- Email: [email protected]
The inhibition of amylase synthesis in the isolated perfused rat liver by cycloheximide and dinitrophenol
BIOCHEMICAL
MEDICINE
20, 353-356
(1978)
The Inhibition of Amylase Synthesis in the Isolated Perfused Rat Liver by Cycloheximide and Dinitrophenol ROBERT
L. MCGEACHIN,
Department
BETTY
ANN
POTTER,
of Biochemistry, University of Louisville, Louisville. Kentucky 40232 Received
May
AND School
EMILY
R. POTTS
of Medicine,
30, 1978
Evidence indicating the synthesis of an cr-amylase (EC 3.2.1.1, (Y-I ,4glucan 4-glucanohydrolase) in the liver of the white rat was presented by two laboratories (l-3). Later, additional evidence supporting this view was provided in studies using puromycin to block amylase synthesis in the liver (45). However, recently arguments against the reality of amylase synthesis by the rat liver have been raised (6,7). Therefore, in this study we have used a different inhibitor of protein synthesis, cycloheximide, and have shown that it, like puromycin, inhibits amylase production in the liver. Additional data on the modifying effects of 2,4-dinitrophenol are also presented. METHODS
The livers used in these experiments were obtained from male, Sprague-Dawley white rats, weighing 400 to 450 g. Cycloheximide (Acti-Dione) was obtained from the Nutritional Biochemical Corporation. The 2,4-dinitrophenol was obtained from the Eastman Organic Chemical Company. The liver perfusion apparatus was identical to that designed by Despopoulos (S), and the procedures used were similar to those previously described (1,2). The perfusing fluid was 55 to 60 ml of heparinized rat blood from normal, fed donors, diluted to 75 to 80 ml with 0.9% NaCl (plasma volume of diluted blood, 45-50 ml). The cycloheximide or dinitrophenol added was dissolved in 5 ml of 0.9% NaCl containing a trace of NaOH. The added compounds were given in the following manner: two-fifths of the dose at zero time and one-fifth at hourly intervals thereafter. Perfusion was continued for 4 hr, withdrawing small samples of blood at 0, 1,2, 3, and 4 hr and removing minor lobes of the liver at 0,2, and 4 hr for amylase determinations. Bile flow was monitored continuously as a rough measure of liver function. 353 OOO6-2944/78/0203-0353$02.OO/O Copyright @ 1978 by Academx Press, Inc. All rights of reproduction in any form reserved.
354
MCGEACHIN,
POTTER,
AND POTTS
Amylase was determined in plasma and liver samples by Van Loon’s method (9) following procedures previously described (10). Liver samples were frozen immediately, stored overnight at - 18”, and thawed before homogenization. The homogenates were centrifuged at 12,000g for 15 min in the SS-1 Sorval centrifuge and the supernatant fluids were analyzed for amylase activity. Amylase levels are expressed as units per 100 ml of plasma or 100 g of liver. Van Loon amylase units are numerically equal to Somogi units. RESULTS AND DISCUSSION
The inhibiting effect of cycloheximide can be clearly seen from the data in Table 1. As little as 10 mg added to the perfusing blood caused 86% inhibition and there was no significantly greater inhibition at doses of 20 and 50 mg. However, when 5 mg of dinitrophenol was added in addition to the cycloheximide, the degree of inhibition was markedly increased. In every case, addition of 5 mg of dinitrophenol caused the net gain in amylase to drop to negative values. It should be noted that amylase synthesis in the perfused rat liver system must be calculated in terms of a net gain (or loss) in amylase in the whole system using both the increase in plasma amylase and the loss in liver amylase during the 4-hr period. However, in cases where the synthesis of amylase in the liver is markedly inhibited, there may be no gain in plasma amylase and a loss in liver amylase probably from the continuing action of intracellular proteolytic enzymes. In such cases, the net change in amylase in the whole system will be a negative value. When 2.5 mg of dinitrophenol is added to 10 mg of cycloheximide, the net gain is lowered very little (from 5.6 to 5.1). However, when the levels of plasma and liver amylase measured during the 4-hr perfusion are studied carefully, it is seen (Table 2) that the plasma TABLE INHIBITION
Cycloheximide added (mg) 0 10 10 10
(IO)” (4) (3) (4) 20 (5) 20 (3) 50 (6) 50 (4)
1
OF RAT LIVER AMYLASE SYNTHESIS AND DINITROPHENOL
Dinitrophenol (mid 0
0 2.5 5 0 5 0 5
added
BY CYCLOHEXIMIDE
Net gain in amylase (units/g of liver) 38.5 5.6 5.1 -6.6 7.3 -7.5 3.3 -8.1
e -c 2 f r f t rt
4.1 1.9 2.1 2.9 2.0 3.1 2.0 3.4
Inhibition (%) 0 86 87 81 91
n The numbers in parentheses indicate the number of animals used in each case.
CYCLOHEXIMIDE
AND AMYLASE TABLE
EFFECT
OF DINITROPHENOL
2
ON LIVER
AMYLASE
Liver Hour 0
1 2 3 4
Control 3760* 3490 2970
SECRETIONS
Plasma Experiment
Experiment I 2 1870 2010 2150
355
SYNTHESIS
2110 2190 2430
3
Control
I
2
3
2660 2820 2990
1310 1970 2470 2800 3000
1340 1290 1270 1350 1400
1610 1600 1600 1460 1690
1440 1460 1540 1490 1490
n These are the data from three experiments and 2.5 mg of dinitrophenol were added to the The data in typical experiments with nothing * Values are expressed as units of amylase
in Table 1 in which IO mg of cycloheximide blood used in perfusing the isolated rat liver. added are given in the control columns. per 100 g of liver or per 100 ml of plasma.
levels remain essentially unchanged and the liver levels increase slightly. This is in contrast to the control experiments (Table 2) or those with cycloheximide alone in which the plasma amylase levels increase and the liver levels decrease. If the net gain in amylase in the isolated perfused rat liver really represents synthesis of a liver amylase and is not an artifact as has been suggested, then an inhibitor of eukaryotic protein synthesis should result in a marked decrease in the net gain seen. This was shown previously using puromycin which inhibits protein synthesis by interfering with peptide chain elongation in both prokaryotic and eukaryotic systems (11). In this paper, we have used a different inhibitor of protein synthesis, cycloheximide. Cycloheximide differs from puromycin in that it inhibits only in eukaryotic systems (11). This provides additional evidence that the increase in amylase seen during our perfusion experiments could not be due to contaminating bacteria, many of which are known to produce amylases. In the data presented here (Table 2), it can again be seen that dinitrophenol (12) can interfere with the secretion of amylase from the liver. However, the limits within which this can be demonstrated are relatively narrow. If as much as 5 mg of dinitrophenol added with 10 mg of cycloheximide, the amylase synthesis is so completely inhibited that no accumulation of amylase within the liver can be seen. With the smaller doses of 2.5 mg of dinitrophenol, some amylase synthesis continues but very little is secreted so small accumulations in the liver may then be noted. It was shown in our previous work (12) that this is an effect specific to dinitrophenol. Other nitrophenols or nitrobenzoic acids do not affect either liver amylase synthesis or excretion. In the previous experiments,
356
MCGEACHIN,
POTTER,
AND POTTS
the addition of 5 to 7.5 mg of dinitrophenol alone led to accumulations of amylase in the liver. At higher doses, amylase synthesis was so inhibited that no such accumulation of amylase in the liver was noted. SUMMARY
In summary, we have shown in this paper that cycloheximide, an inhibitor of eukaryotic but not prokaryotic protein synthesis, markedly inhibits amylase production by the isolated, perfused rat liver. This, with the previous work using puromycin, indicates that the increase in amylase activity noted during the perfusion period represents a true synthesis and is not an artifact. In addition, we have shown that when dinitrophenol is used with cycloheximide at the proper concentrations, blockage of amylase secretion from the liver into the plasma can be demonstrated. REFERENCES 1. McGeachin, R. L., Potter, B. A., and Despopoulos, A., Arch. Biochem. Biophys. 90, 219 (1960). 2. McGeachin, R. L., Potter, B. A., and Despopoulos, A., Arch. Biochem. Biophys. !I&89 (1%2). 3. Rutter, W. J., Arnold, M., Brosemer, R. W., and Miller, J. A., J. Biol. Chem. 236, 1259 (l%l). 4. McGeachin, R. L., Potter, B. A., and Lindsey, A. C.,Arch. Biochem. Biophys. 104,314 (19w. 5. Arnold, M., and Rutter, W. J., J. Biol. Chem. 238, 2760 (1%3). 6. Takeuchi, T.. Matsushima,’ T., and Sugimura, T., Biochim. Biophys. Acta 403, 122 (1975). 7. Karn, R. C., Wise, R. J., and Merritt, A. D., Arch. Biochem. Biophys. 175, 144 (1976). 8. Despopoulos, A., Amer. J. Physiol. 210, 760 (1%6). 9. Van Loon, E. J., Likins, M. R., and Seger, A. J., Amer. J. Clin. Pathol. 22, 1134 (1952). 10. McGeachin, R. L., and Tabler, S. M., Proc. Sot. Exp. Biol. Med. 113, 1003 (1%3). 11. Vasquez, D., FEBS Left. 40, S63 (1974). 12. McGeachin, R. L., Potter, B. A., and Wilson, C. W.. Arch. Biochem. Biophys. 122,265 (1967).
MEDICINE
20, 353-356
(1978)
The Inhibition of Amylase Synthesis in the Isolated Perfused Rat Liver by Cycloheximide and Dinitrophenol ROBERT
L. MCGEACHIN,
Department
BETTY
ANN
POTTER,
of Biochemistry, University of Louisville, Louisville. Kentucky 40232 Received
May
AND School
EMILY
R. POTTS
of Medicine,
30, 1978
Evidence indicating the synthesis of an cr-amylase (EC 3.2.1.1, (Y-I ,4glucan 4-glucanohydrolase) in the liver of the white rat was presented by two laboratories (l-3). Later, additional evidence supporting this view was provided in studies using puromycin to block amylase synthesis in the liver (45). However, recently arguments against the reality of amylase synthesis by the rat liver have been raised (6,7). Therefore, in this study we have used a different inhibitor of protein synthesis, cycloheximide, and have shown that it, like puromycin, inhibits amylase production in the liver. Additional data on the modifying effects of 2,4-dinitrophenol are also presented. METHODS
The livers used in these experiments were obtained from male, Sprague-Dawley white rats, weighing 400 to 450 g. Cycloheximide (Acti-Dione) was obtained from the Nutritional Biochemical Corporation. The 2,4-dinitrophenol was obtained from the Eastman Organic Chemical Company. The liver perfusion apparatus was identical to that designed by Despopoulos (S), and the procedures used were similar to those previously described (1,2). The perfusing fluid was 55 to 60 ml of heparinized rat blood from normal, fed donors, diluted to 75 to 80 ml with 0.9% NaCl (plasma volume of diluted blood, 45-50 ml). The cycloheximide or dinitrophenol added was dissolved in 5 ml of 0.9% NaCl containing a trace of NaOH. The added compounds were given in the following manner: two-fifths of the dose at zero time and one-fifth at hourly intervals thereafter. Perfusion was continued for 4 hr, withdrawing small samples of blood at 0, 1,2, 3, and 4 hr and removing minor lobes of the liver at 0,2, and 4 hr for amylase determinations. Bile flow was monitored continuously as a rough measure of liver function. 353 OOO6-2944/78/0203-0353$02.OO/O Copyright @ 1978 by Academx Press, Inc. All rights of reproduction in any form reserved.
354
MCGEACHIN,
POTTER,
AND POTTS
Amylase was determined in plasma and liver samples by Van Loon’s method (9) following procedures previously described (10). Liver samples were frozen immediately, stored overnight at - 18”, and thawed before homogenization. The homogenates were centrifuged at 12,000g for 15 min in the SS-1 Sorval centrifuge and the supernatant fluids were analyzed for amylase activity. Amylase levels are expressed as units per 100 ml of plasma or 100 g of liver. Van Loon amylase units are numerically equal to Somogi units. RESULTS AND DISCUSSION
The inhibiting effect of cycloheximide can be clearly seen from the data in Table 1. As little as 10 mg added to the perfusing blood caused 86% inhibition and there was no significantly greater inhibition at doses of 20 and 50 mg. However, when 5 mg of dinitrophenol was added in addition to the cycloheximide, the degree of inhibition was markedly increased. In every case, addition of 5 mg of dinitrophenol caused the net gain in amylase to drop to negative values. It should be noted that amylase synthesis in the perfused rat liver system must be calculated in terms of a net gain (or loss) in amylase in the whole system using both the increase in plasma amylase and the loss in liver amylase during the 4-hr period. However, in cases where the synthesis of amylase in the liver is markedly inhibited, there may be no gain in plasma amylase and a loss in liver amylase probably from the continuing action of intracellular proteolytic enzymes. In such cases, the net change in amylase in the whole system will be a negative value. When 2.5 mg of dinitrophenol is added to 10 mg of cycloheximide, the net gain is lowered very little (from 5.6 to 5.1). However, when the levels of plasma and liver amylase measured during the 4-hr perfusion are studied carefully, it is seen (Table 2) that the plasma TABLE INHIBITION
Cycloheximide added (mg) 0 10 10 10
(IO)” (4) (3) (4) 20 (5) 20 (3) 50 (6) 50 (4)
1
OF RAT LIVER AMYLASE SYNTHESIS AND DINITROPHENOL
Dinitrophenol (mid 0
0 2.5 5 0 5 0 5
added
BY CYCLOHEXIMIDE
Net gain in amylase (units/g of liver) 38.5 5.6 5.1 -6.6 7.3 -7.5 3.3 -8.1
e -c 2 f r f t rt
4.1 1.9 2.1 2.9 2.0 3.1 2.0 3.4
Inhibition (%) 0 86 87 81 91
n The numbers in parentheses indicate the number of animals used in each case.
CYCLOHEXIMIDE
AND AMYLASE TABLE
EFFECT
OF DINITROPHENOL
2
ON LIVER
AMYLASE
Liver Hour 0
1 2 3 4
Control 3760* 3490 2970
SECRETIONS
Plasma Experiment
Experiment I 2 1870 2010 2150
355
SYNTHESIS
2110 2190 2430
3
Control
I
2
3
2660 2820 2990
1310 1970 2470 2800 3000
1340 1290 1270 1350 1400
1610 1600 1600 1460 1690
1440 1460 1540 1490 1490
n These are the data from three experiments and 2.5 mg of dinitrophenol were added to the The data in typical experiments with nothing * Values are expressed as units of amylase
in Table 1 in which IO mg of cycloheximide blood used in perfusing the isolated rat liver. added are given in the control columns. per 100 g of liver or per 100 ml of plasma.
levels remain essentially unchanged and the liver levels increase slightly. This is in contrast to the control experiments (Table 2) or those with cycloheximide alone in which the plasma amylase levels increase and the liver levels decrease. If the net gain in amylase in the isolated perfused rat liver really represents synthesis of a liver amylase and is not an artifact as has been suggested, then an inhibitor of eukaryotic protein synthesis should result in a marked decrease in the net gain seen. This was shown previously using puromycin which inhibits protein synthesis by interfering with peptide chain elongation in both prokaryotic and eukaryotic systems (11). In this paper, we have used a different inhibitor of protein synthesis, cycloheximide. Cycloheximide differs from puromycin in that it inhibits only in eukaryotic systems (11). This provides additional evidence that the increase in amylase seen during our perfusion experiments could not be due to contaminating bacteria, many of which are known to produce amylases. In the data presented here (Table 2), it can again be seen that dinitrophenol (12) can interfere with the secretion of amylase from the liver. However, the limits within which this can be demonstrated are relatively narrow. If as much as 5 mg of dinitrophenol added with 10 mg of cycloheximide, the amylase synthesis is so completely inhibited that no accumulation of amylase within the liver can be seen. With the smaller doses of 2.5 mg of dinitrophenol, some amylase synthesis continues but very little is secreted so small accumulations in the liver may then be noted. It was shown in our previous work (12) that this is an effect specific to dinitrophenol. Other nitrophenols or nitrobenzoic acids do not affect either liver amylase synthesis or excretion. In the previous experiments,
356
MCGEACHIN,
POTTER,
AND POTTS
the addition of 5 to 7.5 mg of dinitrophenol alone led to accumulations of amylase in the liver. At higher doses, amylase synthesis was so inhibited that no such accumulation of amylase in the liver was noted. SUMMARY
In summary, we have shown in this paper that cycloheximide, an inhibitor of eukaryotic but not prokaryotic protein synthesis, markedly inhibits amylase production by the isolated, perfused rat liver. This, with the previous work using puromycin, indicates that the increase in amylase activity noted during the perfusion period represents a true synthesis and is not an artifact. In addition, we have shown that when dinitrophenol is used with cycloheximide at the proper concentrations, blockage of amylase secretion from the liver into the plasma can be demonstrated. REFERENCES 1. McGeachin, R. L., Potter, B. A., and Despopoulos, A., Arch. Biochem. Biophys. 90, 219 (1960). 2. McGeachin, R. L., Potter, B. A., and Despopoulos, A., Arch. Biochem. Biophys. !I&89 (1%2). 3. Rutter, W. J., Arnold, M., Brosemer, R. W., and Miller, J. A., J. Biol. Chem. 236, 1259 (l%l). 4. McGeachin, R. L., Potter, B. A., and Lindsey, A. C.,Arch. Biochem. Biophys. 104,314 (19w. 5. Arnold, M., and Rutter, W. J., J. Biol. Chem. 238, 2760 (1%3). 6. Takeuchi, T.. Matsushima,’ T., and Sugimura, T., Biochim. Biophys. Acta 403, 122 (1975). 7. Karn, R. C., Wise, R. J., and Merritt, A. D., Arch. Biochem. Biophys. 175, 144 (1976). 8. Despopoulos, A., Amer. J. Physiol. 210, 760 (1%6). 9. Van Loon, E. J., Likins, M. R., and Seger, A. J., Amer. J. Clin. Pathol. 22, 1134 (1952). 10. McGeachin, R. L., and Tabler, S. M., Proc. Sot. Exp. Biol. Med. 113, 1003 (1%3). 11. Vasquez, D., FEBS Left. 40, S63 (1974). 12. McGeachin, R. L., Potter, B. A., and Wilson, C. W.. Arch. Biochem. Biophys. 122,265 (1967).