Diapositive 1

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Article 1
Localization of opioid receptor antagonist
3H-LY255582 binding sites in mouse brain
Comparison with the distribution of mu, delta and
kappa binding sites S.L. Gackenheimer, , T.M.
Suter, J.E. Pintar, S.J. Quimby, W.J. Wheeler,
C.H. Mitch, D.R. Gehlert and M.A. Statnick
Neuropeptides, 2005 39559-567
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Problème général
Peptides opioïdes
nombreux rôles dans SNC et périphérique
comportement alimentaire, Nociception Récompense
locomotion
µ
?
d
Récepteur
Génétiquement et Pharmacologiquement distincts
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Problème spécifique
Distribution des Récepteurs opioïdes différentes
selon le sous-type
Informations sur leur fonction
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Problème spécifique et hypothèse
LY255582 antagoniste opiacé
µ
?
d
Récepteur
Kd
0.41 nM
5.2 nM
2 nM
(effets comportementaux réduction du
comportement alimentaire contrairement à dautres
antagonistes)
Site (mécanisme) daction différent ?
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But de létude
Etude des sites de liaison du LY255582 dans le
cerveau et comparaison avec la distribution des
autres récepteurs opioïdes
Autoradiographie quantitative
Synthèse dun radiolignad
3H-LY255582
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 Chemical structures of LY255582 and
3H-LY255582.
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3H-LY255582 Kd 0.156 0.07 nM 0.5 nM
µ
?
s
d
Récepteur
3H-naloxone (Kd 1.34 0.4 nM) 2 nM
3H-naltrindole (Kd 0.22 0.09 nM) 0.1
nM
3H-norBNI 0.1 nM
Radioligands
Cte de dissociation
Concentration
Haloperidol (100 nM)
Naltrexone (10 µM)
Non-spécifique
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Representative saturation isotherm generated from
mouse forebrain homogenates. Total binding ().
Non-specific binding () was defined as the
amount of binding remaining after membranes were
incubated in the presence of 10 µM naltrexone.
Specific binding (?) curve fit a single site
binding model using non-linear regression
calculation.
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Representative 12 µm sections of wild-type mouse
brain (129 SVEV6). Column A contains
autoradiograms of sections incubated with 0.5 nM
3H-LY255582. Sections in column B were
incubated with 2 nM 3H-naloxone. Column C
displays sections incubated with 0.1 nM
3H-naltrindole and lastly, column D contains
sections incubated with 0.1 nM 3H-norBNI.
Non-specific binding was near background level
(sections not shown) for each of the radioligands
and was defined as the amount of binding
remaining on sections incubated with radioligand
in the additional presence of 10 µM naltrexone.
The sections are represented in a pseudo-color
scale that is based on gray scale values. Red
represents the highest levels of binding while
green and blue represent the lowest levels.
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Regional distribution of 3H-LY255582,
3H-naloxone, 3H-naltrindole and 3H-norBNI
binding in mouse brain sections (n  4)
PN and fr values based on n  3(), n  2() or
n  1(_at_). Non-specific binding was determined by
including 10 µM naltrexone and 100 nM haloperidol
in the incubation buffer. Density levels are
expressed as nCi/mg plastic  SEM.
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(No Transcript)
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12 µm coronal sections of wild-type mouse brain
(129 SVEV6). Total binding using 0.5 nM
3H-LY255582 (A). Residual binding after
incubation with 10 µM naltrexone (B). Binding in
the presence of 100 nM haloperidol (C). Residual
binding of 3H-LY255582 after incubation with
10 µM naltrexone and 100 nM haloperidol (D). The
sections are represented in a pseudo-color scale
that is based on gray scale values. Red
represents the highest levels of binding while
green and blue represent the lowest levels.
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 Representative 12 µm coronal sections of mouse
brain incubated with 0.5 nM 3H-LY255582. Column
A wild-type mouse (129 SVEV6). Column B triple
KO (mu-, delta-, kappa-opioid receptor) mice.
Column C non-specific binding in wild-type mouse
defined by including 10 µM naltrexone in the
incubation buffer. Residual binding of
3H-LY255582 in mouse brain sections from the
triple KO mice was comparable to background and
non-specific binding. The sections are
represented in a pseudo-color scale that is based
on gray scale values. Red represents the highest
levels of binding while green and blue represent
the lowest levels.