. Each value represents the mean -t SD (number of experiments) (*p < 0.05).

significant change in the activities of phospholipase A and lysophospholipase was detected (Table 3). However, both LPC:acyl-CoA acyltransferase and LPE:acyl-CoA acyl- transferase activities were significantly inhibited when the hearts were perfused with methyl lidocaine. Perfusion with 0.5 mg methyl lidocaine/mL caused a 43 and 30% decrease in LPC:acyl-CoA acyltransferase and LPE:acyl-CoA acyltransferase activities, respectively. Since the majority of the acyltransferase activities are located in the microsomal fraction, the enzyme activities in the microsomes were also determined. The decreases in microsomal enzyme activities were similar to those observed in the homogenate.

The phospholipid composition in the hamster heart per- fused with methyl lidocaine was determined. Subsequent to perfusion with 0.5 mg methyl lidocaine/mL, the lipids were extracted from the perfused heart and the phospholipid com- position of the lipid extract was analyzed by thin-layer chro- matography. The results obtained from the methyl lidocaine perfused hearts were compared with that obtained from the controls (Table 4). No significant difference in phospholipid content and composition was detected between these two groups. Subsequent analysis of the acyl compositions of phosphatidylcholine and phosphatidylethanolamine frac- tions by gas-liquid chromatography indicate that perfusion with methyl lidocaine did not produce any detectable dif- ference in the acyl profiles of these phospholipids (data not shown).

Acylation of lysophosphatidylcholine and lysophosphatidyl- ethanolamine in the isolated heart perfused with methyl lidocaine

Since acyltransferase activities were inhibited in the hearts petfused with methyl lidocaine, the ability of these hearts to acylate exogenous lysophospholipids was examined. Hamster hearts were perfused with 1-[1-"~]palrnitoyl giycerophosphocholine or ~ - [ ~ ~ ~ ] ~ a l r n i t o y l glycerophos- phoethanolamine for 30 min in the presence of 0.5 mg methyl lidocaine/mL. Hearts perfused without methyl lidocaine were used as controls. Subsequent to perfusion, lipids were extracted and the phospholipid classes in the extract were separated by thin-layer chromatography. The total uptake of radioactivity by the hearts were not altered by methyl lido- caine perfusion. Over 95% of the radioactivity taken up by the heart was recovered in the lysophospholipid, phospho- lipid, and neutral lipid fractions. Further analysis of the neutral lipid fraction revealed that the radioactivity was associated with the fatty acids. In the presence of methyl lidocaine, the labelling of phosphatidylcholine or phospha- tidylethanolamine was reduced by 38 and 30%, respectively, with a corresponding increase in the labelling of the fatty acid (Table 5). The labelling of the lysophospholipid was not significantly altered in the methyl lidocaine perfused hearts.

The lysophosphatidylcholine and lysophosphatidyletha- nolamine contents in the methyl lidocaine perfused hearts

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TABLE 4. Phospholipid composition in hamster heart perfused with methyl lidocaine

Composition, ccmol lipid P/g wet weight

Phospholipid class Control Methyl lidocaine

Lysophosphatidylcholine 0.10 + 0.02 0.12+0.03 Lysophosphatidylethanolamine 0.04 + 0.01 0.05 + 0.02 Phosphatidylcholine 11.7+0.80 10.9+ 1.15 Phosphatidylethanolamine 9.81 +0.88 10.2 + 1.00 Phosphatidic acid and

cardiolipin 3.16k0.43 3.55 + 0.57 NOTE: Hamster hearts were perfused in Krebs-Henseleit buffer in the presence and absence of 0.5 mg

methyl lidocaine/mL. Subsequent to perfusion, the hearts were homogenized in chloroform-methanol and the phospholipid compositions of the hearts were analyzed by thin-layer chromatography. Each value represents the mean i SD of four separate experiments.

TABLE 5. Effect of methyl lidocaine on the metabolism of lysophosphatidylcholine and lysophosphatidylethanolamine in the isolated hamster heart

Radioactivity, dpm/g wet weight x lo3

Control Methyl lidocaine

Phosphatidylcholine 840+ 110 521 f 90* Lysophosphatidylcholine 419+75 450 k 94 Fatty acids 910+ 135 1180+ 105*

Phosphatidylethanolamine 75 + 13 52 + 8* Lysophosphatidylethanolamine 84+ 14 79+ 11 Fatty acids 1102 10 138 f 9*

NOTE: Isolated hamster hearts were perfused with 10 pM ~ - [ l - ~ ~ C ] p a l m i t o ~ l glycero-3-phosphocholine (0.5 pCi/mL) or ~ - [ l - ~ ~ ~ ] p a l m i t o y l glycero-3-phosphoethanolamine (0.1 pCi/mL) in the presence or absence of 0.5 mg methyl-lidocaine/mL for 30 min. Subsequent to perfusion, the radioactivity associated with each lipid fraction was determined. Each value represents the mean * SD of four separate experiments (*p < 0.05).

were determined and compared with the hearts perfused without methyl lidocaine. No significant difference was detected between these two groups (data not shown). As documented in a previous report (Savard and Choy 1982), perfusion with 10 pM of lysophospholipids did not cause any significant changes in the lysophospholipid content in the hamster heart. The lysophospholipid content in the heart perfused with 10 pM of lys~pho~pholipids was very similar to that obtained from the heart perfused with Krebs- Henseleit buffer (Table 4).

Discussion The deacylation reaction catalyzed by phospholipase A

is the major route for phospholipid catabolism in mammalian tissues. In the hamster heart, the lysophospholipids formed are further deacylated by lysophospholipase or acylated back to the parent phospholipids by acyl-CoA dependent acyl- transferases (van Golde and van den Bergh 1977). Since lyso- phospholipids are cytolytic at high cellular concentrations, their levels in the cell are normally under rigid control (Weltzein 1979). The regulatory mechanism for the control of lysophospholipid levels in the heart was investigated in rats that were fed diets containing different amounts of vitamin E (Cao et al. 1987). The cardiac lysophosphatidyl- choline level was altered by dietary treatment and the changes were attributed to the modulation of cardiac phos- pholipase A activities by vitamin E. In another study (Choy et al. 1989), lysophosphatidylcholine levels in the heart were altered by ethanol perfusion and the changes were again attributed to the modulation of phospholipase A activity.

These studies clearly indicate the important role phos- pholipase A plays in the regulation of lysophospholipid levels in the heart. However, the role of acyl-CoA dependent acyltransferase in the maintenance of the cardiac lysophos- pholipid levels has not been defined. The ability to inhibit the acyltransferases, but not phospholipase A or lysophos- pholipase activities, by 0.5 mg methyl lidocaine/mL provided us with an excellent approach to examine the contribution of acyltransferases to the regulation of lysophospholipid metabolism.

Kinetic studies revealed that the inhibition of enzyme activity by methyl lidocaine was not at the substrate level, and both LPC:acyl-CoA and LPE:acyl-CoA acyltransferases were inhibited by the same mechanism. One interesting find- ing is that, while the microsomal acyltransferase activities were severely inhibited in the isolated heart perfused with 0.5 mg methyl lidocaine/mL, the phospholipase A and lysophospholipase activities were not affected. At the same time, no accumulation of lysophosphatidylcholine or lyso- phosphatidylethanolamine was detected. Taken together, the activities of the acyltransferases do not seem to have a direct contribution to the maintenance of the lysophospholipid levels in the heart.

The regulation of lysophospholipid levels in the heart was further examined by perfusion with labelled lysophospho- lipids. As shown in earlier studies (Savard and Choy 1982) and substantiated in the present study, perfusion of the heart with 10 pM of lysophospholipid did not significantly perturb the cardiac lysophospholipid content. Hence, it is not sur- prising that perfusion with methyl lidocaine caused a reduc-

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750 BIOCHEM. CELL BIOL. VOL. 68, 1990

tion in the labelling of the phospholipid, but did not change the labelling of the lysophospholipid. Interestingly, there was a n increase in the release of labelled fatty acid. A facile explanation is that the inhibition of the acylation process by methyl lidocaine also resulted in a n increase in the deacylation of the lysophospholipids, which was triggered by a yet undefined mechanism. However, the increase in labelled fatty acid could also be derived from the enhanced deacylation of the labelled phospholipids. If the deacyla- tion of the lysophospholipid was indeed enhanced while the acylation process was inhibited by methyl lidocaine, such enhancement may act as a n important compensatory mech- anism for maintaining the appropriate lysophospholipid levels in the hamster heart.

Acknowledgements This work was supported by the Heart and Stroke

Foundation of Manitoba. We thank Y.-Z. Cao and R. Hurta fo r performing the initial studies. P.C.C. is an M R C Scientist.

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