6J mice

Brain Research, 115 (1976) 345-351

345

© Elsevier Soentific Publishing Company, Amsterdam - Printed an The Netherlands

Corticosterone binding in cytosols from brain regions of mature and senescent male C57BL/6J mice

JAMES F. NELSON, CHRISTIAN F HOLINKA*, KEITH R LATHAM, JANET K. ALLEN and CALEB E. FINCH Andrus Gerontology Center and Department of Biological Sciences, Umverslty of Southern Cahforma, Los ,4ngeles, Cahf 90007, Metabolic Research Umt, Umverstty of Califorma, San Francisco, Calif 94143 (K.R.L.) (U.S .4.) and (J.K.,4.) Department of Biochemistry, University oJ New South Wales, Kensington, New South Wales (Australia)

(Accepted June 2nd, 1976)

Altered responsiveness of glucocorticoid-mediated cell functions occurs in aging mammals 1,7,8,z6, but the sites mediating these changes have not been identified. However, age-related changes in the brain-pituitary-adrenal axis are implicated, since some changes do not appear to be accounted for solely by an altered response of peripheral target tissues to glucocorticoidsl,7, s. It is probable that the regulation of some corticosterone-induced functions is mediated in the brain by macromolecules (receptors) that bind the hormone and translocate into the nucleus to regulate the accumulation of specific gene products 21. Studies supporting this hypothesis have shown that functionally effective sites for corticosterone implants correspond to brain regions, such as the hippocampus and hypothalamus, which selectively accumulate corticosterone 21. Because glucocorticoid receptors appear to be an essential link in the brain-pituitaryadrenal regulatory loop, it is conceivable that age-related changes in receptor levels in these brain regions could account for the altered responses observed. In this study we have measured the effect of aging on in vitro corticosterone binding by cytosols from the hippocampus, hypothalamus and cerebral cortex of male C57BL/6J mice. Although a significant regional difference was observed, no significant effect of aging was detected. C57BL/6J male mice were obtained as retired breeders (8 months old)from the Jackson Laboratory, Bar Harbor, Maine and were maintained as described previously 7. Mature mice were 11-12 months of age and senescent mice were at the age of average longevity in our colony, 28-29 months 7. Mice with gross pathological lesions (e.g., reticulum cell sarcoma of the liver 4, mesenteric lymph node tumors4, v or significant weight change 7) were eliminated at necropsy. [1,2,6,7-3H]Corticosterone ([3H]C, 80 Ci/mmole; New England Nuclear Corp., Boston, Mass.) was 89 ~ pure when checked by thin layer chromatography on silica gel plates (Eastman 1318 l, Rochester, N.Y.) using benzene-ethanol, 3 : l, v/v. Unlabeled steroids were purchased from Sigma Chemical Corp. (St. Louis, Mo.). * Postdoctoral Fellow, Population Council, New York, N.Y., U.S.A.

346 Mice were bilaterally adrenalectomized (ADX) under anesthesia induced by l.p rejection of 0.5-0.6 ml of a 2 3~osolution of 2,2,2-trlbromoethanol (Matheson, Coleman and Bell, Norwood, Ohio) m 0.14 M NaCI. We have previously found that plasma corticosterone in A D X mice of both age groups falls to undetectable levels by 12 h post-ADX, but that a crossreacting substance (based on radioimmunoassay) appears in the plasma of mature mice by 24 h 1¢. Therefore mice were killed 12 h post-ADX to mlmmlze binding of endogenous cortlcosterone to cytosol macromolecules. Following cervical dislocation, brains were quickly removed and dissected on an ice-chilled stage. Cerebral hemispheres were reflected from the central sulcus and cut parallel to and approximately 1 mm medial from the rhmal sulcus. Both horns of the hippocampus were removed by blunt dissection. The hypothalamus was excised rostrally at the optic chlasm, laterally at the lateral hypothalamic sulc~, caudally at the border of the mammlllary bodies, and dorsally about 2 mm above its ventral surface. Brain regions were stored in hquld N2. No loss of cytoplasmic binding capacity wa~ detected in tissue stored as long as 8 months. Frozen brain regions from 2 to 8 mice were pooled and weighed to the nearest milligram. Average weights of hlppocampus, hypothalamus and cortex were 24, 14 and 65 rag, respectively. Tissue was homogenized m 0.1 M sodium phosphate buffer, pH 6.9 at 4"C ( 1ml buffer/0.32 g tissue) with 30 strokes of a 0.5 ml Teflon/glass homogenizer (No. K885480, Kontes Glass Co., Vineland, N.J.). The homogenate was cenrefuged at 64,500 7 g for 60 min at 2 °C in a Beckman Type 40.3 rotor. Cytosol* wa~ removed and mcubated in an ice-bath for 2-7 h in l , 10 -s M [3H]C alone or with 1 ,< 10-G M unlabeled cortlcosterone, dexamethasone, progesterone or estradiol-1713 (Sigma Chem Corp., St. Lores, Mo.). Saturation of specific binding occurred by 2 h and no loss of bmdlng capacity was detected up to the maximum duration ofincubanon. Saturable binding was determined as the difference between the bound 3H m the noncompeted and competed incubations. Repeated extractions of brains with 0.1 M phosphate buffer resulted m less than 5','o additional saturable binding Bound and free :3H were separated by gel chromatography on G100 Sephadex (Pharmacia, Piscataway~ N.J.). A 0.1 ml ahquot of charged cytosol was fractxonated on a 0.8 cm ~ 30 cm column made from a 10 ml glass pipet (bed volume: 14 ml). A pressure head of 35 cm maintained a flow rate of approximately 0.26 ml/min. The excluded volume containing the macromolecular-bound steroids eluted in 21.2:5 0.14 (S.E.M.) mm. Fractions (0.2 ml) were collected and counted at 48-52 o/; efficiency m 5 ml of Aquasol (New England Nuclear, Boston, Mass.). Soluble protein content was determined by the method of Lowry ~9 Results were analyzed by one-way analysis of variance, using Duncan's multiple range test for sigmficance of differences between groups ~j. A typical elution profile of mouse brain cytosol incubated with [3H]corticosterone in the presence and absence of 100-fold excess unlabeled corticosterone ~s shown m Fig. 1. The hatched portion indicates the column fractmns which were used to estimate specifically bound [aH]cortIcosterone. These estimates of binding must be considered The term cytosol Js used by convention, although the possibility of the presence of nuclear proteins can not be excluded

347 void volume

4,

600

500

c~ 4 0 0

J

t

E 300

2O0

I00

A 20

I i i

! /

10

30

i

I

I

50

60

70

fraction no.

Fig. l. Typical elution profile o f [3H]cortlcosterone b o u n d to cytoplasmic macromolecules o f mouse brain. Cytosol (10-20 mg protem/ml) was incubated w]th 1 × 10 -8 M [sH]C with ( ) and w]thout ( . . . . . . ) 1 × 10 -6 M unlabeled cortlcosterone and fractionated by gel filtration on 14 ml columns containing Sephadex G100. The hatched region indicates the area of the profile which is taken as the fraction bound to macromolecules.

as minimal because of possible dissociation of hormone from the binding molecules during elution. In addition, it has been reported for the rat brain that cytosol binding values obtained with incubations of 10-8 M [3H]corticosterone are 30--40 ~ less than the saturation (Bmax) values obtained by Scatchard analysis 3. After completion of the present study, we confirmed this finding for the mouse. Incubations of cytosol from mouse cortex and hippocampus at 1 and 2 × 10-8 M [3H]cort]costerone yielded a 25 difference in saturable binding, comparable to that observed in the rat under similar assay conditions 3. Although binding values in the rat obtained with incubations of 10-8 M corticosterone are lower than B max values, it should be emphasized that stereospecific and brain-regional differences in binding, if expressed proportionally, remain unaltered at least over the range of 5 × 10-a M and 2 × 10-8 M [3H]corticosteroneS. These results indicate that differences that are detectable at saturating incubation concentrations are likely to be observed, within the limits of assay variability, at 10-8 M [3H]corticosterone, the incubation concentration used in the present study. The assay was highly reproducible; the average deviation from the mean of duplicate determinations was 6.4 ~ ± 1.7~ S.E.M. (n = 12). In all brain regions, only a single peak of bound [3H]C, coincident with the void volume, was detected. Previous studies in the rat, using both G10@ 2 and Sepharose 4B 13 gel chromatography, also have found only one peak of corticosterone binding in brain cytosols. Corticosterone binding in mouse brain cytosols differs from that in liver cytosol, which has several peaks of binding that are resolved with G100 chromatography16,17.

348 TABLE 1 Inhibition oJ .saturable t ytoplasmw (orttcosterone binding by competing steroids tn hypothalamus, htppocampus and cerebral corter o / C 5 7 B L / 6 J male rowe Cytosol incubated in 1 10 s M [aH]corUcosterone with and without I 10 " M c o m p e t i n g steroids Per cent inhibition c o m p u t e d as the difference in rad~oactwity b o u n d in the c o m p e t e d a n d n o n - c o m p e t e d incubations, divided by the value so obtained for competition with corticosterone. Values expressed as m e a n s ± S E.M. (n), n n u m b e r o f samples. Each sample comprised regions pooled from 2 8 mice of both age groups. Brain region

Hlppocampus Hypothalamus Cortex All

Pet ~ent inhibition by competing steroid Cottl~osterone

De:~amethasone

Progesterone

100.1 = 0 5 (4) 100.0 = l 0 (4) 1000±07 (4) 100.0±04(12)

962_4_:02 84.4 ± 5.8 99.1±03 932 + 32

960=4.1 80.6 -~ 3 4 9 3 3 j 13 911±28

(2) (2) (2) (6)

17[4-Evtradtol (3) (2) (3) (8)

3 1 . 0 = 6 3 * (6) 3 4 9 _4 16.9"*(3) 28.1± 7.5* (2) 3 1 . 8 ± 6.0 (11)

* Significantly different f r o m other c o m p e t i n g steroids (P -- 0 01L D u n c a n ' s mulUple range test ** Significantly different from other c o m p e t i n g steroids (P - 0 05), D u n c a n ' s multiple range test

In both age groups, cytosol binding of corticosterone in the hypothalamus ~s less than 50 og of that in cortex and hippocampus, but no difference between the latter two regions is detectable (Fig. 2). Mean bmding capacity ± S.E.M., expressed as fmoles bound/mg cytosol protein, for both age groups combined, is: hippocampus, 38.13 4 2.28; cortex, 37.52 ~ 2.70; and hypothalamus, 17.21 ~ 3.19. This regional pattern of cytosol binding differs from that of cell nuclear binding in the rat, which is highest in the hippocampus and markedly lower m both cerebral cortex and hypothatamusl°,z°,

II-12 mo [ ]

40

2 8 - 2 9 mo

o

[]

30

""

I0

CORT

HIPP

HYPO

Fig. 2. Saturable cytoplasmic binding of [~H]corhcostcrone m hlppocampus (HIPP), cortex (CORT) a n d h y p o t h a l a m u s ( H Y P O ) o f l l - 1 2 - m o n t h - o l d a n d 2 8 - 2 9 - m o n t h - o l d C57BL/6J male mice. Mice were adrenalectomized 2 h before assay * (n) where n - n u m b e r o f samples, 2 8 mice per sample. • * Significantly different f r o m other brain regions of respective age groups, P -- 0.01, D u n c a n ' s multiple range test.

349 21,24. Whether this difference is also found in cell nuclear binding in the mouse cannot be inferred from this study, because cytosol binding capacity does not necessarily indicate the amount of receptor which can translocate into the nucleus 11. Inhibition of saturable corticosterone binding by various steroids is shown in Table I. These results are similar to those of earlier studies of rat brain2,13, 22 and other target tissues of glucocorticoids TM,30. Estradiol inhibits corticosterone binding significantly less than dexamethasone and progesterone, which compete only slightly less effectively than corticosterone. No significant difference in the competition patterns is detectable among the 3 brain regions examined. Because the brains were not perfused at sacrifice, cytosol could have been contaminated with transcortin, the serum cortlcosterone binding proteing, 20. However, the effectiveness of the competition with dexamethasone, which does not appreciably bind transcortinl~, 27, indicates that this contamination was minimal, with the possible exception of the hypothalamus, in which competition by dexamethasone was about 10 ~ less effective than in the other brain regions. Cytosol binding capacity of corticosterone remains unaltered during aging in the brain regions examined (Fig. 2). A preliminary report from this laboratory which indicated an age-related decrease of hippocampal corticosterone binding 15 may have differed from the present study because of its small sample sizes. Alternatively, the longer elution time of gel filtration in the preliminary study could have revealed agedifferences in steroid-binder dissociation rate rather than in steroid binding capacity. The slight, although not significant, decrease of binding in mouse cerebral cortex cytosol (Fig. 2), is not inconsistent with the 30 ~ decrease of glucocorticoid binding observed by Roth in cytosols of 'cerebral hemispheres' (presumably cortex) of rats between 12 and 24 months of age 25. Roth's study may have been more sensitive to small differences in binding than ours, because he used Scatchard analysis. As noted earlier, per cent differences between binding values obtained under the subsaturating conditions of our assay should be comparable to those at saturating conditions. However, absolute differences diminish with decreasing incubation concentration, making the detection of small differences more difficult. Alternatively, a species-specific difference in binding would not be surprising: in the mouse, age-related changes of other endocrine parameters vary significantly even between strains~, 6. The absence of any detectable age-related decrease in binding of corticosterone by hippocampal and hypothalamic cytosols in C57BL/6J mice differs from the general trend of post-maturational reductmns of cytosol binding in target tissue of glucocorticoids 17'25'26, androgens 2s,29 and estrogens 14. However, we cannot infer from these results that subsequent steps in the mechanism of corticosterone action, such as nuclear uptake of the corticosterone-receptor complex, remain unimpaired. For example, in a mutant mouse lymphoma cell line which is resistant to killing by glucocorticoids, cytoplasmic binding capacity approximates that of the wild-type strain, whereas nuclear uptake of receptor is markedly diminished11. Moreover, since binding by unfractionated cytosol may represent more than one molecular species with high affinity for steroids 16, 17, the single peak observed in this study (Fig. 1) may contain aggregates or unresolved high molecular weight components, only one of which is the receptor which translocates

350 to the nucleus. C o n s e q u e n t ly , the extent to which the binding assay used in this study ~s a m eas u r e o f translocatable nuclear receptor remains to be determined. W e conclude f r o m these results that, if age-related changes in g l u c o c o r t l c o i d regulation occur m the h l p p o c a m p u s or h y p o t h a l a m u s , some stage o t h er than cytosol binding is revolved This research was s u p p o r t e d by grants to Caleb E. F m c h f r o m N I H (HD-07539 an d HD-07338), by N I H T r a i n i n g G r a n t HD-157, by G e n e r a l Research S u p p o r t G r a n t S05 RR07012-07, by the G l e n n F o u n d a t i o n for Medical Research ( N . Y . C . ) an d by the Orentreich F o u n d a t i o n for the A d v a n c e m e n t o f Science (N.Y.C.)

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