Ryanodine and an iodinated analog: doxorubicin effects on binding and Ca2+ accumulation in cardiac sarcoplasmic...

An 125I-iodinated ryanodine analog, modified by attaching an iodo-Cbz-beta-alanyl group to the C10eq hydroxy of ryanodine (iodo-carbobenzyloxy-beta-alanyl-ryanodine), binds to cardiac sarcoplasmic reticulum Ca2+ release channels with equal affinity as [3H]ryanodine. In the present study, both iodo-Cbz-beta-alanyl-ryanodine and ryanodine bound to canine cardiac microsomal membrane preparations in a Ca2+ dependent manner. At 10 microM free Ca2+ doxorubicin increased specific binding of both ligands, with doxorubicin concentrations of 4.06 +/- 0.44 and 6.22 +/- 1.31 microM inducing 50% maximal enhancement of binding for ryanodine and iodo-Cbz-beta-alanyl-ryanodine, respectively. Effects of ryanodine and iodo-Cbz-beta-alanyl-ryanodine +/- doxorubicin in vitro on cardiac sarcoplasmic reticulum Ca2+ release were compared indirectly by determining Ca2+ accumulation in cardiac microsomal vesicles loaded with 45Ca2+. In the absence of oxalate, neither ryanodine nor iodo-Cbz-beta-alanyl-ryanodine (10 microM) decreased net Ca2+ uptake, whereas doxorubicin reduced Ca2+ accumulation 20 +/- 2%. In the presence of oxalate and 0.4 microM free Ca2+ ("low"), both ryanodine and iodo-Cbz-beta-alanyl-ryanodine modestly decreased (by 19% and 17% at 10 nM, respectively) maximum Ca2+ accumulation. Increasing concentrations of ryanodine (100 nM-100 microM) and iodo-Cbz-beta-alanyl-ryanodine (100 nM-30 microM) had no greater effect, but 100 microM iodo-Cbz-beta-alanyl- ryanodine decreased net Ca2+ uptake 57 +/- 3%. Doxorubicin (30 microM) alone reduced Ca2+ uptake 36%; its effects with 1 nM-10 microM ryanodine or 1 nM-100 microM iodo-Cbz-beta-alanyl-ryanodine were additive.(ABSTRACT TRUNCATED AT 250 WORDS)