127
Biochimica et Biophysico Acta, 380 (1975) 127-131 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56538
COLCHICINE-INDUCED INHIBITION OF PLASMA LIPOPROTEIN LIPASE RELEASE IN THE INTACT RAT
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 August 6th, 1974).
Summary
The release of plasma lipoprotein lipase by heparin was studied in fed and food-deprived rats pretreated with colchicine and vinblastine. Four hours after the administration of either drug the lipoprotein lipase activity released by heparin was ,only half of that found in controls. Colchicine affected the release of both protamine-sensitive and protamine-resistant lipoprotein lipase. It is suggested that colchicine and vinblastine interfere with the transport of lipoprotein lipase from the site of its storage to the vascular cell surface.
Introduction
Lipoprotein lipase, which can be released into the circulation by an intravenous injection of heparin, reaches its maximal activity within several minutes and declines progressively to preinjection values at about 4 h [l] . A second injection of heparin administered one hour after the first, will elicit a lower net release but when the interval between the two injections is increased to 4 h, the second injection will release about the same lipolytic activity as the first [ 11. These results as well as other studies [2,3] have led to the suggestion that the enzyme may be present in two pools, one localized near the vascular cell surface; the other at an intracellular site. The lower response to the second injection of heparin within one hour of the first injection could be due to the time needed for transport of the enzyme from the intracellular pool to the cell surface. Since the release of lipoprotein lipase is somewhat reminiscent of an endocrine secretory process, it seemed of interest to determine whether it will be affected by agents such as colchicine and vinblastine, which, during the recent years have been shown to affect release of various hormones [4-71, glycoproteins [8] , lipoproteins and proteins into the circulation [g-12] .
128
Methods Male rats 250-300 g body weight fed the pelleted diet Am Rod 931, were used. Colchicine, 0.5 mg/lOO g body weight (Sigma Chemical Co., St. Louis, MO.) and vinblastine sulphate, 1.0 mg/lOO g body weight (Eli Lilly Co., Indianapolis, Ind.) dissolved freshly in 0.9% NaCl, were injected intraperitoneally. Control animals were injected with 0.5 ml 0.9% NaCl intraperitoneally Heparin, 10 units/100 g body weight (Evans Medical Corp., Liverpool, G.B.) was administered intravenously and 10 min after the last heparin injection blood was drawn from the aorta into syringes containing 0.05 ml of 10% EDTA and the plasma was separated by centrifugation at 15 000 rev./min for 10 min at 4°C. The activity of lipoprotein lipase was determined according to Schotz et al. [13] using triolein in the form of an emulsion as substrate. To prepare the emulsion the following ingredients were suspended in 15 ml of 0.2 M Tris-HCl buffer, pH 8.6, containing 0.15 M NaCl: 100 nmoles of glyceryl tri-[ 1-l 4 C] oleate (spec. act. 54.9 mCi/mmole, Amersham/Searle Corp., Arlington Heights, Ill.); 22.6 pmoles 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 (BDH Chemicals, Poole, G.B.). The suspension was subjected to ultrasonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany) using a tip of 19 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 rat post-heparin plasma 0.02-0.08 ml; 0.6 ml of the emulsified substrate, and was brought to a 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-3 N HzS04 (40 : 1, v/v) according to Schotz et al. [ 131 and the free fatty acids were adsorbed on Amberlite IRA 400 (BDH Chemicals, Poole, G.B.) as described by Kelley [14]. After the resin was washed four times with 5 ml of hexane, 1 ml of Soluene 100 (Packard Inst. Downers Grove, Ill.) and 15 ml of scintillation fluid (4 g 2,5diphenyloxazole 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 0 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. To measure protamine-inhibited and protamine-resistant lipolytic activity 0.04 ml rat post heparin plasma was incubated for 10 min at 27°C with 0.1 ml of 0.2 M Tris-HCl buffer, pH 8.6 containing 0.5 M NaCl and 3 mg of protamine sulfate (salmine, Sigma Chemical Co., St. Louis, MO.). .Following preincubation, 0.26 ml of 0.15 M NaCl and 0.6 ml of the substrate mixture was added and the incubation was continued for 20 min at 37°C. Results and Discussion The
lipoprotein
lipase
activity
determined
10 min
after
injection
of
129 TABLE
I
EFFECT LIPASE
OF
COLCHiCINE
AND
VINBLASTINE
ON THE
RELEASE
OF PLASMA
LIPOPROTEIN
BY HEPARIN
Rats fed ad libitum were injected intrapenltoneally with colchicine, 0.5 mg/lOO g by wt or with vinblastine 1 mg/lOO g by wt. Control animals were injected with 0.5 ml 0.9% NaCl (intraperitoneally). Heparin 10 u/100 g by wt was injected intravenously. Post heparin plasma was obtained 10 min after the last injection of heparin. Values are means f S.E. Figures in parentheses represent number of rats. a vs b. c, d, P < 0.01; e vs g, h, P < 0.01; e vs a, f, not significant. Percent of control activity
Fatty acid released (#moles/ml
Treatment
pla=afh) Heparin at 0 and 270 min
6.7 f 1.0 (12)a
Heparin at 0 and 270 min Colchicine at 30 min
4.4 f 0.9 (lljb
50
Colchicine at 0 min Heparin at 0 and 270 min
4.3 * 0.7 ( 4)c
49
Vinbiastine at 0 min Heparin at 30 min and 270 min
4.0 + 0.4 ( 7jd
46
100
Heparin at 0 min
10.3 f 1.2 (19)e
100
Colchicine at 0 min Heparin at 30 min
11.1 * 0.2 ( 3)f
100
Colcbicine at 0 min Heparin at 270 min
3.4 * 0.5 ( 3)6
32
Vinblastine at 0 min Heparin at 270 min
5.3 f 0.7 f 3P
51
Colchicine Colchicine
30 pg added to assay 10.5 pmoieslml 60 &g added to assay 11.4 jImoles/ml
plasma/X. plasma/h.
heparin was within the range reported by Aktin and Meng [l] and, in analogy to their experiments, a similar enzymic activity was released by a second injection given 270 min after the first one (Table I). When colchicine was injected 4 h prior to the second injection of heparin the lipoprotein lipase activity did not exceed 50% of the control values. The lower activity of lipoprotein lipase was apparently not due to the presence of colchicine in the circulation as no difference in enzymic activity was detected if colchicine was administered 40 min prior to the termination of the experiment. Addition of 30 and 60 pg colchicine to 1.0 ml of the assay medium did not inter.fere with the detection of enzymic activity. Administration of colchicine resulted in a similar depression in releasable enzymic activity irrespective of whether the drug was given before or after the first injection of heparin. Injection of vinblastine at an equimolar dose to that of colchicine produced the same result, and both drugs had a similar effect. also in rats which received only one injection of heparin, 10 min prior to the termination of the experiment, Since colchicine ~eatment could have affected the food intake of the rats and hence the activity of lipoprotein lipase, the determinations were repeated in rats
130 TABLE
II
EFFECT OF COLCHICINE IN FASTED RATS
ON THE
RELEASE
OF PLASMA
LIPOPROTEIN
LIPASE
BY HEPARIN
Rats were deprived of food for the duration of the experiment. O-270 min (between 8 am and 12.30 pm). Colchicine, 0.5 mg/lOO g by wt was administered intraperitoneally. Control animals were injected with 0.5 ml 0.9% N&l (intraperitoneally). Heparin 10 units/100 g by wt was injected intravenously. Post heparin plasma was obtained 10 min after the last injection of heparin. Values are means k S.E. Figures in parentheses represent number of rats. a vs b, P < 0.001;c vs d, P < 0.001. Treatment
Fatty acid released (pmoles/ml plasma/h)
Heparin at 0 and 210 min Heparin at 0 and 210 min Colchicine at 30 min Heparin at 210 min Colchicine at 0 min Heparin at 270 min
Percent of control activity
8.3 ? 0.4
(4)a
3.5 + 0.3
(4)b
10.9 * 0.3
(4)"
4.0 + 0.35
100 42 100
(4)d
37
deprived of food for the duration of the experiment, between time 0 and 270 min. As seen in Table II, the effect of colchicine was the same in food deprived rats as in rats fed ad libitum (Table I). Since post heparin lipoprotein lipase of plasma is derived most probably from both the liver and from extrahepatic tissues [ 151, an attempt was made to determine whether colchicine does affect enzyme activity derived from the different sources. One way to distinguish between the hepatic and extrabepatic enzyme is by their different resistance to protamine inhibition [15] and thus in the next experiment the enzyme assay was run both in the presence and absence of protamine. As seen in Table III the lipoprotein lipase in plasma of colchicine-treated rats was significantly lower than in controls, both in the absence and in the presence of protamine and the decrease was more pronounced when the enzymic activity was measured in the absence of protamine. If one computes the hepatic and extrahepatic enzyme activity according to Krauss et al. [ 151, the results indicate that the depression
TABLE
III
COMPARISON LIPOPROTEIN
OF THE EFFECT LIPASE
OF COLCHICINE
ON THE RELEASE
OF PROTAMINE
RESISTANT
3 mg of protamine sulfate in 0.1 ml 0.2 M TrisHCl. pH 8.6, 0.5 M NaCl were added to 0.04 ml of plasma and preincubated at 27’C for 10 min prior to addition of substrate. Values are means + S.E. Figures in parentheses represent number of rats. a vs b, P < 0.01; c vs d, P < 0.05. Treatment
Heparin at 0 min Heparin at 0 and 270 min Colchicine at 30 min
Fatty acid released
(~moleslml
plasma/h)
- Protamine
+ Protamine
11.4 * 1.1 (4)a
4.7 f 0.5 (4)C
0.9 (4)b
2.2 + 0.7 (4)d
4.3 +
131
of the extra hepatic enzyme by colchicine is somewhat more marked than that of the hepatic enzyme. The mechanism of action of colchicine and vinblastine on lipoprotein lipase activity remains to be elucidated. It is pertinent to point out that unlike cycloheximide, colchicine in the dose range used does not interfere with protein synthesis [8,10] and hence the lower activity in the plasma did not result from inhibition of enzyme synthesis. Thus it seems plausible that colchicine could affect the transport of enzyme from its storage site to the endbthelial cell surface, as interference with transport and release of various endocrine secretions has been caused by colchicine [4-71. Another possibility to be considered is that colchicine might interfere with the very final step of enzyme release if the latter is transported towards the capillary luminal surface in plasmalemmal vesicles. Since recently colchicine has been shown to bind to various cellular membranes [16], its presence could prevent the fusion of the enzyme carrying plasmalemmal vesicles with the plasma-membrane and hence could reduce the amount of enzyme available for release by heparin. Some support for such a mechanism might be derived also from recent findings that colchicine interferes with the release of free fatty acids from adipose tissue [17] and plasmalemmal vesicles have been implicated to participate in this process [ 181. Acknowledgements The excellent technical help of Mr G. Hollander and Mrs Y. Dabach 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 grants from the Israel Ministry of Health, the Government of Israel and Delegation Generale a la Recherche Scientifique et Technique of the French Government. References 1 Aktin. E. and Meng, H.C. (1972) Diabetes 21. 149-156 2 Robinson, D.S. in Comprehensive Biochemistry, Vol. 18, Lipid Metabolism (1970) (FIorkin, M. and Stotz. E.H., eds.), pp. 51-105. Elsevier Publishing Co. 3 Blanchette-Mackie, E.J. and Scow, R.O. (1971) J. Ceil Biol. 51. l-25. 4 Lacy, P.E.. Howe& S.L., Young, D.A. and Fink, C.J. (1968) Nature 219. 1177-1179 5 Williams, J.A. and Wolff, J. (1970) Rot. Natl. Acad. Sci. U.S. 67, 1901-1908 6 PeIIetier, G. and Bronstein. M.B. (1972) Exp. Ceil Res. 70, 221-223 7 Douglas, W.W. and Sorimachi, M. (1972) Br. J. Pharmacol. 45.129-132 8 Rossignol, B., Herman, G. and Keryer. G. (1972) FEBS Lett. 21, 189-194 9 Stein, 0. and Stein, Y. (1973) Biochim. Biophys. Acta 306.142-147 10 Stein, 0.. Sanger L. and Stein, Y. (1974) J. Ceil Biol. 02, 90-103 11 Orci. L., LeMarchand. Y.. Singh. A., AssimacopoIIos-Jeannet. F.. RouiIIer. Ch. and Jeanrenaud, B. (1973) Nature 244.30-32 12 LeMarchand. Y.. Singh, A.. AssimacopoIIos-Jeannet, F., Orci, L.. RouiIIer. Ch. and Jeanrenaud, B. (1973) J. Biol. Cheni. 248.68624870 13 Schotz, M.C.. Garfinkel. A.S., Huebotter. R.J. and Stewart, J.E. (1970) J. Lipid Res. 11.68-69 14 KeIIey. T.F. (1968) J. Lipid Res. 9. 799-800 16 Krauss. R.M., Windmueller. H.G.. Levy. R.I. and Fredrickson, D.S. (1973) J. Lipid Res. 14, 286-295 16 StadIer. J. and Franke. W.W. (1974) J. CeII Biol. 60, 297’303 17 Schimmel, R.J. (1974) J. Lipid Res. 16, 206-210 18 Cushman, S.W. (1970) J. CeII Biol. 46.342-353
Biochimica et Biophysico Acta, 380 (1975) 127-131 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56538
COLCHICINE-INDUCED INHIBITION OF PLASMA LIPOPROTEIN LIPASE RELEASE IN THE INTACT RAT
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 August 6th, 1974).
Summary
The release of plasma lipoprotein lipase by heparin was studied in fed and food-deprived rats pretreated with colchicine and vinblastine. Four hours after the administration of either drug the lipoprotein lipase activity released by heparin was ,only half of that found in controls. Colchicine affected the release of both protamine-sensitive and protamine-resistant lipoprotein lipase. It is suggested that colchicine and vinblastine interfere with the transport of lipoprotein lipase from the site of its storage to the vascular cell surface.
Introduction
Lipoprotein lipase, which can be released into the circulation by an intravenous injection of heparin, reaches its maximal activity within several minutes and declines progressively to preinjection values at about 4 h [l] . A second injection of heparin administered one hour after the first, will elicit a lower net release but when the interval between the two injections is increased to 4 h, the second injection will release about the same lipolytic activity as the first [ 11. These results as well as other studies [2,3] have led to the suggestion that the enzyme may be present in two pools, one localized near the vascular cell surface; the other at an intracellular site. The lower response to the second injection of heparin within one hour of the first injection could be due to the time needed for transport of the enzyme from the intracellular pool to the cell surface. Since the release of lipoprotein lipase is somewhat reminiscent of an endocrine secretory process, it seemed of interest to determine whether it will be affected by agents such as colchicine and vinblastine, which, during the recent years have been shown to affect release of various hormones [4-71, glycoproteins [8] , lipoproteins and proteins into the circulation [g-12] .
128
Methods Male rats 250-300 g body weight fed the pelleted diet Am Rod 931, were used. Colchicine, 0.5 mg/lOO g body weight (Sigma Chemical Co., St. Louis, MO.) and vinblastine sulphate, 1.0 mg/lOO g body weight (Eli Lilly Co., Indianapolis, Ind.) dissolved freshly in 0.9% NaCl, were injected intraperitoneally. Control animals were injected with 0.5 ml 0.9% NaCl intraperitoneally Heparin, 10 units/100 g body weight (Evans Medical Corp., Liverpool, G.B.) was administered intravenously and 10 min after the last heparin injection blood was drawn from the aorta into syringes containing 0.05 ml of 10% EDTA and the plasma was separated by centrifugation at 15 000 rev./min for 10 min at 4°C. The activity of lipoprotein lipase was determined according to Schotz et al. [13] using triolein in the form of an emulsion as substrate. To prepare the emulsion the following ingredients were suspended in 15 ml of 0.2 M Tris-HCl buffer, pH 8.6, containing 0.15 M NaCl: 100 nmoles of glyceryl tri-[ 1-l 4 C] oleate (spec. act. 54.9 mCi/mmole, Amersham/Searle Corp., Arlington Heights, Ill.); 22.6 pmoles 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 (BDH Chemicals, Poole, G.B.). The suspension was subjected to ultrasonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany) using a tip of 19 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 rat post-heparin plasma 0.02-0.08 ml; 0.6 ml of the emulsified substrate, and was brought to a 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-3 N HzS04 (40 : 1, v/v) according to Schotz et al. [ 131 and the free fatty acids were adsorbed on Amberlite IRA 400 (BDH Chemicals, Poole, G.B.) as described by Kelley [14]. After the resin was washed four times with 5 ml of hexane, 1 ml of Soluene 100 (Packard Inst. Downers Grove, Ill.) and 15 ml of scintillation fluid (4 g 2,5diphenyloxazole 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 0 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. To measure protamine-inhibited and protamine-resistant lipolytic activity 0.04 ml rat post heparin plasma was incubated for 10 min at 27°C with 0.1 ml of 0.2 M Tris-HCl buffer, pH 8.6 containing 0.5 M NaCl and 3 mg of protamine sulfate (salmine, Sigma Chemical Co., St. Louis, MO.). .Following preincubation, 0.26 ml of 0.15 M NaCl and 0.6 ml of the substrate mixture was added and the incubation was continued for 20 min at 37°C. Results and Discussion The
lipoprotein
lipase
activity
determined
10 min
after
injection
of
129 TABLE
I
EFFECT LIPASE
OF
COLCHiCINE
AND
VINBLASTINE
ON THE
RELEASE
OF PLASMA
LIPOPROTEIN
BY HEPARIN
Rats fed ad libitum were injected intrapenltoneally with colchicine, 0.5 mg/lOO g by wt or with vinblastine 1 mg/lOO g by wt. Control animals were injected with 0.5 ml 0.9% NaCl (intraperitoneally). Heparin 10 u/100 g by wt was injected intravenously. Post heparin plasma was obtained 10 min after the last injection of heparin. Values are means f S.E. Figures in parentheses represent number of rats. a vs b. c, d, P < 0.01; e vs g, h, P < 0.01; e vs a, f, not significant. Percent of control activity
Fatty acid released (#moles/ml
Treatment
pla=afh) Heparin at 0 and 270 min
6.7 f 1.0 (12)a
Heparin at 0 and 270 min Colchicine at 30 min
4.4 f 0.9 (lljb
50
Colchicine at 0 min Heparin at 0 and 270 min
4.3 * 0.7 ( 4)c
49
Vinbiastine at 0 min Heparin at 30 min and 270 min
4.0 + 0.4 ( 7jd
46
100
Heparin at 0 min
10.3 f 1.2 (19)e
100
Colchicine at 0 min Heparin at 30 min
11.1 * 0.2 ( 3)f
100
Colcbicine at 0 min Heparin at 270 min
3.4 * 0.5 ( 3)6
32
Vinblastine at 0 min Heparin at 270 min
5.3 f 0.7 f 3P
51
Colchicine Colchicine
30 pg added to assay 10.5 pmoieslml 60 &g added to assay 11.4 jImoles/ml
plasma/X. plasma/h.
heparin was within the range reported by Aktin and Meng [l] and, in analogy to their experiments, a similar enzymic activity was released by a second injection given 270 min after the first one (Table I). When colchicine was injected 4 h prior to the second injection of heparin the lipoprotein lipase activity did not exceed 50% of the control values. The lower activity of lipoprotein lipase was apparently not due to the presence of colchicine in the circulation as no difference in enzymic activity was detected if colchicine was administered 40 min prior to the termination of the experiment. Addition of 30 and 60 pg colchicine to 1.0 ml of the assay medium did not inter.fere with the detection of enzymic activity. Administration of colchicine resulted in a similar depression in releasable enzymic activity irrespective of whether the drug was given before or after the first injection of heparin. Injection of vinblastine at an equimolar dose to that of colchicine produced the same result, and both drugs had a similar effect. also in rats which received only one injection of heparin, 10 min prior to the termination of the experiment, Since colchicine ~eatment could have affected the food intake of the rats and hence the activity of lipoprotein lipase, the determinations were repeated in rats
130 TABLE
II
EFFECT OF COLCHICINE IN FASTED RATS
ON THE
RELEASE
OF PLASMA
LIPOPROTEIN
LIPASE
BY HEPARIN
Rats were deprived of food for the duration of the experiment. O-270 min (between 8 am and 12.30 pm). Colchicine, 0.5 mg/lOO g by wt was administered intraperitoneally. Control animals were injected with 0.5 ml 0.9% N&l (intraperitoneally). Heparin 10 units/100 g by wt was injected intravenously. Post heparin plasma was obtained 10 min after the last injection of heparin. Values are means k S.E. Figures in parentheses represent number of rats. a vs b, P < 0.001;c vs d, P < 0.001. Treatment
Fatty acid released (pmoles/ml plasma/h)
Heparin at 0 and 210 min Heparin at 0 and 210 min Colchicine at 30 min Heparin at 210 min Colchicine at 0 min Heparin at 270 min
Percent of control activity
8.3 ? 0.4
(4)a
3.5 + 0.3
(4)b
10.9 * 0.3
(4)"
4.0 + 0.35
100 42 100
(4)d
37
deprived of food for the duration of the experiment, between time 0 and 270 min. As seen in Table II, the effect of colchicine was the same in food deprived rats as in rats fed ad libitum (Table I). Since post heparin lipoprotein lipase of plasma is derived most probably from both the liver and from extrahepatic tissues [ 151, an attempt was made to determine whether colchicine does affect enzyme activity derived from the different sources. One way to distinguish between the hepatic and extrabepatic enzyme is by their different resistance to protamine inhibition [15] and thus in the next experiment the enzyme assay was run both in the presence and absence of protamine. As seen in Table III the lipoprotein lipase in plasma of colchicine-treated rats was significantly lower than in controls, both in the absence and in the presence of protamine and the decrease was more pronounced when the enzymic activity was measured in the absence of protamine. If one computes the hepatic and extrahepatic enzyme activity according to Krauss et al. [ 151, the results indicate that the depression
TABLE
III
COMPARISON LIPOPROTEIN
OF THE EFFECT LIPASE
OF COLCHICINE
ON THE RELEASE
OF PROTAMINE
RESISTANT
3 mg of protamine sulfate in 0.1 ml 0.2 M TrisHCl. pH 8.6, 0.5 M NaCl were added to 0.04 ml of plasma and preincubated at 27’C for 10 min prior to addition of substrate. Values are means + S.E. Figures in parentheses represent number of rats. a vs b, P < 0.01; c vs d, P < 0.05. Treatment
Heparin at 0 min Heparin at 0 and 270 min Colchicine at 30 min
Fatty acid released
(~moleslml
plasma/h)
- Protamine
+ Protamine
11.4 * 1.1 (4)a
4.7 f 0.5 (4)C
0.9 (4)b
2.2 + 0.7 (4)d
4.3 +
131
of the extra hepatic enzyme by colchicine is somewhat more marked than that of the hepatic enzyme. The mechanism of action of colchicine and vinblastine on lipoprotein lipase activity remains to be elucidated. It is pertinent to point out that unlike cycloheximide, colchicine in the dose range used does not interfere with protein synthesis [8,10] and hence the lower activity in the plasma did not result from inhibition of enzyme synthesis. Thus it seems plausible that colchicine could affect the transport of enzyme from its storage site to the endbthelial cell surface, as interference with transport and release of various endocrine secretions has been caused by colchicine [4-71. Another possibility to be considered is that colchicine might interfere with the very final step of enzyme release if the latter is transported towards the capillary luminal surface in plasmalemmal vesicles. Since recently colchicine has been shown to bind to various cellular membranes [16], its presence could prevent the fusion of the enzyme carrying plasmalemmal vesicles with the plasma-membrane and hence could reduce the amount of enzyme available for release by heparin. Some support for such a mechanism might be derived also from recent findings that colchicine interferes with the release of free fatty acids from adipose tissue [17] and plasmalemmal vesicles have been implicated to participate in this process [ 181. Acknowledgements The excellent technical help of Mr G. Hollander and Mrs Y. Dabach 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 grants from the Israel Ministry of Health, the Government of Israel and Delegation Generale a la Recherche Scientifique et Technique of the French Government. References 1 Aktin. E. and Meng, H.C. (1972) Diabetes 21. 149-156 2 Robinson, D.S. in Comprehensive Biochemistry, Vol. 18, Lipid Metabolism (1970) (FIorkin, M. and Stotz. E.H., eds.), pp. 51-105. Elsevier Publishing Co. 3 Blanchette-Mackie, E.J. and Scow, R.O. (1971) J. Ceil Biol. 51. l-25. 4 Lacy, P.E.. Howe& S.L., Young, D.A. and Fink, C.J. (1968) Nature 219. 1177-1179 5 Williams, J.A. and Wolff, J. (1970) Rot. Natl. Acad. Sci. U.S. 67, 1901-1908 6 PeIIetier, G. and Bronstein. M.B. (1972) Exp. Ceil Res. 70, 221-223 7 Douglas, W.W. and Sorimachi, M. (1972) Br. J. Pharmacol. 45.129-132 8 Rossignol, B., Herman, G. and Keryer. G. (1972) FEBS Lett. 21, 189-194 9 Stein, 0. and Stein, Y. (1973) Biochim. Biophys. Acta 306.142-147 10 Stein, 0.. Sanger L. and Stein, Y. (1974) J. Ceil Biol. 02, 90-103 11 Orci. L., LeMarchand. Y.. Singh. A., AssimacopoIIos-Jeannet. F.. RouiIIer. Ch. and Jeanrenaud, B. (1973) Nature 244.30-32 12 LeMarchand. Y.. Singh, A.. AssimacopoIIos-Jeannet, F., Orci, L.. RouiIIer. Ch. and Jeanrenaud, B. (1973) J. Biol. Cheni. 248.68624870 13 Schotz, M.C.. Garfinkel. A.S., Huebotter. R.J. and Stewart, J.E. (1970) J. Lipid Res. 11.68-69 14 KeIIey. T.F. (1968) J. Lipid Res. 9. 799-800 16 Krauss. R.M., Windmueller. H.G.. Levy. R.I. and Fredrickson, D.S. (1973) J. Lipid Res. 14, 286-295 16 StadIer. J. and Franke. W.W. (1974) J. CeII Biol. 60, 297’303 17 Schimmel, R.J. (1974) J. Lipid Res. 16, 206-210 18 Cushman, S.W. (1970) J. CeII Biol. 46.342-353
