- Email: [email protected]
Cyclic nucleotides and lipolysis in rat fat cells. Interaction between calcium ionophore A23187 and FCCP, uncoupler of oxidative phosphorylation
Life Sciences, Vol. 32, pp. 571-576 Printed in the U.S.A.
Pergamon Pres:
CYCLIC NUCLEOTIDES AND LIPOLYSIS IN RAT FAT CELLS. INTERACTION BETWEEN CALCIUM IONOPHORE A23187 AND FCCP, UNCOUPLER OF OXIDATIVE PHOSPHORYLATION R. Gaion* and G. Krishna
Section on Drug-Tissue Interaction, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood I n s t i t u t e , National Institutes of Health, Bethesda, Md. 20205 (Received in final form October 18, 1982)
Summary
The interaction between calcium ionophore A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) has been studied at the level of cyclic-AMP(cAMP), cyclic-GMP (cGMP) and lipolysis in isolated rat fat cells. Ionophore A23187 (I-10 ~M) stimulated cGMP accumulation and glycerol release without affecting cAMP level. FCCP (I-I00 ~M) inhibited the effect of A23187 on cGMPlevel and glycerol release, but did not affect or increase cAMP. Thus a correlation exists between the changes of cGMPlevels and lipolysis and a dissociation of lipolysis from cAMP. Various hormones have been shown to influence calcium flux in intact adipocytes or its distribution among i n t r a c e l l u l a r pools (I-4). It is also known that calcium plays a permissive role in the l i p o l y t i c effect of hormones (5-8) and in the accumulation of cGMP in fat cells in response to catecholamines and other agents (9,10). We have previously shown that both epinephrine and the calcium ionophore, A23187, cause a very rapid increase of cGMP level associated with an increase of Ca++ f l u x in fat cells (4). Both agents stimulate l i p o l y s i s , even when no change in cAMP level occurs (4), As fat cell guanylate cyclase can be activated by calcium ions (11,12) and cGMPdependent protein kinase is present in adipose tissue (13,14) which directly stimulates hormone-sensitive lipase (15), we suggested that a calcium-cGMP system might exist in fat cells, acting as an intracellular messenger of short-term stimulations caused by low concentrations of catecholamines (4). In order to further c l a r i f y the correlation existing between changes in Ca++ flux, cyclic nucleotide levels and l i p o l y s i s , we have now studied the interaction between the calcium ionophore A23187 and FCCP, an uncoupler of oxidative phosphorylation which lowers the calcium buffering capacity of mitochondria, with a consequent marked increase of cytoplasmic calcium (16). Materials and Methods Male Sprague Dawley rats (140-200 g), fed ad libitum were used for the experiments. The animals were anesthetized with ether and the epididym~ f a t *Present address:
I s t i t u t o di Farmacologia, Largo E. Meneghetti 2, Universita di Padova, 35100 Padova, Italy
0024-3205/83/060571-06503.00/0
572
Lipolysis in Rat Fat Cells
Vol. 32, No. 6, 1983
pads were removed and immediately washed in Krebs-Ringer bicarbonate buffer, pH 7.4, saturated with 95% 02:5% CO2 and containing 3% bovine serum albumin fraction V. The buffer had the following composition: 119 mM NaCl; 4.5 mM KCl; 2.7 mM CaCI2; 1.2 mM KH2P04; 1.2 mM MgSO4 24.2 mM NaHCO3. The tissue was minced and suspended in the same buffer in the presence of crude collagenase (I mg/ml) and fat c e l l s were isolated according to Rodbell and Krishna (17). The cells were counted as described e a r l i e r (17). The final cell suspension was diluted with buffer and 2 ml a l i q u o t s , containing 80,0(10-160,000 c e l l s , were preincubated f o r 15 min at 37°C. The incubation was then started by the addition of the drugs dissolved in i0 ~I of buffer and terminated at the various times by the addition of 0.2 ml of 50% t r i c h l o r o a c e t i c acid (TCA). After mixing and c e n t r i f u g a t i o n of the samples, aliquots of supernatant f l u i d were analyzed for g l y c e r o l , cAMP and cGMP as described below. Estimation of glycerol. Duplicate aliquots (0.5 ml) of the supernatant f l u i d were used for the determination of glycerol. Glycerol was determined according to the method of Lambert and Neish (18) by using the acetyl acetone reagent as proposed by Nash (19). Known amounts of glycerol dissolved in KrebsRinger bicarbonate albumin buffer and treated with TCA served as a standard. Estimation of cAMP and cGMP. TCA was removed from the supernatant f l u i d by four extractions with four volumes of water-saturated ether until a pH 4.3-4.6 was reached. Duplicate aliquots of 50 ~I were then assayed by the radioimmunoassay described by Frandsen and Krishna (20) without f u r t h e r p u r i f i c a t i o n . The samples were succinylated by addition of 5 ~I of succinylation mixture (20). No f u r t h e r p u r i f i c a t i o n of samples by ion exchange chromatography was deemed necessary because s i m i l a r results were obtained when the samples were assayed before or a f t e r p u r i f i c a t i o n by Dowex 1 chromatography. A series of I0 cAMP and cGMP standards (from 1 to I0,000 fmoles) were assayed in t r i p l i c a t e with the samples; the standards also contained 50 ~1 of Krebs-Ringer bicarbonate albumin medium which had been treated with TCA and extracted with ether. Materials. cAMP and cGMP antisera were prepared in our laboratory according to the method of Sterner et a l . (21). lodinated 2'-Osuccinyl cAMP and cGMP tyrosine methyl esters, I ~ P - T M E and 1251 ScGMP-TME were obtained from Collaborative Research Inc., Waltham, Mass. Bovine serum albumin f r a c t i o n V was purchased from Sigma Chemical Co., St. Louis, Mo. lonophore A23187 was a g i f t from Dr. Hamil of L i l l y Research Laboratories, Indianapolis, Ind. Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCPP) was from Pierce Chemical Co., Rockford, I I I . Results Effect of calcium ionophore A23187 on cAMP~ cGMP and l i p o l y s i s . The greatest increase in cGMP caused by the ionophore occurs at 30 sec incubation. For t h i s reason in all the following experiments cGMP levels were measured at this time cAMP was measured at 6 min, the time when maximum accumulation of this cyclic nucleotide takes place in response to catecholamines and other stimulatory agents (4). Glycerol release was measured a f t e r 30 min incubation. As summarized in Table I , in a concentration range from I to 50 ~M, ionophore A23187 did not appreciably a l t e r cAMP levels in f a t c e l l s . However, the greatest increase in cGMP levels occurred at 1 ~M of the ionophore whereas the greatest increase in l i p o l y s i s occurred at I0 ~M. Interactions between A23187 and FCCP. As shown in Fig. 1 FCCP (I-100 pM) did not appreciably a f f e c t cAMP accumulation, e i t h e r in the presence or absence of calcium ionophore, A23187. Even when i00 pM of ionophore were added, cAMP level was increased by only 40%. In the same experiments FCCPcaused a conce~--tration-dependent inhibition of lipolysis (Fig. 2) and cGMPaccumulation (Fig. 3) induced by ionophore A23187. The inhibitory effect of FCCPon basal cGMP level and glycerol release was less evident, but was s t i l l directly proportional to the concentration employed.
Vol. 32, No. 6, 1983
Lipolysis in Rat Fat Cells
573
TABLE I E f f e c t of ionophore; A23187 on cAMP; cGMP and l i p o l y s i s % increase over control A23187 [pM] cAMP cGMP Glycerol rel ease
1
3.6 + 1.5
109.4 + 9.8
i0
13.2 + 1.4
60.8 + 7.2
68.9 +
5.3
126.6 + I0.2
50 25.1 + 2.3 26.7 + 4.3 25.8 + 2.1 Fat c e l l s were incubated in Krebs-Ringer bicarbonate albumin buffer (pH 7.4). A f t e r 15 min of preincubation various concentrations of A23187 were added and the reaction was stopped a f t e r 30 sec, 6 min and 30 min for the determination of cGMP, cAMP and_glycerol, r e s p e c t i v e l y . The basal l e v e l s were: 18.32 + 1.90 pmoles of cAMP/IOb c e l l s , 1.03 + 0.19 pmoles of cGMP/IO6 c e l l s and 74 + 9nmoles of g l y c e r o l / 1 0 u c e l l s . The r e s u l t s are the mean ~ S.E.) of vaTues obtained from 8 incubations in 2 experiments.
FCCP
Io
"=
° "~ 2 0 E
~ <
10-
0
I
0
////I
1
I
I
10
50
A 23187 [pM] FIG. 1 E f f e c t of A23187 and FCCP on cAMP l e v e l . Fat cell s were incubated in KrebsRinger bicarbonate albumin b u f f e r (pH 7.4) at 37°C. A f t e r 15 min preincubat i o n various concentrations of A23187 and FCCP were added and the incubation was stopped 6 min l a t e r by the a d d i t i o n of 0.2 ml of 50% TCA. cAMP was measured in the supernatant f l u i d p r e v i o u s l y treated with water-saturated ether in order to e x t r a c t TCA. Each point represents the mean (+ S.E.) of the values obtained from 8 incubations in 2 d i f f e r e n t experiments.
574
Lipolysis
in Rat Fat Cells
Vol. 32, No. 6, 1983
200 A
FCCP pM]
E
=o o E
0 I I0
100
F,,
100
o >-
0
/~ 0
I
i
I
1
10
50
A 23187 [pM]
FIG. 2 Effect of calcium ionophore A23187 and FCCP on l i p o l y s i s . Fat cells were preincubated for 15 min in Krebs-Ringer bicarbonate albumin buffer (pH 7.4) and the incubation was started by the addition of A23187 and FCCP at the concentration indicated. The reaction was stopped after 30 min by the addition of 0.2 ml of 50% TCA. Glycerol was measured in 0.5 ml aliquots of the supernatant f l u i d . Each value represents the mean (~S.E.) of the results obtained from 8 incubations in 2 different experiments.
3
I
FCCP
"~
[pM]
E Q.
0
~2
10
0
//
0
I,
I
I
1
10
50
A 23187 [pM]
FIG. 3 Effects of FCCP on A23187-induced cGMP accumulation. Fat cells were suspended in Krebs-Ringer bicarbonate albumin buffer (pH 7.4). After 15 min of preincubation at 37°C, FCCP and A23197 were added as indicated and the reaction was stopped 30 sec l a t e r for the determination of cGMP. Each value represents the mean ~T_S.E.) of the results obtained from 8 incubations in 2 separate experiments.
Vol. 32, No. 6, 1983
Lipolysis in Rat Fat Cells
575
Discussion Calcium ionophore A23187 has been extensively used for studying the influence of intracellular calcium changes on cell metabolism. The reports regarding its effects on fat cell metabolism are contradictory. Various groups have reported that i t increases (4,22) or has no affect (10) on glycerol release and that i t reduces (23) or does not affect (4,10,22) cAMPlevels. However, there is common agreement that the ionophore increases cGMP in fat cell (4,9,10) even though i t stimulates phosphodiesterase in hamster adipocytes (9). The present results show that the cGMP reaches its highest levels when A23187 concentrations range from I to 10 ~M. In these conditions the ionophore also caused significant increases of l i p o l y s i s , but did not appreciably alter cR4P levels. Thus calcium ionophore, A23187, appears to e l i c i t i t l i p o l y t i c effect through a mechanism which is independent of changes in cAMP levels. A similar dissociation of lipolysis from the cAMP system has already been observed in the presence of low concentrations of epinephrine, which increase the cGMP levels and the rate of glycerol release, without altering the c~P level (4). Furthermore epinephrine, like A23187, increases the rate of calcium efflux from fat cells (2,4). I t has not been established ~4nether the latter effect is a result of similar changes in intracellular Ca++ pools caused by both agents. However, ionophore A23187 has been reported to increase the total uptake of calcium into fat cells, mostly into extramitochondrial fractions (3). It also causes a massive release of the cation from adipocyte endoplasmic reticulum within seconds (24). Thus A23187-induced release of calcium from intact fat cells (4) may reasonably reflect the i n i t i a l discharge of the cation from endoplasmic reticulure and other intracellular stores into the cytoplasm down the concentration gradient (25-27). As already observed in epididymal spermatozoa (27) calcium would then be promptly pumped out of the cell through the plasma membrane so that the energy dissipating cyclic reuptake of calcium by mitochondria, which leads to uncoupling of oxidative phosphorylation by A23187 (25,27), can be prevented (26,27). This would agree with the fact that the ionophore does not alter ATP content (3,28) and glucose oxidation (29) in fat cells. As calcium ions have been shown to activate guanylate cyclase in fat cell membrane preparations (11,12) and this effect is concentration-dependent, up to about 0.I n~i CaCl2, while no effect or inhibition is obtained by increasing CaCl2 to l m~ (11,12), i t is tempting to assume that the increase in the cGMP levels induced by A23187 is the result of the i n i t i a l increase in cytoplasmic calcium caused by ionophore A23187, which may be the signal for the stimulation of guanylate cyclase and the consequent increase in cGMP level. This can also explain by FCCP decreases cGMPaccumulation. This agent is a well-known uncoupler of oxidative phosphorylation which selectively inhibits the calcium buffering capacity of mitochondria thus causing a release of the cation into the cytoplasm (16) without inducing significnt changes in calcium release from endoplasmic reticulum (30,31). The inhibitory effect of FCCPon cGMPboth in the presence and absence of ionophore A23187 appears to be related to its uncoupling action and/or calcium releasing effect. As an uncoupler of oxidative phosphorylation i t would reduce ATP and hence GTP levels. This lack of substrate for guanylate cyclase would decrease the enzyme activity with a consequent reduction of cGMP level, while the drop in ATP may not affect cAMP format i o n , which is primarily controlled by the availability of ATP produced by cytoplasmic glycolysis (32,33). Alternatively, since FCCP has been shown to cause per s e a massive release of calcium from mitochondria (16) and to potent i a t e the effect of ionophore A23187 on the release of calcium in other systems (30), i t is possible that in the presence of FCCPand the ionophore. A23187 intracellular calcium reaches a high concentration which would inhibit the enzyme (12). However this interpretation will remain speculative until the technical problem of directly measuring cytoplasmic calcium concentrations in intact fat cells has been solved.
576
Lipolysis in Rat Fat Cells
Vol. 32, No. 6, 1983
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
B. AIM, S. EFENDIC AND H. LOW, llorm. Metab. Res. 2 142-146 (1970). A.H. KISSEBAH, P. CLARKE, N. VYDELINGUM, H. HOPE-GILL, B. 1ULLOCHAND T.R. FRASER, Eur. J. Clin. Invest. 5 339-349 (1975). D.L. SEVERSON, R.M. DENTON, B.J. BRIDGES, AND P.J. RANDLE, Biochem. J. 154 209-223 (1976). R.M. GAION AND G. KRISHNA, Obesit,v:pathogenesis and treatment p. 135-148, Academic Press, New York (1981). S. EFENDIC, B. ALM, AND H. LOW, Horm. Metab. Res. 2 282-291 (1970). A.S. KATOCS, E.E. LARGIS, AND D.O. ALLEN, J. Biol. Chem. 249 2000-2204 (1974). R.J. SHIN~IEL, Horm. Metab. Res. 8 195-201 (1976). R.J. SHIMMEL, Horm. Metab. Res. lO 128-134 (1978). N. VYDELINGUM, A.H. KISSEBAH, AND V. WYNN, Horm. Metab. Res. 10 38-46 (1978). J.N. FAIN AND F.R. BUTCHER,J. Cyclic Nucleotide Res. 2, 71-78 (1976). R.M. GAION, AND G. KRISHNA, Life Sci. 3_11, 551-556 (1982). J. LEVILLIERS, F. LECOT, AND J. PAIRAULT, Biochem. Biophys. Res. Commun. 84 727-735 (1978). J.F. KUO, Proc. Natl. Acad. Sci. USA 71 4037-4041 (1974). T.M. LINCOLN, C.L. HALL, C.R. PARK, AND J.D. CORBIN, Proc. Natl. Acad. Sci. USA 73 2559-2563 (1976). J.C. KHO0, P.J. SPERRY, G.N. GILL, AND D. STEINBERG, Proc. Natl. Acad. Sci. USA 74 4843-4747 (1977). F.J. BRINLEY, T. TIFFERET, A. SCARPA, AND L.J. MULLINS, J. Gen. Physiol. 70, 355-384 (1977). M. RODBELLAND G. KRISHNA, Methods in EnRymology, Vol. XXXl - Biomembranes Part A, p. 103-114, Academic Press, N.Y. (1974). N. LAMBERTAND A.C. NEISH, Can. J. Res. (Sec. B) 2__883-89 (1950). T. I~ASH, Biochem. J. 55, 416-421 (1953). E.K. Frandsen, and G. Krishna, Life Sci. 1___8529-542 (1976). A.L. STEINER, C.W. PARKER, AND D.M. KIPNIS, J. Biol. Chem. 247 1106-1113 (1972). I. HIKAWYJ-YEVICH, AND A. SPITZEN, J. Surgical Res. 23, 233-238 (1977). G.M. NEMECEK,J. Cyclic Nucleotide Res. 4 299-309 (1978). J.M. McDONALD, D.E. BURNS, AND L. JARRETT, J. Biol. Chem. 253 3504-3508 (1978). P.W. REED, AND H.A. LARDY, J. Biol. Chem. 247 6970-6977 (1972). D.R. PFEIFFER, R.W. TAYLOR, AND H.A. LARDY, Calcium transport and cell function, Ann. N.Y. Acad. Sci. 307 p. 402-421, New York (1978). D.F. BABCOCK, N.L. FIRST, AND H.A. LARDY, J. Biol. che~,1. 251 3881-3886 (1976). K. SIDDLE, AND C.N. HALES, Bioch. Sco. Trans. 5--, 959-962 (1977) D. BONNE, O. BELHADJ, AND P. COHEN, Eur. J. Biochem. 75 101-105 (1977). M.P. Blaustein, R.W. Ratzlaff, and N.K. Nendrick, Calcium transport and cell function, Ann. N.Y. Acad. Sci. New York 195-211 (1978). A. SCARPAAND G. INESI, FEBS Letters 22, 273-276 (1972). G. FASSINA, P. DORIGOAND R.M. GAION, Pharmacol Res Comm6_, 1-21 (1974). G. FASSINA, P. DORIGO, AND R.M. GAION, Adv. Exp. Biol. Med. 63, 105-122 (1975).
Pergamon Pres:
CYCLIC NUCLEOTIDES AND LIPOLYSIS IN RAT FAT CELLS. INTERACTION BETWEEN CALCIUM IONOPHORE A23187 AND FCCP, UNCOUPLER OF OXIDATIVE PHOSPHORYLATION R. Gaion* and G. Krishna
Section on Drug-Tissue Interaction, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood I n s t i t u t e , National Institutes of Health, Bethesda, Md. 20205 (Received in final form October 18, 1982)
Summary
The interaction between calcium ionophore A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) has been studied at the level of cyclic-AMP(cAMP), cyclic-GMP (cGMP) and lipolysis in isolated rat fat cells. Ionophore A23187 (I-10 ~M) stimulated cGMP accumulation and glycerol release without affecting cAMP level. FCCP (I-I00 ~M) inhibited the effect of A23187 on cGMPlevel and glycerol release, but did not affect or increase cAMP. Thus a correlation exists between the changes of cGMPlevels and lipolysis and a dissociation of lipolysis from cAMP. Various hormones have been shown to influence calcium flux in intact adipocytes or its distribution among i n t r a c e l l u l a r pools (I-4). It is also known that calcium plays a permissive role in the l i p o l y t i c effect of hormones (5-8) and in the accumulation of cGMP in fat cells in response to catecholamines and other agents (9,10). We have previously shown that both epinephrine and the calcium ionophore, A23187, cause a very rapid increase of cGMP level associated with an increase of Ca++ f l u x in fat cells (4). Both agents stimulate l i p o l y s i s , even when no change in cAMP level occurs (4), As fat cell guanylate cyclase can be activated by calcium ions (11,12) and cGMPdependent protein kinase is present in adipose tissue (13,14) which directly stimulates hormone-sensitive lipase (15), we suggested that a calcium-cGMP system might exist in fat cells, acting as an intracellular messenger of short-term stimulations caused by low concentrations of catecholamines (4). In order to further c l a r i f y the correlation existing between changes in Ca++ flux, cyclic nucleotide levels and l i p o l y s i s , we have now studied the interaction between the calcium ionophore A23187 and FCCP, an uncoupler of oxidative phosphorylation which lowers the calcium buffering capacity of mitochondria, with a consequent marked increase of cytoplasmic calcium (16). Materials and Methods Male Sprague Dawley rats (140-200 g), fed ad libitum were used for the experiments. The animals were anesthetized with ether and the epididym~ f a t *Present address:
I s t i t u t o di Farmacologia, Largo E. Meneghetti 2, Universita di Padova, 35100 Padova, Italy
0024-3205/83/060571-06503.00/0
572
Lipolysis in Rat Fat Cells
Vol. 32, No. 6, 1983
pads were removed and immediately washed in Krebs-Ringer bicarbonate buffer, pH 7.4, saturated with 95% 02:5% CO2 and containing 3% bovine serum albumin fraction V. The buffer had the following composition: 119 mM NaCl; 4.5 mM KCl; 2.7 mM CaCI2; 1.2 mM KH2P04; 1.2 mM MgSO4 24.2 mM NaHCO3. The tissue was minced and suspended in the same buffer in the presence of crude collagenase (I mg/ml) and fat c e l l s were isolated according to Rodbell and Krishna (17). The cells were counted as described e a r l i e r (17). The final cell suspension was diluted with buffer and 2 ml a l i q u o t s , containing 80,0(10-160,000 c e l l s , were preincubated f o r 15 min at 37°C. The incubation was then started by the addition of the drugs dissolved in i0 ~I of buffer and terminated at the various times by the addition of 0.2 ml of 50% t r i c h l o r o a c e t i c acid (TCA). After mixing and c e n t r i f u g a t i o n of the samples, aliquots of supernatant f l u i d were analyzed for g l y c e r o l , cAMP and cGMP as described below. Estimation of glycerol. Duplicate aliquots (0.5 ml) of the supernatant f l u i d were used for the determination of glycerol. Glycerol was determined according to the method of Lambert and Neish (18) by using the acetyl acetone reagent as proposed by Nash (19). Known amounts of glycerol dissolved in KrebsRinger bicarbonate albumin buffer and treated with TCA served as a standard. Estimation of cAMP and cGMP. TCA was removed from the supernatant f l u i d by four extractions with four volumes of water-saturated ether until a pH 4.3-4.6 was reached. Duplicate aliquots of 50 ~I were then assayed by the radioimmunoassay described by Frandsen and Krishna (20) without f u r t h e r p u r i f i c a t i o n . The samples were succinylated by addition of 5 ~I of succinylation mixture (20). No f u r t h e r p u r i f i c a t i o n of samples by ion exchange chromatography was deemed necessary because s i m i l a r results were obtained when the samples were assayed before or a f t e r p u r i f i c a t i o n by Dowex 1 chromatography. A series of I0 cAMP and cGMP standards (from 1 to I0,000 fmoles) were assayed in t r i p l i c a t e with the samples; the standards also contained 50 ~1 of Krebs-Ringer bicarbonate albumin medium which had been treated with TCA and extracted with ether. Materials. cAMP and cGMP antisera were prepared in our laboratory according to the method of Sterner et a l . (21). lodinated 2'-Osuccinyl cAMP and cGMP tyrosine methyl esters, I ~ P - T M E and 1251 ScGMP-TME were obtained from Collaborative Research Inc., Waltham, Mass. Bovine serum albumin f r a c t i o n V was purchased from Sigma Chemical Co., St. Louis, Mo. lonophore A23187 was a g i f t from Dr. Hamil of L i l l y Research Laboratories, Indianapolis, Ind. Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCPP) was from Pierce Chemical Co., Rockford, I I I . Results Effect of calcium ionophore A23187 on cAMP~ cGMP and l i p o l y s i s . The greatest increase in cGMP caused by the ionophore occurs at 30 sec incubation. For t h i s reason in all the following experiments cGMP levels were measured at this time cAMP was measured at 6 min, the time when maximum accumulation of this cyclic nucleotide takes place in response to catecholamines and other stimulatory agents (4). Glycerol release was measured a f t e r 30 min incubation. As summarized in Table I , in a concentration range from I to 50 ~M, ionophore A23187 did not appreciably a l t e r cAMP levels in f a t c e l l s . However, the greatest increase in cGMP levels occurred at 1 ~M of the ionophore whereas the greatest increase in l i p o l y s i s occurred at I0 ~M. Interactions between A23187 and FCCP. As shown in Fig. 1 FCCP (I-100 pM) did not appreciably a f f e c t cAMP accumulation, e i t h e r in the presence or absence of calcium ionophore, A23187. Even when i00 pM of ionophore were added, cAMP level was increased by only 40%. In the same experiments FCCPcaused a conce~--tration-dependent inhibition of lipolysis (Fig. 2) and cGMPaccumulation (Fig. 3) induced by ionophore A23187. The inhibitory effect of FCCPon basal cGMP level and glycerol release was less evident, but was s t i l l directly proportional to the concentration employed.
Vol. 32, No. 6, 1983
Lipolysis in Rat Fat Cells
573
TABLE I E f f e c t of ionophore; A23187 on cAMP; cGMP and l i p o l y s i s % increase over control A23187 [pM] cAMP cGMP Glycerol rel ease
1
3.6 + 1.5
109.4 + 9.8
i0
13.2 + 1.4
60.8 + 7.2
68.9 +
5.3
126.6 + I0.2
50 25.1 + 2.3 26.7 + 4.3 25.8 + 2.1 Fat c e l l s were incubated in Krebs-Ringer bicarbonate albumin buffer (pH 7.4). A f t e r 15 min of preincubation various concentrations of A23187 were added and the reaction was stopped a f t e r 30 sec, 6 min and 30 min for the determination of cGMP, cAMP and_glycerol, r e s p e c t i v e l y . The basal l e v e l s were: 18.32 + 1.90 pmoles of cAMP/IOb c e l l s , 1.03 + 0.19 pmoles of cGMP/IO6 c e l l s and 74 + 9nmoles of g l y c e r o l / 1 0 u c e l l s . The r e s u l t s are the mean ~ S.E.) of vaTues obtained from 8 incubations in 2 experiments.
FCCP
Io
"=
° "~ 2 0 E
~ <
10-
0
I
0
////I
1
I
I
10
50
A 23187 [pM] FIG. 1 E f f e c t of A23187 and FCCP on cAMP l e v e l . Fat cell s were incubated in KrebsRinger bicarbonate albumin b u f f e r (pH 7.4) at 37°C. A f t e r 15 min preincubat i o n various concentrations of A23187 and FCCP were added and the incubation was stopped 6 min l a t e r by the a d d i t i o n of 0.2 ml of 50% TCA. cAMP was measured in the supernatant f l u i d p r e v i o u s l y treated with water-saturated ether in order to e x t r a c t TCA. Each point represents the mean (+ S.E.) of the values obtained from 8 incubations in 2 d i f f e r e n t experiments.
574
Lipolysis
in Rat Fat Cells
Vol. 32, No. 6, 1983
200 A
FCCP pM]
E
=o o E
0 I I0
100
F,,
100
o >-
0
/~ 0
I
i
I
1
10
50
A 23187 [pM]
FIG. 2 Effect of calcium ionophore A23187 and FCCP on l i p o l y s i s . Fat cells were preincubated for 15 min in Krebs-Ringer bicarbonate albumin buffer (pH 7.4) and the incubation was started by the addition of A23187 and FCCP at the concentration indicated. The reaction was stopped after 30 min by the addition of 0.2 ml of 50% TCA. Glycerol was measured in 0.5 ml aliquots of the supernatant f l u i d . Each value represents the mean (~S.E.) of the results obtained from 8 incubations in 2 different experiments.
3
I
FCCP
"~
[pM]
E Q.
0
~2
10
0
//
0
I,
I
I
1
10
50
A 23187 [pM]
FIG. 3 Effects of FCCP on A23187-induced cGMP accumulation. Fat cells were suspended in Krebs-Ringer bicarbonate albumin buffer (pH 7.4). After 15 min of preincubation at 37°C, FCCP and A23197 were added as indicated and the reaction was stopped 30 sec l a t e r for the determination of cGMP. Each value represents the mean ~T_S.E.) of the results obtained from 8 incubations in 2 separate experiments.
Vol. 32, No. 6, 1983
Lipolysis in Rat Fat Cells
575
Discussion Calcium ionophore A23187 has been extensively used for studying the influence of intracellular calcium changes on cell metabolism. The reports regarding its effects on fat cell metabolism are contradictory. Various groups have reported that i t increases (4,22) or has no affect (10) on glycerol release and that i t reduces (23) or does not affect (4,10,22) cAMPlevels. However, there is common agreement that the ionophore increases cGMP in fat cell (4,9,10) even though i t stimulates phosphodiesterase in hamster adipocytes (9). The present results show that the cGMP reaches its highest levels when A23187 concentrations range from I to 10 ~M. In these conditions the ionophore also caused significant increases of l i p o l y s i s , but did not appreciably alter cR4P levels. Thus calcium ionophore, A23187, appears to e l i c i t i t l i p o l y t i c effect through a mechanism which is independent of changes in cAMP levels. A similar dissociation of lipolysis from the cAMP system has already been observed in the presence of low concentrations of epinephrine, which increase the cGMP levels and the rate of glycerol release, without altering the c~P level (4). Furthermore epinephrine, like A23187, increases the rate of calcium efflux from fat cells (2,4). I t has not been established ~4nether the latter effect is a result of similar changes in intracellular Ca++ pools caused by both agents. However, ionophore A23187 has been reported to increase the total uptake of calcium into fat cells, mostly into extramitochondrial fractions (3). It also causes a massive release of the cation from adipocyte endoplasmic reticulum within seconds (24). Thus A23187-induced release of calcium from intact fat cells (4) may reasonably reflect the i n i t i a l discharge of the cation from endoplasmic reticulure and other intracellular stores into the cytoplasm down the concentration gradient (25-27). As already observed in epididymal spermatozoa (27) calcium would then be promptly pumped out of the cell through the plasma membrane so that the energy dissipating cyclic reuptake of calcium by mitochondria, which leads to uncoupling of oxidative phosphorylation by A23187 (25,27), can be prevented (26,27). This would agree with the fact that the ionophore does not alter ATP content (3,28) and glucose oxidation (29) in fat cells. As calcium ions have been shown to activate guanylate cyclase in fat cell membrane preparations (11,12) and this effect is concentration-dependent, up to about 0.I n~i CaCl2, while no effect or inhibition is obtained by increasing CaCl2 to l m~ (11,12), i t is tempting to assume that the increase in the cGMP levels induced by A23187 is the result of the i n i t i a l increase in cytoplasmic calcium caused by ionophore A23187, which may be the signal for the stimulation of guanylate cyclase and the consequent increase in cGMP level. This can also explain by FCCP decreases cGMPaccumulation. This agent is a well-known uncoupler of oxidative phosphorylation which selectively inhibits the calcium buffering capacity of mitochondria thus causing a release of the cation into the cytoplasm (16) without inducing significnt changes in calcium release from endoplasmic reticulum (30,31). The inhibitory effect of FCCPon cGMPboth in the presence and absence of ionophore A23187 appears to be related to its uncoupling action and/or calcium releasing effect. As an uncoupler of oxidative phosphorylation i t would reduce ATP and hence GTP levels. This lack of substrate for guanylate cyclase would decrease the enzyme activity with a consequent reduction of cGMP level, while the drop in ATP may not affect cAMP format i o n , which is primarily controlled by the availability of ATP produced by cytoplasmic glycolysis (32,33). Alternatively, since FCCP has been shown to cause per s e a massive release of calcium from mitochondria (16) and to potent i a t e the effect of ionophore A23187 on the release of calcium in other systems (30), i t is possible that in the presence of FCCPand the ionophore. A23187 intracellular calcium reaches a high concentration which would inhibit the enzyme (12). However this interpretation will remain speculative until the technical problem of directly measuring cytoplasmic calcium concentrations in intact fat cells has been solved.
576
Lipolysis in Rat Fat Cells
Vol. 32, No. 6, 1983
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
B. AIM, S. EFENDIC AND H. LOW, llorm. Metab. Res. 2 142-146 (1970). A.H. KISSEBAH, P. CLARKE, N. VYDELINGUM, H. HOPE-GILL, B. 1ULLOCHAND T.R. FRASER, Eur. J. Clin. Invest. 5 339-349 (1975). D.L. SEVERSON, R.M. DENTON, B.J. BRIDGES, AND P.J. RANDLE, Biochem. J. 154 209-223 (1976). R.M. GAION AND G. KRISHNA, Obesit,v:pathogenesis and treatment p. 135-148, Academic Press, New York (1981). S. EFENDIC, B. ALM, AND H. LOW, Horm. Metab. Res. 2 282-291 (1970). A.S. KATOCS, E.E. LARGIS, AND D.O. ALLEN, J. Biol. Chem. 249 2000-2204 (1974). R.J. SHIN~IEL, Horm. Metab. Res. 8 195-201 (1976). R.J. SHIMMEL, Horm. Metab. Res. lO 128-134 (1978). N. VYDELINGUM, A.H. KISSEBAH, AND V. WYNN, Horm. Metab. Res. 10 38-46 (1978). J.N. FAIN AND F.R. BUTCHER,J. Cyclic Nucleotide Res. 2, 71-78 (1976). R.M. GAION, AND G. KRISHNA, Life Sci. 3_11, 551-556 (1982). J. LEVILLIERS, F. LECOT, AND J. PAIRAULT, Biochem. Biophys. Res. Commun. 84 727-735 (1978). J.F. KUO, Proc. Natl. Acad. Sci. USA 71 4037-4041 (1974). T.M. LINCOLN, C.L. HALL, C.R. PARK, AND J.D. CORBIN, Proc. Natl. Acad. Sci. USA 73 2559-2563 (1976). J.C. KHO0, P.J. SPERRY, G.N. GILL, AND D. STEINBERG, Proc. Natl. Acad. Sci. USA 74 4843-4747 (1977). F.J. BRINLEY, T. TIFFERET, A. SCARPA, AND L.J. MULLINS, J. Gen. Physiol. 70, 355-384 (1977). M. RODBELLAND G. KRISHNA, Methods in EnRymology, Vol. XXXl - Biomembranes Part A, p. 103-114, Academic Press, N.Y. (1974). N. LAMBERTAND A.C. NEISH, Can. J. Res. (Sec. B) 2__883-89 (1950). T. I~ASH, Biochem. J. 55, 416-421 (1953). E.K. Frandsen, and G. Krishna, Life Sci. 1___8529-542 (1976). A.L. STEINER, C.W. PARKER, AND D.M. KIPNIS, J. Biol. Chem. 247 1106-1113 (1972). I. HIKAWYJ-YEVICH, AND A. SPITZEN, J. Surgical Res. 23, 233-238 (1977). G.M. NEMECEK,J. Cyclic Nucleotide Res. 4 299-309 (1978). J.M. McDONALD, D.E. BURNS, AND L. JARRETT, J. Biol. Chem. 253 3504-3508 (1978). P.W. REED, AND H.A. LARDY, J. Biol. Chem. 247 6970-6977 (1972). D.R. PFEIFFER, R.W. TAYLOR, AND H.A. LARDY, Calcium transport and cell function, Ann. N.Y. Acad. Sci. 307 p. 402-421, New York (1978). D.F. BABCOCK, N.L. FIRST, AND H.A. LARDY, J. Biol. che~,1. 251 3881-3886 (1976). K. SIDDLE, AND C.N. HALES, Bioch. Sco. Trans. 5--, 959-962 (1977) D. BONNE, O. BELHADJ, AND P. COHEN, Eur. J. Biochem. 75 101-105 (1977). M.P. Blaustein, R.W. Ratzlaff, and N.K. Nendrick, Calcium transport and cell function, Ann. N.Y. Acad. Sci. New York 195-211 (1978). A. SCARPAAND G. INESI, FEBS Letters 22, 273-276 (1972). G. FASSINA, P. DORIGOAND R.M. GAION, Pharmacol Res Comm6_, 1-21 (1974). G. FASSINA, P. DORIGO, AND R.M. GAION, Adv. Exp. Biol. Med. 63, 105-122 (1975).