Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin-permeabilized frog skeletal muscle fibres

1. The effect of caffeine and adenine nucleotides on the sarcoplasmic retic ulum (SR) Ca2+ release mechanism was investigated in permeabilized frog ske letal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca2+ from the SR detected using fura-2 fluorescence. Decreasing the [AT P] from 5 to 0.1 mM reduced the caffeine-induced Ca2+ transient by 89 +/- 4 % (mean +/- S.E.M., n = 16), while SR Ca2+ uptake was unaffected. 2. The dependence of caffeine-induced Ca2+ release on cytosolic [ATP] was u sed to study the relative ability of other structurally related compounds t o substitute for, or compete with, ATP at the adenine nucleotide binding si te. It was found that AMP, ADP and the nonhydrolysable analogue adenylyl im idodiphosphate (AMP-PNP) partially substituted for ATP, although none was a s potent in facilitating the Ca2+-releasing action of caffeine. 3. Adenosine reversibly inhibited caffeine-induced Ca2+ release, without af fecting SR Ca2+ uptake. Five millimolar adenosine markedly reduced the ampl itude of the caffeine-induced Ca2+ transient by 64 +/- 4% (mean +/- S.E.M., n = 11). The degree of inhibition was dependent upon the cytosolic [ATP], suggesting that adenosine may act as a competitive antagonist at the adenin e nucleotide binding site. 4. These data show that (i) the sensitivity of the in situ SR Ca2+ channel to caffeine activation is strongly dependent upon the cytosolic [ATP], (ii) the number of phosphates attached to the 5' carbon of the ribose ring infl uences the efficacy of the ligand, and (iii) removal of a single phosphate group transforms AMP from a partial agonist, to adenosine, which acts as a competitive antagonist under these conditions.