RYANODINE AND AN IODINATED ANALOG - DOXORUBICIN EFFECTS ON BINDING AND CA2-RETICULUM( ACCUMULATION IN CARDIAC SARCOPLASMIC)

An I-125-iodinated ryanodine analog, modified by attaching an iodo-Cbz -beta-alanyl group to the C-10eq hydroxy of ryanodine (iodo-carbobenzy loxy-beta-alanyl-ryanodine) binds to cardiac sarcoplasmic reticulum Ca 2+ release channels with equal affinity as [H-3]ryanodine. In the pres ent study, both iodo-Cbz-beta-alanyl-ryanodine and ryanodine bound to canine cardiac microsomal membrane preparations in a Ca2+ dependent ma nner. At 10 mu M free Ca2+ doxorubicin increased specific binding of b oth ligands, with doxorubicin concentrations of 4.06 +/- 0.44 and 6.22 +/- 1.31 mu M inducing 50% maximal enhancement of binding for ryanodi ne and iodo-Cbz-beta-alanyl-ryanodine, respectively. Effects of ryanod ine and iodo-Cbz-beta-alanyl-ryanodine +/- doxorubicin in vitro on car diac sarcoplasmic reticulum Ca2+ release were compared indirectly by d etermining Ca2+ accumulation in cardiac microsomal vesicles loaded wit h Ca-45(2+). In the absence of oxalate, neither ryanodine nor iodo-Cbz -beta-alanyl-ryanodine (10 mu M) decreased net Ca2+ uptake, whereas do xorubicin reduced Ca2+ accumulation 20 +/- 2%. In the presence of oxal ate and 0.4 mu M free Ca2+ (''low''), both ryanodine and iodo-Cbz-beta -alanyl-ryanodine modestly decreased (by 19% and 17% at 10 nM, respect ively) maximum Ca2+ accumulation. Increasing concentrations of ryanodi ne (100 nM-100 mu M) and iodo-Cbz-beta-alanyl-ryanodine (100 nM-30 mu M) had no greater effect, but 100 mu M iodo-Cbz-beta-alanyl-ryanodine decreased net Ca2+ uptake 57 +/- 3%. Doxorubicin (30 mu M) alone reduc ed Ca2+ uptake 36%; its effects with 1 nM-10 mu M ryanodine or 1 nM-10 0 mu M iodo-Cbz-beta-alanyl-ryanodine were additive. In the presence o f oxalate and 4.0 mu M free Ca2+ (''high''), 10-100 mu M ryanodine res ulted in 27-91% concentration-dependent increases in net Ca2+ uptake. In contrast, iodo-Cbz-beta-alanyl-ryanodine (10-100 mu M) had no effec t. Doxorubicin (30 mu M), alone or in the presence of iodo-Cbz-beta-al anyl-ryanodine, reduced Ca2+ uptake less than 10%, but it decreased by 25% the maximal enhancement of Ca2+ uptake by ryanodine. We conclude that ryanodine and iodo-Cbz-beta-alanyl-ryanodine have similar binding properties, but their effects on cardiac sarcoplasmic reticulum Ca2accumulation may reflect differing actions on the sarcoplasmic reticul um Ca2+ efflux channels.