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Stimulation of fatty acid oxidation in myocytes by phosphodiesterase inhibitors and adenosine analogues
Life Sciences, Vol. 48, pp. PL-97-PL-102 Printed in the U.S.A.
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
STIMULATION OF FATTY ACID OXIDATION IN MYOCYTES BY PHOSPHODIESTERASE INHIBITORS AND ADENOSINE ANALOGUES Salah A b d e l - a l e e m 1, M o s t a f a B a d r 2,
and C r i s t
Frangakis 1
iGlaxo Inc., Department of Pharmacology Five Moore Drive, Research Triangle Park, NC 27709 2University of Missouri - Kansas City, Department of Pharmacology Kansas City, MO 64108 (Submitted December 12, 1990; accepted February 7, 1991; received in final form February 27, 1991) Abstract. The effect of various phosphodiesterase inhibitors, and adenosine analogues on p a l m i t a t e oxidation, w e r e s t u d i e d in isolated rat myocytes. Enoximone, milrinone, and dipyridamole, at a c o n c e n t r a t i o n of 250 ~M, s t i m u l a t e d p a l m i t a t e o x i d a t i o n by 78%, 40%, and 43%, respectively. The s p e c i f i c A l - a g o n i s t , N 6c y c l o p e n t y l a d e n o s i n e , i n c r e a s e d p a l m i t a t e o x i d a t i o n by 56%, at a concentration of 250 ~M. M o r e o v e r , the n u c l e o s i d e transport inhibitor, S-(P-Nitrobenzyl-) 6-thioinosine, increased palmitate oxidation by 40%, at a concentration of I00 ~M. These data suggest that the s t i m u l a t i o n of p a l m i t a t e o x i d a t i o n by e n o x i m o n e and a d e n o s i n e a n a l o g u e s may be m e d i a t e d via the i n h i b i t i o n of the uptake and/or the oxidation of glucose in myocytes.
The role of fatty acids as the major fuel source in the heart is well e s t a b l i s h e d (I). However, the regulation of this pathway in the heart is not fully understood. Evidence has been presented for both simple diffusion and a carrier-mmediated uptake of long-chain fatty acids in cardiac myocytes (2,3). Enoximone, milrinone, a n d a m r i n o n e are r e l a t i v e l y selective i n h i b i t o r s of the h i g h a f f i n i t y c y c l i c AMP, p h o s p h o d i e s t e r a s e (4,5). These compounds mediate elevation in cyclic AMP, causing an incrase in intracellular Ca 2+ concentration and contractile force in cardiac muscle (6,7). Previous studies in this laboratory have shown the stimulation of p a l m i t a t e oxidation in m y o c y t e s by enoximone and milrinone (8). These compounds stimulate fatty acid Correspondence should be directed to: Dr. Salah Abdel-aleem, Inc., Five Moore Drive, Research Triangle Park, NC 27709 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
Glaxo
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Phosphodiesterase on Lipid Metabolism
Vol. 48, No. 18, 1991
o x i d a t i o n , m o s t likely, by i n c r e a s i n g t h e i r c e l l u l a r u p t a k e . However, the exact m o l e c u l a r m e c h a n i s m by w h i c h e n o x i m o n e and milrinone stimulate palmitate oxidation is unknown. The similarities b e t w e e n the actions of amrinone and theophylline in stimulating lipolysis in fat c e l l s (9), a n d t h e a b i l i t y of amrinone, and milrinone to bind to adenosine receptors in different tissues (i0, ii, 12), prompted this study to investigate the effect of various phosphodiesterase inhibitors and adenosine analogues on palmitate oxidation in myocytes, to determine whether the effect of t h e s e c o m p o u n d s on the o x i d a t i o n of p a l m i t a t e is m e d i a t e d by adenosine receptors or some other mechanism. Materials and Methods M a t e r i a l s : [i-14C] p a l m i t i c acid was p u r c h a s e d from N e w E n g l a n d Nuclear. Sigma was the source of bovine serum albumin (essentially fatty acid free), dipyridamole, N6-cyclopentyladenosine, adenosine, S - ) P - N i t r o b e n z y l ) - 6 - t h i o i n o s i n e . Enoximone was kindly p r o v i d e d by Merrell-Dow Research Institute (Cincinnati, OH). M i l r i n o n e , rolaipram, and zaprinast were synthesized at Glaxo Inc. (Research T r i a n g l e Park, NC) . A m r i n o n e was s u p p l i e d by S t e r l i n g W i n t h r o p Research Institute (Renselaer, NY). CI 930 was provided by WarnerLambert (Ann A r b o r , MI) . J o k l i k m i n i m u m e s s e n t i a l m e d i u m was purchased from Gibco Laboratories. Collgenase (type II) was obtained from Worthington. Adult rat myocytes were isolated by the method of Frangakis, et al. (13). Myocytes (2 mg cell protein) suspended in 0.9 ml of Joklik e s s e n t i a l medium, c o n t a i n i n g 25 mM NaHC03, 1.2 mM MgSO 4, 0.i mM CaCI 2 and 10 mM HEPES (pH 7.4), were placed in a 25 ml Erlenmeyer flask. To this cell suspension was added 20 ~i of t h e phosphodiesterase inhibitor solution or adenosine analogue to give the d e s i r e d c o n c e n t r a t i o n of the inhibitor. After incubating the myocytes with these compounds for 5 min at 37°C, 0.i ml of 0.2 mM [l-14C]-palmitic acid (2.5 x 10 5 dpm) was added to c e l l s u s p e n s i o n s . P a l m i t a t e was d i s s o l v e d in a s o l u t i o n of d e f a t t e d bovine serum albumin in the cell suspension buffer. The molar ratio of the palmitate to albumin was 4:1. The Erlenmeyer flask was then c l o s e d w i t h a rubber septum to w h i c h a p l a s t i c center well was attached. Incubations were continued under shaking at 37°C for 30 min and 0.4 ml of IM hyamine hydroxide was injected through the septum into the center well to absorb the released C02, and the reaction was t e r m i n a t e d by injecting 1 ml of 10% perchloric acid through the septum into the incubation medium. Flasks were shaken c o n t i n u o u s l y for 2 hr at 37°C, at which time the plastic center wells were removed, placed into scintillation vials containing i0 ml of S c i n t i v e r s e BD, and c o u n t e d in a l i q u i d s c i n t i l l a t i o n counter. P r o t e i n s were d e t e r m i n e d by the B i o - R a d p r o t e i n assay. Data are presented as the mean ± SD.
Vol. 48, No. 18, 1991
Phosphodiesterase on Lipid Metabolism
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Results The e f f e c t s of zaprinast, a selective phosphodiesterase I (PDE I) inhibitor; dipyridamole, a s e l e c t i v e P D E II i n h i b i t o r ; amrinone, milrinone, a n d enoximone, s e l e c t i v e PDE III inhibitors; rolaipram, PDE IV i n h i b i t o r ; CI 930, IBMX, n o n - s e l e c t i v e PDE i n h i b i t o r s , on palmitate o x i d a t i o n w e r e s t u d i e d in i s o l a t e d rat m y o c y t e s (Table I).
TABLE The e f f e c t s of p h o s p h o d i e s t e r a s e in rat m y o c y t e s . Addition
None
(Control)
I
inhibitors
on p a l m i t a t e
Rate of P a l m i t a t e O x i d a t i o n (nmol/mg p r o t e i n / 3 0 min)
oxidation
% of C o n t r o l
3.47 ± 0.10
(6)
I00
Zaprinast (i00 ~M) (250 ~M)
3.96 ± 0.15 4.06 ± 0.30
(6) (3)
114 120
Dipyridamole (I00 ~M) (250 ~M)
6.12 ± 0 70 5.18 ± 0.40
(6) (3)
176 a 140 a
Amrinone (I00 ~M) (250 ~M)
3.29 ± 0.18 3.52 ± 0.25
(3) (3)
95 i01
Milrinone (I00 ~M) (250 ~M)
4.51 ± 0.12 4.95 ± 0.15
(3) (3)
130 a 143 a
Enoximone (i00 ~iM) (250 ~M)
5.10 ± 0.08 6.20 ± 0.13
(3) (3)
147 a 178 a
3-Isobutyl-l-methylxanthine (IBMX) (250 ~tM) 3.29 ± 0.12
(3)
95
CI 930 (250 ~M)
(2)
i01
3.39
Myocytes were preincubated without (control) or w i t h t h e v a r i o u s c o m p o u n d s l i s t e d a b o v e for 5 m i n at 37°C, a n d the r e a c t i o n s t a r t e d by the a d d i t i o n of 0.2 m M p a l m i t a t e for 30 m i n at 37°C. V a l u e s are m e a n ± SD. N u m b e r s in p a r e n t h e s e s are the number of experiments at w h i c h values are based. a s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l v a l u e s (p<0.05).
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Vol. 48, No. 18, 1991
Enoximone, dipyridamole and milrinone, at a concentration of 250 ~M, stimulated palmitate oxidation by 78%, 40%, and 43%, respectively. Also, these compounds stimulated palmitate oxidation significantly, but to a lesser extent, at a concentration i00 ~M, except dipyridamole which increased palmitate oxidation by 76% at this concentration. FIG. 1 s h o w s the e f f e c t of a d e n o s i n e a n a l o g u e s on p a l m i t a t e oxidation in rat myocytes. N6-cyclopentyladenosine, an Al-agonist , increased palmitate oxidation by 56%, at 250 ~M. B o t h N 6cyclopenty!adenosine (i00 ~M) and e n o x i m o n e (250 ~M) i n c r e a s e d palmitate oxidation by twofold. The nucleoside transport inhibitor, S-(p-Nitrobenzyl-)6-thiolnosine (NBMPR), s t i m u l a t e d p a l m i t a t e oxidation by 40%, at I00 ~M. + 7
+
0
6 0
o 4J.~
5 0
"~ O..
4 0
4J4 J -,'-I 0 3
0
0 ,-.-~ 0
1 0
0 0 FIG.
1
The effect of adenosine analogues on palmitate oxidation in rat myocytes. Myocytes were p r e i n c u b a t e d with the various a d e n o s i n e a n a l o g u e s for 5 min at 37°C, and the r e a c t i o n s t a r t e d by the a d d i t i o n of 0.2 m M palmitate. V a l u e s are mean ± SD of three experiments. + S i g n i f i c a n t l y different from the control values (p<0.05).
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Phosphodiesterase on Lipid Metabolism
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Previous studies from this laboratory have shown that phosphodiesterase III inhibitors, such as enoximone and milrinone, s t i m u l a t e p a l m i t a t e o x i d a t i o n by i n c r e a s i n g its u p t a k e a c r o s s sarcolemmal membranes (8) . In t h e p r e s e n t study, e n o x i m o n e , milrinone and dipyridamole, stimulated palmitate oxidation significantly, at i00 ~M and 250 ~M. Enoximone and milrinone are known to be selective inhibitors of the high affinity cyclic AMP, phosphodiesterase (4,5) . W h e r e a s , d i p y r i d a m o l e is a s e l e c t i v e inhibitor of phospodiesterase II, and a potent nucleoside transport inhibitor as well (14) . S i m i l a r i t i e s in action between phosphodiesterase III i n h i b i t o r s and adenosine analogues in s t i m u l a t i n g lipolysis in fat cells (9), and b i n d i n g to adenosine receptors in different tissues (i0, Ii, 12), prompted this study to investigate t h e e f f e c t of a d e n o s i n e analogues on p a l m i t a t e o x i d a t i o n in m y o c y t e s . N 6 - c y c l o p e n t y l a d e n o s i n e , an A l - a g o n i s t , stimulated p a l m i t a t e oxidation by 56%, at 250 ~tM. The nucleoside transport inhibitor, NBMPR increased palmitate oxidation by 40%, at a concentration of i00 ~tM (FIG. i). T h e r e are a n u m b e r of p o s s i b i l i t i e s w h i c h c o u l d e x p l a i n the s t i m u l a t i o n of l o n g - c h a i n fatty acid o x i d a t i o n by e n o x i m o n e and adenosine analogues. First, the stimulation of palmitate oxidation by these compounds may be mediated via the inhibition of the uptake and/or the oxidation of glucose. This common mechanism is shared by enoximone, adenosine transport inhibitors and adenosine analogues. This hypothesis is s u p p o r t e d by the f i n d i n g s t h a t e n o x i m o n e inhibits pyruvate oxidation in rat myocytes (8), whereas NBMPR and o t h e r a d e n o s i n e a n a l o g u e s inhibit g l u c o s e t r a n s p o r t in v a r i o u s tissues (15, 16). Second, the stimulation of palmitate oxidation by t h e s e c o m p o u n d s may be m e d i a t e d via A l - a d e n o s i n e r e c e p t o r s in myocytes. However, the synergetic effect of N6-cyclopentyladenosine and e n o x i m o n e on the s t i m u l a t i o n of p a l m i t a t e o x i d a t i o n argues against this hypothesis as the common shared mechanism of enoximone and adenosine analogues on the stimulation of plamitate oxidation. In summary, m y o c y t e s by which may be oxidation of
we report the s t i m u l a t i o n of p a l m i t a t e o x i d a t i o n in p h o s p h o d i e s t e r a s e inhibitors and a d e n o s i n e analogues mediated via the inhibition of the uptake and/or the glucose in myocytes.
References I. 2. 3.
J.R. NEELY, and H.E. MORGAN. Ann. Rev. Physiol. 36 413-459 (1974). R.F. DEGRELLA, and R.J. LIGHT. J. Biol. Chem. 255 9739-9745 (1980). B.RAUCH, C. BODE, H.M.PIPER, J.F. HUTTER, R.ZIMMERMANN, E. BRAUNWELL, W. HASSELBACH, and W. KUBLER. J. Mol. Cell. Cardiol.
PL-102
4.
5. 6. 7. 8.
9.
i0 ii 12 13 14 15 16
Phosphodiesterase on Lipid Metabolism
Vol. 48, No. 18, 1991
19 159-165 (1987). A.A. ALOUSI, J.M. CANTER, M.J. MONTENARO, D.J. FORT, and R.A. FERRARI. J. Cardiovasc. Pharm. 5 792-803 (1983). T. KARIYA, L.J. WILLE, and R.C. DAGE. J. Cardiovasc. Pharm. 4 509-514 (1982) . A.E. FARAH. Ann. Rev. Toxicol. 24 275-328 (1984). C.J. FRANGAKIS, C. LANNI, K.P. LASHER, R.G. BENTLEY, and A.E. FARAH. J. Cardiovasc. Pharm. 13 915-924 (1989). S. A B D E L - A L E E M , and C.J. FRANGAKIS. X I n t e r n a t i o n a l S y m p o s i u m on D r u g s A f f e c t i n g L i p i d M e t a b o l i s m . Houston, TX (Abstr.) (1989). P. DORIGO, R.M. GAION, S. Mazzetto, A. MARCOMINI, and I. MARAGMO. Biochem. Pharm. 38 855-858 (1989). P.G.W. PLAGEMANN, a n d R.M. WOHLHUETER, B i o c h e m Pharm. 33 17831788 (1984e). K. TURNHEIM, B. PLANK, and N. KOLASSA. B i o c h e m Pharm. 27 21912197 (1978). E.F. WILLIAMS, P.H. BAKER, and A.S. CLANACHAN. Can. J. Physiol. Pharmacol. 62 31-37 (1984). C.J. FRANGAKIS, J.J. BAHL, H. McDANIEL, and R. BRESSELER. Life Sci. 27 815-825 (1980). S.M. JARVIS, D. McBRIDE, and J.D. YOUNG. J. Physiol. 324 31-46 (1982). S.M. JARVIS, J.D. YOUNG, J.S.R. WU, J.A. BELT, and A.R.P. P A T E R S O N . J. Biol. Chem. 261 1 1 0 7 7 - 1 1 0 8 5 (1986). J.M. MAY. J. Cell Physiol. 135 332-338 (1988).
Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
STIMULATION OF FATTY ACID OXIDATION IN MYOCYTES BY PHOSPHODIESTERASE INHIBITORS AND ADENOSINE ANALOGUES Salah A b d e l - a l e e m 1, M o s t a f a B a d r 2,
and C r i s t
Frangakis 1
iGlaxo Inc., Department of Pharmacology Five Moore Drive, Research Triangle Park, NC 27709 2University of Missouri - Kansas City, Department of Pharmacology Kansas City, MO 64108 (Submitted December 12, 1990; accepted February 7, 1991; received in final form February 27, 1991) Abstract. The effect of various phosphodiesterase inhibitors, and adenosine analogues on p a l m i t a t e oxidation, w e r e s t u d i e d in isolated rat myocytes. Enoximone, milrinone, and dipyridamole, at a c o n c e n t r a t i o n of 250 ~M, s t i m u l a t e d p a l m i t a t e o x i d a t i o n by 78%, 40%, and 43%, respectively. The s p e c i f i c A l - a g o n i s t , N 6c y c l o p e n t y l a d e n o s i n e , i n c r e a s e d p a l m i t a t e o x i d a t i o n by 56%, at a concentration of 250 ~M. M o r e o v e r , the n u c l e o s i d e transport inhibitor, S-(P-Nitrobenzyl-) 6-thioinosine, increased palmitate oxidation by 40%, at a concentration of I00 ~M. These data suggest that the s t i m u l a t i o n of p a l m i t a t e o x i d a t i o n by e n o x i m o n e and a d e n o s i n e a n a l o g u e s may be m e d i a t e d via the i n h i b i t i o n of the uptake and/or the oxidation of glucose in myocytes.
The role of fatty acids as the major fuel source in the heart is well e s t a b l i s h e d (I). However, the regulation of this pathway in the heart is not fully understood. Evidence has been presented for both simple diffusion and a carrier-mmediated uptake of long-chain fatty acids in cardiac myocytes (2,3). Enoximone, milrinone, a n d a m r i n o n e are r e l a t i v e l y selective i n h i b i t o r s of the h i g h a f f i n i t y c y c l i c AMP, p h o s p h o d i e s t e r a s e (4,5). These compounds mediate elevation in cyclic AMP, causing an incrase in intracellular Ca 2+ concentration and contractile force in cardiac muscle (6,7). Previous studies in this laboratory have shown the stimulation of p a l m i t a t e oxidation in m y o c y t e s by enoximone and milrinone (8). These compounds stimulate fatty acid Correspondence should be directed to: Dr. Salah Abdel-aleem, Inc., Five Moore Drive, Research Triangle Park, NC 27709 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
Glaxo
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Phosphodiesterase on Lipid Metabolism
Vol. 48, No. 18, 1991
o x i d a t i o n , m o s t likely, by i n c r e a s i n g t h e i r c e l l u l a r u p t a k e . However, the exact m o l e c u l a r m e c h a n i s m by w h i c h e n o x i m o n e and milrinone stimulate palmitate oxidation is unknown. The similarities b e t w e e n the actions of amrinone and theophylline in stimulating lipolysis in fat c e l l s (9), a n d t h e a b i l i t y of amrinone, and milrinone to bind to adenosine receptors in different tissues (i0, ii, 12), prompted this study to investigate the effect of various phosphodiesterase inhibitors and adenosine analogues on palmitate oxidation in myocytes, to determine whether the effect of t h e s e c o m p o u n d s on the o x i d a t i o n of p a l m i t a t e is m e d i a t e d by adenosine receptors or some other mechanism. Materials and Methods M a t e r i a l s : [i-14C] p a l m i t i c acid was p u r c h a s e d from N e w E n g l a n d Nuclear. Sigma was the source of bovine serum albumin (essentially fatty acid free), dipyridamole, N6-cyclopentyladenosine, adenosine, S - ) P - N i t r o b e n z y l ) - 6 - t h i o i n o s i n e . Enoximone was kindly p r o v i d e d by Merrell-Dow Research Institute (Cincinnati, OH). M i l r i n o n e , rolaipram, and zaprinast were synthesized at Glaxo Inc. (Research T r i a n g l e Park, NC) . A m r i n o n e was s u p p l i e d by S t e r l i n g W i n t h r o p Research Institute (Renselaer, NY). CI 930 was provided by WarnerLambert (Ann A r b o r , MI) . J o k l i k m i n i m u m e s s e n t i a l m e d i u m was purchased from Gibco Laboratories. Collgenase (type II) was obtained from Worthington. Adult rat myocytes were isolated by the method of Frangakis, et al. (13). Myocytes (2 mg cell protein) suspended in 0.9 ml of Joklik e s s e n t i a l medium, c o n t a i n i n g 25 mM NaHC03, 1.2 mM MgSO 4, 0.i mM CaCI 2 and 10 mM HEPES (pH 7.4), were placed in a 25 ml Erlenmeyer flask. To this cell suspension was added 20 ~i of t h e phosphodiesterase inhibitor solution or adenosine analogue to give the d e s i r e d c o n c e n t r a t i o n of the inhibitor. After incubating the myocytes with these compounds for 5 min at 37°C, 0.i ml of 0.2 mM [l-14C]-palmitic acid (2.5 x 10 5 dpm) was added to c e l l s u s p e n s i o n s . P a l m i t a t e was d i s s o l v e d in a s o l u t i o n of d e f a t t e d bovine serum albumin in the cell suspension buffer. The molar ratio of the palmitate to albumin was 4:1. The Erlenmeyer flask was then c l o s e d w i t h a rubber septum to w h i c h a p l a s t i c center well was attached. Incubations were continued under shaking at 37°C for 30 min and 0.4 ml of IM hyamine hydroxide was injected through the septum into the center well to absorb the released C02, and the reaction was t e r m i n a t e d by injecting 1 ml of 10% perchloric acid through the septum into the incubation medium. Flasks were shaken c o n t i n u o u s l y for 2 hr at 37°C, at which time the plastic center wells were removed, placed into scintillation vials containing i0 ml of S c i n t i v e r s e BD, and c o u n t e d in a l i q u i d s c i n t i l l a t i o n counter. P r o t e i n s were d e t e r m i n e d by the B i o - R a d p r o t e i n assay. Data are presented as the mean ± SD.
Vol. 48, No. 18, 1991
Phosphodiesterase on Lipid Metabolism
PL-99
Results The e f f e c t s of zaprinast, a selective phosphodiesterase I (PDE I) inhibitor; dipyridamole, a s e l e c t i v e P D E II i n h i b i t o r ; amrinone, milrinone, a n d enoximone, s e l e c t i v e PDE III inhibitors; rolaipram, PDE IV i n h i b i t o r ; CI 930, IBMX, n o n - s e l e c t i v e PDE i n h i b i t o r s , on palmitate o x i d a t i o n w e r e s t u d i e d in i s o l a t e d rat m y o c y t e s (Table I).
TABLE The e f f e c t s of p h o s p h o d i e s t e r a s e in rat m y o c y t e s . Addition
None
(Control)
I
inhibitors
on p a l m i t a t e
Rate of P a l m i t a t e O x i d a t i o n (nmol/mg p r o t e i n / 3 0 min)
oxidation
% of C o n t r o l
3.47 ± 0.10
(6)
I00
Zaprinast (i00 ~M) (250 ~M)
3.96 ± 0.15 4.06 ± 0.30
(6) (3)
114 120
Dipyridamole (I00 ~M) (250 ~M)
6.12 ± 0 70 5.18 ± 0.40
(6) (3)
176 a 140 a
Amrinone (I00 ~M) (250 ~M)
3.29 ± 0.18 3.52 ± 0.25
(3) (3)
95 i01
Milrinone (I00 ~M) (250 ~M)
4.51 ± 0.12 4.95 ± 0.15
(3) (3)
130 a 143 a
Enoximone (i00 ~iM) (250 ~M)
5.10 ± 0.08 6.20 ± 0.13
(3) (3)
147 a 178 a
3-Isobutyl-l-methylxanthine (IBMX) (250 ~tM) 3.29 ± 0.12
(3)
95
CI 930 (250 ~M)
(2)
i01
3.39
Myocytes were preincubated without (control) or w i t h t h e v a r i o u s c o m p o u n d s l i s t e d a b o v e for 5 m i n at 37°C, a n d the r e a c t i o n s t a r t e d by the a d d i t i o n of 0.2 m M p a l m i t a t e for 30 m i n at 37°C. V a l u e s are m e a n ± SD. N u m b e r s in p a r e n t h e s e s are the number of experiments at w h i c h values are based. a s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l v a l u e s (p<0.05).
PL-]00
Phosphodiesterase on Lipid Metabolism
Vol. 48, No. 18, 1991
Enoximone, dipyridamole and milrinone, at a concentration of 250 ~M, stimulated palmitate oxidation by 78%, 40%, and 43%, respectively. Also, these compounds stimulated palmitate oxidation significantly, but to a lesser extent, at a concentration i00 ~M, except dipyridamole which increased palmitate oxidation by 76% at this concentration. FIG. 1 s h o w s the e f f e c t of a d e n o s i n e a n a l o g u e s on p a l m i t a t e oxidation in rat myocytes. N6-cyclopentyladenosine, an Al-agonist , increased palmitate oxidation by 56%, at 250 ~M. B o t h N 6cyclopenty!adenosine (i00 ~M) and e n o x i m o n e (250 ~M) i n c r e a s e d palmitate oxidation by twofold. The nucleoside transport inhibitor, S-(p-Nitrobenzyl-)6-thiolnosine (NBMPR), s t i m u l a t e d p a l m i t a t e oxidation by 40%, at I00 ~M. + 7
+
0
6 0
o 4J.~
5 0
"~ O..
4 0
4J4 J -,'-I 0 3
0
0 ,-.-~ 0
1 0
0 0 FIG.
1
The effect of adenosine analogues on palmitate oxidation in rat myocytes. Myocytes were p r e i n c u b a t e d with the various a d e n o s i n e a n a l o g u e s for 5 min at 37°C, and the r e a c t i o n s t a r t e d by the a d d i t i o n of 0.2 m M palmitate. V a l u e s are mean ± SD of three experiments. + S i g n i f i c a n t l y different from the control values (p<0.05).
Vol. 48, No. 18, 1 9 9 1
Phosphodiesterase on Lipid Metabolism
PL-101
Previous studies from this laboratory have shown that phosphodiesterase III inhibitors, such as enoximone and milrinone, s t i m u l a t e p a l m i t a t e o x i d a t i o n by i n c r e a s i n g its u p t a k e a c r o s s sarcolemmal membranes (8) . In t h e p r e s e n t study, e n o x i m o n e , milrinone and dipyridamole, stimulated palmitate oxidation significantly, at i00 ~M and 250 ~M. Enoximone and milrinone are known to be selective inhibitors of the high affinity cyclic AMP, phosphodiesterase (4,5) . W h e r e a s , d i p y r i d a m o l e is a s e l e c t i v e inhibitor of phospodiesterase II, and a potent nucleoside transport inhibitor as well (14) . S i m i l a r i t i e s in action between phosphodiesterase III i n h i b i t o r s and adenosine analogues in s t i m u l a t i n g lipolysis in fat cells (9), and b i n d i n g to adenosine receptors in different tissues (i0, Ii, 12), prompted this study to investigate t h e e f f e c t of a d e n o s i n e analogues on p a l m i t a t e o x i d a t i o n in m y o c y t e s . N 6 - c y c l o p e n t y l a d e n o s i n e , an A l - a g o n i s t , stimulated p a l m i t a t e oxidation by 56%, at 250 ~tM. The nucleoside transport inhibitor, NBMPR increased palmitate oxidation by 40%, at a concentration of i00 ~tM (FIG. i). T h e r e are a n u m b e r of p o s s i b i l i t i e s w h i c h c o u l d e x p l a i n the s t i m u l a t i o n of l o n g - c h a i n fatty acid o x i d a t i o n by e n o x i m o n e and adenosine analogues. First, the stimulation of palmitate oxidation by these compounds may be mediated via the inhibition of the uptake and/or the oxidation of glucose. This common mechanism is shared by enoximone, adenosine transport inhibitors and adenosine analogues. This hypothesis is s u p p o r t e d by the f i n d i n g s t h a t e n o x i m o n e inhibits pyruvate oxidation in rat myocytes (8), whereas NBMPR and o t h e r a d e n o s i n e a n a l o g u e s inhibit g l u c o s e t r a n s p o r t in v a r i o u s tissues (15, 16). Second, the stimulation of palmitate oxidation by t h e s e c o m p o u n d s may be m e d i a t e d via A l - a d e n o s i n e r e c e p t o r s in myocytes. However, the synergetic effect of N6-cyclopentyladenosine and e n o x i m o n e on the s t i m u l a t i o n of p a l m i t a t e o x i d a t i o n argues against this hypothesis as the common shared mechanism of enoximone and adenosine analogues on the stimulation of plamitate oxidation. In summary, m y o c y t e s by which may be oxidation of
we report the s t i m u l a t i o n of p a l m i t a t e o x i d a t i o n in p h o s p h o d i e s t e r a s e inhibitors and a d e n o s i n e analogues mediated via the inhibition of the uptake and/or the glucose in myocytes.
References I. 2. 3.
J.R. NEELY, and H.E. MORGAN. Ann. Rev. Physiol. 36 413-459 (1974). R.F. DEGRELLA, and R.J. LIGHT. J. Biol. Chem. 255 9739-9745 (1980). B.RAUCH, C. BODE, H.M.PIPER, J.F. HUTTER, R.ZIMMERMANN, E. BRAUNWELL, W. HASSELBACH, and W. KUBLER. J. Mol. Cell. Cardiol.
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4.
5. 6. 7. 8.
9.
i0 ii 12 13 14 15 16
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19 159-165 (1987). A.A. ALOUSI, J.M. CANTER, M.J. MONTENARO, D.J. FORT, and R.A. FERRARI. J. Cardiovasc. Pharm. 5 792-803 (1983). T. KARIYA, L.J. WILLE, and R.C. DAGE. J. Cardiovasc. Pharm. 4 509-514 (1982) . A.E. FARAH. Ann. Rev. Toxicol. 24 275-328 (1984). C.J. FRANGAKIS, C. LANNI, K.P. LASHER, R.G. BENTLEY, and A.E. FARAH. J. Cardiovasc. Pharm. 13 915-924 (1989). S. A B D E L - A L E E M , and C.J. FRANGAKIS. X I n t e r n a t i o n a l S y m p o s i u m on D r u g s A f f e c t i n g L i p i d M e t a b o l i s m . Houston, TX (Abstr.) (1989). P. DORIGO, R.M. GAION, S. Mazzetto, A. MARCOMINI, and I. MARAGMO. Biochem. Pharm. 38 855-858 (1989). P.G.W. PLAGEMANN, a n d R.M. WOHLHUETER, B i o c h e m Pharm. 33 17831788 (1984e). K. TURNHEIM, B. PLANK, and N. KOLASSA. B i o c h e m Pharm. 27 21912197 (1978). E.F. WILLIAMS, P.H. BAKER, and A.S. CLANACHAN. Can. J. Physiol. Pharmacol. 62 31-37 (1984). C.J. FRANGAKIS, J.J. BAHL, H. McDANIEL, and R. BRESSELER. Life Sci. 27 815-825 (1980). S.M. JARVIS, D. McBRIDE, and J.D. YOUNG. J. Physiol. 324 31-46 (1982). S.M. JARVIS, J.D. YOUNG, J.S.R. WU, J.A. BELT, and A.R.P. P A T E R S O N . J. Biol. Chem. 261 1 1 0 7 7 - 1 1 0 8 5 (1986). J.M. MAY. J. Cell Physiol. 135 332-338 (1988).