Evidence contradicting the notion that gonadal hormones regulate brain opiate receptors

BIOCHEMICAL

Vol. 108, No. 3, 1982 October 15, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1313-1319

EVIDENCE CONTRADICTING THE NOTION THAT GONADAL HORMONES REGULATE BRAIN OPIATE RECEPTORS James A. Dies

and David

L. Roberts

School of Biology Georgia Institute of Technology Atlanta, Georgia 30332 Received

September

7, 1982

SUMMARY : Castration of male rats has been reported to increase brain opiate receptors by nearly 100%. We assayed brain opiate receptors with both naloxone and met-enkephalin, but found no effect of gonadectomy on the Kd or Bmax for either ligand in male or female mice or in male rats. Experiments were performed with 2 strains of mice and 3 strains of rats; mice were gonadectomized 1-7 weeks and rats were castrated 3 weeks before assay. Both washed and unwashed brain membrane preparations were used. Administration of testosterone or estrogen to intact male or female mice did not alter opiate receptors. Castration did not affect the strain or age and brain-region differences found for naloxone binding in male rats.

The mechanisms late

steroid

rent

data

suggest

Physiological inhibit

that

the

secretion

endogenous

opiate

ability

of steroid (3). Data which

action

to up-

workers

and low-

agonists

One attractive is

hypothesis

found

a nearly

from

is

modulate

the

have been receptors

the

for

other

reported

100% increase

opiate

for

receptors

the

hypothesis

a good precedent

regulate

this

could

can

while

agonists

or affinity

or down-

affinity

opiate

cur-

role.

hormones,

to displace

there

to regubut

an important

that

(1,2).

act

uncertain,

releasing

concentration

support

Those high-

indicate

acting

hormones;

hormones directly

(4).

of both

their

systems

still

may play

hypothalamic

receptor

of steroid

ligands

control are

opiates

studies

of several

can block in

control

and Fishman

feedback

in vertebrates

such as naloxone,

receptor, changes

feedback

(Bmax)

that

antagonists

opiate

negative

secretion

and pharmacological

the

opiate

by which

hormone

by Hahn

in the

number

in the brains

of

male rats which had been castrated had been injected with testosterone

for 3 weeks. The Bmax in castrates which propionate for 7 days was the same as

intact

constants

controls.

The dissociation

(Kd)

did

not

differ

This report has been widely cited (e.g., crine status. to provide some of the first and perhaps best evidence

of how the

control

receptor

of steroid

hormones

could

interact

with

opiate

3,5)

with

since

it

endoseems

feedback

regulation.

0006-291X/82/191313-07$01.00/0 1313

Copyrighr 0 1982 by Academic Press, Inc. Ail righrs of reproduction in any form reserved.

Vol. 108, No. 3, 1982 Our original examining mice. receptor by us) we also

the

effects

sites,

of other

different both

thought

steroid

ligands

naltrexone to bind

the

or delta

of gonadal

or in male

(8)

hormones

(used

of Hahn and Fishman on brain

which With

on brain

opiate binding

by Hahn and Fishman)

to the mu receptor

sites.

hormones

findings

RESEARCH COMMUNICATIONS

can show preferential

used methionine-enkephalin,

the mu (7) effect

AND BIOPHYSICAL

aim was to extend

Although are

BIOCHEMICAL

these opiate

site

(6).

receptors

receptors

in

to specific

and naloxone

(used

In some experiments,

may be the physiologic ligands,

by

however, in male

ligand we found or female

for no mice

rats. Materials

and Methods

C57BL/6J and Swiss albino mice were purchased from The Jackson Laboratory and ARS/Sprague Dawley, respectively; the mice were then bred and maintained in our colony under standard laboratoryconditions. Rats were obtained from Charles River Breeding Labs and maintained for at least 2 weeks before use in experiments. Gonadectomies were performed under Nembutal anaesthesia. Testosterone propionate or estradiol benzoate were administered subcutaneously by injection in sesame oil or by implantation of Silastic capsules (1.6&m x 1Omm) which had been packed with hormone and soaked in saline for 24 hrs. Mice were killed by cervical dislocation; rats were decapitated. Regional brain dissections followed the method of Glowinski and Iversen (9). Protein was assayed by the method of Lowry, et al. (10). Brain regions or whole brains minus cerebella were pooled 5-8 per group and homogenized in 10 volumes of cold Tris-HCl buffer (50 mM, pH 7.7) with a Tekmar Tissumizer at high speed. Following centrifugation at 30,000 x g for 20 min, pellets for "unwashed" preparations were resuspended in 75 volumes of Tris buffer and frozen for l-7 days. 'For "washed" membrane preparations, the pellet was resuspended in 75 vol of Tris and incubated at room temperature for 1 hr before recentrifuging and freezing. In some experiments, we followed the procedures outlined by Hahn and Fishman (4): tissue was homogenized in 5 volumes with a Teflon-pestle homogenizer and the pellet from an initial centrifugation at 1000 x g was discarded before the high speed (17,500 x g) pellet was finally resuspended in 60 volumes. In our standard opiate receptor assay, membrane fragments containing approximately 0.2 mg protein were incubated for 3 hr on ice in 1.0 ml of Tris buffer containing 0.16 to 12.0 nM of [N-allyl-2,3-3H]naloxone or [tyrosyl-3,5-3H(N)]met-enkephalin (New England Nuclear; 30-50 Ci/m mol). Bacitracin (50 ug/ml) was added when enkephalin was used. Non-specific binding was determined in the presence of 10 uM morphine or met-enkephalin. Membrane fragments were collected by filtration on Whatman GF/C filters and rapidly washed twice with 5 ml of cold Tris. Vacuum-dried filters were shaken overnight in 10 ml of ACS (Amersham) before liquid scintillation counting. In some experiments, the incubation was performed at room temperature for 1 hr plus 15 min on ice. In both cases, it was determined that binding was at equilibrium and linear beyond the range of tissue concentration used. These methods differed from Hahn and Fishman as follows: they used 2.0 ml of tissue preparation and incubated for 15 min at 37O plus 30 min on ice; they used [3H]naltrexone as the ligand (2 x lo-lo to 7.5 x 10-q M) and non-radioactive naltrexone (low6 M) for non-specific binding; filters (GF/B) were washed twice with 4 ml of Tris. After correction for non-specific binding, the Kd and Bmax were calculated from Scatchard plots using a program written for an Apple II+ computer. For a linear Scatchard plot (i.e., enkephalin binding), all points were included in the least-squares regression analysis. Curved Scatchard plots (i.e., naloxone binding) were split into highand low- affinity 1314

Vol. 108, No. 3, 1982

BIOCHEMICAL

an iterative fragments; binding component from

AND BIOPHYSICAL

procedure the points

was used to remove in the low affinity

Results Naloxone

and enkephalin

sham-operated,

two "unwashed"

preparations

receptors

were

assayed

dectomy,

using

both

We occasionally if

these

found

random

results Since

various

receptors

(11)

naloxone found

A species Fishman's (13). is

of gonadal

already

testosterone of gonadal benzoate

twice

experiment

opiate

The species-difference

preparations experiment, rats;

in

and the

were

with always

we found the second Long Evans

to be

castrates

and

of gonadectomy. in opiate

we also mice,

examined

but

still

induced

by

administered to see if

receptors.

elevations

We found

propionate

Silastic

to achieve

no effect

or estradiol

implants

were

a more persistent

as shown in Figure

used hor-

3, adminis-

to females had no deas measured by [ 3 H]naloxone binding.

receptors,

was then

tissues

no effect (Figure 4). The apparent Kd's binding were similar in the two strains, 1315

by comparing In the

rats. were

used;

castration.

castrated

from for

directly

male

strains

days after between

re-assayed

tested

and intact

two different

made 21-23

experiment,

receptors

increase

mice

and

mice or of estrogen

no difference samples

in rats

of opiate

We therefore

negative;

Hahn and

effects

a further

a week.

of castrated rats,

to confirm level

opiate

hypothesis

in brains

failure

of testosterone

in order

were

to male

on brain

of experiments

albino

or female

a day for

The results

of testosterone

our

male

100 rig/kg

series

(12),

Swiss

be seen.

down-regulate

receptors

as likely

some differences

and that

cannot

would

opiate

effect

the steady-state

to intact

injected

effect

was small,

between

to have different

in which

8 days in another

tectable

explain

"maximum"

hormones

steroids

mone elevation.

25%) in binding,

1 and 2.

administration

known that

or estrogen

were

tration

could

at its

in one experiment

procedures.

and were

comparisons

in outbred

are

We hypothesized

removal

than

gona-

castration.

opiates

in mice

after

the preparations

or reproducible

to opioid

difference

report:

as in

as the

of the 1000 x g pellet);

experiments

in Figures

binding

after

(less

of mice may exhibit

or response

and enkephalin no effect

mice

strains

across

a major

are presented

as well

1 and 7 weeks

in

intact,

in several

and ice-bath'incubation brains

groups

from

mice

fragments

removal

between

differences

of pooled

sham -vs. control We never observed

controls.

times

moderate

were

in preparations C57BL/6

membrane

and without

room temperature

in the

Typical

for

(with

the number

differences

assayed

"washed"

at various

the

were

male and female

standard

observed

particularly but

binding

We used our

the high affinity range, and vice-versa.

and Discussion

and gonadectomized

experiments.

RESEARCH COMMUNICATIONS

Sprague

Dawley

for

high

but

the Bmax's

receptor

In the first

and control

comparison.

first

rats

Again,

and low

Long Evans

affinity

were

were

assayed

castration

had

naloxone

markedly

higher

Vol. 108, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL CONTROL o CASTRATE l

.06

.04

MALES

.024r

A.

\ .o \ '\

RESEARCH COMMUNICATIONS

Y"

B/F .02

.I0

0

.I5

1

Bound

.03

(nM/mg -CONTROL A SHAM novx

.06

.09

protein) FEMALES

.06r

B.

.024r

B/F

0

I

2

1

/

05

I

.I0

1

I

.06

.09

(nM/mg protein) on brain opiate receptors in male mice. Data represent means of two experiments, 21 and 51 days after castration; washed membranes were used. Constants were derived from combined data. A. Naloxone (high- and low- affinity sites): Kd= 0.98 and 17.5 nM; Bmax= 0.04 and 0.25 nM/mg prot. z. Enkephalin: Kd- 5.49 nM; Bmax= 0.12 nM/m8 prot. 1

No effect

of

castration

Fig.

2

No effect mice.

of

ovariectomy

(OVX)

g. Enkephalin:

the Long

since

the Long

might

declines lobe

Evans

an increase After

Evans

rats

in rat

hypothalamus

Compared

with

This

in older

male adult

were

receptors

receptors

in

female

OVX. Treatment groups (high- and low0.22 nM/mg prot.

Naloxone 0.05 and

considerably

young

were

a true

older

CD rats assayed

than

With striatum

strain

difference,

but

the Sprague-Dawley's,

aging,

S-endorphin

and increases

adult male rats, old adults and naloxone was much less

in

content frontal

had higher levels potent in inducing

(15). of gonadal

of different

male

represent

and corpus

no effect

rats

A. B&x=

an age difference.

in LH secretion finding

21 days after

constants. 14.72 nM;

could

met-enkephalin,

in young Opiate

strain.

represent

(14).

and

opiate

Kd= 6.3 nM; Bmax= 0.12 nM/mg prot.

simply

of hypothalamic

on brain

were used,

were combined for calculating affinity sites): Kd= 1.65

it

9

.03

Bound

Fig.

Washed membrane fragments

in

I

.I5

strains, since

these

hormones

we examined were

23 days after 1316

in male

or female

the effect

mice or

of castration

used by Hahn and Fishman castration;

hypothalamus

(4). and the

Vol. 108, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A.

8.

.12-

.12-1

.00-

*CONTROL 0 ESTRAMOL

l0 Control TESTOSTERONE

P

0 .08

l

-1

l =\

B/F

B/F

.04-

-

-

.04

I .I

I

03

I

I

,

.2

.3

.4

Bound

t

(nM

Img

LONG

.05

0

.I0

EVANS 0 CONTROL

.I5

1

I

I

I

.I

.2

.3

.4

protein)

A

I

-

SPRAGUE

CASTRATE

.20

DAWLEY

l

.

.25

.30

.35

Bound ( nM / mg protein)

4

3 No effect in intact

Fig.

of gonadal mice.

steroid

administration

on [3H]naloxone

binding

Assays were performed 8 days after implantation of 10 mm Silastic capsules. A. Males + testosterone: Kd= 1.36 and 20.1 nM; Bmax= 0.12 and 0.60 nM/mg Trot. B.-Females + estradiol: Kd= 1.22 and 34.72 nM; Bmax= 0.12 and 0.85 nM/mg pyot. Fig. 4 No effect of castration on [3H]naloxone Mean body weights: Long Evans= 632 g; Sprague were derived from combined data. Long Evans Sprague nM; Bmax= 0.05 and 0.50 nM/mg prot. 8.27 nM; Bmax= 0.02 and 0.31 nM/mg prot.

rest our

of the brain standard

(minus

washed

membrane

by Hahn and Fishman. Specific and

binding

castrate-

(tissue bound

cerebellum)

tissues

preparation

As shown

tended

preparation-naloxone

hardly

assayed and the

in Figure

to be higher were

were

binding in 2 strains of rat. Dawley= 381 g. Constants (2 expts.): Kd= 0.55 and 10.3 Dawley (1 expt.): Kd= 0.31 and

unwashed

5, no marked

in the washed distinguishable,

concentration)

separately.

We used both preparation

differences

preparation; but

comparisons,

used were

found.

the control-

in 30 of 34 direct the control

samples

more naloxone. Despite

receptors,

our

inability

the original

to find hypothesis

a castration-induced of regulation 1317

increase by a testosterone

in opiate feedback

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 108, No. 3, 1982

CONTROL

CASTRATE Membrane8 WASHED UNWASHED

‘; .

n .

.2Or

.I5 B/F .I0 -

.4 Bound

.5

.6

.?

(nbl / mg protein)

I

C

-

I

C

,250 ,200 nM

ma Prd .I50 t 7 ; L

06

cx

MP

3 2 STR

3 51

7 f L

6.0nM

c

Q6nM

MB

Fig. 5 No effect of castration on [3H]naloxone binding in young CD male rats. Mean body weight= 227 g. A. Hypothalamus: Kd= 1.04 and 23.4 nM; Bmax= 0.20 and 0.70 nM/mg prot. 2. Rest of Brain (minus cerebellum): Kd= 1.15 and 28.7 nM; Bmax= 0.22 and 1.2 nM/mg prot. Fig.

6

Regional

analysis

of

[3H]naloxone

binding

in

castrated

(C)

and

intact

(I) male rats. Long Evans rats (mean weight= 656 g) were used 21 days after castration. Brain regions from 5 (C) and 4 (I) rats were pooled and assayed in triplicate with 0.6 and 6.0 nM naloxone. PIT= pituitary; g= cerebral cortex; g= hypothalamus; D= striatum; MB= midbrain. -MP= medulla-pons;

control

receptor castration

system

still

concentration in 5 rat

show regional

seemed attractive. have been brain regions

differences

Although naltrexone sites (6), the difference

Since

described (16), and pituitary.

but no difference

regional

differences

we looked for The results,

between

control

in

an effect in Figure

opiate

of 6,

and castrated

and naloxone presumably bind to the same receptor between our results and those of Hahn and Fishman 1318

rats.

Vol. 108, No. 3, 1982 could

be due to the

available play

BIOCHEMICAL

commercially;

a significant (17)

to find

bind have

results

that

we wish

increase

in vacant

centrations" binding

assay

ligand

would

decrease washed

act

preparations

rat

results

opiate

receptors;

problem

in

prevents

readily do not

naloxone

brain.

negative

on brain

not

hormones

to which

mouse or male

technical

result

that

the

increase

of an increased

or

Wilkenson a limited

these

attempt

workers

sus-

us from observing

receptors

due to a reduction be pointed

should the

to increase with

Scatchard the

no change

do not

suggest

out,

apparent

opiate

binding

of receptors

in endogenous however,

plot

is affinity

in Bmax.

in

number

It

upon which

the Kd),

gonadal

the receptors

reported

suggested

be the (4).

that

is

the

to see...."

Hahn and Fishman could

RESEARCH COMMUNICATIONS

L3H]Naltrexone

conclude

or female

recently

some purely

observed

used.

regulating

of castration

I'... that

pected

in

in male

also

an effect

ligands

we can only role

met-enkephalin et al.

different

AND BIOPHYSICAL

that

based,

ligand

con-

in the equilibrium in endogenous

of the binding effect

they

or llan

a decrease

Our results

a significant

which

with

(i.e.,

washed

of endogenous

and unligand.

Acknowledgement This work was supported RR07024-16.

by a Biomedical

Research

Support

Grant,

PH.5 SO7-

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Van Vugt, D. A., and Meites, J. (1980) Federation Proc. 39, 2533-2538. Cicero, T. J. (1980) Federation Proc. 39, 2551-2554. McEwen, B. S. (1980) Mol. Cell. Endocrinol. 18, 151-164. Hahn, E. F., and Fishman, J. (1979) Biochem. Biophys. Res. Comm. 90, 819-823. Krieger, D. T. and Martin, J, B. (1981) New Engl. J. Med. 304, 876-885. and Cuatrecasas, P. (1981) Federation Proc. 40, 2729-2734. Chang, K.-J., Goodman, R. R., Snyder, S. H., Kuhar, M. J., and Young, W. S., III. (1980) Proc. Natl. Acad. Sci. 77, 6239-6243. Chang, K.-J., and Cuatrecasas, P. (1979) J. biol. Chem. 254, 2610-2618. Glowinski, J., and Iversen, L. E. (1966) J. Neurochem 13, 655-699. Lowry, 0. H., Rosebrough, N. J., Farr, A. .I., and Randall, R. J. (1951) J. biol. Chem. 193, 265-275. Baran, A., Shuster, L., Eleftheriou, B. E., and Bailey, D. W. (1975) Life Sciences 17, 633-640. Waterfield, A. A., Lord, J.A.H., Hughes, J., and Kosterlitz, H.W. (1978) Eur. J. Pharmacol. 47, 249-250. Kuschinsky, K., and Hornykiewicz, 0. (1974) Eur. J. Pharmacol. 26, 41-50. Gambert, S. R., Garthwaite, T. L., Pontzer, C. H. and Hagen, T. C. (1980) Neuroendo. 31, 252-255. Steger, R. W., Sonntag, W. E., Van Vugt, D. A., Forman, L. J., and Meites, J. (1980) Life Sciences 27, 747-753. Chang, K.-J., Cooper, B. R., Hazum, E., and Cuatrecasas, P. (1979) Mol. Pharmacol. 16, 91-104. C. A. (1981) In Steroid Hormone Wilkenson, M., Herdon, H., and Wilson, Regulation K. Fuxe, J. A. Gustafsson, anntterburg, --of the Brain, eds., Pp 253-263, Pergamon Press, New York. 1319