Effects of dantrolene and its derivatives on Ca2+ release from the sarcoplasmic reticulum of mouse skeletal muscle fibres

Article abstract of DOI:10.1038/sj.bjp.0704307

1. We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca²⁺ 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 Ca²⁺-induced Ca²⁺ release (CICR) under Mg²⁺-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 Ca²⁺ 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 Ca²⁺ release. 7. The magnitudes of inhibition of Clof-induced Ca²⁺ 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-Ca²⁺ release channel may be similar to that of physiological Ca²⁺ release (PCR). 8. These results indicate that the difference in opening modes of the RyR-Ca²⁺ release channel is recognized by certain Dan derivatives.

Full text of DOI:10.1038/sj.bjp.0704307

British Journal of Pharmacology (2001) 134, 729 ± 736
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E€ects of dantrolene and its derivatives on Ca2+ release from the
sarcoplasmic reticulum of mouse skeletal muscle ®bres
*,1Takaaki Ikemoto, 2Takamitsu Hosoya, 2Hiroshi Aoyama, 1Yasutaka Kihara, 2Masaaki Suzuki &
1Makoto Endo
2
1Department of Pharmacology, Saitama Medical School, Moroyama-machi, Saitama 350-0495, Japan and Department of
Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
1
We analysed the e€ect of dantrolene (Dan) and ®ve newly synthesized derivatives (GIFs) on
Ca2+ release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle.
In intact muscles, GIF-0185 reduced the size of twitch contraction induced by electrical
2
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 ®bres, Dan inhibited Ca2+-induced Ca2+ release (CICR) under Mg2+-free conditions
at room temperature. In contrast, GIF-0082 and GIF-0185 showed no inhibitory e€ect on CICR
under the same conditions.
4
Dan-induced inhibition of CICR was not a€ected by the presence of GIF-0082, whereas it was
diminished in the presence of GIF-0185.
GIF-0082 and GIF-0185 signi®cantly inhibited clo®bric acid (Clof)-induced Ca2+ release, as did
Dan.
5
6
Several Dan derivatives other than GIF-0082 and GIF-0185 showed an inhibitory e€ect on twitch
tension but not on the CICR mechanism. All of these derivatives inhibited Clof-induced Ca2+ release.
7
The magnitudes of inhibition of Clof-induced Ca2+ 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-Ca2+ release channel may be similar to that of physiological Ca2+ release
(PCR).
8
recognized by certain Dan derivatives.
British Journal of Pharmacology (2001) 134, 729 ± 736
These results indicate that the di€erence in opening modes of the RyR-Ca2+ release channel is
Keywords: Calcium release; clo®bric acid; dantrolene
Abbreviations: CICR, Ca2+-induced Ca2+ release; Clof, clo®bric acid; Dan, dantrolene; MH, malignant hyperthermia; PCR,
physiological Ca2+ release; RyR, ryanodine receptor; SR, sarcoplasmic reticulum
Introduction
Ryanodine receptor (RyR)-mediated Ca2+ release from the
sarcoplasmic reticulum (SR) is an essential step in excitation-
contraction coupling (E-C coupling) in skeletal and cardiac
muscles. Puri®ed RyR proteins incorporated into lipid bilayers
show properties of the Ca2+-induced Ca2+ release (CICR)
channel (Meissner, 1994). While CICR is thought to be the
mechanism of Ca2+ release in cardiac E-C coupling (Nabauer et
al., 1989; Bers & Perez-Reyes, 1999), it is not considered to be
the mechanism of physiological Ca2+ release (PCR) in skeletal
muscles E-C coupling (Endo, 1985; Schneider, 1994), because,
for example, inhibitors of CICR such as procaine and adenine
(in the presence of ATP) do not inhibit PCR (Thorens & Endo,
1975; Ishizuka et al., 1983). However, since mice with a targeted
mutation of the skeletal muscle RyR (RyR1) gene lack E-C
coupling (Takeshima et al., 1994), it must be concluded that
PCR takes place through RyR1. This means that RyR1 can
open in two di€erent modes, PCR and CICR. In order to
elucidate a molecular or submolecular mechanism of PCR that
is distinct from CICR, speci®c inhibitors of PCR at the level of
RyR1 would be very useful. However, no agents have been
shown to inhibit PCR without also a€ecting CICR. Some
speci®c inhibitors of CICR mentioned above, and other agents
that inhibit both PCR and CICR, such as tetracaine or
ruthenium red, are known (Almers & Best, 1976; Ohnishi, 1979;
Smith et al., 1986).
Dantrolene sodium (Dan) was originally reported to inhibit
E-C coupling of skeletal muscle (Ellis & Bryant, 1972; Ellis &
Carpenter, 1972). It was then found to be an e€ective agent for
the treatment of porcine and human malignant hyperthermia
(MH), a pathophysiological state in susceptible individuals
caused by an excessive release of Ca2+ through the RyR1
activated by inhalation anaesthetics (Harrison, 1975; Kolb et
al., 1982; MacLennan & Phillips, 1992). MH was shown to be a
consequence of an excessive CICR (Endo et al., 1983), and it
was demonstrated that Dan, in addition to inhibiting PCR, can
e€ectively inhibit CICR at 378C (Ohta & Endo, 1986; Ohta et
al., 1989). Interestingly, it was then found that at room
temperature, Dan does not inhibit CICR, although it inhibits
E-C coupling at 378C and at room temperature equally (Ohta &
*Author for correspondence; E-mail: tikemoto@saitama-med.ac.jp

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Dantrolene derivatives and Ca release
Endo, 1986; Kobayashi & Endo, 1988; Ohta et al., 1990). The
results described in this study are somewhat di€erent from
these reports. However, they do indicate that under a certain
experimental condition, Dan somehow preferentially inhibits
PCR at room temperature.
The extensor digitorum longus muscles (EDL) were
excised. One of the four heads of EDL was carefully
isolated in a physiological saline solution (PSS, in mM:
NaCl 150; KCl 4; CaCl2 1.8; MgCl2 1; 2-[4-(2-hydro-
xyethyl)-1-piperazinyl]ethanesulphonic acid (HEPES) 5;
glucose 5.6; pH 7.4). The bundles were tied at both ends
with silk threads and mounted between a pair of stainless
steel rods that attached to a force transducer (AE801,
In the present study, we examined the e€ects of several
newly synthesized Dan derivatives with the goal of ®nding
some agents that more clearly distinguish between PCR and
CICR than Dan. We tested these agents for inhibition of
twitch tension of intact muscle ®bres and inhibition of CICR
in saponin-treated skinned muscle ®bres. We also tested for
their e€ects on clo®bric acid (Clof)-induced Ca2+ release,
which has certain characteristics similar to those of PCR
(Ikemoto & Endo, 2001, accompanying study). We found
that some Dan derivatives indeed distinguish PCR from
CICR more clearly than Dan itself. In addition, the data
demonstrate further similarity of the mode of opening of
RyR-Ca2+ release channels activated by Clof to the mode of
PCR.
Akers, Norway or UL-10GR, NMB, Japan) and
a
micromanipulator (Narishige, Japan). The muscle prepara-
tion was immersed in PSS.
Preparation of skinned ®bres was described previously
(Ikemoto & Endo, 2001, accompanying study). All experi-
ments were carried out at room temperature (21 ± 238C).
Measurement of tension in intact fibres
Supramaximal ®eld electrical stimulation (20 V, 0.5 ms
duration) was delivered by
a pair of platinum wires
placed on both sides of the preparation. After twitch
tension became constant in PSS containing D-tubocurarine
(25 mM) and 0.2% dimethyl sulphoxide (DMSO), the
Methods
external solution was changed to
a
PSS containing
Preparation of muscle fibres
dantrolene (Dan) or its derivatives, stock solutions of
which were made by dissolving the agents in DMSO as
described below. After the twitch tension became constant
during a 15 ± 30 min treatment period, it was compared
Male ICR mice (5 ± 9 weeks) were anaesthetized with 50 ±
80 mg kg71 sodium pentobarbitone (i.p.) and decapitated.
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 photoanity 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 simpli®ed compound of GIF-0082.
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Dantrolene derivatives and Ca release
731
with that measured before treatment. D-tubocurarine was
present throughout these experiments. Statistical signi®-
cance was tested by Dunnett's test for multiple compar-
isons (Figure 2a).
Results
Effects of Dan and GIFs on Ca2+ release in intact fibres
and on CICR in skinned fibres
Measurement of Ca2+ release in skinned fibres
We designed and synthesized N-(3-azido-5-iodo)benzylated
dantrolene, referred to as GIF-0082, which carries an azido
group as a photoreactive unit (Kotzyba-Hibert et al., 1995).
As shown in Figure 2a, GIF-0082 inhibited the twitch tension
of intact skeletal muscle in a dose-dependent manner (0.2 ±
50 mM, open squares), although the extent of inhibition was
less than that produced by Dan (Figure 2a, ®lled circles).
GIF-0185, another Dan derivative, also inhibited the twitch
tension to the same extent as Dan (Figure 2a, open triangles).
Since GIF-0082 and GIF-0185 showed no inhibitory e€ect on
the Ca2+ uptake activity in the SR or on the relationship
between pCa and tension in skinned ®bres (data not shown)
as was the case with Dan (Ohta et al., 1990), these data
indicate that both of these GIFs inhibit the physiological
Ca2+ release (PCR) from the SR in intact muscles.
Figure 2b shows the e€ects of Dan (open circles), GIF-
0082 (®lled squares), and GIF-0185 (®lled triangles) on
the Ca2+-induced Ca2+ release (CICR) in skinned ®bres
of mouse skeletal muscles. The rates of Ca2+ release were
signi®cantly decreased by the application of Dan,
although it exhibited little e€ect on Ca2+ release at high
Ca2+ concentrations (open triangles vs open circles). The
rates of Ca2+ release at pCa 6.0 were decreased by
approximately 50% in the presence of high concentrations
of Dan (20 ± 50 mM, Figure 4a, open circles). These data
indicated that Dan had an inhibitory e€ect on CICR
even at room temperature, in apparent disagreement with
a previous report (Ohta et al., 1990). In contrast, the
rates of CICR were not signi®cantly changed by the
GIFs (50 mM) at any Ca2+ concentration (Figure 2b, open
triangles vs ®lled squares and ®lled triangles). Thus, these
GIFs can distinguish CICR from PCR much more clearly
than Dan.
The Ca2+ released from the skinned ®bres was quanti®ed
using Fura-2 as described previously (Ikemoto & Endo,
2001, accompanying study). Brie¯y, the protocol for the
measurement of the Ca2+ release rate consisted of (1)
loading the SR with Ca2+, (2) test (Ca2+-releasing stimuli
are given), and (3) determination of the Ca2+ remaining in
the SR. During the test period, various concentrations of
Ca2+ bu€ered with 10 mM EGTA, sometimes together with
Clof or CICR activators (ca€eine or AMP), were given, and
the e€ects of further addition of Dan or its derivatives were
examined. The activity of Ca2+ release was expressed in
terms of the decay rate constants based on the amount of
Ca2+ in the SR. Stock solutions of Dan or its derivatives
were dissolved in DMSO to obtain concentrations of 0.1 ±
25 mM (Dan, GIF-0082, and GIF-0185) or 25 mM (other
derivatives). All test solutions with or without these agents
were adjusted to contain 0.2% DMSO except for those used
in the experiments performed to determine the e€ects of
GIFs on Dan-induced inhibition of CICR activity; in those
experiments, the concentration of DMSO in the test solution
was kept at 0.4%. Statistical signi®cance was tested using
the paired t-test.
Synthesis of Dan derivatives
Dan derivatives, which we referred to as GIF-0082 (Figure
1b) and GIF-0163 (Figure 1e), were prepared by coupling
Dan (Figure 1a) with (3-azido-5-iodo)benzyl bromide or
benzyl bromide, respectively. GIF-0146 (Figure 1c), which is
an iodo-congener of dantrolene, was prepared from 4-
iodoaniline by the conventional method reported by Snyder
et al. (1967). The methoxy analogue of Dan, GIF-0185
(Figure 1c), was synthesized by the Stille reaction (Stille,
1986) of 4-iodoanisole and 5-(tri-n-butylstannyl)-2-furalde-
hyde (Denat et al., 1992), followed by condensation with 1-
Effect of Dan and GIF-0082 on Clof-induced Ca2+ release
in skinned fibres
aminohydantoin (Traube
&
Ho€a, 1898). GIF-0149-R
We have reported that the opening mode of the RyR channel
activated by Clof in skinned ®bres has some features similar
to those of PCR, but di€erent from those of CICR (Ikemoto
& Endo, 2001, accompanying study). If the similarity applies
also to GIF-0082 and GIF-0185, they may inhibit Clof-
induced Ca2+ release as well. Therefore, in skinned ®bres, we
examined the e€ect of GIF-0082 on Clof-induced Ca2+
release at pCa48. As shown in Figure 3a, the rates of Clof
(410 mM)-induced Ca2+ release were signi®cantly decreased
by GIF-0082 (50 mM). On the other hand, GIF-0082 exerted
no e€ect on the Ca2+ release rate in the presence or absence
of ca€eine (Figure 3b), in accordance with the absence of
e€ects on CICR (Figure 2b), while Dan signi®cantly
decreased the Ca2+ release rate under the same conditions
(Figure 3b). The results were made clearer by normalizing the
rate with GIF-0082 or Dan by the rates without these
inhibitors in the same ®bres (Figure 3c). Although the AMP-
stimulated Ca2+ release rate was not signi®cantly inhibited by
Dan, its normalized rate may suggest that it is reduced by
Dan. It is also seen that the extent of inhibition by GIF-0082
(Figure 1d) was obtained by condensation of 5-(4-nitrophe-
nyl)-2-furaldehyde with racemic 1-amino-5-(6-methoxycarbo-
nylhexyl)hydantoin (Barraclough et al., 1994). All of the
newly synthesized GIF compounds were puri®ed by
recrystallization (499% purity) and fully characterized by
1H- and 13C-NMR (nuclear magnetic resonance) spectro-
scopy, IR (infrared) spectroscopy, UV (ultraviolet) spectro-
scopy, and combustion analysis. The details of the synthesis
of GIFs will be reported elsewhere (manuscript in prepara-
tion).
Other materials
Dantrolene sodium was a gift from Yamanouchi Pharma-
ceutical Co., Ltd. (Japan) or purchased from Nacalai Tesque,
Inc. (Japan). D-tubocurarine chloride was purchased from
Nacalai Tesque, Inc. All the other chemicals were as
described in the previous paper (Ikemoto & Endo, 2001,
accompanying study).
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Dantrolene derivatives and Ca release
was greater, at higher Clof concentrations. GIF-0185 also
inhibited Clof-induced Ca2+ release, as described in the next
section.
that the PCR from the SR is not mediated by the CICR
mechanism (Endo, 1985; Schneider, 1994). On the other
hand, it is possible that in preparations of skinned ®bres,
molecule(s) responsible for the action of GIFs may have been
lost. This is very unlikely, however, since Clof-induced Ca2+
release in skinned ®bres is well a€ected by GIFs as just
Effects of GIF-0082 and GIF-0185 on Dan-induced
inhibition of Ca2+ release
It is not surprising that while GIF-0082 and GIF-0185 do not
inhibit CICR at all, they inhibit PCR, because it is thought
Figure 2 E€ects of Dan, GIF-0082, and GIF-0185 on Ca2+ release.
(a) In intact ®bres, after treatment with dantrolene (Dan), GIF-0082,
or GIF-0185 in the concentration range of 0.2 ± 50 mM, 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). *P50.05 (Dunnett's test). (b) The rates of Ca2+ release were
obtained in saponin-skinned ®bres with 20 mM Dan (n=5), 50 mM
GIF-0082 (n=3 ± 7) or 50 mM GIF-0185 (n=3 ± 4) in the absence of
Mg2+ and adenine nucleotide at pCa48 ± 4.5. Open triangles show
the rates without these agents (control, n=11 ± 16). The symbols for
GIF-0082 and GIF-0185 were slightly o€set horizontally for clarity
(mean+s.e.mean). *P50.05, **P50.01 (vs control). Error bars
smaller than the symbols are not shown.
Figure 3 E€ects of Dan and GIF-0082 on ca€eine- and clo®bric
acid-induced Ca2+ release in skinned ®bres. The rates of Ca2+ release
with 20 mM Dan or 50 mM GIF-0082 were measured under Mg2+
-
and Ca2+-free conditions (bu€ered with 10 mM EGTA) with 5 ±
20 mM clo®bric acid (Clof, Figure 3a, mean+s.e.mean, n=3 ± 6) or
with or without CICR activators (ca€eine, Caf; AMP; Figure 3b,
n=5 ± 7). Note the di€erence in the scales. (c) The results in a and b
were normalized to the control value (without Dan or GIF-0082)
within each preparation. *P50.05, **P50.01, ***P50.001.
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Dantrolene derivatives and Ca release
733
described. The following experiments also provide support
that this theory is unlikely.
0185 in skinned ®bres clearly shows that CICR in skinned
®bres retains some sensitivity to GIF-0185.
Dan decreased the rates of CICR in skinned ®bres in a
dose-dependent manner (2 ± 50 mM, Figure 4a, open circles).
Although GIF-0082 and GIF-0185 could not inhibit the
CICR as shown in Figure 2b, the inhibitory action of low
concentrations of Dan (2 ± 20 mM) were completely abolished
by the presence of GIF-0185 (Figure 4a, ®lled triangles). On
the other hand, Dan-induced inhibition of CICR was not
a€ected by the presence of GIF-0082 (Figure 4a, open
squares). The abolition of the Dan e€ect on CICR by GIF-
We further studied the e€ect of co-applications of GIF-0082
or GIF-0185 and Dan on Clof-induced Ca2+ release. As shown
in Figure 4b, both GIF-0082 (25 mM) and GIF-0185 (5 mM)
signi®cantly inhibited Clof-induced Ca2+ release. We con-
®rmed that these GIFs showed no inhibitory e€ect on CICR at
the same concentrations (data not shown). Dan (2 and 5 mM)
also inhibited Clof-induced Ca2+ release (Figure 4b). Simulta-
neous applications of GIF-0082 or GIF-0185 and Dan showed
additive inhibitory e€ects (Figure 4b, ®lled column).
Effect of other Dan derivatives on the Ca2+ release
Next we examined the e€ect of Dan derivatives other than
GIF-0082 and GIF-0185. Among the many derivatives we
tested, GIF-0146 and GIF-0149-R inhibited twitch tension in
intact ®bres, while GIF-0163 had little e€ect (Figure 5a). The
magnitude of inhibitory action of these derivatives and Dan
on twitch tension was in the following sequence: Dan
(20 mM)=GIF-0185 (50 mM)4GIF-0082 (50 mM)4GIF-0146
(50 mM)5GIF-0149-R (50 mM)4GIF-0163 (50 mM) (Figures
2a and 5a). On the other hand, in skinned ®bres, the rates of
CICR at pCa 6 were not decreased with any of these
derivatives (Figure 5b).
We also tested the e€ect of these derivatives on the Clof-
induced Ca2+ release in skinned ®bres. In order to mimic the
physiological condition, Clof was applied at pCa 7 in the
presence of 0.5 mM Mg2+ and 10 mM AMP. As shown in
Figure 6a, all of the derivatives that showed the inhibitory
e€ect on twitch contraction decreased the rates of Clof
Figure 4 Di€erent e€ect of GIF-0082 and GIF-0185 on Ca2+
release. (a) In skinned ®bres, the rates of Ca2+ release measured with
Dan (0 ± 50 mM) 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 mM 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 mM,
mean+s.e.mean) and/or either GIF-0185 (5 mM) or GIF-0082
(25 mM, n=4) under Ca2+, Mg2+-free conditions (bu€ered with
10 mM EGTA) were normalized to the control value (without these
agents) within each preparation. *P50.05, **P50.01.
Figure 5 E€ects 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 mM) were compared with those before
treatment in intact muscle. (b) In skinned ®bres, 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).
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T. Ikemoto et al
Dantrolene derivatives and Ca release
(20 mM)-induced Ca2+ release (Figure 6a). On the other
hand, ca€eine-induced Ca2+ release (Caf, 20 mM) was not
signi®cantly inhibited by these derivatives, although the rates
appeared slightly reduced (Figure 6b).
The extent of reduction of the rates of Clof-induced or
ca€eine-induced Ca2+ release in skinned ®bres by Dan and its
derivatives was compared with that of the twitch tension in
intact ®bres (Figure 6c). The magnitude of inhibition of
twitch contraction was in good agreement with that of the
inhibition of Clof-induced Ca2+ release (Figure 6c, ®lled
circles), while no correlation was seen between the twitch
tension and the rate of ca€eine-induced Ca2+ release (Figure
6c, open circles).
In conclusion, we showed that some Dan derivatives
speci®cally inhibited PCR and Clof-induced Ca2+ release
without appreciable inhibition of CICR.
Discussion
We analysed the e€ects of Dan and some newly synthesized
derivatives on twitch tension (indicating PCR) of intact ®bres
and CICR in skinned ®bres of mouse skeletal muscle. We
demonstrated that several Dan derivatives did not inhibit
CICR at all, but de®nitely inhibited twitch tension (Figures 2
and 4). Since twitch inhibition of these Dan derivatives is
most likely to be the result of their action on the Ca2+-release
mechanism of the SR, they appear to distinguish PCR and
CICR clearly.
The notion that these Dan derivatives directly a€ect the
Ca2+ release mechanism of the SR was supported by the
fact that they inhibited Clof-induced Ca2+ release in
skinned ®bres (Figures 3, 4, and 6). In the accompanying
study, we demonstrated that Clof-induced Ca2+ release has
some features similar to those of PCR (Ikemoto & Endo,
2001, accompanying study). The results of the present
report provide
a further similarity: the magnitude of
twitch inhibition by Dan and its derivatives that is most
likely to indicate the magnitude of PCR inhibition is very
closely correlated with the magnitude of inhibition of
Clof-induced Ca2+ release, but not with that of CICR
inhibition (Figure 6).
Difference of Dan effect on CICR at room temperature
between the present and previous results
We showed that the CICR in skinned ®bres from mouse
skeletal muscle was signi®cantly inhibited by Dan (20 mM) at
room temperature, which is in disagreement with the previous
report (Ohta et al., 1990). The inhibitory e€ect of Dan on CICR
was reduced by increasing Mg2+ concentrations, and it was
abolished in the presence of 0.5 mM Mg2+ (unpublished
observation), and some of the previous results were obtained
in presence of Mg2+ (40.5 mM) (Ohta & Endo, 1986; Ohta et
al., 1990). However, Ohta et al. (1990) reported that Dan
(50 mM) showed no inhibitory e€ect on CICR in skinned ®bres
of skeletal muscles from guinea-pigs even in the absence of
Mg2+ at room temperature (208C). The reason for the
discrepancy is presently unknown. It may be a consequence
of the di€erence in animal species used.
Figure 6 Correlation between the rates of Ca2+ release in skinned
®bres and twitch contraction in the presence of Dan derivatives. In
skinned ®bres, the rates of Ca2+ release with Dan (20 mM) or GIFs
(50 mM) were measured at pCa 7.0 with 20 mM Clof (a) or ca€eine
(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 mM Dan or 50 mM other derivatives from Figures 2a
and 5a. The data for Caf or Clof were ®tted by a least square
method, and the correlation coecients were 0.317 and 0.956,
respectively. *P50.05, **P50.01.
Usefulness of Dan and its derivatives on the study of Ca2+
release
Recently, Dan has been used in a wide variety of cells other
than skeletal muscles as an inhibitor of intracellular Ca2+
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Dantrolene derivatives and Ca release
735
release (Hui, 1983; Shoshan-Barmatz et al., 1991; Partridge &
Valenzuela, 1999; Seutin et al., 2000). However, the exact
mechanism of action of Dan has remained unclear, not only
in skeletal muscles, but also in other types of cells. For
example, it is not clear whether the inhibitory action of Dan
on Ca2+ release results from direct binding to the RyR
(Parness & Palnitkar, 1995; Palnitkar et al., 1997; Fruen et
al., 1997). Therefore, it is important to elucidate the
molecular mechanism of Dan-induced inhibition of the
Ca2+ release via the RyR.
Some of the Dan derivatives we synthesized inhibited PCR
without an inhibitory e€ect on CICR. They inhibited Clof-
induced Ca2+ release in skinned ®bres, an e€ect that was
signi®cantly correlated with their inhibitory e€ect on twitch
tension (Figure 6c). In addition, GIF-0185 diminished the
inhibition by Dan on CICR, although it showed no
inhibitory e€ect on CICR (Figures 2b and 3a). These results
indicate that the target(s) of Dan and its derivatives are
clearly retained in skinned ®bres. Among these speci®c PCR
inhibitors at room temperature (21 ± 238C), GIF-0082, with
its azido-functionalized and iodinated structure, is applicable
to photo-anity and its binding experiment on target
molecule(s). Since GIF-0082 does not interfere with the
inhibitory action of Dan on CICR (unlike GIF-0185), it
might well be more speci®c to PCR than GIF-0185. We,
therefore, think that this agent may be a useful probe to
study PCR of skeletal muscles in various preparations
including isolated SR membranes and the puri®ed RyR.
We wish to thank Dr M. Iino (University of Tokyo, Japan) for
reading the manuscript. This work was supported by a Grant-in-
Aid from the Ministry of Education, Science, Sports, and Culture
of Japan.
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