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

0006-291X/79/201355-09$01.00/0 1355

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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

1356

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