11184119911167-172 © 1991 Elsevier Science PublishersB.V. I)005-2760/91/$03.511 ADONIS 00052760910I)199U
167
Biochbnica et Biophysica Acta.
The catabolism of e x o g e n o u s lysophosphatidylcholine in isolated perfused rat and guinea pig hearts: a comparative study Thomas Mock and
Ricks'Y.K. Man
Department of Pharmacology and Therapeutics. UnilersiO' of Manitoba. 140nmpeg(Canada)
(Received I11December 1gO0) (Revised manuscriptleceived4 March 1091)
Key words: Lysophosphatidylcholine:Catabolism:Perfused heart: Lysophosphoiipase:AcylCoA : lysoPCacyltransferase Lysophosphatidylcholine (lysoPC) is an arrhythmogenic phospholipid metabolite which accumulates in the ischemi¢ myocardium. Reduced catabolism of lysoPC has been prooosed to be one of the biochemical mechanisms responsible for the increase in lysoPC content. In this investigation we compared the mierosomal catabolism of exogenous labeled lysoPC in isolated perfused rat and guinea pig hearts. Analysis of the amount of radioactivity in microsomal phosphatidylcholine (PC) and free fatty acid (FFA) was used as an index of the participation in lysoPC clearance by acylation catalyzed by acyI-CoA: lysoPC acyltransi'erase and deacylatlon catalyzed by lysophospholipase, respectively. There was no significant difference in the incorporation of radioactivity into rat and guinea pig heart microsomes; however, the patterns of radioactivity in lysoPC melabolites were notably different. Equal participation by deacylation and reacylation was observed in rat microsnmes, whereas deacylation was clearly the preferred route for lysoPC clearance in guinea pig microsomes. Modulation of enzyme activi~ by treatment of the isolated heart with pHMB, a sulfliydryl agent, was used to probe the relationship among acylation, deacylation and the extent of lysoPC clearance, in guinea pig microsomes impairment of lysoPC acylation was not associated with any change in the amount of radioactivity in lysoPC because of a compensatory increase in deacylation. In contrast, impaired deacylation iu ral mierosomes led to significant elevations in the amount of radioactivity in lysoPC. We conclude, therefore, that in intact per'fused rat and guinea pig hearts the relative participation of aeylation and deac:,'lation in lysoPC clearance differs. Moreover, we propose that the level of deacylation by lysophospholipase is an important factor in the extent of clearance of lysoPC.
Introduction
Lysophospholipids are phospho[;p,.'d metabolites which are normal constituents of virtually all biological membrnne~ [1]. The major lysophospholipid in the mammalian heart is lysophosphatidylcholine (lvsoPC; I-acyl or 2-acyl glycerophosphocholine). Interest in lysoPC as a biochemical mcdiatoi of alrhythmogcncsis during myocardial ischemia was generated by the findings of elevated levels of lysoPC coupled with its ability
Abbreviations: lysoPC, lysophosphatidylcholine;PC. phosphatidylcholine; FFA, free fatty acid; PLA. phospholipaseA; pHMB. parahydroxymercuribenzoieacid; CoA. CoenzymeA; ANOVA, analysis of variance. Correspondence: R.Y.K. Man, Department of Pharmacology and Therapeutics, Universityof Manitoba, Winnipeg,Manitoba, Canada R3E OW3.
to produce electrophysiological derangements typical of those seen in myocardial ischemia [2,3]. Moreover lysoPC may contribute to functional impairment of the ischemic myocardium by virtue of its modulatory effects on other processes such as cyclic nucleotide metabolism [4]. contractility of coronary vasculature [5,6], lipid peroxidatioo [7,8] and Ca -'+ mobilization [9,10]. Cardiac lysoPC is produced via hydrolysis of phosphatidylcholine (PC) by phospholipase A (PLA) [1 1]. LysoPC is cleared by a number of enzymes including, acyl CoA : lysoPC acyl transferase, lysoPC : lysoPC transacylase and lysophospholipase [12,t3]. However, the presence of lysoPC:lysoPC transacylase is speciesspecific [14A5]. Because of its cytolytic nature lysoPC levels in normal tissue are rigidly controlled suggesting precise coordination between production and elimination. Since in vitro acyl CoA:IysoPC acyltransferase activity is much higher than lysophospholipase activity,
168 it has been suggested that acylation catalyzed by acyl CoA: lysoPC acyltransfcrase is the predominant mechanism of lysoPC clearance. However, little information is presently available which provides insight into the relative importance of each enzyme in overall lysoPC catabolism in the intact heart and consequently whether modulation of one enzyme is likely to be of more significance in determining the rate and extent of lysoPC elimination. Earlier studies from this and other laboratories have taemonstrated the ability of the isolated heart to take up labeled lysoPC [16,17] with incorporation into the microsomal (membrane) fraction [17]. The finding that radioactivity is not confined to lysoPC indicates extensive catabolism [17]. In this study we report our findings on the metabolic fate of exogenous lysoPC in the microsomal fraction of isolated perfused rat and guinea pig hearts. Perturbation of enzyme activity with parahydroxymercuribenzoic acid (pHMB) was used as a tool to gain a greater understanding into the interplay between two key catabolic enzymes acyl CoA:lysoPC acyl transferase and lysophospholipase in the regulation of lysoPC catabolism.
Materials and Methods
tions of 2.5/~M to 7.5 # M for the guinea pig heart and 2.5 # M to 15 /aM for the rat heart. Perfusion of the guinea pig heart with 15.0 /~M pHMB led to visible impairment of contraction and was not examined further. Following 30 min of perfusion with either a control (no pHMB) or test solution, the perfusate was switched to one containing 2.5 p.M lysoPC (mixture of 1-~4C-labeled and unlabeled lysoPC) and pHMB for an additional 10 min. The final concentration of isotope was 0.0075/~Ci/ml of perfusate.
Preparation of microsomal fraction Upon termination of perfusion the heart was removed and the vascular space was cleared with 10 ml of ice-cold Kreb's Henseleit buffer. The heart was cut open, blotted dry and weighed. The heart was then quickly homogenized in 16 ml of ice-cold 0.25 M sucrose, 10 m M Tris-HCl (pH 7.4), 1 m M E D T A buffer (approx. 10% homogenate) with a Polytron Homogenizer at a rheostat setting of 7. The homogenate was centrifuged at 20000 X g for 10 min at 4°C. The supernatant thus obtained was then centrifuged at 100000 x g for 60 min at 4°C. The resulting microsomal pellet was reconstituted in a small volume of water and stored at - 80°C. Protein content was measured by the procedure of Lowry et al. [19].
Materials Sprague-Dawley rats were obtained from the Animal Care Facility at the University of Manitoba. Guinea pigs were purchased from Charles River Laboratories (Canada). LysoPC and PC (both from pig liver) were from Serdary Research Products. Thin-layer chromatography plates (SIL G-25) were obtained from Brinkmann. [l-~4C]Palmitoyl IysoPC (58 m C i / m m o l ) was the product of Amersham. Eeolume (liquid scintillant) was obtained from ICN Biomedicals. Linoleoyl CoA and pHMB (sodium salt) were purchased from Sigma. All other chemicals were of reagent grade and obtained from Fisher Scientific. The composition of the Kreb's-Henseleit buffer used throughout this study was as follows: NaCI 120 mM, N a H C O 3 25 mM, dextrose 5.5 mM, KCI 4.76 mM, MgSO4 1.19 mM, NaHPO 4 1.18 mM and CaCI 2 1.27 mM. Stock solutions of pHMB (1 m g / m l water) were prepared fresh daily.
Isolated heart perfusion Heparin (600 units i.p.) was administered to rats and guinea pigs (250-350 g) 1 h before start of experiment. The animals were killed by cervical dislocation and the hearts quickly removed and immersed in ice-cold oxygenated Kreb's-Henselel) buffer. The aorta was cannu!ated for retrograde perfi~slon according to the method of Langendorff [18]. Hearts were perfused at a flow rate of 10 m l / m i n with Kreb's-Henseleit buffer warmed to 37°C and continuously gassed with 95% 0 2 / 5 % CO 2. Test solutions contained pHMB at concentra-
Lipid extraction and analysis Microsomal lipids were extracted according to the method of Bligh and Dyer [20]. The lipid extract was dried under a stream of N2 and then dissolved in a small amount of chloroform/methanol (2:1, v/v). A sample of the lipid extract was then applied to a thin-layer chromatography plate along with unlabeled carrier lysoPC. The plates were developed in one dimension in a solvent containing chloroform/methan o l / water/acetic acid (70: 30: 4: 2, v/v). The bands were visualized with iodine vapor. Those regions corresponding to lysoPC, PC and neutral lipid were identified. and scraped into sc!ntiilation vials. To each vial were added 100 tzl acetic acid, 500 p.I water and 5 ml Ecolume. The vials were vortexed well and analyzed by scintillation spectrometry. Extraction of microsomal lipid by the method described led to virtually 100% recovery of lipid as determined by recovery of labeled marker. The radioactivity in all lipid extracts was confined almost exclusively ( > 99%) to iysoPC, PC, and the neutral lipid fraction. The amount of radioactivity in the PC fraction was used as an index of acyl CoA:lysoPC acyl transferase activity and the amount of radioactivity in the neutral lipid fraction as an index of lysophospholipase activity (i.e., FFA release). Appreciable reutilization of liberated labeled FFA into PC was unlikely due to the short time course in this study. Moreover, no appreciable intrapreparative conversion of lysoPC was observed.
169
Assay of microsomal acyl CoA : lysoPC acyl transferase and lysophospholipase actit~ity Enzyme activities in washed microsomes were assayed in vitro as described elsewhere [12]. Briefly, acyl C o A : l y s o P C acyltransferase activity was d e t e r m i n e d from the rate of production of labeled PC in a mixture containing 100 /.tM [1-14C]palmit%l lysoPC (2.27 ffCi/tzmol), 100 tzM linoleoyl CoA, 50 mM sodium phosphate buffer (pH 7.4) and 5 p,g protein in a volume of 200 /tl. The reaction was initiated by the addition of labeled lysoPC and was carried out at room t e m p e r a t u r e (23°C) for 2.5 min. Lysophospholipase activity was m e a s u r e d by the release of labeled fatty acid (neutral lipid fraction) in a mixture containing 50 p,M [l-~4C]palmitoyl lysoPC (2.27 i z C i / ~ m o l ) , 4 m M MgCI 2, 50 m M sodium phosphate buffer (pH 7.4) and 50 tzg protein in a total volume of 200 ~1. The tubes were incubated a t. 37°C for 15 min. W a t e r was substituted for protein in the reaction blanks. The reaction was stopped by the addition of 1.5 ml of c h l o r o f o r m / methanol ( 2 : 1 , v / v ) . The reaction products were extracted with the addition of 5 5 0 / z l water. After ~x)rtexing and centrifugation an aliquot of the lower phase was drawn and dried u n d e r N 2. Lipids were s e p a r a t e d and analyzed as described above. U n l a b e l e d PC was spotted along with the lipid extract to aid in the identification of the PC band. In o r d e r to assess the effects of p H M B t r e a t m e n t on enzyme activities, reaction mixtures (minus lysoPC substrate) were incubated with the required a m o u n t of p H M B for 30 rain at the desired temperature. T h e reaction was then initiated by the addition of lysoPC substrate as described above. Data analysis Student's t-test was employed to analyze differences in uptake and a m o u n t s of radioactivity in lipid fractions in u n t r e a t e d rat and g u i n e a pig heart microsomes (i.e., controls). Evaluation of statistical significance between u n t r e a t e d and t r e a t e d groups was performed by analysis of variance ( A N O V A ) where indicated in the text. Tukey's test was subsequently used to assess individual differences. Differences with P < 0.05 was considered to be statistically significant. V~lues are expressed as m e a n + S.E. Results LysoPC metabolism by microsomal enzymes" of rat and guinea pig hearts The enzyme activities of acyl C o A : lysoPC acyltransferase and !ysophospholipase were assayed in the microsomal fractions of rat and g u i n e a pig hearts. As illustrated in Table 1 there is substantially more acyl C o A : lysoPC acyltransferase activity than lysophospholipase activity in the microsomal fraction of both rat
TABLE 1 Acticity t¢ mierosomal lysoPC-catabolizing enzTmesmeasuredin I'itro Enzyme activities in rat and guinea pig heart microsomes were assayed as described in Materials and Methods. Values denote mean + S.E. Note that units for enzyme activities differ. Enzyme Acyl CoA : lysoPC acyltransferase (izmol PC/rag protein per h)
Rat 2.01 +_0.65 n=3
Guinea pig 1.69_+0.20 n= 3
Lysophospholipase (nmol FFA/mg protein per h)
44 +_4 n=3
677 4-70 n=3
and guinea pig hearts when m e a s u r e d in vitro u n d e r optimal experimental conditions.
LysoPC handling by isolated rat and guinea pig hearts T h e r e was a m a r k e d difference in the profile of radioactive metabolites of lysoPC in the microsomal fractions obtained from intact rat and guinea pig hearts (Table I1). Less lysoPC remained unmetabolized in g u i n e a pig microsomes than in rat microsomes (a 20% difference). The a m o u n t of radioactivity associated with PC was greater in the rat heart microsomal fraction than in guinea pig microsomes (also 20% greater). In contrast 47% more radioactivity was recovered in the neutral lipid fraction of g u i n e a pig microsomes (indicative of F F A release) compared to rat microsomes. Unlike rat microsomes where no significant difference in the a m o u n t of radioactivity in the PC and neutral lipid fractions was detected, the a m o u n t of radioactivity associated with the neutral lipid fraction of guinea pig microsomes was almost twice as much as that fo: nd with the PC fraction ( P < 0.001). It is also clear that no TABLE 11 Profile of lal~'led lysoPCmetabolites in rat and guinea pig microsomes Isolated rat and guinea pig hearts were perfnsed in the Langendorff mode for 31) min with Kreb's.Henseleit buffer followed by perfusion with buffer containing 2.5 ~M [l-14C]lysoPC(0.0075 u.Ci/ml) for 10 rain. Microsomes were prepared and lipids extracted, separated and analyzed for radioactivity as detailed in Materials and Methods. Values denote mean:i:S.E., n = 7 for rat, n=6 for guinea pig. " P < 0.01 compared to corresponding value in the rat; b p < 0.001 compared to guinea pig PC. Values in parantheses denote average weighted participation in clearance of lysoPC as determined from radioactivity in reaction products. LysoPC (dpm/mg protein)
PC (dpm/mg protein)
FFA + neutral lipid (dpm/mg protein)
Rat
1694+220
4059±494 (48%I
4370=1:317 (52%)
Guinea pig
14085=140
3391 ±309 (35%)
6423+418 ''h 165%)
170 80 T
apparent relationship exists between the activities obtained in vitro under optimal conditions (see Table I) and the relat;ve participation in the intact isolated organ using tile amount of radioactivity in the PC fraction as an index of acyl C o A : I y s o P C acyl transferase activity and the a m o u n t of radioactivity in the neutral lipid fraction as an index of lysophospholipase activity (i.e., FFA release). Deacylation by lysophospholipase is obviously of far greater importance in both preparations than the specific activity m e a s u r e d in vitro would indicate.
60
[[[3 control
[ ~ 2 . 5 ,~M 5.0/zM ~ 7.5/~M E~] 15.0 #M
iinih'itiR
o
PC FFA + Neutral lipid Fig. 1. Profile of labeled lysoPC metabolites in microsomes from untreated and pHMB-treated rat hearts. The isolated rat heart was perfused for 30 lain with Kreb's-Henseleit buffer either without pHMB (i.e. control) or with 2.5 ,uM to 15.0 p.M pHMB (refer to key). Six to eighl hearts were perfused under each experimental condition. Each heart was then perfused for l0 rain with buffer containing both pHMB and labeled lysoPC. Mierosomal lipid analysis was performed as described in Materials and Methods. Data are depicted as % of total radioactivity incorporated into microsomes and represent mean _+S.E. * P < 0.05 compared to control. LysoPC
Effects of pHMB treatment on the amotmt of label Or lipids T r e a t m e n t of rat and guinea pig hearts with p H M B had no effect on the recovery of microsomal protein over the concentration ranges tested as assessed by A N O V A . The average recovery of microsomal protein from control rat heart (no p H M B ) was 1.60 + 0.09 m g / g wet weight (n = 6) and from p H M B - t r e a t e d rat heart 1.41 + 0.08 m g / g wet weight (n = 29). Corresponding recoveries from g u i n e a pig were 1.13 + 0.06 m g / g wet weight (n = 6) and 1.08 + 0.04 m g / g wet weight (n = 16) for control and p H M B - t r e a t e d hearts respectively. U p t a k e of lysoPC by the microsomal fraction did not differ a m o n g control and p H M B - t r e a t e d groups with either the rat or guinea pig heart (by A N O V A ) . Incorporation into rat microsomes was 10120+_800 d p m / m g protein (n = 7 ) and 1 2 9 4 0 + 7 3 2 d p m / m g protein (n = 30) in the absence and presence of pHMB, respectively. Incorporation into g u i n e a pig microsomes was 1123(1 + 671 d p m / m g protein (n = 6) and 11845 + 485 d p m / m g protein (n = 161 in the absence and presence of pHMB, respectively. Moreover, there was no statistically significant difference in uptake of labeled lysoPC by the microsomes between u n t r e a t e d rat and guinea pig hearts. p H M B p r e t r e a t m e n t of the isolated rat heart gave rise to a significant c o n c e n t r a t i o n - d e p e n d e n t increase in the a m o u n t of radioactivity in lysoPC in rat microsomes ( P < 0 . 0 1 ; A N O V A ) (Fig. 1). Control values differed significantly from those obtained at 7.5 # M p H M B (29% increase over control, P < 0.05) and 15.0 tLM p H M B (36% increase over control, P < 0.05). Associated with this increase in the a m o u n t of radioactive lysoPC was a corresponding decrease in the a m o u n t o i radioactivity in neutral lipid ( P < 0 . 0 5 ; A N O V A ) . A level of statistical significance was attained at both 7.5 p,M p H M B (26% decrease c o m p a r e d to control, P < 0.05) and 15.(I/xM p H M B (34% decrease compared to control, P < 0.05). Although there a p p e a r e d to be a trend towards increased a m o u n t of radioactivity in PC, no statistical significance was detected. In contrast, p r e t r e a t m e n t of the isolated guinea pig heart with up to 7.5 ~tM p H M B did not result in any
statistically significant c h a n g e in the a m o u n t of radioactivity in lysoPC ( A N O V A ) (Fig. 2). T h e r e was, however, a c o n c e n t r a t i o n - d e p e n d e n t decrease in the a m o u n t of radioactivity associated with PC ( P < 0.001; A N O V A ) . Values o b t a i n e d at both 5.0 # M p H M B (30% decrease c o m p a r e d to control) and 7.5 p,M p H M B (41% decrease c o m p a r e d to control) were significantly different than those o b t a i n e d with 2.5 p,M p H M B and control (all P < 0.05). A n increase in radioactivity in the neutral lipid fraction was observed over this concentration range ( P < 0.01" A N O V A ) . A level of statistical significance was achieved with 7.5 ,u,M p H M B
80 T
r--3 control
I ~ ;~.5 #aM 60
~
~
5.0/zM 7.5 #M
40
1ii
FFA + Neutral lipid Fig. 2. Profile of labeled lysoPC metabolites in microsomes from untreated and pHMB-treated guinea pig hearts Perfusion of the isolated guinea pig heart and analysis of microsomal iipids were performed according to Materials and Methods. Five to six hearts were perfused under each exgerimental condition. Data presentation as in Fig. 2. * P < 0.05 compared to control and 2.5 gM pHMB. LysoPC
PC
171
3.G1 7.22 10.82 16.23
21,64
3607
ng pHMB/#g protein Fig. 3. Inhibitionof rat and guinea pig heart microsemal lysophospholipase and acyl CoA:lysoPC acyltransferaseby pHMB. Microsomal fractions were prepared and enzymes assayed as described in Materials and Methods. All p,)intsdepict activityobtained relative to control ( n o pHMB pretreatment) with control representing I(lllr;. See Table I for absolute values. Symbols:rat and guinea pig lysophospholipase (zx. a. respectively); rat and guinea pig acyl CoA:lysoPC acyhransferase(c. e. respectively). (26% increase over control) wh:n compared with 2.5 v.M pHMB and control (bo~h P < 0.05).
Inhibition of enzyme activity by p, tMB in vitro In order to determine if there is a qualitative difference in the response of enzyme ,~ctivities to pHMB, microsomal fractions were incub:,:r d with 3.61 to 36.117 ng pHMB/v.g protein for 30 mia prior to the actual assay. As can be seen in Fig. 3 tt ere appears to be no difference in the sensitivity of ra: and guinea pig microsomal lysophospholipases and ; cyl CoA : lysoPC acyl transferase to pHMB-mediated i ahibition in vitro. A slightly lesser amount of p H M B an a per p.g protein basis, was required to produce a level of moderate inhibition of lysophospholipase comparable to that of than acyl CoA:lysoPC acyltransferase under these experimental conditions. However, the enzymes are equally inhibited at higher amounts of pHMB. Discussion Over the last decade numerous published reports have demonstrated the wide-ranging cardiac and vascular effects of lysoPC [2-5,21-24]. Recently, lysoPC has also been implicated as a putative second messenger in some instances based on its likely participation in signal transduction [25]. Despite the ever-increasing awareness of the importance of lysoPC as a modulator of cardiac function, relatively little is known about the mechanisms which govern its level and the dynamics of its clearance in the intact heart. The aim of this study was to assess the importance of acyl CoA : lysoPC acyltransferase and lysophospholipase acttvites in microsomal lysoPC c:ttabolism in a comparative analysis of isolated perfused rat and guinea pig hearts. The intact
isolated perfused heart model used in this study allowed for simultaneous analysis of both acylation and deacylation of lysoPC. In both the rat and guinea pig heart microsomes the capacity for acylation of lysoPC far exceeds that for deacyla~ion based on in vitro analysis, a finding which is in accordance with previous reports [12,26]. It has therefore been proposed that acyl CoA:lysoPC acyl transferase-mediated PC synthesis is the primary mechanism for lysoPC clearance. However, the profile of radioactive metabolites of lysoPC in heart microsomes obtained in this study suggests that deacylation by lysophospholipase is of far greater relative importance than the in vitro data would indicate. Indeed, clearance of lysoPC by deacylation is the preferred pathway for catabolism in guinea pig microsomcs. Deacylation by ly~phospholipase and acyiation by acyl CoA:lysoPC acyltransferase are of equal importance in the clearance of lysoPC in rat microsomes. Thus, the present results suggest that specific activity mea~;'Jred in vitro does not correlate well with relative participation of lysophospholipase and acyl CoA:lysoPC acyltransferase in catabolism of lysoPC in the intact organ, Clearly conditions necessary for optimal expression of activity are not present in the intact organ. There is evidence, however, to indicate that the activity of lysophospholipase measured in vitro is qualitatively associated with thc extent and potential for clearauce by deacylation in the intact organ. The sulfhydryl agent pHMB was used in order to produce enzyme inhibition as a means of assessing the effects of changes in acylation and deacylation on iysoPC clearance. The inhibition of acyl CoA:lysoPC acyltransfcrase and lysophospholipase by pHMB has been previously demonstrated in other models [27,28]. A fortuitous result was the finding that, within the concentration ranges of pHMB evaluated impairment of the microsomal acyl CoA:lysoPC acyltransferase in the guinea pig was manifest in a decrease in the amount of radioactivity associated with PC, whereas in the rat impairment of microsomal lysophospholipase was reflected in a decrease in the amount of radioactivity in the neutral lipid. It was thus possible to measure the metabolic fate of exogenous lysoPC in two species with significantly altered capacities for clearance of lysoPC by these two major routes. Despite decreases in PC synthesis in microsomes from pHMB-treated guinea pig heart there were no differences in the amount of radioactivity in lysoPC because of a compensatory increase in FFA release via lysophospholipase activity. It is probable that a degree of competition for lysoPC substrate exists between the enzymes which enables the lysophospholipase to compensate for diminished acyl CoA:lysoPC acyltransferase-mediated clearance to maintain a constant extent of elimination. Thus, it appears that there is an element of 'crosstalk" bctwcen
172 these enzymes and therefore they are not acting independently of each other. In contrast impairment of deacylation by lysophospholipase in rat microsomes was accompanied by an increase in the level of unmetabolized labeled lysoPC. Evidently rat microsomes are less able to maintain the same level of clearance once lysophospholipase activity has been compromised. Moreover, there appears to be no significant potential for compensatory elimination of lysoPC by acyl C o A : l y s o P C acyltransferase. These findings provide evidence for a link between lysophospholipase activity and the extent of lysoPC catabolism. The inhibition of enzyme activity by p H M B in isolated intact rat and guinea pig hearts, as evidenced in the changes in the amount of label in the respective lipid fractions, cannot be satisfactorily explained by the sensitivity of the microsomal enzymes to p H M B inhibition m e a s u r e d in vitro. It is conceivable, however, that some as yet unknown characteristics of the intact heart are responsible for the a p p a r e n t variance. Myocardial ischemia produces profound alterations in phospholipid homeostasis, an index of which is the rise in lysoPC levels. Increased lysoPC may be brought about by a u g m e n t e d production, reduced catabolism and poor washout or combinations of these. Increased production, as a consequence of e n h a n c e d phospholipase A activity, is generally perceived to contribute to elevated lysoPC in the ischemic heart [29,30]. Several investigators have suggested that impaired catabolism may also factor in the rise of lysoPC [12,31]. In the present study we have clearly shown that inhibition of lysophospholipase activity by p H M B in rat heart microsomes is associated with reduced catabolism of exogenously supplied lysoPC. Several concomitants of myocardial ischemia such as reduced p H and long chain acylcarnitines have been shown to produce potent inhibition of microsomal lysophospholipase [26,32]. Modulation of lysophospholipase activity in vivo during ischemia may thus be a critical d e t e r m i n a n t in the ability to clear lysoPC and maintain basal levels. Acknowledgements This investigation was supported by the Medical Research Counci! of Canada. T.M. is the recipient of a H e a r t and Stroke Foundation of C a n a d a Traineeship. References 1 Wehzien, H.U. (1979) Biochim. Biophys. Acta 559, 259-287. 2 Sobel, B.E., Corr, P.B., Robison, A.K., Goldstein, R.A.,
Witkowski, F.X. and Klein, M.S. (1978) J. Clin. Invest. 62. 546553. 3 Corr, P.B., Cain, M.E., Witkowski, F.X.. Price, D.A. and Sobel, B.E. (1979) Circ. Res. 44, 822-832. 4 Ahumada. G.G.. Bergmann. S.R., Carlson, E.. Corr. P.B. and SoOel, B.E. (1979) Cardiovasc. Res. 13. 377-382. 5 Bergmann. S.R.. Ferguson, T.B. Jr. and Sobel. B.E. (1981) Ara. J. Physiol. 240, H229-H237. 6 Saite, T., Wolf, A., Menon, N.K., Saeed, M. and Bing. R.J. (1988) Proc. Natl. Acad. Sci. USA 85, 8246-8250. 7 Kihlstrom, M., Marjomaki, V. and Salminen. A. (1987) Basic Res. Cardiol. 82 (Suppl. I), 261-269. 8 Mak, I.T.. Kramer, J.H. and Weglicki, W.B. (1986) J. Biol. Chem. 261, 1153-1157. 9 Sedlis, S.P., Corr, P.B., Sobel, B.E. and Ahumada, G.G. (1983) Am. J. Physiol. 244, H32-H38. 10 Ambudkar, LS., Abdallah, E.S. and Shamoo, A.E. (1988) Mol. Cell. Bioehem. 79, 81-89. 11 Nalbone, G. and Hostetler, K.Y. (1985)J. Lipid Kes. 26,104-114. 12 Mock, T. and Man, R.Y.K. (1990) Lipids. 25, 357-362. 13 Gross, R.W. and Sobel, B.E. (1982)J. Biol. Chem. 257, 6702-6708. 14 Grcss, R.W., Drisdel, R.C. and Sobel, B.E. (1983)J. Biol. Chem. 258, 15165-15172. 15 Savard, J.D. and Choy, P.C. (1982) Biochim. Biophys. Acta 711, 40-48. 16 Stein, Y. and Stein, O. (1965) Bioehim. Biophys. Acta 106, 527-539. 17 Giffin, M., Arthur, G., Choy, P.C. and Man, R.Y.K. (1988) Can. J. Physiol. Pharmacol. 66,185-189. 18 Langendofff, O. (1895) Pfluegers Arch. 61, 291-332. 19 Lowry, O.H., Rosebrough, M.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 20 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 912-917. 21 Fink, K.L. and Gross, R.W. (1984) Circ. Rcs. 55, 585-594. 22 Corr, P.B., Snyder, D.W., Lee, B.I., Gross, R.W., Keim, C.R. and Sobel, B.E. (1982) Am. J. Physiol. 243, HI87-H195. 23 Pogwizd, S.M., Onufer, J.R., lrd-amer, J.B., Sobel, B.E. and Corr, P.B. (1986) Circ. Res. 59, 416-426. 24 Corr, P.B., Yamada, K.A., Creer, M.H., Sharma, A.D. and Sobel, B.E. (1987) J. Mol. Cel. Cardiol. 19 (Suppl. V), 45-53. 25 Rustenbecl¢, L and Leozeo, S. (1989) Naunyn-Schmiedeberg's Arch. Pharm~col. 339, 37-41. 26 Severson, D.L. and Fletcher, T. (1985) Can. J. Physiol. Pharmacol. 63, 944-951. 27 Kroner, E.E., Peskar, B.A., Fischer, H. and Ferber, E. (1981) J. Biol. Chem. 256, 3690-3697. 28 Weller, P.F., Bach, D.S. and Austen, K.F. (1984) J. Biol. Chem. 259,15100-15105. 29 Das, D.K., Engelman, R.M., Rousou, J.A., Breyer, R.H., Otani. H. and Lemeshow, S. (1986) Am. J. Physiol. 251, H71-H79. 30 Otani, H., Prasad, M.R., Jones, R.M. and Das, D.K. (1989) Am. J. Physiol. 257, H252-H258. 31 Bentham, J.M., Higgins, A.J. and Woodward, B. (1987) Basic Res. Cardiol. 82 (Suppl. 1), 127-135. 32 Mock, T. and Man, R.Y.K. (1990) Proc. West. Pharmacol. Soc. 33,139-142.
167
Biochbnica et Biophysica Acta.
The catabolism of e x o g e n o u s lysophosphatidylcholine in isolated perfused rat and guinea pig hearts: a comparative study Thomas Mock and
Ricks'Y.K. Man
Department of Pharmacology and Therapeutics. UnilersiO' of Manitoba. 140nmpeg(Canada)
(Received I11December 1gO0) (Revised manuscriptleceived4 March 1091)
Key words: Lysophosphatidylcholine:Catabolism:Perfused heart: Lysophosphoiipase:AcylCoA : lysoPCacyltransferase Lysophosphatidylcholine (lysoPC) is an arrhythmogenic phospholipid metabolite which accumulates in the ischemi¢ myocardium. Reduced catabolism of lysoPC has been prooosed to be one of the biochemical mechanisms responsible for the increase in lysoPC content. In this investigation we compared the mierosomal catabolism of exogenous labeled lysoPC in isolated perfused rat and guinea pig hearts. Analysis of the amount of radioactivity in microsomal phosphatidylcholine (PC) and free fatty acid (FFA) was used as an index of the participation in lysoPC clearance by acylation catalyzed by acyI-CoA: lysoPC acyltransi'erase and deacylatlon catalyzed by lysophospholipase, respectively. There was no significant difference in the incorporation of radioactivity into rat and guinea pig heart microsomes; however, the patterns of radioactivity in lysoPC melabolites were notably different. Equal participation by deacylation and reacylation was observed in rat microsnmes, whereas deacylation was clearly the preferred route for lysoPC clearance in guinea pig microsomes. Modulation of enzyme activi~ by treatment of the isolated heart with pHMB, a sulfliydryl agent, was used to probe the relationship among acylation, deacylation and the extent of lysoPC clearance, in guinea pig microsomes impairment of lysoPC acylation was not associated with any change in the amount of radioactivity in lysoPC because of a compensatory increase in deacylation. In contrast, impaired deacylation iu ral mierosomes led to significant elevations in the amount of radioactivity in lysoPC. We conclude, therefore, that in intact per'fused rat and guinea pig hearts the relative participation of aeylation and deac:,'lation in lysoPC clearance differs. Moreover, we propose that the level of deacylation by lysophospholipase is an important factor in the extent of clearance of lysoPC.
Introduction
Lysophospholipids are phospho[;p,.'d metabolites which are normal constituents of virtually all biological membrnne~ [1]. The major lysophospholipid in the mammalian heart is lysophosphatidylcholine (lvsoPC; I-acyl or 2-acyl glycerophosphocholine). Interest in lysoPC as a biochemical mcdiatoi of alrhythmogcncsis during myocardial ischemia was generated by the findings of elevated levels of lysoPC coupled with its ability
Abbreviations: lysoPC, lysophosphatidylcholine;PC. phosphatidylcholine; FFA, free fatty acid; PLA. phospholipaseA; pHMB. parahydroxymercuribenzoieacid; CoA. CoenzymeA; ANOVA, analysis of variance. Correspondence: R.Y.K. Man, Department of Pharmacology and Therapeutics, Universityof Manitoba, Winnipeg,Manitoba, Canada R3E OW3.
to produce electrophysiological derangements typical of those seen in myocardial ischemia [2,3]. Moreover lysoPC may contribute to functional impairment of the ischemic myocardium by virtue of its modulatory effects on other processes such as cyclic nucleotide metabolism [4]. contractility of coronary vasculature [5,6], lipid peroxidatioo [7,8] and Ca -'+ mobilization [9,10]. Cardiac lysoPC is produced via hydrolysis of phosphatidylcholine (PC) by phospholipase A (PLA) [1 1]. LysoPC is cleared by a number of enzymes including, acyl CoA : lysoPC acyl transferase, lysoPC : lysoPC transacylase and lysophospholipase [12,t3]. However, the presence of lysoPC:lysoPC transacylase is speciesspecific [14A5]. Because of its cytolytic nature lysoPC levels in normal tissue are rigidly controlled suggesting precise coordination between production and elimination. Since in vitro acyl CoA:IysoPC acyltransferase activity is much higher than lysophospholipase activity,
168 it has been suggested that acylation catalyzed by acyl CoA: lysoPC acyltransfcrase is the predominant mechanism of lysoPC clearance. However, little information is presently available which provides insight into the relative importance of each enzyme in overall lysoPC catabolism in the intact heart and consequently whether modulation of one enzyme is likely to be of more significance in determining the rate and extent of lysoPC elimination. Earlier studies from this and other laboratories have taemonstrated the ability of the isolated heart to take up labeled lysoPC [16,17] with incorporation into the microsomal (membrane) fraction [17]. The finding that radioactivity is not confined to lysoPC indicates extensive catabolism [17]. In this study we report our findings on the metabolic fate of exogenous lysoPC in the microsomal fraction of isolated perfused rat and guinea pig hearts. Perturbation of enzyme activity with parahydroxymercuribenzoic acid (pHMB) was used as a tool to gain a greater understanding into the interplay between two key catabolic enzymes acyl CoA:lysoPC acyl transferase and lysophospholipase in the regulation of lysoPC catabolism.
Materials and Methods
tions of 2.5/~M to 7.5 # M for the guinea pig heart and 2.5 # M to 15 /aM for the rat heart. Perfusion of the guinea pig heart with 15.0 /~M pHMB led to visible impairment of contraction and was not examined further. Following 30 min of perfusion with either a control (no pHMB) or test solution, the perfusate was switched to one containing 2.5 p.M lysoPC (mixture of 1-~4C-labeled and unlabeled lysoPC) and pHMB for an additional 10 min. The final concentration of isotope was 0.0075/~Ci/ml of perfusate.
Preparation of microsomal fraction Upon termination of perfusion the heart was removed and the vascular space was cleared with 10 ml of ice-cold Kreb's Henseleit buffer. The heart was cut open, blotted dry and weighed. The heart was then quickly homogenized in 16 ml of ice-cold 0.25 M sucrose, 10 m M Tris-HCl (pH 7.4), 1 m M E D T A buffer (approx. 10% homogenate) with a Polytron Homogenizer at a rheostat setting of 7. The homogenate was centrifuged at 20000 X g for 10 min at 4°C. The supernatant thus obtained was then centrifuged at 100000 x g for 60 min at 4°C. The resulting microsomal pellet was reconstituted in a small volume of water and stored at - 80°C. Protein content was measured by the procedure of Lowry et al. [19].
Materials Sprague-Dawley rats were obtained from the Animal Care Facility at the University of Manitoba. Guinea pigs were purchased from Charles River Laboratories (Canada). LysoPC and PC (both from pig liver) were from Serdary Research Products. Thin-layer chromatography plates (SIL G-25) were obtained from Brinkmann. [l-~4C]Palmitoyl IysoPC (58 m C i / m m o l ) was the product of Amersham. Eeolume (liquid scintillant) was obtained from ICN Biomedicals. Linoleoyl CoA and pHMB (sodium salt) were purchased from Sigma. All other chemicals were of reagent grade and obtained from Fisher Scientific. The composition of the Kreb's-Henseleit buffer used throughout this study was as follows: NaCI 120 mM, N a H C O 3 25 mM, dextrose 5.5 mM, KCI 4.76 mM, MgSO4 1.19 mM, NaHPO 4 1.18 mM and CaCI 2 1.27 mM. Stock solutions of pHMB (1 m g / m l water) were prepared fresh daily.
Isolated heart perfusion Heparin (600 units i.p.) was administered to rats and guinea pigs (250-350 g) 1 h before start of experiment. The animals were killed by cervical dislocation and the hearts quickly removed and immersed in ice-cold oxygenated Kreb's-Henselel) buffer. The aorta was cannu!ated for retrograde perfi~slon according to the method of Langendorff [18]. Hearts were perfused at a flow rate of 10 m l / m i n with Kreb's-Henseleit buffer warmed to 37°C and continuously gassed with 95% 0 2 / 5 % CO 2. Test solutions contained pHMB at concentra-
Lipid extraction and analysis Microsomal lipids were extracted according to the method of Bligh and Dyer [20]. The lipid extract was dried under a stream of N2 and then dissolved in a small amount of chloroform/methanol (2:1, v/v). A sample of the lipid extract was then applied to a thin-layer chromatography plate along with unlabeled carrier lysoPC. The plates were developed in one dimension in a solvent containing chloroform/methan o l / water/acetic acid (70: 30: 4: 2, v/v). The bands were visualized with iodine vapor. Those regions corresponding to lysoPC, PC and neutral lipid were identified. and scraped into sc!ntiilation vials. To each vial were added 100 tzl acetic acid, 500 p.I water and 5 ml Ecolume. The vials were vortexed well and analyzed by scintillation spectrometry. Extraction of microsomal lipid by the method described led to virtually 100% recovery of lipid as determined by recovery of labeled marker. The radioactivity in all lipid extracts was confined almost exclusively ( > 99%) to iysoPC, PC, and the neutral lipid fraction. The amount of radioactivity in the PC fraction was used as an index of acyl CoA:lysoPC acyl transferase activity and the amount of radioactivity in the neutral lipid fraction as an index of lysophospholipase activity (i.e., FFA release). Appreciable reutilization of liberated labeled FFA into PC was unlikely due to the short time course in this study. Moreover, no appreciable intrapreparative conversion of lysoPC was observed.
169
Assay of microsomal acyl CoA : lysoPC acyl transferase and lysophospholipase actit~ity Enzyme activities in washed microsomes were assayed in vitro as described elsewhere [12]. Briefly, acyl C o A : l y s o P C acyltransferase activity was d e t e r m i n e d from the rate of production of labeled PC in a mixture containing 100 /.tM [1-14C]palmit%l lysoPC (2.27 ffCi/tzmol), 100 tzM linoleoyl CoA, 50 mM sodium phosphate buffer (pH 7.4) and 5 p,g protein in a volume of 200 /tl. The reaction was initiated by the addition of labeled lysoPC and was carried out at room t e m p e r a t u r e (23°C) for 2.5 min. Lysophospholipase activity was m e a s u r e d by the release of labeled fatty acid (neutral lipid fraction) in a mixture containing 50 p,M [l-~4C]palmitoyl lysoPC (2.27 i z C i / ~ m o l ) , 4 m M MgCI 2, 50 m M sodium phosphate buffer (pH 7.4) and 50 tzg protein in a total volume of 200 ~1. The tubes were incubated a t. 37°C for 15 min. W a t e r was substituted for protein in the reaction blanks. The reaction was stopped by the addition of 1.5 ml of c h l o r o f o r m / methanol ( 2 : 1 , v / v ) . The reaction products were extracted with the addition of 5 5 0 / z l water. After ~x)rtexing and centrifugation an aliquot of the lower phase was drawn and dried u n d e r N 2. Lipids were s e p a r a t e d and analyzed as described above. U n l a b e l e d PC was spotted along with the lipid extract to aid in the identification of the PC band. In o r d e r to assess the effects of p H M B t r e a t m e n t on enzyme activities, reaction mixtures (minus lysoPC substrate) were incubated with the required a m o u n t of p H M B for 30 rain at the desired temperature. T h e reaction was then initiated by the addition of lysoPC substrate as described above. Data analysis Student's t-test was employed to analyze differences in uptake and a m o u n t s of radioactivity in lipid fractions in u n t r e a t e d rat and g u i n e a pig heart microsomes (i.e., controls). Evaluation of statistical significance between u n t r e a t e d and t r e a t e d groups was performed by analysis of variance ( A N O V A ) where indicated in the text. Tukey's test was subsequently used to assess individual differences. Differences with P < 0.05 was considered to be statistically significant. V~lues are expressed as m e a n + S.E. Results LysoPC metabolism by microsomal enzymes" of rat and guinea pig hearts The enzyme activities of acyl C o A : lysoPC acyltransferase and !ysophospholipase were assayed in the microsomal fractions of rat and g u i n e a pig hearts. As illustrated in Table 1 there is substantially more acyl C o A : lysoPC acyltransferase activity than lysophospholipase activity in the microsomal fraction of both rat
TABLE 1 Acticity t¢ mierosomal lysoPC-catabolizing enzTmesmeasuredin I'itro Enzyme activities in rat and guinea pig heart microsomes were assayed as described in Materials and Methods. Values denote mean + S.E. Note that units for enzyme activities differ. Enzyme Acyl CoA : lysoPC acyltransferase (izmol PC/rag protein per h)
Rat 2.01 +_0.65 n=3
Guinea pig 1.69_+0.20 n= 3
Lysophospholipase (nmol FFA/mg protein per h)
44 +_4 n=3
677 4-70 n=3
and guinea pig hearts when m e a s u r e d in vitro u n d e r optimal experimental conditions.
LysoPC handling by isolated rat and guinea pig hearts T h e r e was a m a r k e d difference in the profile of radioactive metabolites of lysoPC in the microsomal fractions obtained from intact rat and guinea pig hearts (Table I1). Less lysoPC remained unmetabolized in g u i n e a pig microsomes than in rat microsomes (a 20% difference). The a m o u n t of radioactivity associated with PC was greater in the rat heart microsomal fraction than in guinea pig microsomes (also 20% greater). In contrast 47% more radioactivity was recovered in the neutral lipid fraction of g u i n e a pig microsomes (indicative of F F A release) compared to rat microsomes. Unlike rat microsomes where no significant difference in the a m o u n t of radioactivity in the PC and neutral lipid fractions was detected, the a m o u n t of radioactivity associated with the neutral lipid fraction of guinea pig microsomes was almost twice as much as that fo: nd with the PC fraction ( P < 0.001). It is also clear that no TABLE 11 Profile of lal~'led lysoPCmetabolites in rat and guinea pig microsomes Isolated rat and guinea pig hearts were perfnsed in the Langendorff mode for 31) min with Kreb's.Henseleit buffer followed by perfusion with buffer containing 2.5 ~M [l-14C]lysoPC(0.0075 u.Ci/ml) for 10 rain. Microsomes were prepared and lipids extracted, separated and analyzed for radioactivity as detailed in Materials and Methods. Values denote mean:i:S.E., n = 7 for rat, n=6 for guinea pig. " P < 0.01 compared to corresponding value in the rat; b p < 0.001 compared to guinea pig PC. Values in parantheses denote average weighted participation in clearance of lysoPC as determined from radioactivity in reaction products. LysoPC (dpm/mg protein)
PC (dpm/mg protein)
FFA + neutral lipid (dpm/mg protein)
Rat
1694+220
4059±494 (48%I
4370=1:317 (52%)
Guinea pig
14085=140
3391 ±309 (35%)
6423+418 ''h 165%)
170 80 T
apparent relationship exists between the activities obtained in vitro under optimal conditions (see Table I) and the relat;ve participation in the intact isolated organ using tile amount of radioactivity in the PC fraction as an index of acyl C o A : I y s o P C acyl transferase activity and the a m o u n t of radioactivity in the neutral lipid fraction as an index of lysophospholipase activity (i.e., FFA release). Deacylation by lysophospholipase is obviously of far greater importance in both preparations than the specific activity m e a s u r e d in vitro would indicate.
60
[[[3 control
[ ~ 2 . 5 ,~M 5.0/zM ~ 7.5/~M E~] 15.0 #M
iinih'itiR
o
PC FFA + Neutral lipid Fig. 1. Profile of labeled lysoPC metabolites in microsomes from untreated and pHMB-treated rat hearts. The isolated rat heart was perfused for 30 lain with Kreb's-Henseleit buffer either without pHMB (i.e. control) or with 2.5 ,uM to 15.0 p.M pHMB (refer to key). Six to eighl hearts were perfused under each experimental condition. Each heart was then perfused for l0 rain with buffer containing both pHMB and labeled lysoPC. Mierosomal lipid analysis was performed as described in Materials and Methods. Data are depicted as % of total radioactivity incorporated into microsomes and represent mean _+S.E. * P < 0.05 compared to control. LysoPC
Effects of pHMB treatment on the amotmt of label Or lipids T r e a t m e n t of rat and guinea pig hearts with p H M B had no effect on the recovery of microsomal protein over the concentration ranges tested as assessed by A N O V A . The average recovery of microsomal protein from control rat heart (no p H M B ) was 1.60 + 0.09 m g / g wet weight (n = 6) and from p H M B - t r e a t e d rat heart 1.41 + 0.08 m g / g wet weight (n = 29). Corresponding recoveries from g u i n e a pig were 1.13 + 0.06 m g / g wet weight (n = 6) and 1.08 + 0.04 m g / g wet weight (n = 16) for control and p H M B - t r e a t e d hearts respectively. U p t a k e of lysoPC by the microsomal fraction did not differ a m o n g control and p H M B - t r e a t e d groups with either the rat or guinea pig heart (by A N O V A ) . Incorporation into rat microsomes was 10120+_800 d p m / m g protein (n = 7 ) and 1 2 9 4 0 + 7 3 2 d p m / m g protein (n = 30) in the absence and presence of pHMB, respectively. Incorporation into g u i n e a pig microsomes was 1123(1 + 671 d p m / m g protein (n = 6) and 11845 + 485 d p m / m g protein (n = 161 in the absence and presence of pHMB, respectively. Moreover, there was no statistically significant difference in uptake of labeled lysoPC by the microsomes between u n t r e a t e d rat and guinea pig hearts. p H M B p r e t r e a t m e n t of the isolated rat heart gave rise to a significant c o n c e n t r a t i o n - d e p e n d e n t increase in the a m o u n t of radioactivity in lysoPC in rat microsomes ( P < 0 . 0 1 ; A N O V A ) (Fig. 1). Control values differed significantly from those obtained at 7.5 # M p H M B (29% increase over control, P < 0.05) and 15.0 tLM p H M B (36% increase over control, P < 0.05). Associated with this increase in the a m o u n t of radioactive lysoPC was a corresponding decrease in the a m o u n t o i radioactivity in neutral lipid ( P < 0 . 0 5 ; A N O V A ) . A level of statistical significance was attained at both 7.5 p,M p H M B (26% decrease c o m p a r e d to control, P < 0.05) and 15.(I/xM p H M B (34% decrease compared to control, P < 0.05). Although there a p p e a r e d to be a trend towards increased a m o u n t of radioactivity in PC, no statistical significance was detected. In contrast, p r e t r e a t m e n t of the isolated guinea pig heart with up to 7.5 ~tM p H M B did not result in any
statistically significant c h a n g e in the a m o u n t of radioactivity in lysoPC ( A N O V A ) (Fig. 2). T h e r e was, however, a c o n c e n t r a t i o n - d e p e n d e n t decrease in the a m o u n t of radioactivity associated with PC ( P < 0.001; A N O V A ) . Values o b t a i n e d at both 5.0 # M p H M B (30% decrease c o m p a r e d to control) and 7.5 p,M p H M B (41% decrease c o m p a r e d to control) were significantly different than those o b t a i n e d with 2.5 p,M p H M B and control (all P < 0.05). A n increase in radioactivity in the neutral lipid fraction was observed over this concentration range ( P < 0.01" A N O V A ) . A level of statistical significance was achieved with 7.5 ,u,M p H M B
80 T
r--3 control
I ~ ;~.5 #aM 60
~
~
5.0/zM 7.5 #M
40
1ii
FFA + Neutral lipid Fig. 2. Profile of labeled lysoPC metabolites in microsomes from untreated and pHMB-treated guinea pig hearts Perfusion of the isolated guinea pig heart and analysis of microsomal iipids were performed according to Materials and Methods. Five to six hearts were perfused under each exgerimental condition. Data presentation as in Fig. 2. * P < 0.05 compared to control and 2.5 gM pHMB. LysoPC
PC
171
3.G1 7.22 10.82 16.23
21,64
3607
ng pHMB/#g protein Fig. 3. Inhibitionof rat and guinea pig heart microsemal lysophospholipase and acyl CoA:lysoPC acyltransferaseby pHMB. Microsomal fractions were prepared and enzymes assayed as described in Materials and Methods. All p,)intsdepict activityobtained relative to control ( n o pHMB pretreatment) with control representing I(lllr;. See Table I for absolute values. Symbols:rat and guinea pig lysophospholipase (zx. a. respectively); rat and guinea pig acyl CoA:lysoPC acyhransferase(c. e. respectively). (26% increase over control) wh:n compared with 2.5 v.M pHMB and control (bo~h P < 0.05).
Inhibition of enzyme activity by p, tMB in vitro In order to determine if there is a qualitative difference in the response of enzyme ,~ctivities to pHMB, microsomal fractions were incub:,:r d with 3.61 to 36.117 ng pHMB/v.g protein for 30 mia prior to the actual assay. As can be seen in Fig. 3 tt ere appears to be no difference in the sensitivity of ra: and guinea pig microsomal lysophospholipases and ; cyl CoA : lysoPC acyl transferase to pHMB-mediated i ahibition in vitro. A slightly lesser amount of p H M B an a per p.g protein basis, was required to produce a level of moderate inhibition of lysophospholipase comparable to that of than acyl CoA:lysoPC acyltransferase under these experimental conditions. However, the enzymes are equally inhibited at higher amounts of pHMB. Discussion Over the last decade numerous published reports have demonstrated the wide-ranging cardiac and vascular effects of lysoPC [2-5,21-24]. Recently, lysoPC has also been implicated as a putative second messenger in some instances based on its likely participation in signal transduction [25]. Despite the ever-increasing awareness of the importance of lysoPC as a modulator of cardiac function, relatively little is known about the mechanisms which govern its level and the dynamics of its clearance in the intact heart. The aim of this study was to assess the importance of acyl CoA : lysoPC acyltransferase and lysophospholipase acttvites in microsomal lysoPC c:ttabolism in a comparative analysis of isolated perfused rat and guinea pig hearts. The intact
isolated perfused heart model used in this study allowed for simultaneous analysis of both acylation and deacylation of lysoPC. In both the rat and guinea pig heart microsomes the capacity for acylation of lysoPC far exceeds that for deacyla~ion based on in vitro analysis, a finding which is in accordance with previous reports [12,26]. It has therefore been proposed that acyl CoA:lysoPC acyl transferase-mediated PC synthesis is the primary mechanism for lysoPC clearance. However, the profile of radioactive metabolites of lysoPC in heart microsomes obtained in this study suggests that deacylation by lysophospholipase is of far greater relative importance than the in vitro data would indicate. Indeed, clearance of lysoPC by deacylation is the preferred pathway for catabolism in guinea pig microsomcs. Deacylation by ly~phospholipase and acyiation by acyl CoA:lysoPC acyltransferase are of equal importance in the clearance of lysoPC in rat microsomes. Thus, the present results suggest that specific activity mea~;'Jred in vitro does not correlate well with relative participation of lysophospholipase and acyl CoA:lysoPC acyltransferase in catabolism of lysoPC in the intact organ, Clearly conditions necessary for optimal expression of activity are not present in the intact organ. There is evidence, however, to indicate that the activity of lysophospholipase measured in vitro is qualitatively associated with thc extent and potential for clearauce by deacylation in the intact organ. The sulfhydryl agent pHMB was used in order to produce enzyme inhibition as a means of assessing the effects of changes in acylation and deacylation on iysoPC clearance. The inhibition of acyl CoA:lysoPC acyltransfcrase and lysophospholipase by pHMB has been previously demonstrated in other models [27,28]. A fortuitous result was the finding that, within the concentration ranges of pHMB evaluated impairment of the microsomal acyl CoA:lysoPC acyltransferase in the guinea pig was manifest in a decrease in the amount of radioactivity associated with PC, whereas in the rat impairment of microsomal lysophospholipase was reflected in a decrease in the amount of radioactivity in the neutral lipid. It was thus possible to measure the metabolic fate of exogenous lysoPC in two species with significantly altered capacities for clearance of lysoPC by these two major routes. Despite decreases in PC synthesis in microsomes from pHMB-treated guinea pig heart there were no differences in the amount of radioactivity in lysoPC because of a compensatory increase in FFA release via lysophospholipase activity. It is probable that a degree of competition for lysoPC substrate exists between the enzymes which enables the lysophospholipase to compensate for diminished acyl CoA:lysoPC acyltransferase-mediated clearance to maintain a constant extent of elimination. Thus, it appears that there is an element of 'crosstalk" bctwcen
172 these enzymes and therefore they are not acting independently of each other. In contrast impairment of deacylation by lysophospholipase in rat microsomes was accompanied by an increase in the level of unmetabolized labeled lysoPC. Evidently rat microsomes are less able to maintain the same level of clearance once lysophospholipase activity has been compromised. Moreover, there appears to be no significant potential for compensatory elimination of lysoPC by acyl C o A : l y s o P C acyltransferase. These findings provide evidence for a link between lysophospholipase activity and the extent of lysoPC catabolism. The inhibition of enzyme activity by p H M B in isolated intact rat and guinea pig hearts, as evidenced in the changes in the amount of label in the respective lipid fractions, cannot be satisfactorily explained by the sensitivity of the microsomal enzymes to p H M B inhibition m e a s u r e d in vitro. It is conceivable, however, that some as yet unknown characteristics of the intact heart are responsible for the a p p a r e n t variance. Myocardial ischemia produces profound alterations in phospholipid homeostasis, an index of which is the rise in lysoPC levels. Increased lysoPC may be brought about by a u g m e n t e d production, reduced catabolism and poor washout or combinations of these. Increased production, as a consequence of e n h a n c e d phospholipase A activity, is generally perceived to contribute to elevated lysoPC in the ischemic heart [29,30]. Several investigators have suggested that impaired catabolism may also factor in the rise of lysoPC [12,31]. In the present study we have clearly shown that inhibition of lysophospholipase activity by p H M B in rat heart microsomes is associated with reduced catabolism of exogenously supplied lysoPC. Several concomitants of myocardial ischemia such as reduced p H and long chain acylcarnitines have been shown to produce potent inhibition of microsomal lysophospholipase [26,32]. Modulation of lysophospholipase activity in vivo during ischemia may thus be a critical d e t e r m i n a n t in the ability to clear lysoPC and maintain basal levels. Acknowledgements This investigation was supported by the Medical Research Counci! of Canada. T.M. is the recipient of a H e a r t and Stroke Foundation of C a n a d a Traineeship. References 1 Wehzien, H.U. (1979) Biochim. Biophys. Acta 559, 259-287. 2 Sobel, B.E., Corr, P.B., Robison, A.K., Goldstein, R.A.,
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