Download 9-Methyl-7-Bromoeudistomin D, a Caffeine Like

9-Methyl-7-Bromoeudistomin D, a Caffeine Like Ca2+ Releaser, Probably
Binds to Cytochromes P-450 1A1 and 2B in Liver Microsomes
Masae Kakubari1),Masatoshi Adachi1),Masakuni Degawa2) ,Yasushi Ohizumi1,2,3,4)
1)Department
of Pharmaceutical Moleculer Biology, Pharmacetical Insutitute, Tohoku University, Aoba, Aoba-ku,Sendai 980-8578,Japan
2)Department of Molecular Toxicology, School of Pharmaceutical Sciences,23 University of Shizuoka, 52-1
Yada, Suruga-ku, Shizuoka, 422-8526, Japan
3)Department of Anti-dementia Functional Food Development, Research Center of 75 Supercritical Fluid Technology,
Graduate School of Engineering, Tohoku University,76 6-6-07
Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan
4)Laboratory of Kampo Medicines, Yokohama College of 65 Pharmacy, 601 Matano-cho, 66 Totsuka-ku, Yokohama 245-0066, Japan
Received October 3, 2011, Accepted December 7,2011
3
H-labeled 9-metyhl-7-bromoeudistomin D (3H-MBED), a powerful caffeine-like Ca releaser binds to liver
microsomes as well as the caffeine binding site of terminal cisternae of skeletal muscle sarcoplasmic
reticulum(SR)(Fang at al.,1993; Adachi et al.,1994). 3H-MBED binding to rabbit liver microsomes was inhibited by
alpha-naphthoflavone (α-NF), a cytochrome P450 (CYP) 1A1/1A2 inhibitor in a concentration-dependent manner (IC
≒ 3 nM), while that was not affected by alpha-phenyl- alpha-prorylbenzeneacetic acid
N,N-diethylaminoethy1-2,2-diphenylvalerate hydrochloride (SKF525A), an inhibitor for several CYPs
including CYP2B subfamily enzymes. We comparatively examined the binding levels of 3H-MBED to liver
microsomes from rats treated by β-NF, phenobarbital (PB), and 2-methoxy-4-aminoazobenzene (2-MeO-AAB). The
enhanced binding levels were observed in the microsomes from rats treated with by β-NF (CYP1A1/1A2 inducer) and
PB (CYP2B inducer), but not 2-MeO-AAB (CYP1A2-selevtive inducer). The 3H-MBED binding levels to the
microsomes from β-NF-treated rats were inhibited by α-NF but not by SKF525A, whereas the activities to those from
PB-treated rats were decreased by SKF525A but not by α-NF. Furthermore, the enhanced binding levels of 3H-MBED
were inhibited by addition of caffeine. The present findings suggest that 3H-MBED binds to CYP1A1 and CYP2B in
liver microsomes.
Keywords: 9-Methyl-7-bromoeudistomin D / ca releaser / cytochromes P-450 / binding sites
Introduction
It has been shown that 9-methyl-7-bromoeudistomin D
(MBED), a derivative of eudistomin, D isolated from
Cribbean tunicate Eudistoma olivaceum, is a powerful Ca2+
releaser from ryanodine receptor with properties similar to
caffeine (Nakamura et al.,1986; Kobayahshi at al.,1986;
Seino et al., 1989) and that 3H-labeled MBED binds to the
caffeine binding site of skeletal and smooth muscle
sarcoplasmic reticulum (SR) (Fang et al.,1993; Adachi et
al., 1994). 3H-MBED bound to rabbit liver microsomes
with a more than (30)-fold greater receptor density
compared with that of SR (Adachi et al., 1994).
Furthermore,
caffeine non-competitively
inhibited
*Correspondence author: Dr.Yasushi Ohizumi, .
Graduate School of Engineering, Tohoku University, 6-6-07 Aoba, Aramaki,
Aoba-ku, Sendai 980-8579, Japan
Tel: +81-22-795-7248
Fax: +81-22-795-7246
E-mail: [email protected]
3
H-MBED binding to liver microsomes, these results
suggesting that the property of the 3H-MBED binding site
in liver microsomes is distinct from that in skeletal SR
(Adachi et al., 1994; Adachi et al., 1994).
In this paper, to clarify the 3H-MBED binding site in the
liver microsomes, we comparatively examined the
3
H-MBED binding activities to the liver microsomes from
rats treated with different types of cytochorome P450
(CYP) inducers.
Fig.1.
Chemical structure of tritium labeled
9-methyI-7-bromoeudistomin D (3H-MBED)
Materials and Methods
1. Chemicals
3
H-labeled MBED (fig.1) was synthesized as previously
described (Fang et al., 1993) with as specific radioactivity
of 10.2 ci/mmol. 3H-MBED was dissolved in DMSO of
which the final concentration was kept at< 2% (v/v) in all
the experiments. 2-MeO-AAB was synthesized as
previously described (1987). Aprotinin, benzamidine
hydrochloride,
leupetine,
pepstatin
A
and
N,N-diethylaminoethy1-2,2-diphenylvalerate
hydrochloride (SKF525A) were obtained from Wako Pure
Chemical Industries (Tokyo,Japan). α-NF, PB and 30%
polyethleneimine were obtained from Tokyo Chemical
Industries (Tokyo,Japan).
2. Induction of CYP and preparation of liver microsomes.
Seven-week-old male F334 rats were used. The following
drugs were used to induce CYP: PB (0.11 mmol/kg, i.p.,in
corn oil), 2-MeO-AAB (0.22 mmol/kg, i.p., in corn oil and
β-NF 0.22 mmol/kg, i.p., in H2O). The liver was excised
24hr after the administration of the inducers and rat liver
microsomes were prepared as described (Degawa et al.,
1986). Rabbit liver microsomes were prepared from male
Japanese White rabbits weighing about 3kg as previously
described (Adachi et al., 1994). Protein concentrations
were measured by the method of Bradford (Bradford,
1976) using a protein assay kit (Bio-Rad) with BSA as
standard.
3. 3H-MBED binding assay
Fig.2. Effects of CYP inhibitors on 3H-MBED binding to rabbit liver microsomes
Rabbit liver microsomes (25 μg protein) were incubated with 50 nM 3H-MBED in the absence or presence of various
concentrations of α-NF and SKF525A for 45 min at 0 ℃ as described in “ Material and Methods”. The symbols indicate
the means, and the bars represent their standard errors (n=3).
Fig.3. Comparison of the binding level of 3H-MBED to the liver microsomes from the rats treated with different
CYP inducers
2-MeO-AAB-, β-NF- and PB-treated, or untreated rat liver microsomes (25μg) were incubated with 50 nM 3H-MBED
for 45 min at 0 ℃ as described in “ Material and Methods”.
3
H-MBED binding assay was performed as described by
Adachi et al (Adachi et al., 1994). Liver microsomes (25μg
protein) were incubated with 3H-MBED (50nM) at 0℃ for
45 min in a solution containing 0.3 M surcrose, 2 mM
DDT, 0.1 mM p-APMSF and 20 mM Tris-HCl (pH 7.4) in
the presence or absence of drugs. The amount of
3
H-MBED bound was determined by filtration through
polyethyleneimine-treated (3% for 45 min) Whatman
filters (GF/B). The filters were washed twice with 5 ml of
ice-cold 50mM Tris-HCl (pH 7.4).
Specific binding levels were estimated by subtracting
nonspecific binding level in the presence of 5 μM
unlabeled MBED from total binding level.
Results and Discussion
α-NF, a CYP1A1/1A2-selective inhibitor (Murray and
Reidy., 1990; Jenner and Netter., 1972), led to a
concentration-dependent inhibition of 3H-MBED binding
to rabbit liver microsomes with the IC50 value of 3nM,
while SKF525A, an inhibitor for several CYPs including
Fig.4. Effects of CYP inhibitors on 3H-MBED binding to the liver microsomes of β-NF-treated rats
The liver microsomes (25 μg protein) from β-NF-treated rats were incubated with 50 nM 3H-MBED in the absence or
presence of various concentrations of α-NF and SKF25 for 45 min at 0 ℃ as described in “ Material and Methods ”. The
symbols indicate the means (n=3), and the bars represent their standard errors.
Fig.5. Effects of CYP inhibitors on 3H-MBED binding to the liver microsomes of PB-treated rats
The liver microsomes (25 μg protein) from PB-treated rats were incubated with 50 nM 3H-MBED in the absence or
presence of various concentrations of α-NF and SKF25 for 45 min at 0 ℃ as described in “ Material and Methods ”. The
symbols indicate the means (n=3), and the bars represent their standard errors.
CYP2B subfamily enzymes (Enomoto, et al. 2005; Murray
and Reidy.,1990; Jenner and Netter.,1972), has no effect as
shown in fig.2. These results suggest that 3H-MBED
binding site is related to CYP1A1/1A2 in rabbit liver
microsomes. 2-MeO-AAB, β-NF and PB have been
reported to be typical inducers of microsomal CYP1A2,
CYP1A1/1A2 and CYP2B, respectively, in the rat liver
(Hishinuma et al., 1987; Murray and Reidy.,1990).
We examined the effect of treatment of rats with
2-MeO-AAB, β-NF, and PB on the binding levels of
3
H-MBED to the liver microsomes. The 3H-MBED
binding levels were increased to 60 pmol/mg protein and
120 pmol/mg in β-NF- and PB-treated rats, respectively,
whereas no such increase occurred in 2-MeO-AAB-treated
rats (fig.3). Such enhanced 3H-MBED binding was
markedly inhibited by addition of caffeine (3 mM).
Furthermore, 3H-MBED binding to liver microsomes
from β-NF-treated rats was concentration-dependently
inhibited by α-NF (IC≒10 nM), but not by SKF525A
(fig.4). In contrast, SKF525A caused an inhibition of
3
H-MBED binding to liver microsomes from rats treated
with PB, a typical inducer of CYP2B subfamily enzymes,
but α-NF, a typical inhibitor of CYP1A subfamily enzymes,
had no effect on this binding (fig.5). These results suggest
that in rat liver microsomes, 3H-MBED probably binds to
CYP1A1 and CYP2B.
We have previously reported that caffeine can
allosterically inhibit 3H-MBED binding to rabbit liver
microsomes and that the 3H-MBED binding site is distinct
from but related to that of caffeine (Adachi et al., 1994).
Although caffeine has been reported to be a good substrate
for CYP1A2 (Arnaud, 1987; Butler, 1989; Wang and Zhou,
2009), binding level of 3H-MBED to liver microsomes
from rats treated with 2-MeO-AAB, a CYP1A2-selective
inducer, was not increased. This suggests that 3H-MBED
does not necessarily bind at the substrate-binding site of
CYP enzymes.
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