Effects of dantrolene and its derivatives on Ca(2+) release from the sarcoplasmic reticulum of mouse skeletal muscle fibres

Abstract

1. We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca(2+) release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle. 2. In intact muscles, GIF-0185 reduced the size of twitch contraction induced by electrical stimulation to the same extent as Dan. GIF-0082, an azido-functionalized Dan derivative, also inhibited twitch contraction, although the extent of inhibition was less than that of Dan and of GIF-0185. 3. In skinned fibres, Dan inhibited Ca(2+)-induced Ca(2+) release (CICR) under Mg(2+)-free conditions at room temperature. In contrast, GIF-0082 and GIF-0185 showed no inhibitory effect on CICR under the same conditions. 4. Dan-induced inhibition of CICR was not affected by the presence of GIF-0082, whereas it was diminished in the presence of GIF-0185. 5. GIF-0082 and GIF-0185 significantly inhibited clofibric acid (Clof)-induced Ca(2+) release, as did Dan. 6. Several Dan derivatives other than GIF-0082 and GIF-0185 showed an inhibitory effect on twitch tension but not on the CICR mechanism. All of these derivatives inhibited Clof-induced Ca(2+) release. 7. The magnitudes of inhibition of Clof-induced Ca(2+) release by all Dan derivatives were well correlated with those of twitch inhibition. This supports the notion that the mode of Clof-induced opening of the RyR-Ca(2+) release channel may be similar to that of physiological Ca(2+) release (PCR). 8. These results indicate that the difference in opening modes of the RyR-Ca(2+) release channel is recognized by certain Dan derivatives.

Figure 1

Structures of dantrolene and its derivatives. (a) The structure of dantrolene sodium (Dan). (b) N-(3-azido-5-iodo)benzylated Dan derivative, which is referred to as GIF-0082, was designed as a photoaffinity probe by incorporation of azido group as a photoreactive unit and iodine. (c) GIF-0146 and GIF-0185 are Dan analogues, in which the substituent on the phenyl ring is changed from an electron-withdrawing nitro group to electron-donating iodo and methoxy groups, respectively. (d) GIF-0149-R is another type of Dan derivative in which an alkyl chain is anchored on the hydantoin ring. (e) GIF-0163, N-benzyldantrolene, is the structurally more simplified compound of GIF-0082.

Figure 2

Effects of Dan, GIF-0082, and GIF-0185 on Ca²⁺ release. (a) In intact fibres, after treatment with dantrolene (Dan), GIF-0082, or GIF-0185 in the concentration range of 0.2 – 50 μM, the size of twitch contraction was compared with that before the treatment. Relative tensions were plotted against the concentrations. (mean±s.e.mean, n=9 for Dan, n=3 – 6 for GIF-0082, n=3 – 6 for GIF-0185). ^*P<0.05 (Dunnett's test). (b) The rates of Ca²⁺ release were obtained in saponin-skinned fibres with 20 μM Dan (n=5), 50 μM GIF-0082 (n=3 – 7) or 50 μM GIF-0185 (n=3 – 4) in the absence of Mg²⁺ and adenine nucleotide at pCa>8 – 4.5. Open triangles show the rates without these agents (control, n=11 – 16). The symbols for GIF-0082 and GIF-0185 were slightly offset horizontally for clarity (mean±s.e.mean). ^*P<0.05, ^**P<0.01 (vs control). Error bars smaller than the symbols are not shown.

Figure 3

Effects of Dan and GIF-0082 on caffeine- and clofibric acid-induced Ca²⁺ release in skinned fibres. The rates of Ca²⁺ release with 20 μM Dan or 50 μM GIF-0082 were measured under Mg²⁺- and Ca²⁺-free conditions (buffered with 10 mM EGTA) with 5 – 20 mM clofibric acid (Clof, Figure 3a, mean±s.e.mean, n=3 – 6) or with or without CICR activators (caffeine, Caf; AMP; Figure 3b, n=5 – 7). Note the difference in the scales. (c) The results in a and b were normalized to the control value (without Dan or GIF-0082) within each preparation. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 4

Different effect of GIF-0082 and GIF-0185 on Ca²⁺ release. (a) In skinned fibres, the rates of Ca²⁺ release measured with Dan (0 – 50 μM) at pCa 6.0 under Mg²⁺-free conditions were normalized to the control value (without Dan) obtained in the same preparation in the absence (Dan alone, mean±s.e.mean, n=3 – 6) or presence of 50 μM GIF-0082 (n=3) or GIF-0185 (n=5). (b) The rates of Ca²⁺ release by 10 mM Clof in the presence of Dan (2 or 5 μM, mean±s.e.mean) and/or either GIF-0185 (5 μM) or GIF-0082 (25 μM, n=4) under Ca²⁺, Mg²⁺-free conditions (buffered with 10 mM EGTA) were normalized to the control value (without these agents) within each preparation. ^*P<0.05, ^**P<0.01.

Figure 5

Effects of some Dan derivatives on Ca²⁺ release. (a) The sizes of twitch contraction after treatment of GIF-0146, GIF-0149-R, GIF-0163, or GIF-0185 (50 μM) were compared with those before treatment in intact muscle. (b) In skinned fibres, the rates of Ca²⁺ release measured with these derivatives at pCa 6.0 in the absence of Mg²⁺ were normalized to the control value (without the derivatives) within each preparation (mean±s.e.mean, n=3 – 4 for a, n=3 for b).

Figure 6

Correlation between the rates of Ca²⁺ release in skinned fibres and twitch contraction in the presence of Dan derivatives. In skinned fibres, the rates of Ca²⁺ release with Dan (20 μM) or GIFs (50 μM) were measured at pCa 7.0 with 20 mM Clof (a) or caffeine (Caf, b) in the test solution containing 0.5 mM Mg²⁺ and 10 mM AMP (mean±s.e.mean, n=3 – 5 for a, n=3 – 4 for b). (c) These results were normalized to the control value (without Dan or its derivatives) and plotted against the mean value of relative twitch contraction at 20 μM Dan or 50 μM other derivatives from Figures 2a and 5a. The data for Caf or Clof were fitted by a least square method, and the correlation coefficients were 0.317 and 0.956, respectively. ^*P<0.05, ^**P<0.01.