Interconverting forms of the opiate receptor in rat striatal patches

Abstract

The binding of a radiolabeled "mu receptor" prototype opiate, dihydromorphine (H2morphine), and the binding of a "delta receptor" prototype, [D-Ala2,D-Leu5]enkephalin (D-Enk), to slide-mounted rat caudate slices were simultaneously compared quantitatively and visualized by autoradiography. Generally, D-Enk bound to opiate receptors distributed evenly throughout the entire striatum (type 2 pattern), whereas H2morphine labeled discrete islands or patches of receptors (type 1 pattern). In the presence of Mn2+ (3 mM) or other divalent cations, however, Na+ and GTP at 25 degrees C caused an increase in D-Enk binding at the expense of H2morphine binding at striatal opiate receptor patches. Thus, these conditions shifted D-Enk binding from an even pattern to one that included both an even and patchy distribution. These incubation conditions not only promoted D-Enk binding to striatal patches but also enabled the opiate receptor to regulate adenylate cyclase with the same (P less than 0.01) ligand selectivity pattern as that obtained by the displacement of D-Enk binding. The relative affinity of opiate receptors in striatal patches for opiate peptides, naloxone, and morphine appears to be a function of incubation conditions and coupling to adenylate cyclase and is not indicative of distinctly different opiate receptors. We postulate a three-state allosteric model consisting of mu agonist-, mu antagonists-, and adenylate cyclase-coupled delta-agonist-preferring states, whose equilibrium may be regulated by a sulfhydryl group mechanism.

Full text

Proc.
Natl
Acad.
Sci.
USA
Vol.
78,
No.
8,
pp.
4818-4822,
August
1981
Biochemistry
Interconverting
ja
and
6
forms
of
the
opiate
receptor
in
rat
striatal
patches
(type
1
receptor
pattern/divalent
metal
cations/sulfhydryl
effectors/adenylate
cyclase/autoradiography)
WAYNE
D.
BOWEN*,
SUSAN
GENTLEMAN*,
MILES
HERKENHAMt,
AND
CANDACE
B.
PERT*
*Biologica
Psychiatry
Branch,
National
Institute
of
Mental
Health,
Bethesda,
Maryland
20205;
and
tLaboratory
of
Neurophysiology,
National
Institute
of
Mental
Health,
Bethesda,
Maryland
20205
Communicated
by
Edward
V.
Evarts,
April
29,
1981
ABSTRACT
The
binding
of
a
radiolabeled
"l
receptor"
pro-
totype
opiate,
dihydromorphine
(H2morphine),
and
the
binding
of
a
"8
receptor"
prototype,
[D-Ala2,
D-Leu5]enkephalin
(D-Enk),
to
slide-mounted
rat
caudate
slices
were
simultaneously
compared
quantitatively
and
visualized
by
autoradiography.
Generally,
D-
Enk
bound
to
opiate
receptors
distributed
evenly
throughout
the
entire
striatum
(type
2
pattern),
whereas
H2morphine
labeled
dis-
crete
islands
or
patches of
receptors
(type
1
pattern).
In
the
pres-
ence
of
Mn2+
(3
mM)
or
other
divalent
cations,
however,
Na+
and
GTP
at
5LC
caused
an
increase
in
D-Enk
binding
at
the
expense
of
H2morphine
binding
at
striatal
opiate
receptor
patches.
Thus,
these
conditions
shifted
D-Enk
binding
from
an
even
pattern
to
one
that
included
both
an
even
and
patchy
distribution.
These
incu-
bation
conditions
not
only
promoted
D-Enk
binding
to
striatal
patches
but
also
enabled
the
opiate
receptor
to
regulate
adenylate
cyclase
with
the
same
(P
<
0.01)
ligand
selectivity
pattern
as
that
obtained
by
the
displacement
of
D-Enk
binding.
The
relative
af-
finity
of
opiate
receptors
in
striatal
patches
for
opiate
peptides,
naloxone,
and
morphine
appears
to
be
a
function
of
incubation
conditions
and
coupling
to
adenylate
cyclase
and
is
not
indicative
of
distinctly
different
opiate
receptors.
We
postulate
a
three-state
allosteric
model
consisting
of
#
agonist-,
it
antagonist-,
and
aden-
ylate
cyclase-coupled
8-agonist-preferring
states,
whose
equilib-
rium
may
be
regulated
by
a
sulfhydryl
group
mechanism.
After
the
discovery
of
the
benzomorphan
"opiates"
(1),
multiple
opiate
receptors
were
invoked
to
explain
the
qualitative
differ-
ences
these
drugs
produced
(2).
Later,
Kosterlitz
documented
a
dramatic
difference
between
the
",u
receptors"
of
the
guinea
pig
ileum
(where
morphine
is
pharmacologically
more
potent
than
opiate
peptides)
and
the
"8
receptors"
of
the
vas
deferens
(where
the
opposite
rank
order
of
potency
is
apparent)
(3).
The
,u-receptor-like
rank
order
of
potency
of
opiates
in
displacing
[3H]naloxone
binding
from
brain
membranes
correlates
quite
precisely
with
their
ability
to
elicit
"analgesia"
in
rodents
(4,
5)
as
well
as
to
suppress
guinea
pig
ileal
contractions
(6),
while
the
8-receptor-like
rank
order
of
displacement
of
3H-labeled
opiate
peptide
binding
correlates
with
the
suppression
of
mouse
vas
deferens
contractions
(3,
7).
It
has
been
previously
reported
that
GTP
inhibits
"type
1"
opiate
binding,
which
has
a
,-receptor-like
ligand
selectivity
pattern
(8,
9)
and
a
highly
discrete
localization
in
brain
when
visualized
autoradiographically
(10).
By
contrast,
"type
2"
bind-
ing
is
more
sensitive
to
displacement
by
peptides
than
by
al-
kaloids
(8,
9),
has
a
much
more
diffuse
brain
distribution
(11),
and
is
more
resistant
to
GTP
inhibition.
Rat
striatum
shows
this
distinct
difference
in
type
1
and
type
2
labeling
pattern
(12).
We
now
report
that
we
can
alter
the
ligand
selectivity
pattern
of
GTP-sensitive,
type
1,
opiate
binding
to
the
islands
of
rat
stria-
tum
by
manipulating
incubation
conditions
with
cations
and
GTP.
The
diffuse
type
2
labeling
pattern
appears
to
be
unaf-
fected
by
these
manipulations.
Our
findings
suggest
that,
at
temperatures
sufficient
to
permit
membrane
fluidity,
opiate
receptors
located
in
striatal
patches
can
shift
from
a
form
with
a
,-receptor-like
ligand
selectivity
pattern
to
a
form
with
a
8-
receptor-like
ligand
selectivity
pattern
as
they
couple
to
adenyl-
ate
cyclase.
METHODS
Male
Sprague-Dawley
rats
(approximately
200
g)
were
sacri-
ficed
by
decapitation.
Their
brains
were
rapidly
excised,
quick-
frozen
in
isopentane
at
-40°C, and
mounted
on
cryostat
chucks.
The
cortex
was
carefully
shaved
off
down
to
the
level
of
the
corpus
callosum,
and
25-pm-thick
coronal
sections
of
cau-
date-putamen
at
midstriatal
levels
were
cut
at
-
14°C.
Sections
were
thaw-mounted
near
the
edge
of
cleaned
gelatin-coated
slides,
air-dried
on
ice
about
2
hr,
and
then
dried
at
-
14C
for
at
least
48
hr
before
use.
Mounted
sections
could
be
stored
with
desiccant
at
-
80°C
for
at
least
2
months
with
no
apparent
effect
on
ligand
binding.
Approximately
60-70
midstriatal
sections
could
be
obtained
from
one
brain.
Slide-mounted
sections
(one
section
per
slide)
were
incu-
bated
with
3H-labeled
ligand
under
specified
conditions
in
30-
ml
beakers
in
a
volume
of
10
ml
consisting
of
0.05
M
Tris
HCl
buffer
at
pH
7.4
and
1%
bovine
serum
albumin.
3H-Labeled
dihydromorphine
(H2morphine)
(65
Ci/mmol;
1
Ci
=
3.7
X
1010
becquerels)
or
3H-labeled
[D-Ala2,
D-Leu']enkephalin
(D-
Enk)
(25
Ci/mmol)
was
included
at
about
50,000
cpm/ml
(ex-
cept
where
otherwise
indicated)
to
give
a
final
concentration
of
1.0
or
2.5
nM,
respectively.
To
terminate
the
incubation,
the
medium
was
removed
by
suction,
and
the
slide
was
washed
five
times
with
15
ml
of
ice-cold
0.05
M
Tris
HCI
buffer,
pH
7.4,
with
or
without
100
mM
NaCl.
Each
wash
lasted
15-20
sec
and
the
medium
was
removed
by
suction.
Those
sections
incubated
in
the
presence
of
100
mM
NaCl
were
washed
in
buffer
con-
taining
100
mM
NaCl.
Binding
of
3H-labeled
ligand
to
the
tissue
slice
was
quantitated
by
measuring
the
radioactivity
of
the
tis-
sue-laden
slide
fragment
in
10
ml
of
Aquassure
scintillation
cocktail
(New
England
Nuclear)
after
vigorously
agitating
the
vial
contents
for
30
min.
Specific
binding
was
calculated
as
the
difference
in
radioactivity
bound
in
the
presence
and
absence
of
1
pM
etorphine.
Preparation
of
incubated
slides
for
autora-
diography
and
microscopic
visualization
was
carried
out
as
de-
scribed
(10).
Grain
densities
in
selected
areas
of
sections
were
quantified
at
high
power
with
the
aid
of
a
reticle.
Abbreviations:
H2morphine,
dihydromorphine;
D-Enk,
[D-Ala2,
D-
Leu5]enkephalin.
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertise-
ment"
in
accordance
with
18
U.
S.
C.
§1734
solely
to
indicate
this
fact.
4818

Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
4819
Table
1.
Incubation
temperature,
Mn2+,
GTP,
Na-,
and
preincubation
with
Na'
+
GTP
differentially
alter
[3HlH2morphine
and
D-[3H]Enk
binding
to
slide-mounted
sections
of
rat
striatum
%
of
control
binding
in.
Tris
buffer
(no
additions)
Preincubation
No
preincubation
with
Na
+
GTP
250C
00C
25°C
Conditions
H2morphine
D-Enk
H2morphine
D-Enk
H2morphine
D-Eiik
No
additions
13.62*
15.68*
4.97*
4.97*
8.88*
15.91*
GTP
-51
-42
-61
-45
-66
-43
Na
-74
-43
-88
-84
-80
+10
Na
+
GTP
-94
-89
-97
-98
-98
-70
Mn
+35
-7
-65
-31
+11.
+8
Mn
+
GTP
-2
-14
-85
-41
-13
+13
Mn
+
Na
-17
+56
-82
-52
-41
+79
Mn
+
Na
+
GTP
-62
+30.
-91
-65
-69
+37
Slide-mounted
striatal
sections
were
incubated
with
[3H]Hmorphine
or
D-[3H]Enk
in
the
presence
of
NaCl
(100
mM),
GTP
(2
,uM),
or
Mn(OAc)2
(3
mM)
as
specified
in
the
table.
For
"no
preincubation,"
in-
cubation
was
carried
out
at
25°C
for
30
min
and
at
00C
for
3
hr.
For
preincubation
experiments,
slides
were
preincubated
batchwise
in
racks
holding
30
slides
for
15
min
at
250C
in
500
ml
of
medium
containing
0.05
M
Tris-HCl
buffer
at
pH
7.4,
1%
bovine
serum
albumin,
100
mM
NaCl,
and
2
,MM
GTP.
The
slides
were
then
washed
three
times-in
500-ml
changes
of
0.05
M
Tris
HCl
buffer,
pH
7.4,
at
250C,
for
30
sec
with
mild
agitation.
After
preincubation,
slides
were
incubated
for
30
min
at
250C
under
the
conditions.
specified.
Values
are
expressed
as
percent
change
from
control
binding
("no
additions")
and
are
taken
from
the
av-
erage
results
of
two
experiments,
each
with
duplicate
incubations.
Within
a
single
experiment,
duplicate
values
were
within
5%.
Absolute
values
for
fmol
bound
per
slice
varied
less
than
25%
between
experi-
ments,
and
the
percent
effect
of
addition
varied
less
than
10%.
Nonspecific
binding.(1
AM
etorphine)
was
determined
under
each
condition
and
was
found
to
be
essentially
unaltered
from
"no
additions."
*
Control
values
(100%),
fmol
per
slice.
RESULTS
Table
1
shows
a
comparison
of
the
effects
of
several
manipu-
lations
on
the
binding
of
H2morphine,
a
a
prototype
opiate,
with
the
binding
of
the
8
prototype
opiate,
D-Enk.
GTP
and
Na+
reduce
the
binding
of
both
H2morphine
and
D-Enk
to
slide-
mounted
rat
caudate
sections.
At
250C,
Mn2+
alone
had
a
slight
enhancing
effect
on
H2morphine
binding
and
no
significant
effect
on
D-Enk
binding.
However,
Na+
in
the
presence
of
Mn2+
markedly
decreased
the
binding
of
the
,u
ligand
while
enhancing
the
binding
of
the
8
ligand.
When
the
incubations
were
conducted
at
0WC,
no
Na+-induced
enhancement
of
D-Enk
binding
could
be
demonstrated
in
the
presence
of
Mn2
.
Fur-
thermore,
preincubation
of
the
tissue
with
Na+
and
GTP
ap-
peared
to
augment
the
differential
effects
on
the
"A"
and
"8"
binding,
especially
under
conditions
of
Na+
alone
and
Mn2+
plus
Na+.
We
chose
the
two
extreme
(alkaloid-promoting
or
peptide-
promoting)
conditions
for
further
study.
In
the
presence
of
Mn2+
alone
(alkaloid-promoting
conditions)
the
ratio
of
D-Enk
to
H2morphine
bound
per
striatal
slice
was
0.8,
whereas
the
presence
of
Mn2+,
Na+,
and
GTV
(peptide-promoting
condi-
tions)
gave
a
ratio
of
3.9.
Scatchard
analysis
of
these
experiments
revealed
that
the
peptide-promoting
conditions
caused
a
6-fold
increase
in
the
apparent
affinity
for
D-Enk
(from
48
to
8
nM
for
the
dissociation
constant),
but
reduced
the
apparent
affinity
of
H2morphine
to
approximately
1/4th
(from
1.5
to
5.7
nM
for
the
dissociation
constant).
Evidence
that
alterations
in
incubation
conditions
alter
the
ligand
selectivity
pattern
of
opiate
receptors
in
the
rat
striatum
are
shown
in
Fig.
1.
Under
the
alkaloid-pro-
moting
incubation
conditions
(Fig.
1B),
morphine
became
an
order
of
magnitude
more
potent
in
displacing
D-Enk
binding
than
under
the
peptide-promoting
conditions
(Fig.
1D).
The
inverse
was
also
true,
because
the
peptide-promoting
condi-
tions
(Fig.
1C)
enhanced
[Leu]enkephalin's
ability
to
compete
for
H2morphine
binding
about
an
order
of
magnitude
over
its
potency
under
alkaloid-promoting
conditions
(Fig.
1A).
Fig.
2A
shows
autoradiographs
of
the
binding
of
these
two
ligands
to
rat
striatum
under
peptide-promoting
(GTP
+
Na+
+
Mn2+)
or
alkaloid-promoting
(Mn2+)
conditions.
Like
[3H]-
naloxone
(10),
H2morphine
bound
to
striatal
patches
(13),
and
the
addition
of
Na+
and
GTP
appeared
to
decrease
the
intensity
of
the
labeling
but
did
not
change
the
distribution
pattern.
By
bIt
._
~a
._
x
0.
csm
3._
0
0
._
._
._
A
D
=
10
E0
50
ra
90
w
-
m
0
-
10
0
.:
50
;:
90
.a
10
9
8
7
-log[drugl,
M
6
B
10
9
8
7
-log[drug],
M
6
FIG.
1.
Slide-mounted
caudate
sections
were
incubated
with
[3H]-
H2morphine
or
D-[3H]Enk
in
the
presence
of
Mn(OAc)2
(3
mM)
(A
and
B)
or
Mn(OAc)2
(3
mM),
NaCl
(100
mM),
and
GTP
(2
AM)
(C
and
D)
in
the
presence
of
various
concentrations
of
unlabeled
morphine
(.)
or
[Leulenkephalin
(o).
Incubations
were
at
2500
for
30
min.
All
in-
cubation
mixtures
contained
bacitracin
at
100
,ug/ml.
Values
are
ex-
pressed
as
percent
inhibition
of
specific
binding
of
3H-labeled
ligand
and
are
plotted.
as
log-probit
plots.
Under
conditions
of
Mn2'
alone
(A
and
B),
[3H]H2morphine
and
D-[3HlEnk
were
bound
(in
the
absence
of
displacingligand)
at
11.0
and
6.78
fmol
per
slicej
respectively.
Under
conditions
of
Mn2+
+
Na+
+
GTP
(C
and
D),
[3HlHI2morphine
and
D-
[3H]Enk
were
bound
(in
the.
absence
of
displacing
ligand)
at
7.47
and
13.30
fmol
per
slice,
respectively.
Values
are
averages
of
duplicate
determinations.
Biochemistry:
Bowen
et
al.

4820
Biochemistry:
Bowen
et
al.
FIG.
2.
Effect
of
incubation
conditions
on
the
autoradiographic
la-
beling
pattern
of
tritiated
peptide
and
alkaloid
binding
in
rat
striatum.
(A)
Sections
were
incubated
30
min
at
25°C
with
[3HlH2morphine
(DHM)
(10
nM)
or
D-[3H]Enk
(D-ENK)
(25
nM)
under
the
specified
conditions.
(B)
Sections
were
incubated
with
[3H]H2morphine
(1.0
nM)
or
D-[3H]Enk
(2.5
nM)
for
30
min
at
2500
either
with
or
without
prior
preincubation
(Preinc)
with
NaCl
(100
mM)
and
GTP
(2
,M).
(C)
Sections
were
incubated
with
D-[3H]Enk
(25
nM)
under
the
specified
conditions
for
48
min
at
2500.
In
all
cases
concentrations
were:
Mn-
(OAc)2
and
Co(OAc)2
(3
mM),
NaCl.
(100
mM),
and
GTP
(2
,uM).
Incu-
bations
were
carried
out
as.
described
in
Materials
and
Methods,
and
sections
were,
prepared
for
autoradiography
as
described
in
ref.
10.
contrast,
the
addition
of
Na+
and
GTP
appeared
to
generate
new
D-Enk
binding
sites
within
the
patchy
areas,
thus
changing
a
diffiuse
D-Enk
pattern
into
a
more
patchy
one.
Fig.'
2B
shows
that
mere
preincubation
with
Na+
and
GTP
with
subsequent
incubation
in
tritiated
ligand
and'
Tris
buffer
alone
dramatically
increased
the
patchiness
of
D-Enk
but
reduced
the
patchiness
of
H2morphine.
Grain
density
analyses
(number
of
grains
per
256
tm
2,
mean
±
SD)
of
autoradiographs
of
all
manipulations
reported
in
Table1
showed
that
conditions
that
enhanced
bind-
ing
of
the
tritiated
peptide
and
diminished
the
binding
of
the
tritiated
opiate
alkaloid
did
so
by
increasing
binding
of the
pep-
tide
to
patchy
areas
(from
5.9
±
0.8
to
13.4
±
1.2)
with
a
con-
commitant
decrease
in
binding
of
H2morphine
to
those
patches
(from
15.6
±
0.9
to
7.2
±
0.6).
By
contrast,
the
striatal
areas
outside
of
the
patches,
which
were
twice
as
high
for
D-Enk
com-
pared
to
Hgmorphine
(3.2
±
0.3
versus
1.6
±
0.1),
were
un-
affected
by
these
manipulations
(3.4
±
0.4
versus
1.4
±
0.3).
Because
Na'
requires
manganese
ion
to
promote
D-Enk
binding
while
reducing
H2morphine
binding,
we
examined
other
divalent
transition
metals,
for
this
property.
Table
2
shows
that,
at
the
same
concentration
(3
mM),
both
Fe2+
and,
even
-more
strongly,
Co2+
accentuated
the
difference
between
D-Enk
and
H2morphine
binding
when
Na+
was
present.
Cu2+
and
Zn2+
(3
mM)
each-reduced
both
H2morphine
and
D-Enk
bind-
ing
to
undetectable
levels
(data
not
shown).
The
sulfhydryl-pro-
tecting
reagent
-dithiothreitol
also
increased
peptide
and
re-
duced
alkaloid
binding
in
the
absence
of
GTP
and
Na+.
Fig.
2C,
in
agreement
with
grain
count
analyses
and
quan-
titation
of
bound
tritium,
reveals
that
Co2+,
even
more
than
Mn2
,
alters
the
distribution
of
D-Enk
binding
from
diffuse
to
patchy
in
the
presence
of
Na+
and
GTP.
In
contrast
to
the
aL-receptor-like
ligand
selectivity
pattern
(4-6)
obtained
with
displacement
of
[3H]naloxone
from
striatal
slices
(10),
the
displacement
of
D-Enk
under
the
peptide-pro-
moting
conditions
(+
Mn2
,
+
GTP,
+
Na+)
was
characterized
by
the
8-receptor-like
pattern
in
which
opiate
peptides
are
more
potent than
alkaloids.
This
ligand
selectivity
pattern
was
the
same
as
(P
<
0.01)
that
obtained
when
the
same
six
opiates
were
tested
for
their
ability
to
inhibit
GTP-stimulated
adenylate
cy-
clase
activity
in
striatal
membranes
(Fig.
3)
in
the
presence
of
Mn2+,
GTP,
and
Na+.
Furthermore,
opiate
inhibition
of
-do-
pamine-stimulated
adenylate
cyclase
activity
was
undetectable
Table
2.
Differential
effect
of
dithiothreitol
and
divalent
metal
cations
on
binding
of
[3H]H2morphine
and
D-[3H]Enk
to
slide-
mounted
rat
striatal
sections
%
of
control
binding
(no
additions)
Conditions
[3H]H2morphine
D-[3H]Enk
No
additions
5.73*
17.49*
Na
-66
-20
GTP
-63
-26
Fe
(3
mM)
-7
+15
Fe
+
Na
-46
+35
Fe
+
GTP
-2
+10
Co
(3
mM)
-99
-24
Co
+
'Na
-100
+27
Co
+
GTP
-100
-29
Dithiothreitol
(3
mM)
-10
+46
Dithiothreitol
+
Na
-77
-3
Dithiothreitol
+
GTP
-66
-1
Slide-mounted
striatal
sections
were
incubated
with
[3HlHm2norphine
or
D[3H]Enk
in
the
presence
of
Co(OAc)2
or
FeSO4
(3
mM),
NaCl
(100
mM),
GTP
(2
,MM),
or
dithiothreitol
(3
mM)
as
specified.
Incubations
were
at
25°C
for
48
min.
Values
are
expressed
as
percent
change
from
control
binding
("no
additions")
and
are
the
averagesof
duplicates
(duplicates
are
all
within'5%)
from.two
separate
experiments.
*
Control
values
(100%),
fmol
per
slice.
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
I

Proc.
NatL
Acad.
Sci.
USA
78
(1981)
4821
100
bB
._
s
80
._
w
60
-
0
40
._
0
~Q
._
4._
=
2
u:
Morphine
/,&3Endorphin
[Leu]Enk/
Levorphanol
-
Etorphine
IKD-Ala,
Met]Enk
amide
0
20
40 60 80
100
IC50
of
cyclase
inhibition,
nM
FIG.
3.
IC50
values
(concentrations
at
which
the
reaction
is
inhib-
ited
by
50%)
for
inhibition
of
D-[3H]Enk
binding
by
various
unlabeled
alkaloids
and
peptides
were
determined
under
conditions
of
Mn(OAc)2
(3
mM),
NaCl
(100
mM),
GTP
(2
uM),
and
bacitracin
(100
,ug/ml)
by
the
procedure
described
in
the
legend
of
Fig.
1.
Membranes
from
rat
striata
were
prepared
on
a
discontinuous
sucrose
gradient
and
assayed
for
adenylate
cyclase
activity
as
described
(14).
The
adenylate
cyclase
assay
solution
contained
50
mM
imidazole
HCl
at
pH
7.2,
100
mM
NaCl,
5
mM
MnCl2,
1
mM
ATP,
3
mM
phosphoenolpyruvate,
pyruvate
kinase
at
20
units/ml,
1
mM
ethylene
glycol
bis(,-aminoethyl
ether)-
N,N,N',N'-tetraacetic
acid
(EGTA),
1
mM
dithiothreitol,
0.1
mM
iso-
butylmethylxanthine,
0.01
mM
GTP,
and
0.1-0.2
mg
of
membrane
protein
per
ml.
The
incubation
was
at
37TC
for
10
min.
Each
inhibition
curve
was
determined
in
quadruplicate.
The
line
was
fit
by
the
method
of
least
squares
analysis,
r2
=
0.84;
P
<
0.01.
in
membranes
prepared
from
the
posterior
third
of
the
striatum,
where
type
1
opiate
receptor
patches
are
sparse
(12)
(data
not
shown).
DISCUSSION
Fresh
frozen
sections
of
rat
striatum
thaw-mounted
on
slides
robustly
display
ion-
and
nucleotide-induced
alterations
in
op-
iate
receptor
binding
similar
to
those
characterized
previously
in
homogenates.
Thus,
these
effects
can
be
simultaneously
ana-
lyzed
quantitatively
and
visualized
autoradiographically
on
ad-
jacent
sections.
The
diffuse
labeling
pattern
(type
2)
produced
by
the
radiolabeled
enkephalin
analog
was
not
altered
by
the
effects
of
Na'
and
GTP.
Divalent
cation-dependent
Na'
en-
hancement
of
peptide
binding
at
the
expense
of
morphine
bind-
ing
occurs
exclusively
at
type
1
receptors
located
in
patches
and
in
a
streak
under
the
corpus
callosum.
The
binding
of
opiate
receptor
agonists
in
vitro
is
decreased
by
GTP
(15)
in
a
fashion
similar
to
that
of
agonists
at
other
mem-
brane-bound
receptors
(16-20).
This
GTP
sensitivity
could
re-
flect
allosteric
modulation
of
receptors
via
membrane
proteins
("N"
subunits)
that
couple
receptor
molecules
to
adenylate
cy-
clase
molecules
in
a
regulatory
mode
(21).
Sodium
ion
is
another
allosteric
effector
at
opiate
receptors:
it
is
required
for
adenylate
cyclase
inhibition
(22,
23),
it
selectively
renders
opiate
antag-
onist
binding
resistant
to
GTP
(24),
alterations
in
its
membrane
conductance
mediate
specific
opiate
electrophysiological
effects
(25),
and
it
selectively
enhances
opiate
antagonist
binding
in
vitro
while
decreasing
opiate
agonist
binding
(26).
In
this
study,
we
have
observed
still
another
selective
effect
of
Na+:
its
ability
to
enhance
opiate
peptide
binding
at
the
expense
of
opiate
al-
kaloid
binding
when
Mn2+,
Fe2",
or
Co2`
is
present
at
room
temperature.
The
presence
of
opiate
receptor
binding
sites
in
brain
mem-
branes
with
different
ligand
selectivity
patterns
has
been
in-
terpreted
as
evidence
for
"distinct"
or
"multiple"
opiate
recep-
tors
(3,
7,
27).
However,
our
study
suggests
that
the
ligand
selectivity
pattern
of
opiate
binding
at
distinct,
well-defined,
autoradiographically
visualized
sites
in
patches
of
rat
striatum
is
labile-it
can
be
dramatically
altered
by
allosteric
effectors
with
appropriate
incubation
conditions.
A
parsimonious
model
for
explaining
all
these
data
is
to
assume
that
a
single
opiate
receptor
protein
in
these
patches
alters
its
relative
affinities
for
opiate
peptides,
morphine,
and
naloxone
as
it
undergoes
func-
tionally
significant
but
subtle
transformations
while
"floating"
in
the
fluid
membrane
(Fig.
4).
This
scheme
fuses
contemporary
notions
of
hormone
regulation
of
adenylate
cyclase
(21,
28)
with
evidence
(20,
29)
that
different
ligand
selectivity
patterns
ac-
company
different
states
of
adenylate
cyclase-receptor
linkage.
In
fact,
opiate
receptors
solubilized
to
date
from
rat
brain
and
neuroblastoma
glioma
appear
in
preliminary
studies
to
display
but
a
single
8-receptor-like
ligand
selectivity
pattern
(30),
a
finding
reminiscent
of
muscarinic
receptors,
whose
heteroge-
neity
also
disappears
after
solubilization
(31).
It
appears
possible
in
vitro
to
artificially
stabilize
one
of
the
three
postulated
receptor
states
[e.g.,
promoting
the
formation
of
the
antagonist
state
by
lowering
the
incubation
temperature
(32)
and
adding
Na'].
Presumably,
in
vivo
interconversions
oc-
cur
rapidly
and
the
status
of
the
equilibrium,
which
varies
among
organs
and
species,
may
be
a
function
of
the
availability
of
other
membrane
components-e.g.,
adenylate
cyclase
or
the
thiol-disulfide
redox
potential
of
the
cell.
Disruption
by
di-
thiothreitol
or
divalent
cations
[in
the
Irving-Williams
(33)
or-
der
of
general
chelating
potency]
promotes
the
formation
of
the
3-ligand-preferring
state.
We
surmise
that
the
breakage
and
reformation
of
disulfide
bonds
may
be
critical
for
this
confor-
mational
transformation.
This
notion
is
consistent
with
the
am-
ply
documented
disruption
of
opiate
alkaloid
binding
by
several
sulfhydryl
reagents
(34,
35)
and
the
demonstration
that
opiate
receptor
membrane
mobility
is
altered
by
sulfhydryl
reagents
(36).
Can
type
2
opiate
receptors,
which
appear
"fixed"
in
a
pep-
tide-preferring
state,
also
regulate
adenylate
cyclase?
Opiate
receptor-mediated
inhibition
of
adenylate
cyclase
(as
in
Fig.
3)
cannot
be
observed
by
using
membranes
derived
from
the
pos-
terior
third
of
caudate,
where
type
1
opiate
receptor
patches
are
small
and
few
(data
not
shown)
and
type
2
opiate
receptors
,-ANTAGONIST
FIG.
4.
A
model
of
the
interconverting
forms
of
type
1
opiate
re-
ceptors
of
striatal
patches;
R,
receptor;
N,
GTP-regulatory
subunit;
MOR,
morphine;
ENK,
enkephalin;
Nal,
naloxone.
The
status
of
the
sodium
channel
in
the
cyclase-coupled
8
form
cannot
be
surmised
from
existing
electrophysiological
data.
The
location of
the
critical
sulfhy-
dryl
group(s)
is
unknown
and
thus
they
are
drawn
ambiguously
at
the
interface
between
N
and
R.
Type
2
opiate
receptors,
by
contrast,
seem
incapable
of
assuming
the
,u
conformations.
Biochemistry:
Bowen
et
al.

4822
Biochemistry:
Bowen
et
al.
predominate
(12).
Thus,
type
2
opiate
receptors
of
rat
caudate
seem
unable
to
inhibit
adenylate
cyclase
under
the
conditions
described
here.
Finally,
this
model
makes
it
superfluous
to
seek
the
endog-
enous
ligand
for
the
g
receptor,
because
the
enkephalins
are
quite.
capable
of
regulating
adenylate
cyclase
and, indeed,
are
more
efficient
than
the
neurotoxin
morphine.
Indeed,
the
/L
conformation
may
represent
an
adenylate
cyclase-uncoupled,
"desensitized"
state
(37).
If
the
elegantly
characterized
acetyl-
choline
receptor,
the
"prototype
allosteric
membrane
protein"
(38),
is
at
all
relevant,
then
the
physiological
function
of
the
proposed
conformational
oscillations
of
the
opiate
receptor
may
be
to
regulate
Na'
flux,
as
well
as
adenylate
cyclase.
We
are
grateful
to
Mr.
Rufus
Gunn
for
preparation
of
striatal
slices.
1.
Eddy,
N.
B.
&
May,
E.
L.
(1966)
in
Synthetic
Analgesics,
Inter-
national
Series
of
Monographs
in
Organic
Chemistry,
eds.
Bar-
ton,
D.
H.
R.
&
van
Doering,
W.
(Pergamon,
Oxford), Vol.
8,
pp.
113-182.
2.
Martin,
W.
R.
(1967)
Pharmacol
Rev.
19,
463-521.
3.
Lord,
J.
A.,
Waterfield,
A.
A.,
Hughes,
J.
&
Kosterlitz,
H.
W.
(1977)
Nature
(London)
267,
495-499.
4.
Wilson,
R.,
Rogers,
M.,
Pert,
C.
&
Snyder,
S.
(1975)1J.
Med.
Chem.
18,
240-242.
5.
Pert,
C.
B.,
Snyder,
S.
H.
&
Portoghese,
P.
S.
(1976)
J.
Med.
Chem.
19,
1248-1250.
6.
Kosterlitz,
H.
W.,
Leslie,
F.
M.
&
Waterfield,
A.
A.
(1975)
J.
Pharm.
PharmacoL.27,
73-78.
7.
Bedell,
C.
R.,
Clark,
R.
B.,
Hardy,
G.
W.,
Lowe,
L.
A.,
Uba-
tuba,
F.,
Vane,
J.
R.,
Wilkenson,
S.,
Miller,
R.
J.,
Chang,
J.-K.
&
Cuatrecasas,
P.
(1977)
Proc.
R.
Soc.
London
Ser.
B
198,
.249-265.
8.
Pert,
C.
B.
&
Taylor,
D.
(1980)
in
Endoganous
and
Exogenous
Opiate
Agonists
and
Antagonists,
ed.
Way,
E.
L.
(Pergamon,
New
York),
pp.
87-90.
9.
Zukin,
R.
'S.
&
Gintzler,
A.
R.
(1980)
Brain
Res.
186,
486-491.
10.
Herkenham,
M.
&
Pert,
C.
B.
(1980)
Proc.
Natl.
Acad.
Sci.
USA
77,
4469-4473.
11.
Pert,
C.
B.,
'Taylor,
D.
P.,
Pert,
A.,
Herkenham,
M.
A.
&
Kent,
J.
L.
(1980)
in
Advances
in
Biochemical
Psychopharmacology,
Neural
Peptides
and
Neuronal
Communication,
eds.
Costa,
E.
&
Trabucci,
M.
(Raven,
New
York),
pp.
581-589.
12.
Herkenham,
M.
&
Pert,
C.
B.
(1981)
Nature
(London)
291,
415-418.
13.
Young,
W.
S.,
III
&
Kuhar,
M.
J.
(1979)
Brain
Res.
179,
255-270.
14.
Gentleman,
S.,
Parenti,
M.,
Commissiong,
J.'W.
&
Neff,
N.
H.
(1981)
Brain
Res.
210,
271-275.
15.
Blume,
A.
J.
(1978)
Proc.
NatL
Acad.
Sci.
USA
75,
1713-1717.
16.
Williams,
L.
T.
&
Lefkowitz,
R.
J.
(1977)
J.
Biol
Chem.
252,
7207-7213.
17.
Rodbell,
M.,
Lin,
M.
C.
&
Salomon,
Y.
(1974)J.
BioL
Chem.
249,
59-65.
18.
Moody,
T.,'Taylor,
D.
&
Pert,
C.
(1981)J.
SupramoL
Struct. 15,
153-159.
19.
U'Prichard,
D.
C.
&
Snyder,
S.
H.
(1978)
J.
BioL
Chem.
253,
3444-3452.
20.
Creese,
I.
&
Sibley,
D.
R.
(1980).
in
Psychopharmacology
and
Biochemistry
of
Neurotransmitter
Receptors,
eds.
Yamamura,
H.
I.,
Olsen,
R.
W.
&
Usdin,
E.
(Elsevier,
Amsterdam),
pp.
387-410.
21.
Rodbell,
M.
(1980)
Nature
(London)
284,
17-22.
22.
Wilkening,
'D.,
Sabol,
S.
L.
&
Nirenberg,
M.
(1980)
Brain
Res.
189,
459-466.
23.
Blume,
A.
H.
(1980)
in
Psychopharmacology
and
Biochemistry
of
Neurotransmitter
Receptors,
eds.
Yamamura,
H.
I.,
Olsen,
R.
W.
&
Usdin,
E.
(Elsevier,
Amsterdam),
pp.
369-384.
24.
Childers,
S.
R.
&
Snyder,
S.
H.
(1978)
Life
Sci.
23,
759-762.
25.
Zieglgansberger,
W.
&
Bayerl,
H.
(1976)
Brain
Res.
115,
1.1-128.
26.
Pert,
C.
&
Snyder,
S.
(1974)
MoL
PharmacoL
10,
868-879.
27.
Childers,
S.
R.,
Snowman,
A.
M.
&
Snyder,
S.
H.
(1979)-Eur.J.
PharmacoL
55,
11-18.
28.
'Jacobs,
S.
&
Cuatrecasas,
P.
(1976).
Biochim.
Biophys.
Acta
433,
482-495.
29.
Kebabian,
J.
&
Kalne,
D.
(1979)
Nature
(London)
277,
93-99.
30.
Simonds,
W.
F.,
Koski,
G.,
Streaty,
R.
A.,!Hjelmeland,
L.
M.
&
Klee,
W.
A.
(1980)
Proc.
Natl
Acad.
Sci.
USA
77,4623-4627.
31.
Birdsall,
N.
J.
M.,
Burgen,
A.
S.
V.
&
Hulne,
E.
C.
(1980)
Proc.
R.
Soc.
London
Ser.
B
207,
1-12.
32.
Creese,
I.,
Pasternak,
G.
W.,
Pert,
C.
B.
&
Snyder,
S.
H.
(1975)
Life
Sci.
16,
1837-1842.
33.
Irving,
H.
&
Williams,
R.
J.
P.
(1953)J.
Chem.
Soc.,
3192.
34.
Pasternak,
G.
W.,
Wilson,
H.
A.
&
Snyder,
S.
H.
(1975)
MoL
PharmacoL
11,
340-351.
35.
Simon,
E.
J.
&
Groth,
J.
(1975)
Proc.
NatL
Acad.
Sci.
USA
72,
2404-2407.
36.
Hazum,
.E.,
Chang,
K.-J.
&
Cuatrecasas,
P.
(1975)
Nature
(Lon-
don)
282,
626-628.
37.
Lefkowitz,
R.
J.
(1980)
in
Psychopharmacology
and
Biochemistry
of
Neurotransmitter
Receptors,
eds.
Yamamura,
H.
I.,
Olsen,
R.
W.
&
Usdin,
E.
(Elsevier,
Amsterdam),
pp.
155-170.
38.
Changeux,
J.-P.
(1981)
Harvey
Lect.,
in
press.
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)