260
Biochimica et Biophysics Acta, 388 (1975) 260-267 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56604
INTERFERENCE WITH THE TRANSPORT OF HEPARIN-RELEASABLE LIPOPROTEIN LIPASE IN THE PERFUSED RAT HEART BY COLCHICINE AND VINBLASTINE
T. CHAJEK, 0. STEIN and Y. STEIN Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital, and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem (Israel) (Received December 9th, 1974).
Summary
The effect of pretreatment with colchicine or vinblastine on the lipoprotein lipase activity of rat heart was studied. Administration of colchicine or vinblastine 4 h prior to perfusion of the heart caused a very marked reduction in lipoprotein lipase activity released into the perfusate within 1 min of heparin perfusion. At the same time an increase in residual heartlipase occurred so that total lipoprotein lipase content of the heart (heparin releasable plus residual) did not change. The colchicine effect was dose and time dependent; no decrease in heparin-releasable enzyme activity occurred after only 30 min of pretreatment or upon addition of colchicine into the perfusate. .These results indicate that colchicine did not impede enzyme synthesis or its release from the cell surface, but may have interfered with the transport of lipoprotein lipase from the site of its synthesis to the endothelial cell surface.
Introduction
In a previous study it was shown that in colchicine- or vinblastine-treated rats less lipolytic activity is released by heparin than in the control animals [l] . Recent studies have provided evidence that heparin-releasable lipolytic activity is derived from the liver and peripheral tissues and that the two are inhibited by protamine to a different extent [2]. On the basis of this difference in sensitivity to protamine it seemed that the peripheral lipase might be more affected by the treatment with the antimicrotubular drugs [ 11. The heart is known as a rich source of lipoprotein lipase which can be readily released into the perfusate by heparin in an in vitro system [ 3,4]. The aim of the present study was to investigate the effect of colchicine on peripheral lipoprotein lipase in an
261
isolated perfused rat heart. This system enabled also to determine whether the previously reported [l] fall in heparin-releasable lipase might be due to the inhibition of its transport to the endothelial cell surface, or could also be affected by inhibition of enzyme synthesis. Materials and Methods Animals Female rats of 180-200 g body weight of the Hebrew University strain kept in constant temperature rooms and fed the pelleted Am-Rod 931 diet [ 51, were used. For the duration of the experiment, between 8 a.m. and 12 noon, the rats were deprived of food. Colchicine (Sigma Chemical Co., St. Louis, MO.) 0.05 or 0.5 mg/lOO g body weight and vinblastine sulphate (Eli Lilly. Co., Indianapolis, Ind.) 1.0 mg/lOO g body weight dissolved freshly in 0.9% NaCl were injected intraperitoneally in a volume of 0.5 ml at time zero; control animals were injected with 0.5 ml, 0.9% NaCl. Perfusion of heart Rat hearts were excised under ether anaesthesia, freed of residual blood with 0.9% NaCl at room temperature and perfused as previously described [6] in a non-recirculatory system with Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 4% bovine serum albumin (fatty acid poor, Miles Lab., Kankakee, Ill.) and 200 mg/dl glucose. After the passage of lo-12 ml of perfusion medium and reestablishment of a regular heart beat, the flow rate was about 8-10 ml/min. Heparin (Evans Medical Corp., Liverpool, U.K.) was injected into the reservoir to give a final concentration of 5 units/ml and the perfusion fluid which passed through the heart was collected in ice-cooled tubes between O-l min, l-3 min, and 3-5 min. At the end of perfusion the heart was blotted. the cleaned ventricles were weighed and aliquots were removed for homogenization. 160-180-mg aliquots of ventricular muscle were coarsely minced with scissors and homogenized in ice-cold 8 ml of medium using the Polytron (Kinematica GmbH Luzern, Switzerland) homogenizer with the pt 10-11 probe at maximum speed for 2 min at 0°C. The homogenization medium consisted of 0.025 M NH3 /NH4 Cl buffer (pH 8.1) and heparin at final concentration of 1 unit/ml; aliquots of the homogenate were taken for the determination of lipoprotein lipase. To separate “soluble” and bound enzyme activity in hearts which had not undergone perfusion the homogenates were centrifuged for 30 min at 105 000 X g at 4°C in a Spinco L50 ultracentrifuge. The residue was resuspended in the original volume of homogenization medium and lipoprotein lipase activity was determined on both the supematant and resuspended residue fractions. Determination of lipoprotein lipase activity The lipoprotein lipase activity was determined on aliquots of perfusates and homogenates according to Schotz et al. [7] using triolein in the form of an emulsion as substrate. To prepare the emulsion the following ingredients were suspended in 16 ml of 0.2 M Tris * HCl buffer, pH 8.6, containing 0.15 M NaCl; 100 nmol of glyceryl tri[l- ’ ‘C] oleate (spec. act. 54.9 Ci/mol, Amersham/Sear-
262
ly Corp., Arlington Heights, Ill.); 22.6 E.cmol carrier triolein (Sigma Chemical Co., St.Louis, MO.), 14.4 mg bovine serum albumin (fatty acid poor, Miles Lab., Kankakee, Ill.) and 0.36 ml of 1 : 1000 solution of Triton X-100 (B.D.H. Chemicals, Poole, U.K.). The suspension was subjected to ultrasonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany) using a probe of 10 mm in diameter, at maximal scale for 4 min at 4°C. 4.8 ml of fasting human serum were added to 15 ml of the substrate emulsion which was used within 30 min of preparation. The assay system consisted of heart perfusate or homogenate, 0.2-0.4 ml; 0.6 ml of the emulsified substrate and was brought to final volume of 1.0 ml with 0.15 M NaCl. Incubations were carried out in duplicate for 20 min at 37°C in a shaking incubator. The reaction was terminated by addition of 4 ml of isopropanol/l.5 M Hz SO4 (40 : 1, v/v) according to Schotz et al. [7] and the free fatty acids were adsorbed on Amberlite IRA 400 (B.D.H. Chemicals, Poole, U.K.) as described by Kelley [8]. After the resin was washed four times with 5 ml of hexane, 1 ml of Soluene 100 (Packard Inst. Co., Downers Grove, Ill.) and 15 ml of scintillation fluid (4 g 2,5-diphenyloxazole and 100 mg 1,4-bis-[2-(4-methyl-5-phenyloxazolyl)] -benzene in 1000 ml toluene) were added and the samples were counted in a Tricarb liquid p scintillation spectrometer 3380 equipped with an absolute activity analyzer, model 544. The radioactivity present in the hexane phase in samples incubated without the enzyme was subtracted from all experimental values. The activity in perfusates was expressed per g heart weight. To measure the effect of protamine and NaCl on the lipolytic activity, 0.2 ml of rat heart homogenate or perfusate were pre-incubated for 10 min at 27°C with 0.1 ml of 0.2 M Tris * HCl buffer, pH 8.6 containing 6 mg/ml of protamine sulfate (salmine, Sigma Chemical Co., St. Louis, MO.) or with 0.2 ml of 2 M NaCl. Control assays were performed also in the,absence of serum. Results In order to determine whether injection of colchicine affects the content of lipoprotein lipase in the heart, enzyme activity was determined 4 h after injection of either colchicine or 0.9% NaCl to female rats, which were deprived of food between 8 a.m. and 12 noon. The total enzyme activity recovered in the heart was not affected by the injection of colchicine and there was no TABLE
I
LIPOPROTEIN LIPASE ACTIVITY HOMOGENATES
IN SUPERNATANT
AND RESIDUE
FRACTIONS
OF RAT HEART
All rats were deprived of food at zero time and were injected with either 0.9% NaCl or 0.5 mg/lOO g body weight of colchicine; at 4 h hearts were removed, homogenized, centrifuged and enzyme assay performed on samples of 105 000 X g supematant and on the residue. Figures in parentheses represent number of experiments; values are means kS.E., fatty acid released. wmol/g tissue/h. Treatment
0.9% N&l Colchicine
Distribution of enzyme activity
(6) (6)
supematant
Residue
66.5 k I.2 61.3 f 2.6
97.1 + 9.1 90.4 * 7.2
263
TABLE
II
EFFECT OF COLCHICINE AND VINBLASTINE LIPASE IN PERFUSATES OF RAT HEART
ON THE
HEPARIN-RELEASABLE
AII rats were deprived of food at zero time and were injected with the indicated moved and perfused 4 h after the injection. Figures in parentheses represent values are means +S.E., for fatty acid released, pmol/g tissue/h. Treatment
Perfusate* &-1
0.9% NaCl (15) Colchicin? 0.5 mg* * (5) Colchicine 0.05 mg* * (5) Vinblastine 1.0 mg** (4) * The activity in the perfusate ** Per 100 g body weight.
28.1 5.0 16.4 9.5
(min after heparin injection) l-3
* ? f f
1.9 0.6 4.0 1.7
3-5
10.1 9.1 9.8 11.5
was expressed
? * + +
1.4 1.5 1.6 2.0
5.1 5.5 3.3 4.3
+ f * *
0.9 1.6 1.0 0.9
LIPOPROTEIN
materials; hearts were renumber of experiments:
Heart at end of perfusion
Total enzyme activity
56.7 85.1 60.3 85.1
100.6 + 5.1 105.3 * 6.4 89.8 * 7.0 110.6 + 4.2
+ f + k
5.5 6.3 6.4 6.0
per g heart weight.
significant difference in the distribution of enzyme activity between the supernatant and residue fractions (Table I). In the next series of experiments the effect of pretreatment of the rats with two antimicrotubular agents, colchicine and vinblastine, was tested on the lipoprotein lipase which can be released into a non-recirculatory perfusion system by the introduction of heparin. The results presented in Table II show that the release of the enzyme occurs almost immediately after introduction of heparin and that between O-l min a sharp peak of enzyme activity appears in the perfusion medium. The rate of lipoprotein lipase release decreases sharply during the subsequent time intervals studied. Thus, between l-3 min of perfusion, the activity releasable per min decreases by more than 80% and during the next 2 min less than 10% of the initially releasable activity appeared in the perfusate per min. Pretreatment of the rats with 0.5 mg/lOO g body weight of colchicine 4 h prior to perfusion resulted in a very sharp decrease (P < 0.001) in heparin-releasable activity during the first min of perfusion. The enzyme activity which appeared in the perfusate during the subsequent two periods of perfusion (1-3 min and 3-5 min) did not differ significantly from that in the control group (Table II). As in the colchicine-treated rat more lipoprotein lipase was found in the heart than in the saline injected rat (P < O.OOl), it is evident that the total enzyme activity (which is the sum total of perfusate and homogenate activities) was the same in the experimental and control groups. A similar degree of decrease of the rapidly releasable enzyme activity was found also after pretreatment of the rats with vinblastine, given at an equimolar concentration to that of colchicine. Pretreatment of the rats with colchicine 0.05 mg/lOO g body weight also caused a decrease (p < 0.02) in the rapidly releasable enzyme activity (Table II). In order to learn more about the effect of colchicine in this system, the drug was added to the perfusate at a concentration which might have been reached after injection of colchicine in vivo, assuming a homogeneous distribution in body water. As seen in Table III the presence of 2.5 * 10e5 M colchicine in the perfusate did not lower the enzyme release by heparin and did not affect the enzyme activity recovered in the heart homogenate. The time course of colchitine action is shown also in Table III. No effect was seen after 30 min of
264 TABLE
III
HEPARIN-RELEASABLE LIPOPROTEIN LIPASE IN PERFUSATES TIME INTERVALS AFTER PRETREATMENT WITH COLCHICINE
OF RAT
HEART
AT VARIOUS
AII rats were deprived of food throughout the experimental period. Colchicine in perfusate was 2.5 . low5 M. the dose of colchicine in viva was 0.5 mg/lOO g body weight. Figures in parentheses represent number of experiments; values are means *S.E., fatty acid released, Iumol/g tissue/h. Treatment
Perfusate*
0.9% NaCl (6) Colchicine added to perfusate (4) Colchicine in viva 30 min (3) 120 min (3) 240 min (3) * The activity
in the perfusate
(min after heparin
e-1
l-3
3-5
Heart at end of perfusion
31.7 * 0.5 37.9 + 2.5
14.3 + 3.1 14.7 * 1.5
4.6 k 0.9 3.7 + 0.2
52.6 ? 3.8 56.5 f 6.7
102.5 111.7
+ 6.7 + 8.2
33.4 + 2.7 21.9 * 2.9 4.8 f 0.6
11.5 + 2.7 11.3 k 0.3 9.7*3
4.0 k 1.3 7.0 * 1.5 -
72.1 -r 4.8 88.3 C 3.3 99.3 f 6.7
120.1 128.6 113.9
c 3.3 f 4.7 + 5.6
was expressed
injection)
Total enzyme activity
per g heart weight.
pretreatment and a smaller, but statistically significant fall (p < 0.02) in the Pretreatrapidly releasable enzyme peak was observed 2 h after pretreatment. ment with colchicine for 4 h resulted in a very marked decrease in the lipoprotein lipase activity releasable during the first min of perfusion. The comparison of the activity recovered in the heart at the end of perfusion shows that in rats pretreated with colchicine for 2 or 4 h more enzyme activity (p < 0.001) was recovered in the heart homogenate than in the hearts of the 0.9% NaCl-injected rats. To define more closely the lipolytic activity measured, assays were performed also on samples which had been preincubated with protamine or 1 M NaCl. As shown in Table IV, protamine and high NaCl concentration inhibited the activity of the enzyme released into the perfusate, as well as that determined in homogenates of hearts at the end of perfusion. No enzyme activity in either perfusate or heart could be measured in the absence of serum in the assay system.
TABLE
IV
EFFECT OF INHIBITORS RAT HEART Values expressed Enzyme
source
Heart Perfusate * 0-lmin l-3 min 3-5 min * The activity
ON LIPOPROTEIN
as fatty acid released. Control 44.6
pmol/g
LIPASE
AND
HOMOGENATES
tissue/h.
1 M NaCl
* 1.6
0.5 f 0.1
32.0 k 0.8 12.2 * 3.0 3.5 i 0.2
0.6 f 0.1 >0.5 >0.5
in the perfusate
IN PERFUSATES
was expressed
Protamine >0.5 >o. 5 >0.5 >0.5
per g heart weight.
Minus serum 2.0 + 0.6 1.1 f 0.3 >0.5 >0.5
OF
265
Discussion Antimicrotubular agents, colchicine and vinblastine have been shown to arrest processes which involve cellular transport of various products in secretory vesicles or vacuoles [g--13] . Since impedance with secretion of proteins could also be due to the interference with their synthesis, protein synthesis was determined in colchicine-treated rat [14] and mouse [15] liver and was found to be normal. However, some reduction of proline incorporation into collagen by colchicine at a concentration of 1 - 10e4 M was reported recently by Ehrlich et al. [16] and hence it became important to determine whether under our experimental conditions (estimated colchicine concentration 2 * lo-’ M) colchitine does affect the synthesis of the lipoprotein lipase. Since the total lipoprotein lipase activity in hearts of colchicine-treated rats was not lower than that of the controls, it seems that in the intact rat, colchicine did not affect enzyme production, in analogy to serum lipoproteins and serum albumin [ 14,151. Neither did colchicine affect the fraction of the enzyme which remained in the supematant after homogenization of the heart and centrifugation. The most pronounced effect of colchicine in the present study was on the so-called “heparin-releasable activity”, a term used by Rogers and Robinson [17] for the enzymic activity released into the perfusate during the first min of heparin perfusion of the rat heart. These authors have included the activity appearing in the perfusate after the first min in the non-releasable enzyme fraction, the bulk of which consisted of the enzyme recovered in the heart homogenate at the end of perfusion. Colchicine or vinblastine did not affect the enzyme activity in the perfusate which appeared between 1 and 5 min of perfusion, while the activity in the heart homogenate tended to be higher after longer periods of pretreatment. As the effect of colchicine was limited to the heparin-releasable enzyme, attempts were directed to elucidate the mode of its action. Direct interference with the release of the enzyme by heparin was ruled out by the experiments in which colchicine was present in the perfusate. These results as well as the lack of effect of colchicine after 30 min of pretreatment do not support the possibility that binding of colchicine to plasma membrane is involved in the fall of heparin-releasable enzyme activity. The increase of the colchicine effect with the time of pretreatment indicates that the drug has influenced the renewal of the releasable enzyme, which is apparently localized on the endothelial cell surface. Aktin and Meng [4] have shown that the repletion of the heparinreleasable enzyme in plasma and heart is completed during 3 h after heparin injection. In our previous study a reduction of about 80% in the release of very low density lipoproteins from the liver cell into the circulation was found 4 h after the injection of colchicine (0.5 mg/lOO g body weight). Thus if in the heart a comparable depression in transport processes occurs as in the liver, the decline of heparin-releasable enzyme activity between 0 and 4 h by about 80% indicates that the TV of the releasable enzyme is about 2 h. This estimate is quite close to the reported t ,,+ of lipoprotein lipase activity in adipose tissue [IS]. The exact localization of the heparin-releasable enzyme is not known, however, the very rapidity of its release after heparin injection indicates that it is situated on the endothelial cell surface. Several possibilities may be envisaged as to the mode of transport of the enzyme from the site of its synthesis to the
266
site of its action, i.e. the cell surface. If the enzyme can be considered as an “exportable” enzyme which is synthesized in the capillary endothelium, it would follow the usual path of secretory proteins and reach the cell surface in the lumen of Golgi-derived secretory vesicles. Alternately, it might be considered as a part of the so-called “cell coat” or membrane glycoproteins, the glycosidation of which is completed in the Golgi apparatus [19]. Since both modes of transport involve the Go&derived secretory vesicles, colchicine could have caused the fall in releasable lipoprotein lipase activity by interfering with the transport of Golgi vesicles. Yet another possibility has to be considered, namely that the enzyme is not synthesized in the endothelial cell itself, but is transported to the endothelial cell surface via the plasmalemmal vesicles, which abound in those cells on both the lumenal and ablumenal surface and which are known to participate in macromolecular transport [20]. In such a case one could postulate two possible sites of action of colchicine, one involving the secretory pathway in the synthesizing cell, the other could affect the transporting plasmalemmal vesicles proper. It has not been conclusively proven whether colchicine affects these transport processes through its known action on microtubules, but one of the more attractive theories is that it might affect the fusion of the vesicles with the plasma membrane [14]. As an intact microtubular system might be necessary for an ordered process of membrane fusion, colchinine could affect the last by its binding of tubuline [21]. It seems of interest to point out that accumulation of heparin-non-releasable lipoprotein lipase activity in the hearts of rats exposed to 4°C [17] might be related to the known effects of hypothermia on the reassembly of microtubules in various cellular systems [ 221 . Acknowledgements The excellent technical help of Mrs Y. Dabach and Mr G. Hollander is gratefully acknowledged. This study was supported in part by a grant from the Joint Research Foundation of the Hebrew University and Hadassah for Dr T. Chajek, and by a grant from the Delegation Generale a la Recherche Scientifique et Technique of the French Government. Dr. Y. Stein is an Established Investigator of the Ministry of Health. References 1 Chajek, T.. Stein, 0. and Stein. Y. (1975) Biochim. Biophys. Acta 380.127-131 KLAUS, R.m, Windmueller, H.G., Levy, R.L. and Fredrickson, D.S. (1973) J. Lipid Res. 14, 286-295 3 Robinson, D.S. and Jennineo. M.A. (1966) J. Lipid Res. 6, 222-227 4 Aktin, E. and Mew. H.C. (1972) Diabetes 21.149-166 5 Rachmilewitz, D.. Stein, 0.. Roheim. P.S. and Stein, Y. (1972) Biochim. Biophys. Acta 270, 414424 6 Stein, 0. and Stein, Y. (1963) Biochim. Biophys. Acta 70, 617-630 7 Schots, M.C., Garfinkel, A.S.. Huebotter. R.J. and Stewart, J.E. (1970) J. Lipid Res. 11, 68-69 8 Kelley. T.F. (1968) J. Lipid Res. 9. 799-800 9 Lacy, P.E., Howell, S.L., Young, D.A. and Fink, C.J. (1968) Nature 219.1177-1179 10 Williams, J.A. and Wolff, J. (1970) Proc. Natl. Acad. Sci. U.S. 67.1901-1908 11 Pelletier, G. and Bronstein. M.B. (1972) EXP. Cell Res. 70.221-223 12 Douglas, W.W. and Sorimachi, M. (1972) Br. J. PharmacoL 46.129-132 13 Rossignol. B., Herman, G. and Keryer. G. (1972) FEBS L&t. 21.189-194 2
267 14 15
Stein, 0.. Sanger. L. and Stein, Y. (1974) J. Cell BioL 62.90-103 Le Marchand. Y.. Patzelt, C., Assbnacopoulos-Jeannet. F., Loten. E.G. and Jeanrenaud, B. (1974)
16
Clin. Invest. 53.1512-1517 Ehrlich, H.P., Ross, R. and Bomstein, P. (1974)
17 18 19 20 21 22
J.
J. Cell BioL 62, 390-405
Rogers. M.P. and Robinson, D.S. (1974) J. Lipid Res. 15.263-272 Robinson, D.S. (1970) in Comprehensive Biochemistry (Florkin, M. and Stotz, Metabolism, Vol. 18. pp_ 51-105, Elsevier, Amsterdam Bennett, G., Leblond. C.P. and Haddad. A. (1974) J. Cell Biol. 60, 258-284 Bruns. R.R. and Palade, G.E. (1968) J. Cell Biol. 37, 277-299 Ohnsted, J.B. and Borisy, G.G. (1973) Annu. Rev. Biochem. 42,507-540 Tilney. L.G. and Porter, K.R. (1967) J. Cell Biol. 34.327-343
E.H.. eds). Lipid
Biochimica et Biophysics Acta, 388 (1975) 260-267 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56604
INTERFERENCE WITH THE TRANSPORT OF HEPARIN-RELEASABLE LIPOPROTEIN LIPASE IN THE PERFUSED RAT HEART BY COLCHICINE AND VINBLASTINE
T. CHAJEK, 0. STEIN and Y. STEIN Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital, and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem (Israel) (Received December 9th, 1974).
Summary
The effect of pretreatment with colchicine or vinblastine on the lipoprotein lipase activity of rat heart was studied. Administration of colchicine or vinblastine 4 h prior to perfusion of the heart caused a very marked reduction in lipoprotein lipase activity released into the perfusate within 1 min of heparin perfusion. At the same time an increase in residual heartlipase occurred so that total lipoprotein lipase content of the heart (heparin releasable plus residual) did not change. The colchicine effect was dose and time dependent; no decrease in heparin-releasable enzyme activity occurred after only 30 min of pretreatment or upon addition of colchicine into the perfusate. .These results indicate that colchicine did not impede enzyme synthesis or its release from the cell surface, but may have interfered with the transport of lipoprotein lipase from the site of its synthesis to the endothelial cell surface.
Introduction
In a previous study it was shown that in colchicine- or vinblastine-treated rats less lipolytic activity is released by heparin than in the control animals [l] . Recent studies have provided evidence that heparin-releasable lipolytic activity is derived from the liver and peripheral tissues and that the two are inhibited by protamine to a different extent [2]. On the basis of this difference in sensitivity to protamine it seemed that the peripheral lipase might be more affected by the treatment with the antimicrotubular drugs [ 11. The heart is known as a rich source of lipoprotein lipase which can be readily released into the perfusate by heparin in an in vitro system [ 3,4]. The aim of the present study was to investigate the effect of colchicine on peripheral lipoprotein lipase in an
261
isolated perfused rat heart. This system enabled also to determine whether the previously reported [l] fall in heparin-releasable lipase might be due to the inhibition of its transport to the endothelial cell surface, or could also be affected by inhibition of enzyme synthesis. Materials and Methods Animals Female rats of 180-200 g body weight of the Hebrew University strain kept in constant temperature rooms and fed the pelleted Am-Rod 931 diet [ 51, were used. For the duration of the experiment, between 8 a.m. and 12 noon, the rats were deprived of food. Colchicine (Sigma Chemical Co., St. Louis, MO.) 0.05 or 0.5 mg/lOO g body weight and vinblastine sulphate (Eli Lilly. Co., Indianapolis, Ind.) 1.0 mg/lOO g body weight dissolved freshly in 0.9% NaCl were injected intraperitoneally in a volume of 0.5 ml at time zero; control animals were injected with 0.5 ml, 0.9% NaCl. Perfusion of heart Rat hearts were excised under ether anaesthesia, freed of residual blood with 0.9% NaCl at room temperature and perfused as previously described [6] in a non-recirculatory system with Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 4% bovine serum albumin (fatty acid poor, Miles Lab., Kankakee, Ill.) and 200 mg/dl glucose. After the passage of lo-12 ml of perfusion medium and reestablishment of a regular heart beat, the flow rate was about 8-10 ml/min. Heparin (Evans Medical Corp., Liverpool, U.K.) was injected into the reservoir to give a final concentration of 5 units/ml and the perfusion fluid which passed through the heart was collected in ice-cooled tubes between O-l min, l-3 min, and 3-5 min. At the end of perfusion the heart was blotted. the cleaned ventricles were weighed and aliquots were removed for homogenization. 160-180-mg aliquots of ventricular muscle were coarsely minced with scissors and homogenized in ice-cold 8 ml of medium using the Polytron (Kinematica GmbH Luzern, Switzerland) homogenizer with the pt 10-11 probe at maximum speed for 2 min at 0°C. The homogenization medium consisted of 0.025 M NH3 /NH4 Cl buffer (pH 8.1) and heparin at final concentration of 1 unit/ml; aliquots of the homogenate were taken for the determination of lipoprotein lipase. To separate “soluble” and bound enzyme activity in hearts which had not undergone perfusion the homogenates were centrifuged for 30 min at 105 000 X g at 4°C in a Spinco L50 ultracentrifuge. The residue was resuspended in the original volume of homogenization medium and lipoprotein lipase activity was determined on both the supematant and resuspended residue fractions. Determination of lipoprotein lipase activity The lipoprotein lipase activity was determined on aliquots of perfusates and homogenates according to Schotz et al. [7] using triolein in the form of an emulsion as substrate. To prepare the emulsion the following ingredients were suspended in 16 ml of 0.2 M Tris * HCl buffer, pH 8.6, containing 0.15 M NaCl; 100 nmol of glyceryl tri[l- ’ ‘C] oleate (spec. act. 54.9 Ci/mol, Amersham/Sear-
262
ly Corp., Arlington Heights, Ill.); 22.6 E.cmol carrier triolein (Sigma Chemical Co., St.Louis, MO.), 14.4 mg bovine serum albumin (fatty acid poor, Miles Lab., Kankakee, Ill.) and 0.36 ml of 1 : 1000 solution of Triton X-100 (B.D.H. Chemicals, Poole, U.K.). The suspension was subjected to ultrasonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany) using a probe of 10 mm in diameter, at maximal scale for 4 min at 4°C. 4.8 ml of fasting human serum were added to 15 ml of the substrate emulsion which was used within 30 min of preparation. The assay system consisted of heart perfusate or homogenate, 0.2-0.4 ml; 0.6 ml of the emulsified substrate and was brought to final volume of 1.0 ml with 0.15 M NaCl. Incubations were carried out in duplicate for 20 min at 37°C in a shaking incubator. The reaction was terminated by addition of 4 ml of isopropanol/l.5 M Hz SO4 (40 : 1, v/v) according to Schotz et al. [7] and the free fatty acids were adsorbed on Amberlite IRA 400 (B.D.H. Chemicals, Poole, U.K.) as described by Kelley [8]. After the resin was washed four times with 5 ml of hexane, 1 ml of Soluene 100 (Packard Inst. Co., Downers Grove, Ill.) and 15 ml of scintillation fluid (4 g 2,5-diphenyloxazole and 100 mg 1,4-bis-[2-(4-methyl-5-phenyloxazolyl)] -benzene in 1000 ml toluene) were added and the samples were counted in a Tricarb liquid p scintillation spectrometer 3380 equipped with an absolute activity analyzer, model 544. The radioactivity present in the hexane phase in samples incubated without the enzyme was subtracted from all experimental values. The activity in perfusates was expressed per g heart weight. To measure the effect of protamine and NaCl on the lipolytic activity, 0.2 ml of rat heart homogenate or perfusate were pre-incubated for 10 min at 27°C with 0.1 ml of 0.2 M Tris * HCl buffer, pH 8.6 containing 6 mg/ml of protamine sulfate (salmine, Sigma Chemical Co., St. Louis, MO.) or with 0.2 ml of 2 M NaCl. Control assays were performed also in the,absence of serum. Results In order to determine whether injection of colchicine affects the content of lipoprotein lipase in the heart, enzyme activity was determined 4 h after injection of either colchicine or 0.9% NaCl to female rats, which were deprived of food between 8 a.m. and 12 noon. The total enzyme activity recovered in the heart was not affected by the injection of colchicine and there was no TABLE
I
LIPOPROTEIN LIPASE ACTIVITY HOMOGENATES
IN SUPERNATANT
AND RESIDUE
FRACTIONS
OF RAT HEART
All rats were deprived of food at zero time and were injected with either 0.9% NaCl or 0.5 mg/lOO g body weight of colchicine; at 4 h hearts were removed, homogenized, centrifuged and enzyme assay performed on samples of 105 000 X g supematant and on the residue. Figures in parentheses represent number of experiments; values are means kS.E., fatty acid released. wmol/g tissue/h. Treatment
0.9% N&l Colchicine
Distribution of enzyme activity
(6) (6)
supematant
Residue
66.5 k I.2 61.3 f 2.6
97.1 + 9.1 90.4 * 7.2
263
TABLE
II
EFFECT OF COLCHICINE AND VINBLASTINE LIPASE IN PERFUSATES OF RAT HEART
ON THE
HEPARIN-RELEASABLE
AII rats were deprived of food at zero time and were injected with the indicated moved and perfused 4 h after the injection. Figures in parentheses represent values are means +S.E., for fatty acid released, pmol/g tissue/h. Treatment
Perfusate* &-1
0.9% NaCl (15) Colchicin? 0.5 mg* * (5) Colchicine 0.05 mg* * (5) Vinblastine 1.0 mg** (4) * The activity in the perfusate ** Per 100 g body weight.
28.1 5.0 16.4 9.5
(min after heparin injection) l-3
* ? f f
1.9 0.6 4.0 1.7
3-5
10.1 9.1 9.8 11.5
was expressed
? * + +
1.4 1.5 1.6 2.0
5.1 5.5 3.3 4.3
+ f * *
0.9 1.6 1.0 0.9
LIPOPROTEIN
materials; hearts were renumber of experiments:
Heart at end of perfusion
Total enzyme activity
56.7 85.1 60.3 85.1
100.6 + 5.1 105.3 * 6.4 89.8 * 7.0 110.6 + 4.2
+ f + k
5.5 6.3 6.4 6.0
per g heart weight.
significant difference in the distribution of enzyme activity between the supernatant and residue fractions (Table I). In the next series of experiments the effect of pretreatment of the rats with two antimicrotubular agents, colchicine and vinblastine, was tested on the lipoprotein lipase which can be released into a non-recirculatory perfusion system by the introduction of heparin. The results presented in Table II show that the release of the enzyme occurs almost immediately after introduction of heparin and that between O-l min a sharp peak of enzyme activity appears in the perfusion medium. The rate of lipoprotein lipase release decreases sharply during the subsequent time intervals studied. Thus, between l-3 min of perfusion, the activity releasable per min decreases by more than 80% and during the next 2 min less than 10% of the initially releasable activity appeared in the perfusate per min. Pretreatment of the rats with 0.5 mg/lOO g body weight of colchicine 4 h prior to perfusion resulted in a very sharp decrease (P < 0.001) in heparin-releasable activity during the first min of perfusion. The enzyme activity which appeared in the perfusate during the subsequent two periods of perfusion (1-3 min and 3-5 min) did not differ significantly from that in the control group (Table II). As in the colchicine-treated rat more lipoprotein lipase was found in the heart than in the saline injected rat (P < O.OOl), it is evident that the total enzyme activity (which is the sum total of perfusate and homogenate activities) was the same in the experimental and control groups. A similar degree of decrease of the rapidly releasable enzyme activity was found also after pretreatment of the rats with vinblastine, given at an equimolar concentration to that of colchicine. Pretreatment of the rats with colchicine 0.05 mg/lOO g body weight also caused a decrease (p < 0.02) in the rapidly releasable enzyme activity (Table II). In order to learn more about the effect of colchicine in this system, the drug was added to the perfusate at a concentration which might have been reached after injection of colchicine in vivo, assuming a homogeneous distribution in body water. As seen in Table III the presence of 2.5 * 10e5 M colchicine in the perfusate did not lower the enzyme release by heparin and did not affect the enzyme activity recovered in the heart homogenate. The time course of colchitine action is shown also in Table III. No effect was seen after 30 min of
264 TABLE
III
HEPARIN-RELEASABLE LIPOPROTEIN LIPASE IN PERFUSATES TIME INTERVALS AFTER PRETREATMENT WITH COLCHICINE
OF RAT
HEART
AT VARIOUS
AII rats were deprived of food throughout the experimental period. Colchicine in perfusate was 2.5 . low5 M. the dose of colchicine in viva was 0.5 mg/lOO g body weight. Figures in parentheses represent number of experiments; values are means *S.E., fatty acid released, Iumol/g tissue/h. Treatment
Perfusate*
0.9% NaCl (6) Colchicine added to perfusate (4) Colchicine in viva 30 min (3) 120 min (3) 240 min (3) * The activity
in the perfusate
(min after heparin
e-1
l-3
3-5
Heart at end of perfusion
31.7 * 0.5 37.9 + 2.5
14.3 + 3.1 14.7 * 1.5
4.6 k 0.9 3.7 + 0.2
52.6 ? 3.8 56.5 f 6.7
102.5 111.7
+ 6.7 + 8.2
33.4 + 2.7 21.9 * 2.9 4.8 f 0.6
11.5 + 2.7 11.3 k 0.3 9.7*3
4.0 k 1.3 7.0 * 1.5 -
72.1 -r 4.8 88.3 C 3.3 99.3 f 6.7
120.1 128.6 113.9
c 3.3 f 4.7 + 5.6
was expressed
injection)
Total enzyme activity
per g heart weight.
pretreatment and a smaller, but statistically significant fall (p < 0.02) in the Pretreatrapidly releasable enzyme peak was observed 2 h after pretreatment. ment with colchicine for 4 h resulted in a very marked decrease in the lipoprotein lipase activity releasable during the first min of perfusion. The comparison of the activity recovered in the heart at the end of perfusion shows that in rats pretreated with colchicine for 2 or 4 h more enzyme activity (p < 0.001) was recovered in the heart homogenate than in the hearts of the 0.9% NaCl-injected rats. To define more closely the lipolytic activity measured, assays were performed also on samples which had been preincubated with protamine or 1 M NaCl. As shown in Table IV, protamine and high NaCl concentration inhibited the activity of the enzyme released into the perfusate, as well as that determined in homogenates of hearts at the end of perfusion. No enzyme activity in either perfusate or heart could be measured in the absence of serum in the assay system.
TABLE
IV
EFFECT OF INHIBITORS RAT HEART Values expressed Enzyme
source
Heart Perfusate * 0-lmin l-3 min 3-5 min * The activity
ON LIPOPROTEIN
as fatty acid released. Control 44.6
pmol/g
LIPASE
AND
HOMOGENATES
tissue/h.
1 M NaCl
* 1.6
0.5 f 0.1
32.0 k 0.8 12.2 * 3.0 3.5 i 0.2
0.6 f 0.1 >0.5 >0.5
in the perfusate
IN PERFUSATES
was expressed
Protamine >0.5 >o. 5 >0.5 >0.5
per g heart weight.
Minus serum 2.0 + 0.6 1.1 f 0.3 >0.5 >0.5
OF
265
Discussion Antimicrotubular agents, colchicine and vinblastine have been shown to arrest processes which involve cellular transport of various products in secretory vesicles or vacuoles [g--13] . Since impedance with secretion of proteins could also be due to the interference with their synthesis, protein synthesis was determined in colchicine-treated rat [14] and mouse [15] liver and was found to be normal. However, some reduction of proline incorporation into collagen by colchicine at a concentration of 1 - 10e4 M was reported recently by Ehrlich et al. [16] and hence it became important to determine whether under our experimental conditions (estimated colchicine concentration 2 * lo-’ M) colchitine does affect the synthesis of the lipoprotein lipase. Since the total lipoprotein lipase activity in hearts of colchicine-treated rats was not lower than that of the controls, it seems that in the intact rat, colchicine did not affect enzyme production, in analogy to serum lipoproteins and serum albumin [ 14,151. Neither did colchicine affect the fraction of the enzyme which remained in the supematant after homogenization of the heart and centrifugation. The most pronounced effect of colchicine in the present study was on the so-called “heparin-releasable activity”, a term used by Rogers and Robinson [17] for the enzymic activity released into the perfusate during the first min of heparin perfusion of the rat heart. These authors have included the activity appearing in the perfusate after the first min in the non-releasable enzyme fraction, the bulk of which consisted of the enzyme recovered in the heart homogenate at the end of perfusion. Colchicine or vinblastine did not affect the enzyme activity in the perfusate which appeared between 1 and 5 min of perfusion, while the activity in the heart homogenate tended to be higher after longer periods of pretreatment. As the effect of colchicine was limited to the heparin-releasable enzyme, attempts were directed to elucidate the mode of its action. Direct interference with the release of the enzyme by heparin was ruled out by the experiments in which colchicine was present in the perfusate. These results as well as the lack of effect of colchicine after 30 min of pretreatment do not support the possibility that binding of colchicine to plasma membrane is involved in the fall of heparin-releasable enzyme activity. The increase of the colchicine effect with the time of pretreatment indicates that the drug has influenced the renewal of the releasable enzyme, which is apparently localized on the endothelial cell surface. Aktin and Meng [4] have shown that the repletion of the heparinreleasable enzyme in plasma and heart is completed during 3 h after heparin injection. In our previous study a reduction of about 80% in the release of very low density lipoproteins from the liver cell into the circulation was found 4 h after the injection of colchicine (0.5 mg/lOO g body weight). Thus if in the heart a comparable depression in transport processes occurs as in the liver, the decline of heparin-releasable enzyme activity between 0 and 4 h by about 80% indicates that the TV of the releasable enzyme is about 2 h. This estimate is quite close to the reported t ,,+ of lipoprotein lipase activity in adipose tissue [IS]. The exact localization of the heparin-releasable enzyme is not known, however, the very rapidity of its release after heparin injection indicates that it is situated on the endothelial cell surface. Several possibilities may be envisaged as to the mode of transport of the enzyme from the site of its synthesis to the
266
site of its action, i.e. the cell surface. If the enzyme can be considered as an “exportable” enzyme which is synthesized in the capillary endothelium, it would follow the usual path of secretory proteins and reach the cell surface in the lumen of Golgi-derived secretory vesicles. Alternately, it might be considered as a part of the so-called “cell coat” or membrane glycoproteins, the glycosidation of which is completed in the Golgi apparatus [19]. Since both modes of transport involve the Go&derived secretory vesicles, colchicine could have caused the fall in releasable lipoprotein lipase activity by interfering with the transport of Golgi vesicles. Yet another possibility has to be considered, namely that the enzyme is not synthesized in the endothelial cell itself, but is transported to the endothelial cell surface via the plasmalemmal vesicles, which abound in those cells on both the lumenal and ablumenal surface and which are known to participate in macromolecular transport [20]. In such a case one could postulate two possible sites of action of colchicine, one involving the secretory pathway in the synthesizing cell, the other could affect the transporting plasmalemmal vesicles proper. It has not been conclusively proven whether colchicine affects these transport processes through its known action on microtubules, but one of the more attractive theories is that it might affect the fusion of the vesicles with the plasma membrane [14]. As an intact microtubular system might be necessary for an ordered process of membrane fusion, colchinine could affect the last by its binding of tubuline [21]. It seems of interest to point out that accumulation of heparin-non-releasable lipoprotein lipase activity in the hearts of rats exposed to 4°C [17] might be related to the known effects of hypothermia on the reassembly of microtubules in various cellular systems [ 221 . Acknowledgements The excellent technical help of Mrs Y. Dabach and Mr G. Hollander is gratefully acknowledged. This study was supported in part by a grant from the Joint Research Foundation of the Hebrew University and Hadassah for Dr T. Chajek, and by a grant from the Delegation Generale a la Recherche Scientifique et Technique of the French Government. Dr. Y. Stein is an Established Investigator of the Ministry of Health. References 1 Chajek, T.. Stein, 0. and Stein. Y. (1975) Biochim. Biophys. Acta 380.127-131 KLAUS, R.m, Windmueller, H.G., Levy, R.L. and Fredrickson, D.S. (1973) J. Lipid Res. 14, 286-295 3 Robinson, D.S. and Jennineo. M.A. (1966) J. Lipid Res. 6, 222-227 4 Aktin, E. and Mew. H.C. (1972) Diabetes 21.149-166 5 Rachmilewitz, D.. Stein, 0.. Roheim. P.S. and Stein, Y. (1972) Biochim. Biophys. Acta 270, 414424 6 Stein, 0. and Stein, Y. (1963) Biochim. Biophys. Acta 70, 617-630 7 Schots, M.C., Garfinkel, A.S.. Huebotter. R.J. and Stewart, J.E. (1970) J. Lipid Res. 11, 68-69 8 Kelley. T.F. (1968) J. Lipid Res. 9. 799-800 9 Lacy, P.E., Howell, S.L., Young, D.A. and Fink, C.J. (1968) Nature 219.1177-1179 10 Williams, J.A. and Wolff, J. (1970) Proc. Natl. Acad. Sci. U.S. 67.1901-1908 11 Pelletier, G. and Bronstein. M.B. (1972) EXP. Cell Res. 70.221-223 12 Douglas, W.W. and Sorimachi, M. (1972) Br. J. PharmacoL 46.129-132 13 Rossignol. B., Herman, G. and Keryer. G. (1972) FEBS L&t. 21.189-194 2
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Rogers. M.P. and Robinson, D.S. (1974) J. Lipid Res. 15.263-272 Robinson, D.S. (1970) in Comprehensive Biochemistry (Florkin, M. and Stotz, Metabolism, Vol. 18. pp_ 51-105, Elsevier, Amsterdam Bennett, G., Leblond. C.P. and Haddad. A. (1974) J. Cell Biol. 60, 258-284 Bruns. R.R. and Palade, G.E. (1968) J. Cell Biol. 37, 277-299 Ohnsted, J.B. and Borisy, G.G. (1973) Annu. Rev. Biochem. 42,507-540 Tilney. L.G. and Porter, K.R. (1967) J. Cell Biol. 34.327-343
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