European Journal of Pharmacology, 175 (1990) 203-205
203
Elsevier E,JP 20533
Short communication
Caffeine inhibits forskolin-stimulated cyclic A M P accumulation in rat brain S a a k w a M a n t e a n d K e n n e t h P. M i n n e m a n Department of Pharmacology, Ernory University Medical School Atlanta, GA 30322, U.S.A, Received 9 November 1989, accepted 14 November 1989
Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex. The ICs0 for this inhibition was 21 + 2.9 /tM, and 200 /~M caffeine almost completely blocked the forskolin response. Theophylline which is a behavioural stimulant mimicked the effect of caffeine. However, there was only a slight inhibition of the forskolin response by theobromine and IBMX (3-isobutyl-l-methylxanthine), both of which lack behavioral stimulant effects. Caffeine; Forskolin; cAMP; Cortex; (Rat)
1. Introduction
Caffeine (1,3,7-trimethylxanthine) is one of the most widely consumed behaviorally active drugs in the world, being present in high concentrations in tea, coffee and many commercial beverages (Snyder and Sklar, 1984). In high doses, caffeine can cause nervousness, anxiety a n d / o r insomnia (Goldstein et al., 1965). Several molecular mechanisms have been suggested to underlie the behavioral effects of caffeine, but none of these have proven entirely satisfactory in explaining its effects. Caffeine was shown to competitively inhibit the breakdown of cyclic AMP through the inhibition of the enzyme 3'5'-cyclic nucleotide phosphodiesterase (Klainer et al., 1962). However, millimolar concentrations of caffeine are required to inhibit the enzyme (Beavo et al., 1970), making it unlikely that this is the primary mechanism of action of caffeine in the central nervous system. Caffeine is known to re-
Correspondence to: K,P. Minneman, Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322, U.S.A.
lease stored Ca 2 + from the sarcoplasmic reticulum of skeletal, cardiac and smooth muscle (Endo, 1977). Again, concentrations required for release of Ca 2+ are very high (millimolar). Another proposed mechanism was through blockade of benzodiazepine receptors (Skolnick et al., 1980), however, there is no correlation between the behavioral stimulant potencies of methylxanthines and their potencies at benzodiazepine receptors (Snyder et al., 1981). The most widely accepted mechanism for the behavioral stimulant effects of the methylxanthines is that these compounds act as competitive antagonists at adenosine receptors in brain and other tissues (Snyder et al., 1981). Although there is generally a good correlation between the potencies of methylxanthines as adenosine receptor antagonists and their ability to cause behavioral stimulation (Snyder et al., 1981), there are some exceptions. For example IBMX (3-isobutyl1-methylxanthine) is more potent than caffeine in blocking adenosine receptors but does not cause behaviour stimulation; it actually decreases locomotor activity in mice (Snyder et al., 1981). While studying the ability of caffeine to antagonize adenosine receptor binding in brain, we
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
204
have identified a potent and pharmacologically specific action of caffeine on cyclic A M P accumulation.
< /g
2. Materials and methods
2.1. Tissue preparation Male Sprague-Dawley rats (200-400 g; Zivic Miller) were killed by cervical dislocation, decapitated and brains removed. Cerebral cortices were dissected and transferred to a beaker of cold Krebs-Ringer bicarbonate buffer (KRB; (mM) 120 NaC1, 5.5 KC1, 2.5 CaCI2, 1.2 NaH2PO4, 1.2 MgC12, 20 N a H C O 3, 11 glucose and 0.019 C a N a 2 EDTA) which had been equilibrated with 95% 02-5% CO 2.
2.2. Cyclic AMP accumulation in slices Cyclic A M P accumulation in slices of rat cerebral cortex was measured by the [3H]adenine prelabelling technique (Shimizu et al., 1969) as previously described (Bazil and Minneman, 1986). Cerebral cortices were chopped into 350 × 350 t~m trapezoids on a McIlwain tissue chopper and dispersed in K R B at 37 o C. The buffer was decanted and the slices were resuspended in warm K R B and incubated in a shaking water bath a 3 7 ° C under 95% 0 2 : 5 % CO 2 for 15 rain. The medium was decanted and slices from a single hemicortex added to 15 ml warm KRB containing 30 /~Ci [3H]adenine and 6 /~M unlabelled adenine. The mixture was preincubated at 3 7 ° C with shaking for 40 min under O2-CO 2, slices were collected on nylon mesh, washed with warm KRB, and resuspended in the same buffer. An aliquot of 50 /~1 gravity packed slices were added to each incubation tube. Incubations were carried out in a final volume of I ml K R B containing appropriate drugs for 15 min at 3 7 ° C in a shaking water bath. Incubations were terminated by addition of 100/~1 of 77% trichloroacetic acid; after which 50 /xl of 10 m M cyclic A M P was added. Each sample was homogenized briefly with a Polytron, and centrifuged at 19000 × g for 15 rain. An aliquot of 50 t~l supernatant was removed from each sample for
(XX),
o
Basal
Forsk
6
5
4
- l o g CAFFEINE (M)
Fig. 1. Effect of caffeine on forskolin-stimulated cyclic A M P accumulation. Cyclic A M P accumulation was determined in the absence (Basal) and presence of 0.5 ~ M forskolin (Forsk) with or without the indicated concentrations of caffeine. Each value represents the mean_+S.E.M, of data from two experiments, each performed in triplicate.
determination of the total radioactivity incorporated into the tissue. [3H]Cyclic A M P formed was isolated by sequential D O W E X and alumina chromatography as previously described (Bazil and Minneman, 1986). Recovery was determined on parallel columns with authentic [3H]cyclic A M P and the yield ranged from 50-95%.
3. Results
3.1. Inhibition of forskolin-stimulated cyclic A M P accumulation by caffeine Forskolin, 0.5 /~M, caused a 6- to 7-fold increase in cyclic A M P accumulation in cerebral cortical slices. Caffeine dose dependently inhibits the forskolin response as shown in fig. 1. The ICs0 for the inhibition was 21 +_ 2.9 laM. At a concentration of 200 ~M, caffeine inhibited 82 +_ 3.8% of the response to 0.5 t~M forskolin.
3.2. Pharmacological specificity Other methylxanthines were tested to determine whether they mimicked the effect of caffeine. Figure 2 shows that caffeine and theophylline (200/~M) caused an 80-90% inhibition of the response to 0.5 t~M forskolin, while IBMX and
205
o_
P > 0.05). * * P < 0.001.
theobromine (200 ~M) caused a much smaller inhibition (30%) of the forskolin response.
4. Discussion These results demonstrate that caffeine potently inhibits forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex. The concentration of caffeine required to inhibit forskolin-stimulated cyclic AMP accumulation are within the range of whole brain levels of caffeine found to be associated with behavioral stimulation in mice (Snyder et al., 1981). The effect of caffeine is mimicked by the behaviorally active analog theophyUine, but less effectively by the behaviorally inactive analog theobromine, or the locomotor depressant IBMX. Forskolin is known to directly activate the catalytic subunit of adenylate cyclase, markedly increasing the rate of conversion of ATP to cyclic AMP (Seaman and Daly, 1981). However, forskolin also has a variety of other actions, and it is not yet clear how caffeine acts to inhibit the forskolin response. Since caffeine does not inhibit forskolin-stimulated adenylate cyclase activity in membrane preparations from brain (unpublished results), caffeine does not appear to block direct
Acknowledgement This work was supported by United States Public Health Service Grant DA 03413.
References Bazil, C.W. and K.P. Minneman, 1986, An investigation of the low intrinsic activity of adenosine and its analogs at low affinity (A2) adenosine receptors in rat cerebral cortex, J. Neurochem. 47, 547. Beavo, J.A., N.L. Rogers, O.B. Crofford, J.G. Hardman, E.W. Sutherland and E.V. Newman, 1970, Effects of xanthine derivatives on lipolysis and on adenosine 3',5' monophosphate phosphodiesterase activity, Mol. Pharmacol. 6, 597. Endo, M., 1977, Calcium release from the sarcoplasmic reticulure, Physiol. Rev. 57, 71. Goldstein, A., S. Kaizer and R. Warren, 1965, Psychotropic effects of caffeine in man. II. Alertness, psychomotor coordination, and mood, J. Pharmacol. Exp. Ther. 150, 146. Klainer, L.M., Y.-M. Chi, S.L. Freidberg, T.W. Rail and E.W. Sutherland, 1962, Adenyl cyclase. IV. The effects of neurohormones on the formation of adenosine 3',5'-phosphate by preparations from brain and other tissues, J. Biol. Chem. 237, 1239. Seaman, K. and J.W. Daly, 1981. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein, J. Biol. Chem. 256, 9799. Shimizu, H., J.W. Daly and C.R. Creveling, 1969, A radio-isotopic method for measuring the formation of adenosine 3',5' cyclic monophosphate in incubated slices of brain, J. Neurochem. 16, 1609. Skolnick, P., S.M. Paul and P.J. Marangos, 1980, Purines as endogenous ligands of the benzodiazepine receptor, Fed. Proc. 38, 3050. Snyder, S.H., J.J. Katims, Z. Annau, R.F. Bruns and J.W. Daly, 1981, Adenosine receptors and behavioral actions of methylxanthines, Proc. Natl. Acad. Sci. 78, 3260. Snyder, S.H. and P. Sklar, 1984, Behavioral and molecular actions of caffeine: Focus on adenosine, J. Psychiat. Res. 18, 91.
203
Elsevier E,JP 20533
Short communication
Caffeine inhibits forskolin-stimulated cyclic A M P accumulation in rat brain S a a k w a M a n t e a n d K e n n e t h P. M i n n e m a n Department of Pharmacology, Ernory University Medical School Atlanta, GA 30322, U.S.A, Received 9 November 1989, accepted 14 November 1989
Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex. The ICs0 for this inhibition was 21 + 2.9 /tM, and 200 /~M caffeine almost completely blocked the forskolin response. Theophylline which is a behavioural stimulant mimicked the effect of caffeine. However, there was only a slight inhibition of the forskolin response by theobromine and IBMX (3-isobutyl-l-methylxanthine), both of which lack behavioral stimulant effects. Caffeine; Forskolin; cAMP; Cortex; (Rat)
1. Introduction
Caffeine (1,3,7-trimethylxanthine) is one of the most widely consumed behaviorally active drugs in the world, being present in high concentrations in tea, coffee and many commercial beverages (Snyder and Sklar, 1984). In high doses, caffeine can cause nervousness, anxiety a n d / o r insomnia (Goldstein et al., 1965). Several molecular mechanisms have been suggested to underlie the behavioral effects of caffeine, but none of these have proven entirely satisfactory in explaining its effects. Caffeine was shown to competitively inhibit the breakdown of cyclic AMP through the inhibition of the enzyme 3'5'-cyclic nucleotide phosphodiesterase (Klainer et al., 1962). However, millimolar concentrations of caffeine are required to inhibit the enzyme (Beavo et al., 1970), making it unlikely that this is the primary mechanism of action of caffeine in the central nervous system. Caffeine is known to re-
Correspondence to: K,P. Minneman, Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322, U.S.A.
lease stored Ca 2 + from the sarcoplasmic reticulum of skeletal, cardiac and smooth muscle (Endo, 1977). Again, concentrations required for release of Ca 2+ are very high (millimolar). Another proposed mechanism was through blockade of benzodiazepine receptors (Skolnick et al., 1980), however, there is no correlation between the behavioral stimulant potencies of methylxanthines and their potencies at benzodiazepine receptors (Snyder et al., 1981). The most widely accepted mechanism for the behavioral stimulant effects of the methylxanthines is that these compounds act as competitive antagonists at adenosine receptors in brain and other tissues (Snyder et al., 1981). Although there is generally a good correlation between the potencies of methylxanthines as adenosine receptor antagonists and their ability to cause behavioral stimulation (Snyder et al., 1981), there are some exceptions. For example IBMX (3-isobutyl1-methylxanthine) is more potent than caffeine in blocking adenosine receptors but does not cause behaviour stimulation; it actually decreases locomotor activity in mice (Snyder et al., 1981). While studying the ability of caffeine to antagonize adenosine receptor binding in brain, we
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
204
have identified a potent and pharmacologically specific action of caffeine on cyclic A M P accumulation.
< /g
2. Materials and methods
2.1. Tissue preparation Male Sprague-Dawley rats (200-400 g; Zivic Miller) were killed by cervical dislocation, decapitated and brains removed. Cerebral cortices were dissected and transferred to a beaker of cold Krebs-Ringer bicarbonate buffer (KRB; (mM) 120 NaC1, 5.5 KC1, 2.5 CaCI2, 1.2 NaH2PO4, 1.2 MgC12, 20 N a H C O 3, 11 glucose and 0.019 C a N a 2 EDTA) which had been equilibrated with 95% 02-5% CO 2.
2.2. Cyclic AMP accumulation in slices Cyclic A M P accumulation in slices of rat cerebral cortex was measured by the [3H]adenine prelabelling technique (Shimizu et al., 1969) as previously described (Bazil and Minneman, 1986). Cerebral cortices were chopped into 350 × 350 t~m trapezoids on a McIlwain tissue chopper and dispersed in K R B at 37 o C. The buffer was decanted and the slices were resuspended in warm K R B and incubated in a shaking water bath a 3 7 ° C under 95% 0 2 : 5 % CO 2 for 15 rain. The medium was decanted and slices from a single hemicortex added to 15 ml warm KRB containing 30 /~Ci [3H]adenine and 6 /~M unlabelled adenine. The mixture was preincubated at 3 7 ° C with shaking for 40 min under O2-CO 2, slices were collected on nylon mesh, washed with warm KRB, and resuspended in the same buffer. An aliquot of 50 /~1 gravity packed slices were added to each incubation tube. Incubations were carried out in a final volume of I ml K R B containing appropriate drugs for 15 min at 3 7 ° C in a shaking water bath. Incubations were terminated by addition of 100/~1 of 77% trichloroacetic acid; after which 50 /xl of 10 m M cyclic A M P was added. Each sample was homogenized briefly with a Polytron, and centrifuged at 19000 × g for 15 rain. An aliquot of 50 t~l supernatant was removed from each sample for
(XX),
o
Basal
Forsk
6
5
4
- l o g CAFFEINE (M)
Fig. 1. Effect of caffeine on forskolin-stimulated cyclic A M P accumulation. Cyclic A M P accumulation was determined in the absence (Basal) and presence of 0.5 ~ M forskolin (Forsk) with or without the indicated concentrations of caffeine. Each value represents the mean_+S.E.M, of data from two experiments, each performed in triplicate.
determination of the total radioactivity incorporated into the tissue. [3H]Cyclic A M P formed was isolated by sequential D O W E X and alumina chromatography as previously described (Bazil and Minneman, 1986). Recovery was determined on parallel columns with authentic [3H]cyclic A M P and the yield ranged from 50-95%.
3. Results
3.1. Inhibition of forskolin-stimulated cyclic A M P accumulation by caffeine Forskolin, 0.5 /~M, caused a 6- to 7-fold increase in cyclic A M P accumulation in cerebral cortical slices. Caffeine dose dependently inhibits the forskolin response as shown in fig. 1. The ICs0 for the inhibition was 21 +_ 2.9 laM. At a concentration of 200 ~M, caffeine inhibited 82 +_ 3.8% of the response to 0.5 t~M forskolin.
3.2. Pharmacological specificity Other methylxanthines were tested to determine whether they mimicked the effect of caffeine. Figure 2 shows that caffeine and theophylline (200/~M) caused an 80-90% inhibition of the response to 0.5 t~M forskolin, while IBMX and
205
o_
P > 0.05). * * P < 0.001.
theobromine (200 ~M) caused a much smaller inhibition (30%) of the forskolin response.
4. Discussion These results demonstrate that caffeine potently inhibits forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex. The concentration of caffeine required to inhibit forskolin-stimulated cyclic AMP accumulation are within the range of whole brain levels of caffeine found to be associated with behavioral stimulation in mice (Snyder et al., 1981). The effect of caffeine is mimicked by the behaviorally active analog theophyUine, but less effectively by the behaviorally inactive analog theobromine, or the locomotor depressant IBMX. Forskolin is known to directly activate the catalytic subunit of adenylate cyclase, markedly increasing the rate of conversion of ATP to cyclic AMP (Seaman and Daly, 1981). However, forskolin also has a variety of other actions, and it is not yet clear how caffeine acts to inhibit the forskolin response. Since caffeine does not inhibit forskolin-stimulated adenylate cyclase activity in membrane preparations from brain (unpublished results), caffeine does not appear to block direct
Acknowledgement This work was supported by United States Public Health Service Grant DA 03413.
References Bazil, C.W. and K.P. Minneman, 1986, An investigation of the low intrinsic activity of adenosine and its analogs at low affinity (A2) adenosine receptors in rat cerebral cortex, J. Neurochem. 47, 547. Beavo, J.A., N.L. Rogers, O.B. Crofford, J.G. Hardman, E.W. Sutherland and E.V. Newman, 1970, Effects of xanthine derivatives on lipolysis and on adenosine 3',5' monophosphate phosphodiesterase activity, Mol. Pharmacol. 6, 597. Endo, M., 1977, Calcium release from the sarcoplasmic reticulure, Physiol. Rev. 57, 71. Goldstein, A., S. Kaizer and R. Warren, 1965, Psychotropic effects of caffeine in man. II. Alertness, psychomotor coordination, and mood, J. Pharmacol. Exp. Ther. 150, 146. Klainer, L.M., Y.-M. Chi, S.L. Freidberg, T.W. Rail and E.W. Sutherland, 1962, Adenyl cyclase. IV. The effects of neurohormones on the formation of adenosine 3',5'-phosphate by preparations from brain and other tissues, J. Biol. Chem. 237, 1239. Seaman, K. and J.W. Daly, 1981. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein, J. Biol. Chem. 256, 9799. Shimizu, H., J.W. Daly and C.R. Creveling, 1969, A radio-isotopic method for measuring the formation of adenosine 3',5' cyclic monophosphate in incubated slices of brain, J. Neurochem. 16, 1609. Skolnick, P., S.M. Paul and P.J. Marangos, 1980, Purines as endogenous ligands of the benzodiazepine receptor, Fed. Proc. 38, 3050. Snyder, S.H., J.J. Katims, Z. Annau, R.F. Bruns and J.W. Daly, 1981, Adenosine receptors and behavioral actions of methylxanthines, Proc. Natl. Acad. Sci. 78, 3260. Snyder, S.H. and P. Sklar, 1984, Behavioral and molecular actions of caffeine: Focus on adenosine, J. Psychiat. Res. 18, 91.
