The specific binding site of 9-[3H]methyl-7-bromoeudistomin D, a caffeine-like Ca2+ releaser, in liver microsomes in distinct from that in skeletal sarcoplasmic reticulum.
3H-labeled 9-methyl-7-bromoeudistomin D ([3H]MBED), a powerful caffeine-like Ca2+ releaser, binds to the caffeine binding site of terminal cisternae (TC) of skeletal muscle sarcoplasmic reticulum (SR) (Fang, Y-I., Adachi, M., Kobayashi, J., and Ohizumi, Y. (1993). J. Biol. Chem. 268, 18622-18625.) and activates Ca(2+)-induced Ca2+ release (CICR). [3H]MBED, however, bound to rabbit hepatic microsomes with a comparable affinity (Kd = 50 nM) and with a more than 30-fold greater receptor density (Bmax = 350 pmol/mg of protein), compared with those in SR. Caffeine (0.1-10 mM) caused a concentration dependent inhibition of [3H]MBED binding to hepatic microsomes with the IC50 value of 0.3 mM. The mode of inhibition by caffeine was allosteric, indicating that the binding site of the ligand is distinct from but related to that of caffeine. Procaine (1-10 mM), a representative inhibitor of CICR, which suppresses [3H]MBED binding to TC-SR, inhibited ligand binding to hepatic microsomes only slightly. Moreover, ligand binding to the hepatic binding site was not affected by adenosine 5'-(beta, gamma-methylene) triphosphate (AMP-PCP) (10-100 microM), which is an activator of CICR and potentiates [3H]MBED binding to TC-SR. Inhibitors of [3H]MBED binding to liver microsomes other than caffeine were nucleotides such as ADP, ATP, GTP, UTP (1 mM), while CTP, cAMP, AMP, adenosine (1 mM), ryanodine (0.1-100 mM) and inositol 1,4,5-trisphosphate (1 microM) were not effective. These features of the hepatic microsomal [3H]MBED binding site distinguish it from that of skeletal muscle SR. [3H]MBED, which binds to the different sites which are both sensitive to caffeine, is useful as a probe to investigate the actions of caffeine at the molecular level.
Adachi
M
M
Department of Pharmaceutical Molecular Biology, Tohoku University, Sendai, Japan.
Kakubari
M
M
Ohizumi
Y
Y
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002243:Carbolines / Q000493:pharmacokinetics* / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009711:Nucleotides / Q000494:pharmacology; D011343:Procaine / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
D002243:Carbolines; D009711:Nucleotides; C060741:9-methyl-7-bromoeudistomin D; D002110:Caffeine; D011343:Procaine; D002118:Calcium