Effects of ivermectin and midecamycin on ryanodine receptors and the Ca2+-ATPase in sarcoplasmic reticulum of rabbit and rat skeletal muscle

1. Ryanodine receptor (RyR) Ca2+ channels in the sarcoplasmic reticulum (SR ) of skeletal muscle are regulated by the 12 kDa FK506- (or rapamycin-) bin ding protein (FKBP12). Rapamycin can also activate RyR channels with FKBP12 removed, suggesting that compounds with macrocyclic lactone ring structure s can directly activate RyRs. Here are tested this hypothesis using two oth er macrocyclic lactone compounds, ivermectin and midecamycin. 2. Rabbit skeletal RyRs were examined in lipid bilayers. Ivermectin (cis, 0 .66-40 mu M) activated six of eight native, four of four control-incubated and eleven of eleven FKBP12-'stripped' RyR channels. Midecamycin (cis, 10-3 0 mu M) activated three of four single native channels, six of eight contro l-incubated channels and six of seven FKBP12-stripped channels. Activity de clined when either drug was washed out. 3. Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blo ts of terminal cisternae (TC), incubated for 15 min at 37 degrees C with 40 mu M ivermectin or midecamycin, showed normal amounts of FKBP12. In contra st, no FKBP12 was detected after incubation with 40 mu M rapamycin. 4. Ivermectin reduced Ca2+ uptake by the SR Ca2+-Mg2+-ATPase. Ca2+ uptake b y TC fell to similar to 40% in the presence of ivermectin (10 mu M), both w ith and without 10 mu M Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to similar to 40% with 10 mu M ivermectin. Midecamycin (10 mu M) reduc ed Ca2+ uptake by TC vesicles to similar to 76% without Ruthenium Red and t o similar to 90% with Ruthenium Red. 5. The rate of rise of extravesicular [Ca2+] increased similar to 2-fold wh en 10 mu M ivermectin was added to TC vesicles that had been partially load ed with Ca2+ and then Ca2+ uptake blocked by 200 nM thapsigargin. Ivermecti n also potentiated caffeine-induced Ca2+ release to similar to 140% of cont rol. These increases in Ca2+ release were not seen with midecamycin. 6. Ivermectin, but not midecamycin, reversibly reduced Ca2+ loading in four of six skinned rat extensor digitorum longus (EDL) fibres to similar to 90 %, and reversibly increased submaximal caffeine-induced contraction in five of eight fibres by similar to 110% of control. Neither ivermectin nor mide camycin altered twitch or tetanic tension in intact EDL muscle fibres withi n 20 min of drug addition. 7. The results confirm the hypothesis that compounds with a macrocyclic lac tone ring structure can directly activate RyRs. Unexpectedly, ivermectin al so reduced Ca2+ uptake into the SR. These effects of ivermectin on SR Ca2handling may explain some effects of the macrolide drugs an mammals.