Properties of the binding sites of [3H]9-methyl-7-bromoeudistomin D in bovine aortic smooth muscle microsomes.

[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), a powerful caffeine-like Ca2+ releaser, binds to the caffeine binding site of terminal cisternae of skeletal muscle sarcoplasmic reticulum and activates Ca(2+)-induced Ca2+ release. Properties of the binding site of [3H]MBED were investigated in aortic smooth muscle. The specific activity was higher in microsomes than in other fractions. [3H]MBED binding sites in smooth muscle microsomes were of a single class with a high affinity (KD 50nM), comparable with that in skeletal muscle sarcoplasmic reticulum. Caffeine competitively inhibited [3H]MBED binding, indicating MBED shares the same binding site with caffeine. Solubilization and fractionation of the microsomes gave two fractions of [3H]MBED binding activities. These results suggest that, in smooth muscle, there are multiple binding sites of [3H]MBED and caffeine, which might correspond to different pharmacological actions of caffeine on smooth muscle. Therefore, [3H]MBED, which binds to the different binding sites of caffeine, is useful as a probe for investigation of the actions of caffeine at the molecular level.