209 Biochirnica et Biophysica Acta, 500 (1977) 209--212
© Elsevier/North-Holland Biomedical Press
BBA Report BBA 21458 COLCHICINE INCREASES HEPATIC A L K A L I N E PHOSPHATASE ACTIVITY
G. WILFRED Department of Biochemistry, Christian Medical College, Vellore 632002 (India)
(Received June 20th, 1977)
Summary In vivo administration of colchicine increases the activity of alkaline phosphatase significantly in the livers of rats. Prior treatment with cycloheximide prevented the induction of the enzyme by colchicine suggesting that de novo protein synthesis was essential for the effect of colchicine on alkaline phosphatase activity. Bilateral adrenalectomy did not affect the response of alkaline phosphatase following the administration of colchicine. This indicates that the rise in the level of alkaline phosphatase in liver caused by colchicine is not secondary to the release of glucocorticoids.
Colchicine inhibits the functions of microtubules due to its binding with tubulin [1] which results in the d~isruption of the assembly of microtubules [ 2]. Several in vitro studies have also suggested interaction of this alkaloid with isolated membranes [3,4]. In its presence, the properties and functions of membranes are affected. Thus, colchicine alters the redistribution of lectin binding sites on the membranes of leukocytes [5] and the distribution of intramembrane particles of T e t r a h y m e n a p y r i f o r m i s [6] and inhibits the transport of nucleosides in a number of mammalian cell lines [ 7,8]. However, studies in animals of the effect of in vivo administration of colchicine and other antimicrotubular chemicals on membranes are lacking. This prompted me to study the effect of in vivo administration of colchicine on the plasma membrane of livers of rats. The present communication reports the results of in vivo injection of colchicine on the activity of alkaline phosphatase, an enzyme of the plasma membrane in rat liver. Female albino rats weighing 120--190 g were fasted overnight for the experiments. Animals were anesthetised with light ether, and killed by drawing blood from the heart. The livers were quickly removed. Suitable portions (2 g) of livers were minced with scissors, washed with 0.25 M
210 sucrose and homogenised in 5 vols. of ice-cold 0.25 M sucrose. The enzyme alkaline phosphatase was then extracted from the homogenates according to the m e t h o d of Morton [9] by stirring the homogenates with n-butanol (18% v/v) at 0°C for 30 min and centrifuging at 22 500 X g for 30 min at 0°C in a refrigerated centrifuge (International Model PR-2). The aqueous layer was removed carefully and used for measuring the enzyme activity. The activity of the enzyme was estimated by measuring the phenol liberated from phenylphosphate [10,11]. The assay system contained 52.5 pmoles Na: CO3/NaHCO3 buffer (pH 10.2), 10.5 pmoles disodium phenylphosphate, 2.5 pmoles MgC12 and enzyme in a total volume of 1.05 ml. Incubation was carried out at 37°C for 15 min. The phenol released was measured by reacting with aminoantipyrine and ferricyanide. The activity of the enzyme is expressed as units per mg of protein. One unit of enzyme activity is defined as 1 nmol of phenylphosphate hydrolysed at 37°C per min. Protein was estimated by the colorimetric m e t h o d of Lowry et al. [12] using bovine albumin as the standard. In the first set of experiments, colchicine (Eli Lilly & Co., U.S.A.), dissolved in 0.9% NaC1 was administered intraperitoneally in a single dose of 10, 25, 50, 1 0 0 , 2 5 0 , 500 and 1000 ug per 100 g body weight to different groups of animals. Control animals were treated with normal saline. All the animals were killed 8 h later and the activity of alkaline phosphatase in livers was estimated. The results of this study are presented in Table I. It is clear that colchicine significantly increases the activity of hepatic alkaline phosphatase. The stimulatory effect of colchicine on the enzyme activity was evident with the dose of 25 pg, which increased the enzyme activity by 98% when compared to that of saline-treated control rats. Higher doses of 50, 1 0 0 , 2 5 0 , 5 0 0 and 1000 pg of colchicine increased the enzyme activity 3.0-, 5.7-, 7.6-, 8.2- and 9.4-fold, respectively. A time study of the induction of the enzyme was carried out by the intraperitoneal injection of a single dose of 125 #g of colchicine to rats and sacrificing groups of animals after 2, 4, 6, 10, 16, 24, 34 and 48 h. Saline-treated animals were used as controls. The results of the study revealed that colchicine produced a rise TABLE
I
HEPATIC ALKALINE COLCHICINE
PHOSPHATASE
ACTIVITY
AFTER
IN V I V O A D M I N I S T R A T I O N
OF
E x p e r i m e n t a l d e t a i l s are g i v e n in t h e t e x t . N u m e r a l s in p a r e n t h e s e s r e f e r to t h e n u m b e ~ o f a n i m a l s u s e d . E a c h v a l u e is t h e m e a n ± S.E. T r e a t m e n t and dose of c o l c h i c i n e (t~g/100 g body weight) Saline Colchicine 10 25 5O 100 25O 5O0 1000
Hepatic alkaline p h o s p h a t a s e activity (units/mg protein) 4 . 0 4 -+ 0 . 6 5 ( 1 0 ) 3.85 8.03 12.24 23.02 30.70 33.13 38.14
-+ 1.04 ± 1.62 +- 2.19 + 2.30 +- 3.29 -+ 2.03 + 4.77
(8) (7) (8) (8) (6) (7) (7)
211
in the level of alkaline phosphatase in liver by 4 h. The enzyme activity reached the m a x i m u m value at 24 h (5.32 -+ 0.75 units of enzyme activity per mg of protein in control animals and 47.6 + 6.86 units of enzyme activity per mg of protein in colchicine-treated animals) and started declining thereafter at 34 and 48 h. Studies with cycloheximide indicated that the colchicine-induced increase in alkaline phosphatase activity was due to de novo synthesis of enzyme in the liver. In these experiments, cycloheximide in doses of 100 or 150 pg per 100 g body weight were administered intramuscularly 30 min prior to treatment with 125 pg of colchicine (per 100 g body weight) and the animals were killed 6 h after the injection of colchicine. It was observed that the increase in hepatic alkaline phosphatase activity due to the administration of 125 pg of colchicine was prevented to the extent of 73 and 84% in animals treated with 100 and 150 pg of cycloheximide, respectively. These results clearly suggest that de novo protein synthesis is essential for the induction of hepatic alkaline phosphatase by colchicine. Temple and Wolff [ 13 ] have reported that colchicine stimulates the release of steroids in cultured adrenal t u m o r cells. Pekarthy et al. [ 14] has demonstrated that hepatic alkaline phosphatase is induced by intravenous infusion of cortisol. Based on these findings, it can be suggested that the stimulation of the activity of hepatic alkaline phosphatase after the administration of colchicine is probably secondary to the release of glucocorticoids. The studies on the effect of colchicine on alkaline phosphatase activity in adrenalectomized rats are not, however, in agreement with the above suggestion. Bilateral adrenalectomy was performed in one group of animals and the second group of animals were sham operated. In the case of adrenalectomised animals, normal saline was given in the place of drinking water and all the animals were used for the experiments 6 days later. Each group of animals received intraperitoneally either saline or 50/~g of colchicine per 100 g body weight and the hepatic alkaline phosphatase activity was estimated after sacrificing the animals 6 h later. The results of this study are presented in Table II. The increase in alkaline phosphatase activity d~e to the administration of colchicine in adrenalectomised animals is not different from that obtained in sham-operated animals. From these results it is evident that glucocorticoids are not involved in the induction of alkaline phosphatase by colchicine. TABLE n E F F E C T O F A D R E N A L E C T O M Y ON T H E I N D U C T I O N O F L I V E R A L K A L I N E P H O S P H A T A S E BY C O L C H I C I N E E x p e r i m e n t a l d e t a i l s are g i v e n in t h e t e x t . N u m e r a l s in p a r e n t h e s e s i n d i c a t e t h e n u m b e r o f a n i m a l s u s e d . E a c h v a l u e is t h e m e a n -+ S.E. 5 0 / a g c o l c h i c i n c / 1 0 0 g b o d y w e i g h t w e r e u s e d . Treatment
Hepatic alkaline phosphatase activity (units/rag protein)
S h a m operation plus saline S h a m operation plus colchicine
4 . 2 4 -+ 1 . 0 4 (5) 1 2 . 8 5 -+ 3 . 2 4 (8)
Adrenalectomy plus saline Adrenalectomy plus colehicine
4 . 8 4 +- 0 . 4 8 (5) 1 1 . 5 3 -+ 1.94 (8)
212
It is interesting to find that alkaline phosphatase activity is significantly elevated in liver following the administration of colchicine. From the present studies, however, it cannot be understood whether the stimulation of alkaline phosphatase activity by colchicine is related specifically to the depolymerisation of microtubules or interaction of colchicine with the plasma membrane resulting in alteration in its surface topography which may be a trigger for increased biosynthesis of alkaline phosphatase. That colchicine acts through some as yet unknown mechanism also cannot be ruled out. This work was supported by a grant from Fluid Research Fund of Christian Medical College, Vellore. I am grateful to Professor T.N.S. Varma for his interest and encouragement in this study. I also wish to thank Mr. T.A. Perumal for his help in performing the adrenalectomies. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
B o r i s y , G . G . and Taylor, E.W. ( 1 9 6 7 ) J. CeU. Biol. 3 4 , 5 2 5 - - 5 3 4 Wilson, L., B a m b u r g , J . R . , Mizel, S.B., G r i s h a m , L.M. and Creswcll, K.M. ( 1 9 7 4 ) F e d . P r o c . 33, 158--166 S t a d l e r , J. and Franke, W,W. ( 1 9 7 4 ) J. CeU. Biol. 6 0 , 2 9 7 - - 3 0 3 F e l t , H. and Barondes, S. ( 1 9 7 0 ) J. N e u . r o c h e m . 1 7 , 1 3 5 5 - - 1 3 6 5 Oliver, J . M . , U k e n a , T.E. and Berlin, R . D . ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 7 1 , 3 9 4 W u n d e r l i c h , F., MueIIer, R . and Speath, V. ( 1 9 7 3 ) S c i e n c e 1 8 2 , 1 1 3 6 - - 1 1 3 9 U k e n a , T . E . and Berlin, R . D . ( 1 9 7 2 ) J. E x p . Med. 1 3 6 , 1 - - 7 Mizel, S.B. and Wilson, L. ( 1 9 7 2 ) B i o c h e m i s t r y 11, 2 5 7 3 - - 2 5 7 8 M o r t o n , R . K . ( 1 9 5 4 ) B i o c h e m . J. 5 7 , 5 9 5 - - 6 0 3 K i n d , P . R . N . and King, E.J. ( 1 9 5 4 ) J. Clin. Pathol. 7, 3 2 2 - - 3 2 6 K i n g , E.J. and Jegatheesan, K , A . ( 1 9 5 9 ) J. Clin. Pathol. 1 2 , 8 5 - - 8 9 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . and Randall, R . J . ( 1 9 5 1 ) J. Biol. Chem. 1 9 3 , 265--275 T e m p l e , R. a n d W o l f f , J. ( 1 9 7 3 ) J. Biol. Chem. 2 4 8 , 2 6 9 1 - - 2 6 9 8 P e k a r t h y , J . M . , S h o r t , J., L a n s i n g , A.I. and L i e b e r m a n n , I. ( 1 9 7 2 ) J. Biol. Chem. 2 4 7 , 1767--1774
© Elsevier/North-Holland Biomedical Press
BBA Report BBA 21458 COLCHICINE INCREASES HEPATIC A L K A L I N E PHOSPHATASE ACTIVITY
G. WILFRED Department of Biochemistry, Christian Medical College, Vellore 632002 (India)
(Received June 20th, 1977)
Summary In vivo administration of colchicine increases the activity of alkaline phosphatase significantly in the livers of rats. Prior treatment with cycloheximide prevented the induction of the enzyme by colchicine suggesting that de novo protein synthesis was essential for the effect of colchicine on alkaline phosphatase activity. Bilateral adrenalectomy did not affect the response of alkaline phosphatase following the administration of colchicine. This indicates that the rise in the level of alkaline phosphatase in liver caused by colchicine is not secondary to the release of glucocorticoids.
Colchicine inhibits the functions of microtubules due to its binding with tubulin [1] which results in the d~isruption of the assembly of microtubules [ 2]. Several in vitro studies have also suggested interaction of this alkaloid with isolated membranes [3,4]. In its presence, the properties and functions of membranes are affected. Thus, colchicine alters the redistribution of lectin binding sites on the membranes of leukocytes [5] and the distribution of intramembrane particles of T e t r a h y m e n a p y r i f o r m i s [6] and inhibits the transport of nucleosides in a number of mammalian cell lines [ 7,8]. However, studies in animals of the effect of in vivo administration of colchicine and other antimicrotubular chemicals on membranes are lacking. This prompted me to study the effect of in vivo administration of colchicine on the plasma membrane of livers of rats. The present communication reports the results of in vivo injection of colchicine on the activity of alkaline phosphatase, an enzyme of the plasma membrane in rat liver. Female albino rats weighing 120--190 g were fasted overnight for the experiments. Animals were anesthetised with light ether, and killed by drawing blood from the heart. The livers were quickly removed. Suitable portions (2 g) of livers were minced with scissors, washed with 0.25 M
210 sucrose and homogenised in 5 vols. of ice-cold 0.25 M sucrose. The enzyme alkaline phosphatase was then extracted from the homogenates according to the m e t h o d of Morton [9] by stirring the homogenates with n-butanol (18% v/v) at 0°C for 30 min and centrifuging at 22 500 X g for 30 min at 0°C in a refrigerated centrifuge (International Model PR-2). The aqueous layer was removed carefully and used for measuring the enzyme activity. The activity of the enzyme was estimated by measuring the phenol liberated from phenylphosphate [10,11]. The assay system contained 52.5 pmoles Na: CO3/NaHCO3 buffer (pH 10.2), 10.5 pmoles disodium phenylphosphate, 2.5 pmoles MgC12 and enzyme in a total volume of 1.05 ml. Incubation was carried out at 37°C for 15 min. The phenol released was measured by reacting with aminoantipyrine and ferricyanide. The activity of the enzyme is expressed as units per mg of protein. One unit of enzyme activity is defined as 1 nmol of phenylphosphate hydrolysed at 37°C per min. Protein was estimated by the colorimetric m e t h o d of Lowry et al. [12] using bovine albumin as the standard. In the first set of experiments, colchicine (Eli Lilly & Co., U.S.A.), dissolved in 0.9% NaC1 was administered intraperitoneally in a single dose of 10, 25, 50, 1 0 0 , 2 5 0 , 500 and 1000 ug per 100 g body weight to different groups of animals. Control animals were treated with normal saline. All the animals were killed 8 h later and the activity of alkaline phosphatase in livers was estimated. The results of this study are presented in Table I. It is clear that colchicine significantly increases the activity of hepatic alkaline phosphatase. The stimulatory effect of colchicine on the enzyme activity was evident with the dose of 25 pg, which increased the enzyme activity by 98% when compared to that of saline-treated control rats. Higher doses of 50, 1 0 0 , 2 5 0 , 5 0 0 and 1000 pg of colchicine increased the enzyme activity 3.0-, 5.7-, 7.6-, 8.2- and 9.4-fold, respectively. A time study of the induction of the enzyme was carried out by the intraperitoneal injection of a single dose of 125 #g of colchicine to rats and sacrificing groups of animals after 2, 4, 6, 10, 16, 24, 34 and 48 h. Saline-treated animals were used as controls. The results of the study revealed that colchicine produced a rise TABLE
I
HEPATIC ALKALINE COLCHICINE
PHOSPHATASE
ACTIVITY
AFTER
IN V I V O A D M I N I S T R A T I O N
OF
E x p e r i m e n t a l d e t a i l s are g i v e n in t h e t e x t . N u m e r a l s in p a r e n t h e s e s r e f e r to t h e n u m b e ~ o f a n i m a l s u s e d . E a c h v a l u e is t h e m e a n ± S.E. T r e a t m e n t and dose of c o l c h i c i n e (t~g/100 g body weight) Saline Colchicine 10 25 5O 100 25O 5O0 1000
Hepatic alkaline p h o s p h a t a s e activity (units/mg protein) 4 . 0 4 -+ 0 . 6 5 ( 1 0 ) 3.85 8.03 12.24 23.02 30.70 33.13 38.14
-+ 1.04 ± 1.62 +- 2.19 + 2.30 +- 3.29 -+ 2.03 + 4.77
(8) (7) (8) (8) (6) (7) (7)
211
in the level of alkaline phosphatase in liver by 4 h. The enzyme activity reached the m a x i m u m value at 24 h (5.32 -+ 0.75 units of enzyme activity per mg of protein in control animals and 47.6 + 6.86 units of enzyme activity per mg of protein in colchicine-treated animals) and started declining thereafter at 34 and 48 h. Studies with cycloheximide indicated that the colchicine-induced increase in alkaline phosphatase activity was due to de novo synthesis of enzyme in the liver. In these experiments, cycloheximide in doses of 100 or 150 pg per 100 g body weight were administered intramuscularly 30 min prior to treatment with 125 pg of colchicine (per 100 g body weight) and the animals were killed 6 h after the injection of colchicine. It was observed that the increase in hepatic alkaline phosphatase activity due to the administration of 125 pg of colchicine was prevented to the extent of 73 and 84% in animals treated with 100 and 150 pg of cycloheximide, respectively. These results clearly suggest that de novo protein synthesis is essential for the induction of hepatic alkaline phosphatase by colchicine. Temple and Wolff [ 13 ] have reported that colchicine stimulates the release of steroids in cultured adrenal t u m o r cells. Pekarthy et al. [ 14] has demonstrated that hepatic alkaline phosphatase is induced by intravenous infusion of cortisol. Based on these findings, it can be suggested that the stimulation of the activity of hepatic alkaline phosphatase after the administration of colchicine is probably secondary to the release of glucocorticoids. The studies on the effect of colchicine on alkaline phosphatase activity in adrenalectomized rats are not, however, in agreement with the above suggestion. Bilateral adrenalectomy was performed in one group of animals and the second group of animals were sham operated. In the case of adrenalectomised animals, normal saline was given in the place of drinking water and all the animals were used for the experiments 6 days later. Each group of animals received intraperitoneally either saline or 50/~g of colchicine per 100 g body weight and the hepatic alkaline phosphatase activity was estimated after sacrificing the animals 6 h later. The results of this study are presented in Table II. The increase in alkaline phosphatase activity d~e to the administration of colchicine in adrenalectomised animals is not different from that obtained in sham-operated animals. From these results it is evident that glucocorticoids are not involved in the induction of alkaline phosphatase by colchicine. TABLE n E F F E C T O F A D R E N A L E C T O M Y ON T H E I N D U C T I O N O F L I V E R A L K A L I N E P H O S P H A T A S E BY C O L C H I C I N E E x p e r i m e n t a l d e t a i l s are g i v e n in t h e t e x t . N u m e r a l s in p a r e n t h e s e s i n d i c a t e t h e n u m b e r o f a n i m a l s u s e d . E a c h v a l u e is t h e m e a n -+ S.E. 5 0 / a g c o l c h i c i n c / 1 0 0 g b o d y w e i g h t w e r e u s e d . Treatment
Hepatic alkaline phosphatase activity (units/rag protein)
S h a m operation plus saline S h a m operation plus colchicine
4 . 2 4 -+ 1 . 0 4 (5) 1 2 . 8 5 -+ 3 . 2 4 (8)
Adrenalectomy plus saline Adrenalectomy plus colehicine
4 . 8 4 +- 0 . 4 8 (5) 1 1 . 5 3 -+ 1.94 (8)
212
It is interesting to find that alkaline phosphatase activity is significantly elevated in liver following the administration of colchicine. From the present studies, however, it cannot be understood whether the stimulation of alkaline phosphatase activity by colchicine is related specifically to the depolymerisation of microtubules or interaction of colchicine with the plasma membrane resulting in alteration in its surface topography which may be a trigger for increased biosynthesis of alkaline phosphatase. That colchicine acts through some as yet unknown mechanism also cannot be ruled out. This work was supported by a grant from Fluid Research Fund of Christian Medical College, Vellore. I am grateful to Professor T.N.S. Varma for his interest and encouragement in this study. I also wish to thank Mr. T.A. Perumal for his help in performing the adrenalectomies. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
B o r i s y , G . G . and Taylor, E.W. ( 1 9 6 7 ) J. CeU. Biol. 3 4 , 5 2 5 - - 5 3 4 Wilson, L., B a m b u r g , J . R . , Mizel, S.B., G r i s h a m , L.M. and Creswcll, K.M. ( 1 9 7 4 ) F e d . P r o c . 33, 158--166 S t a d l e r , J. and Franke, W,W. ( 1 9 7 4 ) J. CeU. Biol. 6 0 , 2 9 7 - - 3 0 3 F e l t , H. and Barondes, S. ( 1 9 7 0 ) J. N e u . r o c h e m . 1 7 , 1 3 5 5 - - 1 3 6 5 Oliver, J . M . , U k e n a , T.E. and Berlin, R . D . ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 7 1 , 3 9 4 W u n d e r l i c h , F., MueIIer, R . and Speath, V. ( 1 9 7 3 ) S c i e n c e 1 8 2 , 1 1 3 6 - - 1 1 3 9 U k e n a , T . E . and Berlin, R . D . ( 1 9 7 2 ) J. E x p . Med. 1 3 6 , 1 - - 7 Mizel, S.B. and Wilson, L. ( 1 9 7 2 ) B i o c h e m i s t r y 11, 2 5 7 3 - - 2 5 7 8 M o r t o n , R . K . ( 1 9 5 4 ) B i o c h e m . J. 5 7 , 5 9 5 - - 6 0 3 K i n d , P . R . N . and King, E.J. ( 1 9 5 4 ) J. Clin. Pathol. 7, 3 2 2 - - 3 2 6 K i n g , E.J. and Jegatheesan, K , A . ( 1 9 5 9 ) J. Clin. Pathol. 1 2 , 8 5 - - 8 9 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . and Randall, R . J . ( 1 9 5 1 ) J. Biol. Chem. 1 9 3 , 265--275 T e m p l e , R. a n d W o l f f , J. ( 1 9 7 3 ) J. Biol. Chem. 2 4 8 , 2 6 9 1 - - 2 6 9 8 P e k a r t h y , J . M . , S h o r t , J., L a n s i n g , A.I. and L i e b e r m a n n , I. ( 1 9 7 2 ) J. Biol. Chem. 2 4 7 , 1767--1774
