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Selective inhibition of hepatic peroxisomal fatty acid β-oxidation by enoximone
Life Sclences, Vol. Printed in the USA
51,
pp.
53-57
Pergamon
Press
SELECTIVE I N H I B I T I O N OF HEPATIC P E R O X I S O M A L FATTY ACID B-OXIDATION BY ENOXIMONE S alah Abdel-aleern 1, J~han Youssef 2, Crxst Frangakas 1, and Mostafa Badr 2 IGlaxo Research Insmute, Glaxo Inc. Five Moore Drxve, Research Triangle Park, NC 27709 2Umverslty of Mlssotm-Kansas City, Division of Pharmacology Kansas City, MO 64108 (Received
an flnal
form April
27,
1992)
Summarv Although fl-oxldation of fatty acids occurs m both peroxlsomes and mitochondrla, B-oxidizing enzymes in these organelles have distinct differences m their speclfity and sensmwty to mhibltors. In this study, the effects of the phosphodiesterase lnhib~tor enoxlmone on hepauc peroxisomal and mltochondnal goxidation were investigated In hver homogenates from control rats, cyanidelnsensiuve peroxlsomal g-oxldauon of palmltoyl-CoA was inhibited progressively by increasing concentrations of enox~mone. Slmdar results were obtained in hver homogenates from rats pretreated w~th the known peroxlsomal prohferator diethylhexylphthalate. In contrast, m~tochondrlal ILoxldatxon of palmltoyl-CoA was not inhibited by enoxlmone. These data show that enox~mone selecuvely inhibits basal as well as induced peroxlsomal, but not mltochondnal, B-ox~dat,on of the CoA thloester of long-chain fatty acids. The avadabdlty of specific mhibltors of peroxlsomal g-oxidation should prove useful m eluc,datmg regulatory mechanisms operauve in this pathway m normal as well as in prohferated peroxlsomes. Peroxlsomes are subcellular organelles which are capable of 13-oxidizing fatty acids (1-3). The I~-oxldatlon reactions in peroxlsomes are slmdar to those m mltochondna, except for the initial dehydrogenation step. In peroxlsomes, this reaction is catalyzed by an FAD-hnked acyl-CoA oxldase (2), an enzyme which transfers two hydrogens from the CoA-thloester substrates to reduce oxygen into H202 (3). Furthermore, because peroxlsomes lack Tricarboxyhc Acid Cycle enzymes, the products of peroxlsomal B-oxidation are transported to mltochondria to be completely oxidized (4). Very long-chain fatty acids, more than 22 carbons, are poorly metabolized in mltochondna, yet they are efficiently oxld,zed by peroxisomes. Therefore, ~t is assumed that an ~mportant function of peroxlsomal 6-oxidation ~s to degrade these acids into shorter derivatives which are subsequently metabohzed by mitochondrla (4). Correspondence" Dr Mostafa Badr, Umvers,ty of Missouri-Kansas City, D,vision of Pharmacology Kansas City, MO 64108 Copyrlght
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
54
Enoxlmone
on P e r o x i s o m a l
~-ox~dat~on
Vol.
51, No.
i, 1992
R e c e n t l y , we r e p o r t e d that the high affinity p h o s p h o d l e s t e r a s e inhibitor, e n o x l m o n e , had no effect on the m l t o c h o n d r l a l oxidation of p a l m l t o y l - C o A and p a l m l t o y l c a r m t m e (5, 6). Because B-oxidizing e n z y m e s in m l t o c h o n d r i a and p e r o x l s o m e s have exhibited selective s e n s i t i v i t y to lnhlbltors (7), this study was undertaken to compare the effect of e n o x l m o n e on h e p a n c p e r o x l s o m a l and m l t o c h o n d r l a l B-oxidation in order to further characterize the regulation of fatty acid m e t a b o l i s m in the h v e r
M~);erial~ and M e t h o d s Animals and Matermls M a l e S p r a g u e - D a w l e y rats ( 2 0 0 - 2 5 0 g) were used in this study. E n o x l m o n e was k i n d l y p r o v i d e d by the M e r r e l l - D o w R e s e a r c h I n s t i t u t e ( C x n c m n a n , OH) and all o t h e r c h e m i c a l s were from commercial sources. M l t o c h o n d r l a l M e t a b o h s m of P a l m l t o y l - C o A M i t o c h o n d r l a were isolated from rat livers by the differential centrifugatlon method of Parsons et al (8) M i t o c h o n d r i a l m e t a b o h s m was evaluated p o l a r o g r a p h l c a l l y at 30°C by m e a s u r i n g o x y g e n uptake using a Clark type oxygen electrode as described p r e v i o u s l y (9), using p a l m i t o y l C o A (40 g M ) in the presence of L-carnltlne (100 l.tM) as a substrate D e t e r m i n a t i o n s of Peroxlsoma113-oxldauon Liver h o m o g e n a t e s from control rats as well as from those pretreated with the known p e r o x l s o m e p r o h f e r a t o r s d l e t h y l h e x y l p h t h a l a t e (1 2 gm/Kg/d, 1 g for 7 days) were used to e x a m i n e the effect of e n o x l m o n e on p e r o x l s o m a l 13-oxidation of p a l m l t o y l - C o A (50ktM) C y a n i d e - l n s e n SlUVe p a l m i t o y l - C o A oxidation was used as a measure of p e r o x l s o m a l 13-ox~danon and was a s s a y e d by m o n i t o r i n g the rate of N A D ÷ reduction s p e c t r o p h o t o m e t n c a l l y at 340 nm using the method of Lazarow and DeDuve (1) Statlst~cal A n a l y s i s Data were a n a l y z e d for statistlcal significance (p < 0 05) using the student t test
Results Using p a l m i t o y l - C o A as a substrate, rates of m l t o c h o n d r i a l oxygen uptake were identical in the a b s e n c e and presence o f 250 ~tM e n o x l m o n e (Fxg, 1) In contrast, p e r o x l s o m a l f3-oxldation of p a l m l t o y l - C o A was inhibited by over 95% in the presence of s i m i l a r concentrations of e n o x l m o n e (Fig. 1) The lnhlbiUon of p e r o x , s o m a l 6 - o x l d a u o n caused by enoximone was c o n c e n t r a n o n - d e p e n d e n t (Fig. 2). I n c r e a s i n g concentrations of e n o x l m o n e resulted m a p r o g r e s s i v e inhibition of the c y a n i d e - i n s e n s i t i v e p e r o x i s o m a l 8-oxidation, with 43% inhibition o b s e r v e d at 50 p.M, and a c o m p l e t e inhibition (97 + 3%) at 250 g M e n o x i m o n e (Fig. 2).
Vol.
51, No.
i, 1992
Enoximone
on P e r o x i s o m a l
E-oxidation
55
[] Control [] Enoximone o
,v,-I
T
T
10o
o o
© d~
50
Mitochondria
Peroxisomes
F,g. 1 Selective Inh,blUon of Peroxlsomal I]-Oxldation By Enoxlmone. Experiments were performed as described under Materials arrt Methods m the absence and presence of enoxlmone (250 ~tM). Data are means + SEM from 3-5 untreated rats. The values of 100% for the m l t o c h o n d n a l and perox,somal B-oxidation were 28 + 1.3 n m o l / m m / m g protein and 1 + 0.04 ~tmol/mln/g tissue, respectively.
100
80 o
-~ E
60
o
40
o
o
~ ~
20
0
-I
0
b
I
50
t
I
I
I
100 150 Enoximone (~tM)
I
I
200
I
I
250
Fig. 2 C o n c e n t r a t i o n - D e p e n d e n t Inhibitxon of Peroxlsomal l]-Oxldanon by E n o x l m o n e . Peroxlsomal B-oxidation was determined as described under Materials and Methods in the absence and presence of enoxlmone (0-250 ~tM). Data are means + SEM from three untreated rats. The control value (100%) is 1 + 0.04 ~tmol/mln/ g tissue.
56
Enoximone
on P e r o x ~ s o m a l
~-oxidat~on
Vol.
51, No.
i, 1992
When rats were treated wxth the perox~some prohferator dlethylhexylphthalate (DEHP), h e p a u c peroxlsomal B-oxidanon was enhanced from basal levels of 1.02 + 0 04 ~tmole/g/mln to 5.6 + 0 23 txmole/g/man in 7 days (Fig. 3). Enoxlmone also inhibited peroxlsomal B-oxldatmn in h v e r homogenates from DEHP-treated rats m a concentration-dependent m a n n e r similar to that observed in control animals (Figs. 2 and 3).
[ ] Corn Oil [ ] DEHP
w
o o
Z o ~
5-
o
3-
2-
1-
I
q
0
0
25 Enoximone (~tM)
100
250
Fig 3 E f f e c t of E n o x l m o n e on P e r o x l s o m a l B - O x i d a t i o n in L i v e r s F r o m D l e t h y l h e x y l p h t b a l a t e - P r e t r e a t e d Rats Rats were pretreated with dxethylhexylphthalate (1 2 gm/Kg/d, 1.g ) or corn oll for 7 days prior to experiments. Data are means + SEM from 3-5 experiments. *P < 0.05 compared with corn oil **P < 0 05 compared to dlethylhexylphthalate in the absence of enoxlmone.
Discussion The lnhlbltaon of peroxlsomal B-oxldataon of p a l m l t o y l - C o A by enoxlmone and the lack of an i n h i b i t o r y effect on its mltochondrml metabolism mdacates that e n o x l m o n e is a selective mhxbxtor of peroxlsomal B-oxadatlon an the rat laver This lnhabltion of peroxasomal l]-oxldataon by e n o x l m o n e is c o n c e n t r a t i o n - d e p e n d e n t m laver homogenates from control as well as DEHPtreated rats.
Vol.
51, No.
i, 1992
E n o x i m o n e on P e r o x i s o m a l fl-oxidation
57
With the exception of the first reaction, B-oxidation reactions in peroxlsomes are the same as in the mltochondnal system. However, these reactions are catalyzed by enzymes with different physical properties (12). In peroxisomes, the oxldase catalyzing the initial reaction is ratelimiting and appears to determine the chain length specificity for the entire reaction sequence (1 O,11), while the rest of the peroxlsomal B-oxidation spiral is catalyzed by a blfunction al protein in addition to a thlolase (13) The exact mechanism by which enoxlmone inhibits nroxisomal 6oxidation is not yet clear. Because peroxisomal and mltochondrlal B-oxidizing systems exhibit considerable overlap in substrate specificity (14), the efforts to quantltate the exact contribution of each system to the process of fatty acid oxidation have been hindered. While several compounds are known to specifically inhibit mltochondrlal B-oxidation (15,16), only very few specific mhlbltors of peroxlsomal B-oxidation are available (7,17). Discovering specific lnhlbltors of peroxisomal oxidation of fatty acid should provide a useful tool to evaluate the physiological importance and investigate the regulation of this pathway in normal and in proliferated peroxlsomes.
References 1. 2 3. 4. 5. 6. 7 8. 9. 10 11. 12. 13 14 15. 16. 17
P LAZAROW and C DeDUVE. Proc Natl. Acad. Scl (U.S A.) 73 20432046 (1976) N TOLBERT. Ann Rev Blochem. 50 133-157 (1981) N. INESTROSA, M. BRONFMAN, and F. LEIGHTON. Bxochem. J. 182 779-788 (1979) G. MANNAERTS, L. DEBEER, J THOMAS, and P. DESCHEPPER J. Blol Chem 254 4585-4595 (1979). S. ABDEL-ALEEM and C. FRANGAKIS J Cardlovasc. Pharmacol. 18 293-297 (1991). M. BADR, S ABDEL-ALEEM, C. FRANGAKIS, and J. YOUSSEF. The Toxicologist 1 2 4 1 7 (1992) F. LEIGHTON, R. PERSICO, and C NECOCHEA Blochem Blophys. Res. Comm. 120 505-511 (1984). D. PARSONS, G WILLIAMS, and B. CHANCE. Ann N Y. Acad Scl. 137 643666 (1966). G. HOKE, H-Y. CHENG, C. MIRABELLI and G. RUSH. J. Pharmacol Exp. Ther. 2~2 908-914 (1990). T. HASHIMOTO Ann. N Y. Acad Scl. 386 5-12 (1979). P LAZAROW and Y. FUJIKI. Ann. Rev. Cell Blo. 1489-530 (1985). J. REDDY and N. LALWANI. Crxt. Rev. Toxlcol. 12 1-58 (1983). R. BRESSLER, C. CORREDOR, and K. BRENDEL Pharmacol. Rev. 21 105-130 (1969). C SKORIN, C. NECOCHEA, V. JOHOW, U. SOTO, A. GRAU, J. BREMER and F. LEIGHTON Blochem J. 281 561-567 (1992) S. ABDEL-ALEEM and H. SCHULZ Blochem. Phamacol. 364307-4312 (1987). H SCHULZ. Life Sc,. 40 1443-1449 (1987). C. VAN DEN BRANDEN and F. ROELS. FEBS Lett. 187 331-333 (1985).
51,
pp.
53-57
Pergamon
Press
SELECTIVE I N H I B I T I O N OF HEPATIC P E R O X I S O M A L FATTY ACID B-OXIDATION BY ENOXIMONE S alah Abdel-aleern 1, J~han Youssef 2, Crxst Frangakas 1, and Mostafa Badr 2 IGlaxo Research Insmute, Glaxo Inc. Five Moore Drxve, Research Triangle Park, NC 27709 2Umverslty of Mlssotm-Kansas City, Division of Pharmacology Kansas City, MO 64108 (Received
an flnal
form April
27,
1992)
Summarv Although fl-oxldation of fatty acids occurs m both peroxlsomes and mitochondrla, B-oxidizing enzymes in these organelles have distinct differences m their speclfity and sensmwty to mhibltors. In this study, the effects of the phosphodiesterase lnhib~tor enoxlmone on hepauc peroxisomal and mltochondnal goxidation were investigated In hver homogenates from control rats, cyanidelnsensiuve peroxlsomal g-oxldauon of palmltoyl-CoA was inhibited progressively by increasing concentrations of enox~mone. Slmdar results were obtained in hver homogenates from rats pretreated w~th the known peroxlsomal prohferator diethylhexylphthalate. In contrast, m~tochondrlal ILoxldatxon of palmltoyl-CoA was not inhibited by enoxlmone. These data show that enox~mone selecuvely inhibits basal as well as induced peroxlsomal, but not mltochondnal, B-ox~dat,on of the CoA thloester of long-chain fatty acids. The avadabdlty of specific mhibltors of peroxlsomal g-oxidation should prove useful m eluc,datmg regulatory mechanisms operauve in this pathway m normal as well as in prohferated peroxlsomes. Peroxlsomes are subcellular organelles which are capable of 13-oxidizing fatty acids (1-3). The I~-oxldatlon reactions in peroxlsomes are slmdar to those m mltochondna, except for the initial dehydrogenation step. In peroxlsomes, this reaction is catalyzed by an FAD-hnked acyl-CoA oxldase (2), an enzyme which transfers two hydrogens from the CoA-thloester substrates to reduce oxygen into H202 (3). Furthermore, because peroxlsomes lack Tricarboxyhc Acid Cycle enzymes, the products of peroxlsomal B-oxidation are transported to mltochondria to be completely oxidized (4). Very long-chain fatty acids, more than 22 carbons, are poorly metabolized in mltochondna, yet they are efficiently oxld,zed by peroxisomes. Therefore, ~t is assumed that an ~mportant function of peroxlsomal 6-oxidation ~s to degrade these acids into shorter derivatives which are subsequently metabohzed by mitochondrla (4). Correspondence" Dr Mostafa Badr, Umvers,ty of Missouri-Kansas City, D,vision of Pharmacology Kansas City, MO 64108 Copyrlght
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
54
Enoxlmone
on P e r o x i s o m a l
~-ox~dat~on
Vol.
51, No.
i, 1992
R e c e n t l y , we r e p o r t e d that the high affinity p h o s p h o d l e s t e r a s e inhibitor, e n o x l m o n e , had no effect on the m l t o c h o n d r l a l oxidation of p a l m l t o y l - C o A and p a l m l t o y l c a r m t m e (5, 6). Because B-oxidizing e n z y m e s in m l t o c h o n d r i a and p e r o x l s o m e s have exhibited selective s e n s i t i v i t y to lnhlbltors (7), this study was undertaken to compare the effect of e n o x l m o n e on h e p a n c p e r o x l s o m a l and m l t o c h o n d r l a l B-oxidation in order to further characterize the regulation of fatty acid m e t a b o l i s m in the h v e r
M~);erial~ and M e t h o d s Animals and Matermls M a l e S p r a g u e - D a w l e y rats ( 2 0 0 - 2 5 0 g) were used in this study. E n o x l m o n e was k i n d l y p r o v i d e d by the M e r r e l l - D o w R e s e a r c h I n s t i t u t e ( C x n c m n a n , OH) and all o t h e r c h e m i c a l s were from commercial sources. M l t o c h o n d r l a l M e t a b o h s m of P a l m l t o y l - C o A M i t o c h o n d r l a were isolated from rat livers by the differential centrifugatlon method of Parsons et al (8) M i t o c h o n d r i a l m e t a b o h s m was evaluated p o l a r o g r a p h l c a l l y at 30°C by m e a s u r i n g o x y g e n uptake using a Clark type oxygen electrode as described p r e v i o u s l y (9), using p a l m i t o y l C o A (40 g M ) in the presence of L-carnltlne (100 l.tM) as a substrate D e t e r m i n a t i o n s of Peroxlsoma113-oxldauon Liver h o m o g e n a t e s from control rats as well as from those pretreated with the known p e r o x l s o m e p r o h f e r a t o r s d l e t h y l h e x y l p h t h a l a t e (1 2 gm/Kg/d, 1 g for 7 days) were used to e x a m i n e the effect of e n o x l m o n e on p e r o x l s o m a l 13-oxidation of p a l m l t o y l - C o A (50ktM) C y a n i d e - l n s e n SlUVe p a l m i t o y l - C o A oxidation was used as a measure of p e r o x l s o m a l 13-ox~danon and was a s s a y e d by m o n i t o r i n g the rate of N A D ÷ reduction s p e c t r o p h o t o m e t n c a l l y at 340 nm using the method of Lazarow and DeDuve (1) Statlst~cal A n a l y s i s Data were a n a l y z e d for statistlcal significance (p < 0 05) using the student t test
Results Using p a l m i t o y l - C o A as a substrate, rates of m l t o c h o n d r i a l oxygen uptake were identical in the a b s e n c e and presence o f 250 ~tM e n o x l m o n e (Fxg, 1) In contrast, p e r o x l s o m a l f3-oxldation of p a l m l t o y l - C o A was inhibited by over 95% in the presence of s i m i l a r concentrations of e n o x l m o n e (Fig. 1) The lnhlbiUon of p e r o x , s o m a l 6 - o x l d a u o n caused by enoximone was c o n c e n t r a n o n - d e p e n d e n t (Fig. 2). I n c r e a s i n g concentrations of e n o x l m o n e resulted m a p r o g r e s s i v e inhibition of the c y a n i d e - i n s e n s i t i v e p e r o x i s o m a l 8-oxidation, with 43% inhibition o b s e r v e d at 50 p.M, and a c o m p l e t e inhibition (97 + 3%) at 250 g M e n o x i m o n e (Fig. 2).
Vol.
51, No.
i, 1992
Enoximone
on P e r o x i s o m a l
E-oxidation
55
[] Control [] Enoximone o
,v,-I
T
T
10o
o o
© d~
50
Mitochondria
Peroxisomes
F,g. 1 Selective Inh,blUon of Peroxlsomal I]-Oxldation By Enoxlmone. Experiments were performed as described under Materials arrt Methods m the absence and presence of enoxlmone (250 ~tM). Data are means + SEM from 3-5 untreated rats. The values of 100% for the m l t o c h o n d n a l and perox,somal B-oxidation were 28 + 1.3 n m o l / m m / m g protein and 1 + 0.04 ~tmol/mln/g tissue, respectively.
100
80 o
-~ E
60
o
40
o
o
~ ~
20
0
-I
0
b
I
50
t
I
I
I
100 150 Enoximone (~tM)
I
I
200
I
I
250
Fig. 2 C o n c e n t r a t i o n - D e p e n d e n t Inhibitxon of Peroxlsomal l]-Oxldanon by E n o x l m o n e . Peroxlsomal B-oxidation was determined as described under Materials and Methods in the absence and presence of enoxlmone (0-250 ~tM). Data are means + SEM from three untreated rats. The control value (100%) is 1 + 0.04 ~tmol/mln/ g tissue.
56
Enoximone
on P e r o x ~ s o m a l
~-oxidat~on
Vol.
51, No.
i, 1992
When rats were treated wxth the perox~some prohferator dlethylhexylphthalate (DEHP), h e p a u c peroxlsomal B-oxidanon was enhanced from basal levels of 1.02 + 0 04 ~tmole/g/mln to 5.6 + 0 23 txmole/g/man in 7 days (Fig. 3). Enoxlmone also inhibited peroxlsomal B-oxldatmn in h v e r homogenates from DEHP-treated rats m a concentration-dependent m a n n e r similar to that observed in control animals (Figs. 2 and 3).
[ ] Corn Oil [ ] DEHP
w
o o
Z o ~
5-
o
3-
2-
1-
I
q
0
0
25 Enoximone (~tM)
100
250
Fig 3 E f f e c t of E n o x l m o n e on P e r o x l s o m a l B - O x i d a t i o n in L i v e r s F r o m D l e t h y l h e x y l p h t b a l a t e - P r e t r e a t e d Rats Rats were pretreated with dxethylhexylphthalate (1 2 gm/Kg/d, 1.g ) or corn oll for 7 days prior to experiments. Data are means + SEM from 3-5 experiments. *P < 0.05 compared with corn oil **P < 0 05 compared to dlethylhexylphthalate in the absence of enoxlmone.
Discussion The lnhlbltaon of peroxlsomal B-oxldataon of p a l m l t o y l - C o A by enoxlmone and the lack of an i n h i b i t o r y effect on its mltochondrml metabolism mdacates that e n o x l m o n e is a selective mhxbxtor of peroxlsomal B-oxadatlon an the rat laver This lnhabltion of peroxasomal l]-oxldataon by e n o x l m o n e is c o n c e n t r a t i o n - d e p e n d e n t m laver homogenates from control as well as DEHPtreated rats.
Vol.
51, No.
i, 1992
E n o x i m o n e on P e r o x i s o m a l fl-oxidation
57
With the exception of the first reaction, B-oxidation reactions in peroxlsomes are the same as in the mltochondnal system. However, these reactions are catalyzed by enzymes with different physical properties (12). In peroxisomes, the oxldase catalyzing the initial reaction is ratelimiting and appears to determine the chain length specificity for the entire reaction sequence (1 O,11), while the rest of the peroxlsomal B-oxidation spiral is catalyzed by a blfunction al protein in addition to a thlolase (13) The exact mechanism by which enoxlmone inhibits nroxisomal 6oxidation is not yet clear. Because peroxisomal and mltochondrlal B-oxidizing systems exhibit considerable overlap in substrate specificity (14), the efforts to quantltate the exact contribution of each system to the process of fatty acid oxidation have been hindered. While several compounds are known to specifically inhibit mltochondrlal B-oxidation (15,16), only very few specific mhlbltors of peroxlsomal B-oxidation are available (7,17). Discovering specific lnhlbltors of peroxisomal oxidation of fatty acid should provide a useful tool to evaluate the physiological importance and investigate the regulation of this pathway in normal and in proliferated peroxlsomes.
References 1. 2 3. 4. 5. 6. 7 8. 9. 10 11. 12. 13 14 15. 16. 17
P LAZAROW and C DeDUVE. Proc Natl. Acad. Scl (U.S A.) 73 20432046 (1976) N TOLBERT. Ann Rev Blochem. 50 133-157 (1981) N. INESTROSA, M. BRONFMAN, and F. LEIGHTON. Bxochem. J. 182 779-788 (1979) G. MANNAERTS, L. DEBEER, J THOMAS, and P. DESCHEPPER J. Blol Chem 254 4585-4595 (1979). S. ABDEL-ALEEM and C. FRANGAKIS J Cardlovasc. Pharmacol. 18 293-297 (1991). M. BADR, S ABDEL-ALEEM, C. FRANGAKIS, and J. YOUSSEF. The Toxicologist 1 2 4 1 7 (1992) F. LEIGHTON, R. PERSICO, and C NECOCHEA Blochem Blophys. Res. Comm. 120 505-511 (1984). D. PARSONS, G WILLIAMS, and B. CHANCE. Ann N Y. Acad Scl. 137 643666 (1966). G. HOKE, H-Y. CHENG, C. MIRABELLI and G. RUSH. J. Pharmacol Exp. Ther. 2~2 908-914 (1990). T. HASHIMOTO Ann. N Y. Acad Scl. 386 5-12 (1979). P LAZAROW and Y. FUJIKI. Ann. Rev. Cell Blo. 1489-530 (1985). J. REDDY and N. LALWANI. Crxt. Rev. Toxlcol. 12 1-58 (1983). R. BRESSLER, C. CORREDOR, and K. BRENDEL Pharmacol. Rev. 21 105-130 (1969). C SKORIN, C. NECOCHEA, V. JOHOW, U. SOTO, A. GRAU, J. BREMER and F. LEIGHTON Blochem J. 281 561-567 (1992) S. ABDEL-ALEEM and H. SCHULZ Blochem. Phamacol. 364307-4312 (1987). H SCHULZ. Life Sc,. 40 1443-1449 (1987). C. VAN DEN BRANDEN and F. ROELS. FEBS Lett. 187 331-333 (1985).