The use of caged adenine nucleotides and caged phosphate in intact skeletal muscle fibres of the mouse

The effects of 1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), NPE-cag ed ADP, NPE-caged phosphate (P-i) and desoxybenzoinyl phosphate (desyl-cage d P-i) on mouse skeletal muscle function were studied. All these caged comp ounds, when microinjected into intact single mouse muscle fibres, reduced t he myoplasmic calcium during a tetanus (tetanic [Ca2+](i)) and reduced forc e. Flash photolysis partially reversed this reduction of tetanic [Ca2+](i) and force. In fibres fatigued by repeated tetani, flash photolysis of NPE-c aged ATP, ADP and P-i also caused a transient recovery of tetanic [Ca2+](i) and force. Because photolytic release of ATP, ADP and P-i produced compara ble effects it seems that the mechanism of action is the reduction in conce ntration of the caged compound rather than the release of the biologically active molecule. Experiments on mechanically skinned rat skeletal muscle fi bres with intact T-tubular/sarcoplasmic reticulum coupling showed that 1 Mm NPE-caged ATP had no effect on depolarization-induced force. This result s uggests that the depressant effects of the NPE-caged compounds are neither on voltage-activated Ca2+ release from the sarcoplasmic reticulum nor on my ofibrillar function. Thus all the caged compounds tested inhibit excitation -contraction coupling in muscle by an unknown mechanism and this limits the ir Value as sources of biologically important molecules. This inhibitory ef fect was smallest for desyl-caged P-i and under conditions of maximal activ ation photolytic release of P-i caused a direct inhibition of the contracti le proteins. This inhibition amounted to a 1% reduction of maximum force wi th an increase of [P-i] of about 0.3 Mm. The mean rate of force decline und er these conditions was 55 s(-1), which reflects the rate of cross-bridge c ycling during a maximal tetanus.