Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SUBCELLULAR ACTIVITY
Gary Queen,
IN
LOCALIZATION OF BOVINE PITUITARY
Carl Pinsky,
ENDORPHINE AND BRAIN
i
and Frank LaBella 2
Department of Pharmacology and Therapeutics University of Manitoba, Faculty of Medicine Winnipeg, Manitoba, Canada R3E OW3
Received August 13,1976 SUMMARY The e n d o r p h i n e c o n t e n t o f b o v i n e a n t e r i o r (AP), posterior (PP), intermediate (IP) pituitary, brain, and s u b c e l l u l a r fractions of these tissues was e s t i m a t e d in an opiate radioreceptor assay. I P was m o s t c o n centrated in activity, containing 7, 15, 780, and 9,100 times as much as PP, AP, midbrain, and cortex, respectively. More than 90 per cent of total endorphine activity in all lobes of the pituitary sedimented during eentrifugation at 12,000 x g for i0 min. Density-gradient centrifugation gave almost identical sedimentation patterns for all pituitary lobes. In brain, endorphine activity sediments similarly but not identically to that of pituitary. Endorphine activity in the pituitary is localized to a secretory granule presumably derived from a cell type common to all lobes. Brain endorphine is localized to a similar granule, but derived at least in part from nerve endings.
The development
of a sensitive
and specific
for opiate drugs led to the identification exert morphine-like enkephalin decapeptide
(tyr-gly-gly-phe-met-OH) (~-endorphine),
(2).
from whole pituitaries
Supported Associate
protein, (3).
by the Medical o f t h e MRC.
peptide,
for endorphine
Research
Copyright © 1976 by Academic Press, Inc. ,'Ill rights o/reproduction in any/orm reserved.
Methionine-
in brain (I) and a hexasequence,
beta-lipotropic
Continuation
Council
1021
from
is contained
hormone
(LPH),
of the sequence to the
C-fragment
(a generic
assay
peptides which
The sequence of peptides
yields a potent opiate-like
may serve as a prohormone
1
was identified
with this same N-terminal
within a 91 amino acid residue
C-terminus
of endogenous
actions both in vitro and in vivo.
hypothalamus-neurohypophysis
isolated
radioreceptor
(4).
Thus,
LPII
term for all opiate
a n d NMUD/Health a n d W e l f a r e
Canada
Vol. 72, No. 3, 1976
peptides: suggested
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enkephalin, earlier
tion,
C-fragment),
as well as for ~-MSH as
(5).
Elucidation and endorphines
~-endorphine,
of the tissue and i n t r a c e l l u l a r
should c o n t r i b u t e
to an u n d e r s t a n d i n g
localization
of the origin,
and fate of both the brain and p i t u i t a r y o p i a t e - l i k e
this report we present as estimated
results
on the d i s t r i b u t i o n
by an opiate r a d i o r e c e p t o r
of LPH
peptides.
of e n d o r p h i n e
funcIn
activity
assay.
EXPERIMENTAL S e v e n t e e n per cent h o m o g e n a t e s of fresh, bovine tissues in water were adjusted to pH 2.0 with 0 . 1 N HC1, placed in a b o i l i n g water bath for 15 min, cooled and TRIS added in a final c o n c e n t r a t i o n of 0.15 M, pH 7.7. The n e u t r a l i z e d s u s p e n s i o n was c e n t r ~ u g e d at 900,000 g-min, and the supernatant decanted and stored in the cold for subsequent assay. For pituitary tissue, a thin central wedge of the i n t e r m e d i a t e lobe was d i s s e c t e d out, as was an interior portion of the anterior or posterior lobe, well away from the i n t e r m e d i a t e zone. B r a i n tissues included a portion of the cortex and one from the "midbrain" (cortex and c e r e b e l l u m removed). The r a d i o r e c e p t o r assay was based on the method of Pert and Snyder (6),and P a s t e r n a k et al. (7), but c e n t r i f u g a t i o n r a t h e r than f i l t r a t i o n was used to isolate brain membranes. Brains ( c e r e b e l l u m removed) from male, S p r a g u e - D a w l e y rats were h o m o g e n i z e d in 30 volumes of 0.05 M TRIS, pH 7.7 and c e n t r i f u g e d for 750,000 g-min. The pellet was r e s u s p e n d e d in an equal volume of TRIS, incubated at 37 ° for 30 min, and recentrifuged. The pellet was resuspended in TRIS to yield 60 mg brain e q u i v a l e n t per ml. One-half ml aliquots were combined w i t h 0.3 ml TRIS and 0.I ml of c o m p e t i n g extract or standard opiate drug in TRIS and incubated for 30 min at 0 °. 3 H - n a l o x o n e (2.6 x 10 -9 M; 33 Ci/mmol, New England Nuclear) in 0.1 ml TRIS was added, and the tubes incubated for 30 min at 0 °. The tubes were c e n t r i f u g e d to sediment membranes, and the s u p e r n a t a n t a s p i r a t e d and discarded. The pellet was dissolved in 0.3 ml of 2N K O H at 70 ° for lO min, and a 0.2 ml aliquot added to a s c i n t i l l a t i o n counting medium contained methylcellosolve. N o n - r a d i o a c t i v e levorphanol, l0 -6 M, d e p l e t e d the counts by 6 0 - 7 0 % whereas its p h a r m a c o l o g i c a l l y inactive isomer, dextrorphan, caused no depletion. For d i f f e r e n t i a l centrifugation, a 10% h o m o g e n a t e of p i t u i t a r y or whole brain tissue in 0.25 M sucrose was prepared w i t h a teflon pestle fitted to a glass vessel. Nuclei and debris were s e d i m e n t e d at 7,000 g-min and discarded. All procedures were carried out at room t e m p e r a t u r e to avoid d e p l e t i o n of stored hormones (8). Pellets obtained at 30 thousand, 120 thousand, and 480 thousand g-min and the r e s u l t i n g s u p e r n a t a n t were extracted w i t h HC1 for e n d o r p h i n e as described for whole tissue. For d e n s i t y - g r a d i e n t centrifugation, the 7,000 g-min s u p e r n a t a n t was applied to the top of a 35 ml plastic tube, c o n t a i n i n g sucrose or a s u c r o s e - f i c o l l gradient, and c e n t r i f u g e d in a S p i n c o model L at 4 °. The s e d i m e n t a t i o n bands were isolated by means of a S p i n c o tubes l i c i n g devi@e. Each p a r t i c u l a t e band was d i l u t e d with i0 volumes of 0.25 M sucrose and c e n t r i f u g e d at 12000 thousand g-min to pellet s u s p e n d e d material. The pellets were extracted w i t h HC1 for e n d o r p h i n e estimation. Methionineand l e u c i n e - e n k e p h a l i n were purchased from Peninsula Co., and ~-endorphine was provided by Dr. R. G u i l l e m i n to Dr. V. Havlicek, Dept. of Physiology. L e v o r p h a n o l and d e x t r o r p h a n were provided by Dr. G. Krip, H o f f m a n n - L a R o c h e Ltd., Montreal, and n a l o x o n e by Mr. J. Beaulac, E n d o Laboratories, Montreal.
1022
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
22
A.
20
18
L
A
O O 16 x E
II. 14 U
\ I
I
1.0
t-
X
~
1J
20
Z
I
100
1000
nM Opiate
"¢~ 22 e" 0
I
10
b Post.Pit. ~ '~
Midbrain 'k~k
\ x~°rtex
I
"I-
18
16
14
12 I
'
0.01
0.1
1.0
!
I
10
I
100
1000
mg Tissue Equiv. FIGURE 1. (A) D o s e - r e s p o n s e curves for synthetic (B) D o s e - r e s p o n s e curves of acid extracts of beef opiate radioreceptor assay.
peptides pituitary
and opiate and brain
drugs. in the
RESULTS AND DISCUSSION Opiate peptides cleavage
of the putative
31-residue
prohormone,
LPH.
peptide have been reported
in brain (I). tides
of varying size may be potentially
in pituitary
only a 16-residue
The relative
potencies
only total "endorphine" of several synthetic
1023
and a
(2,4) and a pentapeptide
Until the number and relative amounts of pituitary
in these tissues are determined,
be estimated.
However,
derived from
opiate pep-
activity can
peptides
in the
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
opiate radioreceptor
assay are shown in Figure
less potent than the opiate drugs,
].A.
and ~-endorphine
All three peptides are considerably
than the enkephalins.
The deviation from parallelism
curves for levorphanol
and U-endorphine
assays
performed
in the absence
The endorphine and brain,
as estimated
and contains
of the dose-response
is a consistent
observation
in
of sodium.
content
of various
by the competitive
It can be seen that the intermediate activity,
less potent
lobes of the bovine pituitary radioassay,
is shown in Figure
lobe is most concentrated
lB.
in endorphine
about 7, 15, 780 and 9,100 times as much as the pos-
terior pituitary,
anterior
pituitary,
midbrain,
direct comparison
of potency between brain and pituitary may not be valid,
since only the pentapeptide,
enkephalin,
tissue and only ~-endorphine
and C-fragment
.ug 5
50
and cortex,
respectively.
has been identified in the latter.
A
in the former Dose-response
Tissue Equiv.
500
5
50
500
i
i
i
i
.
40 A
0 0 x
35
-Na+
E
n
Pit.
n U
~~t-
"o 30 e-::} o nn = O x
Pit.
"X~evor. 25
o
o
Z ! I
20
15 I
4.6
I
13.7
I
41
I
I
123
4.6 nM
I
13.7
I
I
41
123
Opiate
FIGURE 2. Dose-response curves for endorphine and opiate drugs in the radioreceptor assay in the absence (A) and presence (B) of 100 mM NaCl.
1024
Vol. 72, No. 3, 1976
curves tary
for
the
extracts
and b r a i n s
character
of
effect
ED50 f o r
the
of
did
the
not
opiate
receptor,
is
the
active
endorphines)
during
substance
a secretory
granule.
fractions
again,
most sediments in
for
three
granules,
in
pituitary 3).
because
supernatant
only
from brain the
only
1 to
after
granules,
negligible
e.g.
menting which is
genates
cytoplasmic a fairly
are
the
well
defined
virtually
distinct
in the agonist
of endorphine
g for
pellet.
10 m i n ,
activity in
presumably
among p a r t i -
pituitary,
although
By d e n s i t y - g r a d i e n t
as
is is
of the
in
endorphine
the
total
is
particulates.
centrif-
sedimentation
distinctive
strongly
activ-
indicating
organelle,
pattern
bound to recovered
Also,
for
the in
the
manipulation
centrifugation,
that
within
Our r e s u l t s
show that
the
identically
of the
and presumably is
secretory
granules,
beef
That
in all
lobe
opiate
contained
opiate.
intermediate
endogenous
entirely
region
of endogenous
o f a common g r a n u l e , of
12,000
density-gradient
indicate
granules.
practically
tion
conformation
of
liberates
amounts of activity.
and brain
concentration
and tissue
(nhloxone)
90 p e r c e n t
peptide(s)
during
o f NaC1 t h e
number of sites
similari£y
of
by
(9).
apparent,
removal
pitui-
was i n d i c a t e d
addition
somewhat from that
3 per cent
These observations pituitary
is
for
The o p i a t e - a g o n i s t
levorphanol
of endorphine
the
The e n d o r p h i n e
fraction
isolated
lobes
for
the
at
low s p e e d
systems,
the
to a subcellular
differs
curves
tissues
antagonist
more than
be l o c a l i z e d
two different
(Figure
lobes
The d i s t r i b u t i o n
culate
ugation
conformation
the
with
diminishing
centrifugation
to
in
2):
the
but
one another.
increased
promotes
pituitary
sedimented
all
substance
but
thereby
one another,
parallel
(Figure
was d e c r e a s e d
For all
the
binding
Sodium apparently
ity
parallel
consistently
receptor
naloxone
(levorphanol,
brain
pituitary
of sodium on binding
extracts. of
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
the
three
pituitary, activity lobes
a common c e l l
made up o f and c o r d s
1025
rapidly
contains sediments
type.
parenchymal cells
lobe,
the highest f r o m hom-
the
presence
The l a r g e cells
in
sedi-
intermediate
indicates
of these
substances
with
penetrate
propornumerous into
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCONTINUOUS DENSITY GRADIENT
CONTINUOUS DENSITY GRADIENT 75
60
~
BRAIN
50
40 25
20
60
>, ,,i.-
75
ANT. PIT.
50
40
.¢U