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

摘要

class="enumerated" style="list-style-type:decimal">The effect of caffeine and adenine nucleotides on the sarcoplasmic reticulum (SR) Ca²⁺ release mechanism was investigated in permeabilized frog skeletal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca²⁺ from the SR detected using fura-2 fluorescence. Decreasing the [ATP] from 5 to 0.1 mm reduced the caffeine-induced Ca²⁺ transient by 89 ± 1.4 % (mean ± s.e.m., n = 16), while SR Ca²⁺ uptake was unaffected.The dependence of caffeine-induced Ca²⁺ release on cytosolic [ATP] was used to study the relative ability of other structurally related compounds to substitute for, or compete with, ATP at the adenine nucleotide binding site. It was found that AMP, ADP and the non-hydrolysable analogue adenylyl imidodiphosphate (AMP-PNP) partially substituted for ATP, although none was as potent in facilitating the Ca²⁺-releasing action of caffeine.Adenosine reversibly inhibited caffeine-induced Ca²⁺ release, without affecting SR Ca²⁺ uptake. Five millimolar adenosine markedly reduced the amplitude 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 adenine nucleotide binding site.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 influences 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.