[3H] corticosterone binding activity of adrenal incubation media in response to in vivo and in vitro ACTH stimulation

BIOCHEMICAL

Vol. 90, No. 4, 1979 October

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

29, 1979

[I3H

Pages

CORTICOSTERONE BINDING ACTIVITY

1355-1363

OF ADRENAL INCUBATION MEDIA

IN RESPONSE TO --IN VIVO AND IN VITRO ACTH STIMULATION J.

Departments Received

F. Pritchett*, W. L. Harper, D. N. Marple .I. T. Bradley, and M. L. Till

of Zoology-Entomology Agricultural Experiment

September

20,

and Animal Science; Auburn University Station; Auburn, Alabama 36830

1979 SUMMARY

ad nistration of ACTH upon --in vitro The influence of --in vivo or --in vitro binding adrenal corticosterone production as well as % ] corticosterone ACTH, regardless of the activity of incubation media have been examined. route of administration, significantly elevated corticosterone during an initial incubation period. During initial incubation periods ACTH also elevatedPI%] corticosterone binding activity of the incubation media. Ensuing incubations were characterized by either a marked decline in binding activity (in -- vivo ACTH administration) or a marked decline with sn attendant negative correlation between binding activity and ACTH level. Corticosteroidogenic response to ACTH remained intact during ensuing incubations. The data suggest the existence of an ACTH-sensitive intraglandular corticosterone binding ligand(s), the activity or concentration of which is dramatically altered by incubation protocol and time of sacrifice. INTRODUCTION Competitive

protein

binding

coids

routinely

involve

an initial

might

otherwise

compete

with

tated.

Such extractions

dichloromethane Since cortical were

our laboratory

expected

modify

plasma

corticoid *To whom all

values

rather

obtained

correspondence

assay

performed

involved

tedious

with

for

the steroid

in our

laboratory

determination

plasma

corti-

proteins being

which

quanti-

utilizing

incubation

corticoid

of --in vitro

concentrations

of plasma

media,

extraction

step.

proteins

in incubation However

samples

adreno-

we attempted

quantification

from non-extracted should

for

(1).

in adrenal for

procedures

to remove binding

protein

to ACTH and since

CPB procedures the

the

by Murphy is

to be minimal

by eliminating

radioassay

extraction

have been

as described

responsiveness

(CPB)

were

comparisons consistently

to media of lower

be addressed

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@ I979

by Academic Press, Inc. in any form reserved

of reproduction

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

than those obtained from the samesamples extracted Furthermore these differences in vitro gland.

RESEARCH COMMUNICATIONS

with dichloromethane.

appeared to be related not only to the specific

incubation method employed but also to the functional These findings prompted us to investigate

tissue in incubation may liberate and/or activity

status of the

the possibility

a corticoid-binding

ligand,

that adrenal

the concentration

of which is dependent not only upon incubation protocol but

also upon exposure of the tissue to ACTH. MATBHIALSANDMETHODS Animals and incubation

procedures

Male Sprague-Dawley rats 85 to 90 days of age were utilized. Prior to experimental procedures all animals were housed 3 per cage at an ambient temperature of 24 + .5OC with a photoperiod of 14 hours light:10 hours darkness Animals were provided with Purina Laboratory Chow (lights on at 0730 hours). and tap water -ad libitum. Wo hours prior to sacrifice, animals were transferred to individual cages. For determination of --in vitro and -in vivo responses to ACTUadministered in vivo, animals were injected subcutaneously 60 minutes prior to sacrifice with either 0.5 ml 0.9% NaCl or 0.5 ml 0.9% NaCl containing either 1 or 3 IU AC'JX (Nutritional Biochemicals). Animals utilized for study of in vitro response to --in vitro administration of ACTHor for determination of time of sacrifice upon corticosterone binding activity were allow to remain undisturbed until time of sacrifice. All animals were decapitated between either 0945-1015 or 1945-2015 hours. Trunk blood was collected from only the latter group for subsequent determination of plasma corticosterone. Adrenal glands from each animal were rapidly removed to a dish of cold (3-5OC) Krebs Ringer Bicarbonate solution (KRBC) and trimmed of extraneous tissue. Glands were then quartered, rinsed thoroughly in KRBC, and incubated in a Dubnoff metabolic incubator (37OC, 60 oscillations/ min., 95% 02-5% CO2atmosphere). To assess the influence of -in vivo ACTHon --in vitro corticosterone secretion and binding activity, glands were incubated for an initial 60 minutes in 2 ml KRBCcontaining 2 mg glucose/ml (KRBG). Following the initial incubation periods, glands were transferred to a second 60 minute incubation in 2 ml fresh KRBG. For study of the influence of time of sacrifice upon corticosterone binding activity, an initial incubation only was performed. Following each incubation period, media were decanted into glass vials and frozen (-2OoC). To determine the influence of --in vitro administration of ACTUupon in vitro corticosterone secretion and media binding activity, gland quartersfrom each animal were incubated for an initial 60 minutes in 2 ml KRBGor in a like volume of KKBGfortified with either 50, 200 or 400 milliunits/ml ACTU (Sigma At the end of the first incubation period, glands not initially Biochemicals). exposed to ACTUwere transferred to 2 ml fresh KRBG containing 50, 200, or 400 milliunits/ml ACTS and incubated for an additional 60 minute period. All incubation media were collected and frozen as before. Corticosterone

determination

in plasma and incubation media

Assay of incubation media and plasma samples for corticosterone was accomplished via the CPB radioassay described by Murphy (1, 2) utilizing rabbit serum as the source of the binding ligand and 1, 2 PH] corticosterone

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Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Both plasma samples and aliquots of (New England Nuclear) as the radiolabel. incubation media were extracted with dichloromethane l/10 (v/v) prior to corticosterone determination. Separation of bound from free corticoid was accomplished by charcoal adsorption. The bound fraction was counted (Searle Isocap/ 300) in a scintillation cocktail of toluene-Triton X (2:l) with Omnifluor (6 grams/liter). Interand intra-assay variabilities were less than 7% and 5% respectively. Incubation

medium binding

activity

Aliquots of incubation medium were filtered utilizing a Millipore apparatus To remove endogenous corticosterone and filters (type HA, pore size = 0.45 um). from the samples, 500 ul portions of media were each added to an equal volume of 2.5% dextran-coated charcoal in phosphate buffered saline (PBS), and with constant agitation, were brought to and maintained at 45'C for 30 minutes. Samples were then centrifuged (2-3'C) for 30 minutes (2000 X g) and the supernatants were filtered as before, twice. Removal of endogenous corticosterone was confirmed via CPB assay of portions of the filtered fraction. Binding studies were performed in duplicate upon 500 pl aliquots of each sample. Tritiated corticosterone (specific activity = 136.9 mCi/mg) was added in 100 pl of PBS; DPM added per assay tube are indicated in the tables and figure. The mixture was vortexed and then incubated at 45'C for 10 minutes. Samples were then cooled to 2-3OC and maintained for a 10 minute period. Separation of free from bound isotope was via charcoal adsorption (500 pl added) followed by centrifugation (2000 X g for 20 minutes, 2-3OC). Bound isotope in 500 ~1 of supernatant was counted as before. Coefficients of determination for intraand inter-assay variability was less than 5%. To test the hypothesis that corticosterone binding activity of incubation' medium was influenced by extraction procedures, aliquots of corticosterone free incubation medium were incubated with isotope and then mixed l/10 (v/v) with dichloromethane and vortexed. The organic layer was removed. Free isotope from 400 ~1 aliquots of these samples as well as from 400 ul quantities of non-dichloromethane treated media samples and KRBG was removed as before and bound activity was quantitated as described above. RESULTS The effect activity

of incubation

previously

removed

a mean binding treated

compared

media is

activity,

to the KRBC-glucose

The action corticosterone 2.

of injected production

Both

upon

endogenous

in Table

1.

Although

greater

remained

terone

initial

had been

samples (p

<

exhibited

.05)

extraction in

binding

than

either

significantly

the treated

samples

as

blanks. ACTB upon --in vivo

1 and 3 IU levels concentration

corticosterone

corticosterone

dichloromethane

some activity

PI

Non-treated

was significantly

and corticosterone

corticosterone values,

from which

which

or blanks.

binding

extraction

illustrated

activity

samples

diminished

Table

of dichloromethane

within

incubation

produced

binding significant

one hour. levels

1357

plasma

were

corticosterone, activity elevations

Considering also

is

--in vitro

significantly

&vitro presented

in

in plasma corticoshigher

in the

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Effect

of

dichloromethane binding activity

1

treatment upon of incubation

[&I] corticosterone media

Treatment

N

DPM Addeda

DPM Boundb

Nontreated

6

45750

894 t

Treated

6

45750

171 2 15d

Blank

6

45750

4Sc

75+ -

6e

aDPM added per determination; specific activity = 136.9 mCi/mg. bAll values represent DPM in 500 pl of supematant (means + SEM). cs ds eWithin the indicated column, values with different superscripts differ significantly (p ( 0.05).

TABLE Effect of secretion

Treatment

exogenous and[3H]

ACTH administered corticosterone

2

--in binding

vivo upon activity

corticosterone --in vitro of incubation mediaa.

Corticosterone Secretion (micrograms/hr)

DPM Boundb

N

Plasma Corticosterone (nanograms/ml)

Initial Incubation

Second Incubation

Initial Incubation

Second Incubation

Saline

Inject

6

195.9 + 34.1c

4.28 + .56c

4.71 + .52c

2379 f. 444c

1 Unit

ACTH

6

309.3 + 36.5d

6.22 + .4Bd

5.32 + .83c

3348 2 468d

6

321.5 + 48.5d

7.22 + .56d

4.13 f. .43c

3744 + 48gd

3 Units aAll

ACTH values

represent

means

group

of the second

as compared

to the

incubation,

no ACTH treatment

corticosterone incubation second

in response incubation,

in each below

binding

of the

that

activity

observed

saline-injected

837 + 153cse

the

significantly (p < 0.05). than DPM bound in

effects

However,

at the end

were

detected.

Tritiated

elevated

during

ACTH administration. on binding

activity

initial

incubation

1358

the

initial

At the end of the was apparent.

[‘HI corticosterone

incubations, during

9gcBe

of supematant;

group.

was significantly

to --in vivo

no ACTH effect second

1104 +

f. SEM.

b62250 DPM added per determination; DPM bound are per 500 nl specific activity = 136.9 mCi/mg. CsdWithin columns, values with different superscripts differ eDPM bound in second incubation significantly less (p < 0.01) initial incubation.

treated

642 + 135cBe

binding period.

Moreover,

was significantly

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

TABLE Effect activity

of

upon PH] corticosterone binding during an initial incubationa.

Time

No.

Trial 1 2 3

2 2 2

2691 2475 2745

+ 156= T 144' z 135c

8:00

p.m.

1 2 3

2 2 2

3891 3993 3939

+ 144d + 204d + 60d

The influence

a single

media

exhibited

DPM bound are = 136.9 mCi/mg. values with (p < 0.05).

of sacrifice 60 minute

associated

significantly

killed

means + SEM. per determination; specific activity

the indicated colwm, differ significantly

of time

during

trials,

DPM Boundb

a.m.

cp dWithin scripts

all

N

1O:OO

aValues represent b60800 DPM added of supematsnt;

activity

3

time of sacrifice incubation media

of

Sacrifice

RESEARCH COMMUNICATIONS

incubation

with

less

(morning

animals

binding

per

super-

evening)

upon binding

is presented

activity

during than

pl

different

vs.

killed

500

in Table

3.

the morning

did

media

from

In

hours animals

at night. Regression

costerone

analyses

secretion

an initial without

upon

ACTH are given

in Figure

content

corticosterone

during

correlated

regression

line

Corticosterone

and negatively

for

ACTH level,

observed

for

equivalent

1.

Both were

an initial

less

of binding

Media

was also the slope

than

to --in vitro

activity during

1359

either incubation

activity

and

that

the initial

content

of

of and

the second

of the first

incu-

incubation.

incubation

ACTH level.

was significantly

corre-

positively

at the end of the second

correlated

ACTH levels

during

by an initial

period.

incubation although

corti-

and significantly

incubation

activity

binding

preceded

positively

second

and --in vitro

ACTH exposure

the degree

was significantly

significantly any given

incubation

to ACTH level

binding

binding

of --in vitro

of the media

at the end of the

significantly

corticosterone

the level

or a second

to ACTH level

bation

[3~]

incubation

corticosterone lated

of

was

Additionally less

incubation

than period.

that

Vol. 90, No. 4, 1979

BIOCHEMICAL

-0

5 ‘r

8

0

‘;^ 4am &E $i? a8 /‘:-

.Y

Incubation

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

i

-Incubation1 -

.---*Incubation

2 i

+l

2.

r s 50

400

200

In Vitro

ACTH

Dose

Level

(Milliunits/ml)

Figure 1. [%-I] Corticosterone binding activity of incubation media and corticosterone secretion in response to in vitro ACTH administration. Each point represents the mean with attendant SEM of 6 observations. Each line For represents the regression of the indicated variable upon ACTH dose level. each regression, an absolute value of the correlation coefficient (r)k .468 the slope of the regression line represents p < .05. For either variable, representing incubation 1 differs significantly (p < .025) from the corresponding value for incubation 2. *For either variable and within ACTH dose level, incubation 1 value differs significantly (p c.05) from incubation 2 value. For each determination of binding activity, 62250 DPMPH] corticosterone were added; DPM bound are per 500 pl of supematant; specific activity = 136.9 mCi/mg.

DISCUSSION Results activity

presented

of adrenal

of whether is presented

and refined

strongly

incubation

media

the ACTH is which

upon AC!JX and is --In vitro

herein

administered

suggests related

adrenal primarily

that

indicate in --in

response viva

the degree

to the specific incubation for

of binding

activity protocol

and protocols

binding

ACRID, regardless

Additionally

or in vitro.

of ACTH or for

1360

corticosterone

to exogenous

incubation

procedures

the bioassay

altered

is

evidence dependent

utilized. have been

studies

developed

on the mechanisms

Vol. 90, No. 4, 1979

BIOCHEMICAL

of corticosteroidogenesis. --in vitro Others

adrenal (4,

reliable

index

e,

provided

since

been

total

--in vivo

within

of both that

animal

out

degree

they

for

and constant

the gland

at the

medium shortly

tion)

periods

ACTH-induced

of response incubation (6)

systems

time

of sacrifice

as from

alterations

possible

physiological

isolated

cell

in which

study ensuing

observed

or constant

flow

systems

8).

incubations

were

would

present

have

into

cell within

out prior

initial

this

to

(preincubaConsequently,

whereas

rendered

-in

the incubation

period

be considered

the

recently

analyzed.

the preincubation

with-

to isolated

be washed from

of the

discarded

More

fluid

media

initial

altered

evolved

incubation

could

were

released

would

minute

which

extracellular materials

during

implications)

media

have

has or

by transfer

ACTH (7,

tissue

it

present

a 30-60

properties

to exogenous

-in

adrenocortical

employed

of the tissue

In the present as well

of --in vitro

to possess

and/or

preparation

factors

Preincubation

of adrenal

flow

after

ACTH treatment.

found

activity

from partial

of ACTH followed

medium.

can be a

However

obtained

therefore

the absence

--in vitro

immediately.

studies

elevated

of ACTS.

cortical

by extra-adrenal

ACTH have

were

adrenal

of data

Subsequently,

in

described

production

incubated

may be confounded

and uniformity

vi&protocols (9)

interpretation

ACTH-fortified

since

corticoid

is

first

administration

and ACTH-stimulated

that

period

(3)

to --in vitro

adrenal

tissue

to exogenous

to fresh analysis

that

adrenal

(6).

or pre-incubation

and coworkers

response

basal

systems

responsiveness

in

reported

suggested

the

tissue

Saffran

secretion

5) have

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(and

the

the use of approach

unten-

able. Considering istration present media direct in

--in vivo

and --in vitro

of ACTH, a significant

stimulatory

as indicated levels

by elevated

of corticosterone

interest

the initial

injection

both

was the incubation

of the animal

plasma during

observation media, with

adrenal

response

corticosteroidogenic corticosterone

the initial

levels incubation

of corticosterone the magnitude

of which

ACTH and B) presumably

1361

binding

to in vivo admin-effect was and elevated period. activity

was A) related

dependent

upon the

Of more present to prior activity

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

of the gland at or shortly

after

--in vivo glandular activity

and binding activity

binding activity

of initial

at night was significantly

RESEARCH COMMUNICATIONS

the time of sacrifice.

The relationship

of

is amplified by the fact that

incubation media associated with animals killed greater than that observed in samples obtained

during the mid-morning hours and hence corresponds to the time of maximal natural

activity.

Transfer of the tissue to and subsequent incubation without

in fresh medium

ACTHresulted in a moderate decline in corticosterone

ACTHtreated

groups to levels not different

from the saline injected

and may be due to the decay of the biological sharp decline in corticosterone

high level of binding activity to A) the functional

in the initial

of ACT&

group,

However, a

of all media occurred in both

It is therefore

possible that the relatively

incubation may have been related

status of the gland --in vivo with subsequent alterations

the composition of the extracellular

fluids

of death or B) the possible introduction mediumas a result

activity

binding activity

saline and ACTHtreated groups.

levels in the

in

of the gland before or at the time

of an intracellular

of tissue damageor secretion,

factor(s)

into the

the concentration(s)

of which

were AClX-dependent. To determine whether similar

alterations

obtained in response to ACTHadded directly

in binding activity

to the incubates (Figure l),

tissue was exposed to different

ACTHlevels either

immediately following

or during second incubations

incubations without

sacrifice

ACTS. In terms of corticoid

content of the media increased with AC'JI-Ilevel cating a continuing

capability

ACTH. During the initial was also positively

correlated

period,

to ACTHlevel.

the absence of ACTHand subsequent transfer only was binding activity

during initial

significantly

incubation but was also negatively

incubations

following

production,

initial

corticosterone

during either

in terms of corticoid

incubation

could be

incubation,

indi-

release in response to

corticosterone

binding activity

However, after

preincubation

to fresh ACID-fortified

medium, not

lower than observed during the initial

correlated

1362

in

to the level of ACTHexposure.

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

Results obtained from these initial in binding activity not be directly

due to ACTRis an intraglandular

in response to ACTB after

than observed without preincubation seemslikely

incubations suggest that the elevation

dependent upon extraglandular

binding activity

phenomenonand thus may

ACTS actions -in vivo.

preincubation

and negatively

that a component essential

RESEARCH COMMUNICATIONS

ligand(s)

its possible physiological

was substantially

correlated

less

to ACIH level,

it

to the development of binding activity

of glandular origin was removed during the preincubation of the specific

Since

phase.

Characterization

responsible for the observed binding phenomenonand significance

are subjects of current studies in our

laboratory. ACKNOWLEDGEMENTS The authors express their appreciation to Mrs. Charlotte Butler for her assistance in preparation of the manuscript. This study was supported in part by Auburn University Grant-in-Aid 2275-01-7424. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Murphy, B. E. P. (1967) J. Clin. Endocr. 27, 973-990. Murphy, B. E. P. in Odell, W. D. and Daughaday, W. II., eds. (1971) Principles of competitive protein-binding assays, pp 108-127, Lippincott, Philadelphia. Saffran, M., Grad, B., and Bayliss, M. J. (1952) Endocrinology 50, 639643. Van Goch, J. J., DeWied, D., snd Schonbaum,E. (1963) Am. J. Physiol. 205, 1083-1088. DeWied, D., Van Der Wal, B., and Van Goch, J. J. (1964) Excerpta Medica Int. Congr. Ser. 83(1):64-69. Schulster, D., Tait, S. A. S., Tait, J. F., and Mrotek, J., (1970) Endocrinology 86, 487-502. Bakker, R. F. M., and DeWied, D. (1961) Can. J. Biochem. Physiol. 39, 23-29. Birmingham, M. K., and Kurlents, E. (1958) Endocrinology 62, 47-60. Sayers, G., Swallow, R. L., and Giordano, N. D. (1971) Endocrinology 88, 1063-1068.

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and