Biochem. J. (1994) 300, 81-84 (Printed in Great Britain) 81 Inhibition of inositol trisphosphate-induced calcium release by caffeine is prevented by ATP Ludwig MISSIAEN,* Jan B. PARYS, Humbert DE SMEDT, Bernard HIMPENS and Rik CASTEELS Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium We have investigated the effect of various methylxanthines on specific, since isocaffeine was not effective. The inhibition oc- the basal and InsP -stimulated unidirectional 45Ca2+ efflux from curred similarly in the absence or presence of extravesicular Ca2+ permeabilized A7r5 cells under different experimental conditions. and was not associated with a decrease in the [3H]InsP3 binding We report that caffeine and theophylline inhibit the InsP3- to the receptor. ATP and MgATP (5 mM) prevented the in- induced Ca2+ release, whereas the basal Ca2+ leak remained hibition, suggesting that caffeine may interact with an ATP- largely unaffected. The effect on InsP3-induced Ca2+ release was binding site on the InsP3 receptor or some associated protein. INTRODUCTION 30 mM imidazole/HCl (pH 6.8), 2 mM MgCl2, 1 mM ATP, 1 mM EGTA and 20,g/ml saponin at 25 'C. The non-mito- some, not types Caffeine stimulates Ca2l release through but all, chondrial Ca2+ stores were then loaded with 45Ca2" for 40 min in as a tool of ryanodine receptors [1-3] and has been widely used a medium containing 120 mM KCl, 30 mM imidazole/HCl for demonstrating the presence of a ryanodine receptor in a (pH 6.8), 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM variety of cells. In these studies, it became apparent that caffeine NaN3 and 238 nM free Ca2+ (23 ,tCi/ml) at 25 'C. The wells were concentrations in the range 10-25 mM inhibited Ca2l responses then washed twice in efflux medium A containing 120 mM KCI, induced by inositol 1,4,5-trisphosphate (InsPJ)-producing 30 mM imidazole/HCl (pH 6.8), 2 mM MgCl2, 1 mM ATP, agonists [4-11]. It has been proposed that millimolar concen- 1 mM EGTA and 5 mM NaN3 at 25 'C. In those experiments trations of caffeine inhibit the InsP3 receptor [8,12-14]. However, where the effect of changing the ATP concentration was tested, in other experimental systems, caffeine concentrations up to the efflux medium only contained 120 mM KCl, 30 mM 20 mM [10] and 50 mM [15] did not affect the InsP3 receptor. imidazole/HCl (pH 6.8), 1 mM EGTA and the indicated ATP This discrepancy indicates that the inhibitory effect of caffeine concentration (efflux medium B). The efflux media A and B had must be strongly dependent on experimental conditions and/or a calculated free [Ca2+] of less than 10 nM. The medium was on cell type. replaced every 2 min for a 16 min period. Methylxanthines were We have investigated the effect of caffeine on the basal and added to the efflux medium for 4 min from min 2 to 6 of efflux. unidirectional 45Ca2l efflux from the non-mito- InsP3-stimulated InsP3 at the indicated concentration or A23187 (10,uM) was chondrial Ca2l stores of permeabilized A7r5 smooth-muscle added for 2 min between min 4 and 6 of efflux. cells. The two were specifically addressed: following questions InsP3 and ATP were from Boehringer, Mannheim, Germany. (1) does caffeine inhibit the InsP3-induced Ca2l release and, if so, Caffeine, saponin, NaN3, thapsigargin, A23187 and the various does this inhibition represent a specific action of caffeine? (2) is methylxanthines were from Sigma Chemical Co., St. Louis, MO, the extent of inhibition of the release dependent on experimental U.S.A. 45Ca2+ was from Amersham International, Amersham, conditions? Bucks, U.K. All other reagents were of the highest purity We report that caffeine does inhibit InsP3-mediated Ca2l commercially available. release and that this inhibition must represent a specific inter- action. The inhibition occurs independently of the cytosolic RESULTS [Ca2+], and the presence of ATP or MgATP in the assay medium prevents this inhibitory effect of caffeine. Effect of caffeine on the passive Ca2+ leak Permeabilized A7r5 cells were loaded to steady state with 45Ca2+ MATERIALS AND METHODS and then exposed for 4 min to caffeine (1,3,7-trimethylxanthine), isocaffeine (1,3,9-trimethylxanthine) or theophylline (1,3- A7r5 aortic smooth-muscle cells were cultured as described dimethylxanthine) in the absence of added InsP3 (Figure 1). The previously [16]. The cells were used between passages 10 and 20 highest concentrations tested (50 mM for caffeine and isocaffeine after receipt from the American Type Culture Collection (Beth- and 25 mM for the less soluble theophylline) induced a slight esda, MD, U.S.A.), unless otherwise indicated. They were seeded decrease in the Ca2+ content of the stores. This represented a in 12-well clusters at a density of approx. 104 cells/cm2. non-specific effect on the passive Ca2+ leak and not the activation Experiments were carried out with confluent monolayers of a possible ryanodine receptor, for the following reasons: (1) of cells on day 7 after plating, at an average cell density of isocaffeine, which is about 5 times less effective than caffeine in 7.5 x 104 cells/cm2. activating the ryanodine receptor [17], was equally potent as Permeabilization with saponin was performed by incubating caffeine in decreasing the Ca2+ content of the store; (2) the effect the cells for O min in a solution containing 120 mM KCl, of the methylxanthines also occurred in the presence of 1O ,uM Abbreviation used: InsP3, inositol 1,4,5-trisphosphate. * To whom correspondence and reprint requests should be addressed. 82 L. Missiaen and others 100 - 100 (a) Caffeine Isocaffeine Theophylline aU, Isocaffeine L- c 50- (U 50- 0 0-ax 04 as (U Theophylline 0 +- Caffeine IF. I- ...I O 11 .. III 0 1 10 100 0 1 10 100 [Xanthinel (mM) [Xanthinel (mM) Figure 1 Effect of methylxanthines on the passive Ca2+ leak in permeabilized A7r5 cells The non-mitochondrial Ca2+ stores were first loaded to steady state with 45Ca2+ and then exposed to efflux medium A, to which the indicated concentration of caffeine (X), isocaffeine (X) or theophylline (0) was added for 4 min. The Ca2+ content of the stores after this application was expressed as a percentage of the Ca2+ content of the stores in the absence of these methylxanthines. The Ca2+ contents were corrected for non-specific binding, which was XI 50/ taken as the residual Ca2+ after addition of 10 MM A23187. Non-specific binding amounted on (U average to 3% of total Ca2+ uptake. For clarity, the S.E.M. (n = 4) is only shown for the data at 50 mM methylxanthine. The S.E.M. was always lower than 5%. Caffeine Ruthenium Red, which blocks the ryanodine receptor (results 0u 0.1 1 10 100 not shown); (3) the extent of inhibition was independent of the [InsP31 (MM) cytosolic free [Ca2+] in the range 10 nM-1 MuM (results not shown); (4) no evidence for a ryanodine receptor in A7r5 cells was found [9,16]. A similar non-specific but more pronounced Figure 2 Effect of methylxanthines on the insP3-lnduced Ca2+ release increase in the passive Ca2+ leak has also been observed in permeabilized hepatocytes [15]. (a) Effect of caffeine (-), isocaffeine (U) and theophylline (0) on the Ca2+ release induced by a sub-maximal dose of InsP3 (3.2,M). The methylxanthines were added to the efflux medium A 2 min before the InsP3 challenge as described in the Materials and methods section. The release induced by 3.2 ,uM InsP3 is expressed as a percentage of the release induced by 10,uM A23187. (b) Stores were challenged with the indicated InsP3 concentration in efflux Effect of caffeine on the InsP3-induced Ca2+ release medium A in the absence (-) or in the presence (-) of 50 mM caffeine. The results shown InsP3 is an ubiquitous messenger that releases Ca2+ from internal are typical for three independent experiments. stores [18]. In A7r5 cells, more than 95 % of the releasable Ca2+ is mobilized by InsPJ [16]. We investigated the effect of caffeine on the Ca2+ release induced by 3.2 MuM InsP, in efflux medium A, with passage number. This effect was particularly evident at which contained 1 mM ATP, after loading the stores to steady passage numbers higher than 30. For example, the cells of state with 45Ca2+ (Figure 2a). Ca2+ release induced by this sub- passage 35 were much less InsP -sensitive than those of passage maximal InsP3 concentration, expressed as a percentage of the 15: the EC50 shifted from 1.4MtM InsP3 (passage 15) to 13.1 ,uM ionophore-releasable Ca2 , was decreased by caffeine. It is InsP3 (passage 35) (results not shown). No significant difference noteworthy that a significant inhibition of the release occurred in the amount of Ca2+ accumulated in the stores was observed with caffeine concentrations that are usually applied to intact between these two groups of cells. The change in EC50 in high- cells (up to 25 mM) to activate pharmacologically a possible passage-number cells may be related to the type of InsP3 receptor ryanodine receptor [4-11]. Inhibition of phosphodiesterases was expressed in these cells. We have observed by reverse-tran- not responsible for these effects, since Ca2+ release could not be scriptase polymerase chain reaction a slight increase in the inhibited by 10 MM cyclic AMP (results not shown). The in- relative amount of the type I receptor as compared with the other hibitory effect of caffeine was observed over a whole range of isoforms (results not shown). InsP3 concentrations (Figure 2b).