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

Abstract

1. Ryanodine receptor (RyR) Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal muscle are regulated by the 12 kDa FK506- (or rapamycin-) binding protein (FKBP12). Rapamycin can also activate RyR channels with FKBP12 removed, suggesting that compounds with macrocyclic lactone ring structures can directly activate RyRs. Here we tested this hypothesis using two other macrocyclic lactone compounds, ivermectin and midecamycin. 2. Rabbit skeletal RyRs were examined in lipid bilayers. Ivermectin (cis, 0.66-40 microM) activated six of eight native, four of four control-incubated and eleven of eleven FKBP12-'stripped' RyR channels. Midecamycin (cis, 10-30 microM) activated three of four single native channels, six of eight control-incubated channels and six of seven FKBP12-stripped channels. Activity declined when either drug was washed out. 3. Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blots of terminal cisternae (TC), incubated for 15 min at 37 C with 40 microM ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 microM rapamycin. 4. Ivermectin reduced Ca2+ uptake by the SR Ca2+-Mg2+-ATPase. Ca2+ uptake by TC fell to approximately 40% in the presence of ivermectin (10 microM), both with and without 10 microM Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to approximately 40% with 10 microM ivermectin. Midecamycin (10 microM) reduced Ca2+ uptake by TC vesicles to approximately 76% without Ruthenium Red and to approximately 90 % with Ruthenium Red. 5. The rate of rise of extravesicular [Ca2+] increased approximately 2-fold when 10 microM ivermectin was added to TC vesicles that had been partially loaded with Ca2+ and then Ca2+ uptake blocked by 200 nM thapsigargin. Ivermectin also potentiated caffeine-induced Ca2+ release to approximately 140% of control. 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 approximately 90%, and reversibly increased submaximal caffeine-induced contraction in five of eight fibres by approximately 110% of control. Neither ivermectin nor midecamycin altered twitch or tetanic tension in intact EDL muscle fibres within 20 min of drug addition. 7. The results confirm the hypothesis that compounds with a macrocyclic lactone ring structure can directly activate RyRs. Unexpectedly, ivermectin also reduced Ca2+ uptake into the SR. These effects of ivermectin on SR Ca2+ handling may explain some effects of the macrolide drugs on mammals.

Figure 5. Ivermectin releases Ca²⁺ from partially loaded TC vesicles in the absence and presence of thapsigargin

Records of OD changes at 710-790 nm following changes in extravesicular [Ca²⁺], using antipyrylazo III (500 μm) as the Ca²⁺ indicator. Extravesicular [Ca²⁺] increased by ≈4 μm at the start of each record when TC vesicles were added to the cuvette and then declined as Ca²⁺ was sequestered by the TC vesicles. The following four positive deflections indicate addition of four aliquots of CaCl₂ (7.5 μm final concentration). A, ivermectin added with ethanol vehicle (e + i, 10 μm). B, thapsigargin (th, 200 nM) added before ethanol control (e, 0.2 % v/v). C, thapsigargin (200 nM) added before ivermectin (e + i, 10 μm). In A-C, Ruthenium Red (rr, 10 μm) was added before the ionophore A23187 (a, 3 μg ml⁻¹) at the end of each record. The vertical calibration bar shows [Ca²⁺] (see legend to Fig. 4). In this experiment an increase in [Ca²⁺] of 25 μm caused an increase of 0.3 OD units.

Figure 1. Activation of RyRs by ivermectin

A, continuous channel activity of a single RyR incorporated from native TC vesicles. Upper traces, control recording. Lower traces, recording after addition of 1.3 μm ivermectin. B, continuous activity from an FKBP12-stripped RyR, incorporated from FKBP12-stripped TC vesicles. Upper traces, control activity. Lower traces, activity after addition of 1.3 μm ivermectin. Bilayer potential was +40 mV and solutions contained 250/50 mM CsCl and 1/1 mM Ca²⁺ (cis/trans). In A and B the continuous lines show the zero current level, maintained when the channel is closed. The upper dotted line shows the maximal single channel conductance, and the lower dotted line in B shows the lowest subconductance level, S1. The mean current, I (in pA), was calculated from at least 60 s of activity including that in A and B.

Figure 2. Reversible activation of native RyR channels by midecamycin

Channel activity obtained from one bilayer containing at least two RyR channels. Two sequential additions of 10 μm midecamycin were followed by cis chamber perfusion and then 10 μm midecamycin was added once again, followed by 13 μm ryanodine. One channel was active in the bilayer in the top four records; two channels were active in the final two records. Channel activity was recorded at +40 mV with 1 mM cis Ca²⁺. The maximum open level for a single channel is indicated by the first dotted line in each record. The summed conductance for the two channels is indicated by the upper dotted line in the last two records. The mean current (I) recorded from > 80 s of continuous activity under each condition is shown beside each record (in pA).

Figure 3. Ivermectin and midecamycin do not remove FKBP12 from TC vesicles, in contrast to the effect of rapamycin

Western blot of TC vesicles which had been pre-incubated with rapamycin (0.5-40 μm), ivermectin (Iv, 40 μm) or midecamycin (Mi, 40 μm). The blot was immunostained with an affinity-purified antiFKBP12-peptide antibody. The positions of molecular mass markers are shown on the left. The immunostaining at ≈12 kDa in vesicles treated with ivermectin or midecamycin was not decreased compared with the control vesicles. No detectable FKBP12 was observed in SR vesicles preincubated with 10-40 μm rapamycin.

Figure 4. The effect of ivermectin on Ca²⁺ uptake by SR vesicles

A and C, records of changes in OD at 710 nm in response to changes in extravesicular [Ca²⁺], using antipyrylazo III (200 μm) as a Ca²⁺ indicator. A, Ca²⁺ uptake by TC vesicles after addition of 50 μm Ca²⁺ to the cuvette. Slope a is the initial rate of Ca²⁺ uptake before adding 5 μm Ruthenium Red (rr) (indicated by the discontinuity in the decline in OD). Slope b is the initial rate of Ca²⁺ uptake after adding Ruthenium Red. B, the initial rate of Ca²⁺ uptake by TC as a function of ivermectin concentration, expressed as a percentage of the control rate, before (○) and after (•) adding Ruthenium Red. Data points are means ±s.e.m. (n= 3); vesicles were from two separate SR preparations. C, Ca²⁺ uptake by LSR vesicles after adding 50 μm Ca²⁺ to the cuvette. Slope a is the initial rate of Ca²⁺ uptake. D, the initial rate of Ca²⁺ uptake by LSR as a function of ivermectin concentration, expressed as a percentage of the control rate. Data points are means ±s.e.m. (n= 4); vesicles were from three SR preparations. The control records in A and C were obtained after addition of ethanol (0.5 % v/v). The ivermectin records were obtained after addition of 10 μm ivermectin. The vertical calibration bar shows [Ca²⁺], which was routinely determined from the OD response to step increases in [Ca²⁺] and was linear over the range of [Ca²⁺] values shown in the figure. In this case an increase in [Ca²⁺] of 42 μm caused an increase of 0.1 OD units.

Figure 6. Ivermectin released Ca²⁺ from TC vesicles and enhanced caffeine-induced Ca²⁺ release

A, control records. B, Ca²⁺ release in the presence of ivermectin. A and B show records of OD changes at 710-790 nm in response to changes in extravesicular [Ca²⁺], using antipyrylazo III (500 μm) as a Ca²⁺ indicator. TC vesicles were partially loaded with Ca²⁺ by adding four aliquots of CaCl₂ (each 7.5 μm final concentration) as shown in Fig. 5. The scaling of the records has been adjusted (as indicated by the calibration bars for 20 s and 20 μm Ca²⁺) to best display the response to each addition (note the changes in the calibration bars with caffeine and A23187). First record, increase in extravesicular [Ca²⁺] with 200 nM thapsigargin. Second record, reduced rate of Ca²⁺ release when MgCl₂ was added to the solution (to give a total Mg²⁺ concentration of 9 mM). Third record, Ca²⁺ release was not altered by addition of 0.25 % (v/v) ethanol (A), but was enhanced by 10 μm ivermectin (B). Fourth record, enhanced rate of release following 5 mM caffeine addition. Final record, all remaining Ca²⁺ was released from TC vesicles by A23187. C, ivermectin potentiates caffeine-induced Ca²⁺ release from TC vesicles. Initial rates of Ca²⁺ release (nmol (mg TC protein)⁻¹ min⁻¹) induced by caffeine (0.6-5 mM) in control (ethanol, 0.25 % v/v, ○) and with ivermectin (10 μm, •). The rate of release shown in C is the initial rate in caffeine minus the preceding rate with ethanol (A) or with ethanol plus ivermectin (B). Data points are means ±s.e.m. (n= 7) using TC vesicles from two SR isolations. The vertical calibration bar shows [Ca²⁺] (see legend to Fig. 4). In this experiment an increase in [Ca²⁺] of 2 μm caused an increase of 0.02 OD units.

Figure 7. Ivermectin (40 μm) reduces Ca²⁺ uptake by SR in skinned EDL muscle fibres and increases caffeine-activated Ca²⁺ release

The records show tension as a function of time. A, records obtained from one fibre during maximal Ca²⁺ release (30 mM caffeine, 0.05 mM Mg²⁺), after submaximal Ca²⁺ loading (see text and Methods). The bracketing control records were obtained with the solvent vehicle DMSO (0.4 % v/v) in the low relaxing solution used immediately before loading, and in the load solution. Ivermectin (40 μm: 8 μl of 10 mM ivermectin, i.e. 0.4 % v/v) was added to identical solutions in order to obtain the ivermectin record. B, records obtained from one fibre during exposure to the submaximal Ca²⁺ release solution (15 mM caffeine, 0.25 mM Mg²⁺). The bracketing control records were obtained with the solvent vehicle DMSO (0.4 % v/v) in the low relaxing solution used immediately before release, and in the release solution. The record labelled ivermectin was obtained with 40 μm ivermectin added to identical solutions.