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