Heparin-independent release of lipoprotein lipase activity from perfused rat hearts

Biochimica et Biophysics Acta, 153 (1983) 41-52

47

Elsevier

BBA 5 1460

HEPARIN-INDEPENDENT RAT HEARTS GREGORY

RELEASE

OF LIPOPROTEIN

LIPASE ACTIVITY

FROM PERFUSED

J. BAGBY

Department of Physiology, Louisiana State Unioersiiy Medical Center, New Orleans, LA 70112 (U.S.A.) (Received December (Revised manuscript

30th. 1982) received May 2Oth, 1983)

Key words: Lipoprotein lipase; Heparin; Creatine kinase; (Rat heart)

Heparin-independent release of lipoprotein lipase activity from isolated perfused rat hearts was measured and related to the rapid turnover of the enzyme. Hearts consistently released lipoprotein lipase activity (2.1 * 0.2 U/g released per min) during 60 min of nonrecirculating perfusion without heparin. This rate of release did not significantly differ from that measured in heparin-perfused hearts after the first 10 min of perfusion (2.2 + 0.2 U/g released per min). The fractional release rate of lipoprotein lipase activity during nonheparin perfusion was 1.3% per min, which was higher than that calculated for alkaline phosphatase (0.002%) and creatine kinase (0.03%) activities. The lipase activity released was activated 4-fold by serum and inhibited 94 and 88% by 0.5 M NaCl and 3 mg/ml protamine sulfate, respectively. Lipoprotein lipase activity in the 1-min heparin-releasable (extracellular) and residual (intracellular) compartment remained stable during the last 40 min of nonheparin perfusion. During this period total heart, intracellular and extracellular enzyme t,,, were calculated to be 52, 42 and 10 min, respectively. The results are consistent with the postulate that continuous release of lipoprotein lipase into the vascular compartment may be an important determinant of its rapid turnover in the heart, and possibly other tissues.

Introduction Several investigators have studied the release of lipoprotein lipase activity from hearts perfused with heparin-containing media [l-4]. The portion released during the first minute of perfusion is believed to be primarily associated with the luminal surface of capillary endothelial cells and to function in the hydrolysis of lipoprotein triacylglycerol [5-71. Some investigators have detected lipoprotein lipase activity in coronary effluents from hearts perfused with medium not containing heparin [1,3,5]. Neither the quantitation nor the significance of this release has been addressed experimentally even though a slow continuous release of lipoprotein lipase may be important to the rapid turnover of this enzyme [4,8,9]. 00052760/83/$03.00

0 1983 Elsevier Science Publishers

B.V.

Thus, the present study was initiated to examine heparin-independent release of lipoprotein lipase activity from isolated perfused hearts and to determine if this release could contribute in a major way to the overall turnover of the enzyme. Methods Experimental procedures Adult male Sprague-Dawley rats (300400 g range) purchased from Charles River Breeding Laboratories were maintained for l-3 weeks in a controlled environment with a 12 h light/l2 h dark cycle and allowed access to Purina rat chow and water ad libitum. Rats were fasted for 24 h prior to experiments which were conducted 4-6 h into the light cycle. Rats were anesthetized with

48

diethyl ether so that 4-5 ml of blood could be obtained from abdominal aortas before hearts were rapidly excised. Serum was pooled from rats on a given experimental day and used to activate lipoprotein lipase. Hearts were quickly rinsed in room-temperature 0.9% NaCl prior to being mounted by the aortic stump to a flanged micropipette tip connected to a nonrecirculating Langendorff-type perfusion apparatus. Hearts were perfused for 1 min with a medium consisting of Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 10 mM glucose, 10 mM pyruvate and 1% bovine serum albumin (termed nonheparin medium) to remove trapped blood from the coronary vasculature. All perfusion media were continuously gassed (5% CO, in 95% 0,) and filtered through a 5 pm polycarbonate membrane. Perfusions were conducted at a pressure of 60 cmH,O and 37°C. After the initial 60 s of perfusion, hearts (n = 56) were subjected to one of seven conditions. One group of hearts was terminated in order to obtain the initial total lipoprotein lipase activity. A second group of hearts was immediately transferred to a second perfusion apparatus containing the identical medium supplemented with 5 U heparin/ml (termed heparin medium) and perfused for 1 min. Four subgroups of hearts were enclosed in a 37°C chamber and perfused with nonheparin medium for 5, 20, 40 or 60 mm. At the end of these selected times, these hearts were perfused for 1 min with the heparin medium. The seventh group of hearts was perfused with the heparin medium for 60 min. Coronary effluents were collected at designated intervals during perfusion in tubes immersed in ice in order to minimize exposure time of released enzyme to the 37°C perfusion temperature. Enzyme activities were stable for at least 6 h when samples were kept at 0-4°C while assays were completed within 4 h of the collection time. Volume was determined so that coronary flow could be calculated and enzyme activities expressed on a heart weight basis. At the termination of the respective perfusion conditions, hearts were removed from the perfusion apparatus, blotted, trimmed of atria1 tissue and weighed. Hearts were then minced prior to weighing an atiquot, upon wmch enzyme activities were measured.

Assay procedures Lipoprotein lipase activity was measured under optimal conditions in hearts homogenized in heparin medium and in coronary effluents [ 10,111. Heparin was added to those coronary effluents not containing heparin so that the same heparin concentration (5 U/ml) was present in all samples. Heart tissue was homogenized in heparin medium (1 : 40) to match the assay conditions of the coronary effluent. Preliminary experiments indicated that enzyme activity measured in this medium did not differ from that measured in more commonly used buffers, viz., 1.O M ethylene glycol in 0.05 M Tris-HCl or 0.05 M NH,OH/HCl buffer [lO,ll]. Assays were initiated by adding a 50-~1 sample of coronary effluents or 50 ~1 of a 1 : 40 homogenate to a reaction mixture containing 100 ~1 glycerol-based stable triolein emulsion, 25 ~1 heated serum (60°C for 20 min) and 75 ~1 Ringer solution. The reaction was conducted at pH 8.4 and 37°C for 40 min. Termination of the reaction, extraction of released fatty acids and counting in a liquid scintillation spectrometer were accomplished as described by Nilsson-Ehle and Schotz [ 111. The inter-assay coefficient of variability was 9.6% as determined from stock postheparin plasma lipase, stored at - 50°C. Lipoprotein lipase activities for coronary effluents and tissue homogenates are expressed as pmol free fatty acids liberated/h (unit) per g wet weight of heart. Tissue and coronary effluent alkaline phosphatase [ 121 and creatine kinase [ 131 activities were also determined. The alkaline phosphatase assay was conducted on 0.7 ml of coronary effluent or 25 ~1 of a 1 : 40 dilution of the heart homogenate added to 0.675 ml of the heparin medium in a final volume of 1.0 ml at 30°C and pH 10.3. Creatine kinase activity was measured on 0.5 ml of coronary effluent or 10 ~1 of a 1 : 800 dilution of the heart homogenate added to 0.49 ml of the heparin medium in a final reaction volume of 1.0 ml at 30°C and pH 6.7. Alkaline phosphatase and creatine kinase activities are expressed as pmol substrate/min (unit) per g wet weight. Myocardial ATP, creatine phosphate and glycogen concentrations were determined on some hearts perfused for 60 min with the nonheparin or heparin medium. At the end of perfusion, hearts were clamped in tongs precooled in liquid nitrogen. The

49

tissue was powdered at the temperature of liquid nitrogen. For ATP and creatine phosphate, a portion of the powder was deproteinized with 2 M perchloric acid and neutralized with K&O,. The protein-free extract was used for the determination of ATP and creatine phosphate [ 141.Glycogen was extracted from a second aliquot subsequent to analysis with the anthrone reagent [ 151.

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Nonhewrin

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Statistical analysis Data were evaluated by two-way analysis of variance with each experimental day serving as a block [ 161. Individual means were compared using Duncan’s new multiple range test [ 161.Means were considered significantly different from one another if P < 0.05. Minutes

Results

Coronary flow rates were 12.5 * 0.5 and 11.1 + 0.8 ml/mm per g wet weight (n = 8) in nonheparin- and heparin-perfused hearts, respectively, after 10 mm and did not change significantly during the subsequent 60 min perfusion period (11.5 * 0.6 and 12.8 * 0.9 ml/mm per g, respectively). Heart rates declined from 234 * 4 beats/mm after 10 min of perfusion to 218 f 4 after 60 min. No differences existed between heparin- and nonheparin-perfused hearts in ATP, creatine phosphate or glycogen tissue content after 60 min. Glycogen content was 21.2 + 1.5 pmol glucose/g wet weight (n = 9) which is similar to values reported in the intact rat heart [ 171. Likewise, ATP (4.1 + 0.18 pmol/g, n = 9) and creatine phosphate (6.66 k 0.48 pmol/g, n = 9) contents also resembled levels in the intact heart [18], indicating that the perfusion conditions employed did not cause meaningful deterioration of these hearts during the perfusion period. Lipoprotein lipase activity was continuously present in coronary effluents of hearts perfused for 60 mm with the nonheparin medium (Fig. 1). The rate of release did not significantly change over the 60 mm perfusion period. The enzyme activity present in the coronary effluent of nonheparin-perfused hearts was quite low due to the slow rate of release and the large dilution. However, this problem could be minimized by concentrating these samples with 25 000 molecular weight ultrafiltra-

Fig. 1. Lipoprotein hpase activity released per mm from hearts perfused for 60 min with or without 5 U heparin/ml in the perfusion medium. Data are expressed as X f S.E. (n = 8). From 10 to 60 min of perfusion the rates of enzyme release between heparin- and nonheparin-perfused hearts were not statistically different.

tion membranes cones. Using this procedure 100% of the lipase activity was recovered and exhibited characteristics of lipoprotein lipase in that it was activatable by serum (AO-fold), and inhibited 94%

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Fig. 2. Lipase activity in coronary effluent of hearts perfused without heparin as a function of serum concentration and the inhibition of serum-activated lipase by 0.5 M NaCl and 3 mg/ml protamine sulfate (P.S.). Data are expressed as f f S.E. (n = 6). Coronary effluent used in these assays was collected between 20 and 22 min of in vitro perfusion.

50

by 0.5 M NaCl and 88% by 3 mg/ml protamine sulfate (Fig. 2). Perfusion with the heparin medium resulted in a rapid release of lipoprotein lipase activity during the first minute followed by a slower rate of release at later time periods (Fig. 1). After the tenth minute of perfusion, the rate of release in heparin-perfused hearts (2.2 + 0.4 U/g released per min) did not differ significantly from that measured in nonheparin-perfused hearts (2.1 ~fr0.2 U/g released per min). The release of lipoprotein lipase activity during nonheparin perfusion may have resulted from a net loss of enzyme activity from the heart. Thus, the effect of nonheparin perfusion on total lipoprotein lipase activity and its distribution between I-min heparin-releasable (extracellular) and heparin-residual (intracellular) compartments were determined at selected times during the perfusion. In hearts perfused for 1 min with the nonheparin medium followed by 1 min with the heparin medium the summation of 1-min heparin-releasable and -residual lipoprotein lipase activities (205 k 20 U/g, n = 8) was not significantly different from enzyme activity measured in hearts perfused

only with the nonheparin medium (188 * 24 U/g, n = 8). Thus, the sum of these two fractions may be taken as total enzyme activity in the heart at the time a I-min heparin perfusion was initiated. Fig. 2 summarizes the effect of perfusion time with nonheparin medium on total, heparin-releasable and -residual lipoprotein lipase activity in the heart. During the first 20 min of perfusion with the nonheparin medium a 25% decrease of total heart lipoprotein lipase activity took place (Fig. 2). Furthermore, this decrease was predominantly confined to the I-min heparin-releasable compartment which declined from 69.1 + 11.6 to 36.3 & 3.1 U/g during this period. During the final 40 min of nonheparin perfusion enzyme activity in the 1-min heparin-releasable compartment did not change further. Residual lipoprotein lipase activity did not significantly decline during the 60 min period of perfusion with the nonheparin medium. Creatine kinase and alkaline phosphatase activities were also detected in the coronary effluents of nonheparin- and heparin-perfused hearts (Fig. 3) but their respective rates of release were not affected by the presence of heparin in the medium (data not presented). The release of creatine kinase activity declined as perfusion continued. During

21

30 7

20

40

Alkaline

phosphatase activity

Creatine kinase activity

60

Minutes

Fig. 3. Distribution of lipoprotein lipase activity between I-min heparin-releasable and -residual compartments. Hearts were perfused with nonheparin medium for O-60 mm followed by a l-mm perfusion with heparin medium. Total enzyme activity is the sum of enzyme activities measured in the two compartments. Data are expressed as H f SE. (n = 8). * Significant difference from hearts after 1 min preperfusion washout (P -C 0.05).

Minutes Fig. 4. Alkaline phosphatase and creatine kinase activities released per min from hearts perfused for 60 min. Data are expressed as X f S.E. (n = 6). * Significant difference from the initial rate of release (P -c 0.05).

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the 60 min perfusion period 0.12% (728 f 9 mu/g) and 1.8% (43.6 f 12.2 mu/g) of heart creatine kinase and alkaline phosphatase activities were released. This release was considerably less than the 61% (126 U/g) of the initial lipoprotein lipase activity released from hearts during 60 min of nonheparin perfusion. Discussion

TABLE

I

FRACTIONAL RELEASE RATE CONSTANT (K,,,) AND HALF-LIFE (t,,,) FOR LIPOPROTEIN LIPASE ACTIVITIES IN THE HEART AND ITS INTRACELLULAR AND EXTRACELLULAR COMPARTMENTS. Estimates are based on the rate of release of lipoprotein lipase during the final 40 min of nonheparin perfusion and the respective enzyme activities in the heart or the two compartments. Compartment

Heparin-independent release of lipoprotein lipase activity took place throughout the 60 min nonheparin perfusion period. Some investigators have reported low lipase activities in coronary effluents from hearts perfused with a nonheparin medium [ 1,3,5,17]. In general, this release has been dismissed as unimportant or inconsequential. However, Crass and Meng [l] suggested that a continuous release may have physiological significance especially because endogenously produced heparin may be sufficient to augment enzyme release into the circulation. Recently, Wallinder et al. [ 191described the rapid removal of 125I-labelled lipoprotein lipase by the liver and suggested that this process may have an important physiological purpose of keeping the circulating levels of this enzyme low. Although lipoprotein lipase activity in the heparin-releasable compartment (extracellular) of the heart declined during the first 20 min of perfusion, this activity, together with that associated with the heparin-residual compartment (intracellular), remained stable during the subsequent 40 min of perfusion. Despite this stable distribution of lipoprotein lipase activity between these two compartments, enzyme activity was continually being released into the nonbeparin perfusion medium. This indicates that a steady state of heart lipoprotein lipase activity was achieved during the final 40 min of nonheparin perfusion. During this period the heart contained 157 U/g of lipoprotein lipase activity divided between the intracellular (126 U/g) and extracellular (31 U/g) compartments. At the same time 2.1 U of activity were released per min from the heart. This rate of release represents the minimal turnover of the enzyme in the heart, since it does not take into account enzyme degraded or inactivated within the heart. Existing evidence suggests that the intracellular enzyme fraction is the

Intracellular

&

Extracellular

$

Total

&

= 0.0166 (1.7%) = 0.0686 (6.9%) = 0.0134 (1.3%)

41.7 10.1 51.8

precursor to the extracellular enzyme [4,6,20]. Furthermore, it is likely that enzyme appearing in the coronary effluent during nonheparin perfusion is released from the extracellular compartment. Thus, the half-life of the enzyme in the heart and the two compartments, accounted for by this rate of release, can be estimated from the equation t,,, = In 2/K,,,, where Kre, is the fractional release rate constant (Table I). Due to the larger size of the intracellular pool of lipoprotein lipase activity, it necessarily has a slower half-life (42 min) than that estimated for the extracellular compartment (10 mm). The results indicate that the flux of lipoprotein lipase activity through the extracellular compartment is extremely rapid. Borensztajn et al. [4] estimated the half-life to be about 2 h for extracellular lipoprotein lipase following the administration of cycloheximide. Because treatment with this protein synthesis inhibitor does not preclude the secretion of existing intracellular enzyme into the extracellular compartment, their procedure may lead to an overestimate of the half-life for the extracellular compartment. The calculated half-life of total heart lipoprotein lipase activity is at the lower end of previously reported estimates for this enzyme [4,8,21]. Previous half-life estimates have been calculated from exponential decay curves of enzyme activity following protein synthesis inhibition with either cycloheximide or puromycin. Use of these inhibi-

52

tors has demonstrated the importance of protein synthesis in maintaining myocardial lipoprotein lipase activity. That release of the enzyme into the coronary effluent of the perfused heart gives a similar estimate of its turnover suggests that release maybe an important determinant of the loss of this enzyme from the heart. Other investigators have been unable to detect lipoprotein lipase activity in coronary effluents of nonheparin-perfused hearts [20,22]. Although an explanation for this discrepancy is not evident, a number of methodological differences exist between heart perfusion systems and enzyme assay conditions. For example, Fielding and Higgins [20] assayed for the enzyme at a lower than optimal pH of 7.5. The consistent release of lipoprotein lipase activity observed during nonheparin perfusion and the decline in 1-min heparin-releasable enzyme activity seen during the first 20 min of nonheparin perfusion were not likely caused by deterioration of the hearts during perfusion, since they remained relatively stable in other respects. Also, the fractional release rate of lipoprotein lipase activity (1.3%/min) was substantially higher than that measured for creatine kinase (O.O02%/min) and alkaline phosphatase (O.O3%/min) activities. The present study has demonstrated that lipoprotein lipase activity is released from in vitro perfused hearts with a nonheparin medium at rates sufficient to account for the recognized rapid turnover of this enzyme. The presented calculations depend on the accuracy of the enzyme activity assay to reflect enzyme protein mass. Furthermore, rates of enzyme release may have been influenced by the perfusion conditions used in the present series of experiments. In this regard, the perfusion medium used in these experiments was distinctly different from blood; however, no evidence is currently available to indicate that the medium used would preferentially release lipoprotein lipase from the heart in a way that blood would not release the enzyme. In any event, additional studies will need to be performed to determine if heparin-independent release of lipoprotein lipase activity has physiological significance to the overall turnover of this enzyme.

Acknowledgments

I would like to express sincere appreciation to the tireless and proficient technical assistance of Constance Corll. Valuable suggestions were also received from P.S. Roheim and R.A. Davis. This study was supported by National Institutes of Health grant HL 23329. References I Crass, M.F. and Meng, H.C. (1964) Am. J. Physiol. 206, 610-614 2 Robinson, D.S. and Jennings, M.A. (1965) J. Lipid Res. 6, 222-227 M. and Torii, S. (1961) Proc. Sot. 3 Nakatani, M., Nakamura, Exp. Biol. Med. 107, 853-856 4 Borensztajn, J., Rone, MS. and Sandros, T. (1975) Biochim. Biophys. Acta 398, 394-400 5 Enser, M.B., Kunz, F., Borensztajn, J., Opie, L.H. and Robinson, D.S. (1967) Biochem. J. 104, 306-317 6 Borensztajn, J. and Robinson, D.S. (1970) J. Lipid Res. 11, Ill-117 7 Kompiang, I.P., Bensadoun, A. and Yang, M.W.W. (1976) J. Lipid Res. 17, 498-505 8 Wing, D.R., Fielding, C.J. and Robinson, D.S. (1967) Biothem. J. 104, 45C-46C Bio9 Chajeck, T., Stein, 0. and Stein, Y. (1978) B&him. phys. Acta 528, 456-465 10 Bagby, G.J. and Spitzer, J.A. (1980) Am. J. Physiol. 238, H325-H330 P. and Schotz, M. (1976) J. Lipid Res. 17, 11 Nilsson-Ehle, 536-541 12 Bowers, G.N. and McComb, R.B. (1966) Clin. Chem. 12, 70-89 M., Magid, E., Pitkanen, E., Harkonen, M., 13 Horder, Stromme, J.H., Theodorsen, L., Gerhardt, W. and Waldenstrom, J. (1979) Stand. J. Clin. Lab. Invest. 39, l-5 J.V. (1972) A Flexible Sys14 Lowry, O.H. and Passonneau, tem of Enzymatic Analysis, pp. 15 1- 154, Academic Press, New York 15, 15 Roe, J.H. and Dailey, R.E., (1966) Anal. Biochem. 245-250 16 Steel, R.G.D. and Torrie, J.H. (1960) Principles and Procedures of Statistics, McGraw-Hill, New York 17 Daw, J.C., Lefer, A.M. and Berne, R.M. (1968) Circ. Res. 22, 639-647 J.G., Schwab, G.E., Ross, J. and Mayer, S.E. 18 Dobson, (1974) Am. J. Physiol. 227, 1452-1457 L., Bengtsson, G. and Olivecrona, T. (1979) 19 Wallinder, B&him. Biophys. Acta 575, 166-173 13, 20 Fielding, C.J. and Higgins, J.M, (1974) Biochemistry 4324-4330 D.S. (1968) Biochem. J. 106, 21 Wing, D.R. and Robinson, 667-676 J., Rone, M.S. and Kotlar, T.T. (1976) Bio22 Borensztajn, them. J. 156, 539-543