Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca2+ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca2+, Mg2+, MgATP2-, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500 X g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein X ml-1 SR was loaded actively with 0.02 mM 45CaCl2, resulting in greater than 90 nmol X mg protein -1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested. It was found that (1) caffeine induces a transient, dose-dependent release of Ca2+, (2) the absolute amount of Ca2+ released by caffeine increases with the Ca2+ load of the SR, (3) increasing the ionic strength (mu) from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca2+ release increases with increasing (a) external Ca2+ concentrations up to 5 microM total Ca2+ (or 3 microM free Ca2+) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca2+ release is not affected by changes of either the Mg2+ or the MgATP2- concentration.