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Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland
Brain Research, 542 (1991) 155-158 Elsevier
155
BRES 24544
Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland Jeffrey A. Heroux, Dimitri E. Grigoriadis and Errol B. De Souza Laboratory of Neurobiology, Neuroscience Branch, Addiction Research Center, National Institute on Drug Abuse, Baltimore, MD 21224 (U.S.A.)
(Accepted 13 November 1990) Key words: Aging; Adrenocorticotropic hormone; Corticotropin-releasing factor; Corticosterone; Hypothalamic-pituitary-adrenocortical axis; Hypothalamus; Proopiomelanocortin; Receptor down-regulation
Corticotropin-releasing factor (CRF) receptors were measured in discrete areas of brain and in anterior pituitary of 4-, 12-, 18-, and 24-month-old male Fischer rats. No significant age-related alterations in [~25I]ovineCRF binding were observed in the olfactory bulb, cerebral cortex, hippocampus, brainstem, and cerebellum; there was a trend for CRF binding to decrease in the striatum as a consequence of aging. Significant age-related decreases were observed in 125I-ovineCRF binding in the anterior pituitary and hypothalamus with maximal reductions of 60 and 27%, respectively. Saturation analysis in the anterior pituitary indicated an age-related reduction in the density of CRF receptors (i.e. Bmax)without an alteration in the affinity (i.e. Kd) of CRF for its binding site. Northern analysis of proopiomelanocortin (POMC) mRNA in the anterior pituitary indicated no significant differences in the levels of POMC mRNA between 4- and 24-month-old rats. These and other data suggest that the age-related decrease in anterior pituitary CRF receptors may be due to hypersecretion of hypothalamic CRF rather than a loss of corticotropes in the anterior pituitary.
The aging process has been previously shown to have a profound impact on the normal functioning of the hypothalamic-pituitary-adrenocortical ( H P A ) axis in the rat 15. Aged rats have been reported to have increased basal levels of circulating corticosterone as well as an impaired ability to recover from their adrenocortical stress response 13. Following exposure to a stressor, aged rats possess a diminished ability to terminate corticosterone secretion 13. In addition, the aged pituitary and adrenal gland have a dampened response to corticotropin-releasing factor (CRF) 9 and adrenocorticotropic hormone ( A C T H ) a,ls, respectively. In addition to the key role of C R F in the physiologic regulation of the H P A axis 2'19, C R F has been strongly implicated as a neurotransmitter in the central nervous system (CNS) as evidenced by its ability to produce a broad spectrum of autonomic and behavioral effects 1°,19. Recent studies have suggested that C R F may be important in the manifestation of certain dementias associated with several neurodegenerative disorders 7. For example, decreases in CRF-like immunoreactivity accompanied by reciprocal increases in C R F receptor binding have been documented in postmortem cerebral cortex samples of Alzheimer's patients L4,7,2°. In addition, alterations in the levels of C R F in other brain regions have been reported
in Parkinson's disease, Huntington's chorea, and progressive supranuclear palsy 5'7'2°. In view of the alterations in the H P A axis as a consequence of aging and changes in C R F accompanying several neurodegenerative disorders which occur to a greater degree in the aged, the present study was undertaken to determine the effect of the aging process on levels of C R F receptors in the rat CNS and pituitary gland. Male Fischer rats (Harlan Industries, Indianapolis, IN) were sacrificed by decapitation, and brain and pituitary regions of interest were dissected on dry ice and immediately frozen in liquid nitrogen. Samples were stored at -70 °C until assayed. On the day of assay, brain tissues were weighed and homogenized in 10 vols. of ice-cold 50 m M "Iris containing 10 m M MgCI2, 2 mM E G T A , 0.1% bovine serum albumin, 0.15 m M bacitracin, and 1.0 mM phenylmethylsulfonylfluoride, p H 7.2, at 22 °C using a Polytron (setting 6.0) for 20 s. Membranes were washed twice by centrifugation (40,000 g for 12 min at 4 °C) in the same buffer and resuspended for assay at a concentration of 40 mg original wet wt. tissue/ml buffer. Anterior pituitary tissue was homogenized in 1.5 ml microfuge tubes by 10 passes of a plastic pestle homogenizer in a volume of 1.0 ml buffer. Membranes were washed twice and resuspended at a
Correspondence: E.B. De Souza, The Du Pont Merck Pharmaceutical Co., Experimental Station, E400/4352, P.O. Box 80400, Wilmington, DE 19880-0400, U.S.A.
0006-8993/91/$03.50 t~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
156 tissue concentration of 10 mg/ml of buffer. Determination of CRF binding to membranes was performed as previously described 6 using a final concentration of [125I]Tyr°-ovine CRF ([]zSI]oCRF) of 100 pM (Du PontNew England Nuclear, Boston, MA; specific activity, 2200 Ci/mmol) and 900 pM unlabeled ovine CRF (oCRF; Peninsula Laboratories, Inc, Belmont, CA) for single point binding assays. For saturation studies, [125I]oCRF was used at various concentrations as indicated. Nonspecific binding was determined by the addition of 1 pM unlabeled rat/human CRF (Peninsula). The regional levels of CRF receptor binding as a function of age are shown in Fig. 1. Of the CNS areas assayed, only the hypothalamus showed a significant change with a 27% reduction in CRF binding from the youngest to the oldest rats. The mechanism of this specific regional decrease of CRF receptors is unknown, however previous studies have indicated that the aged hypothalamus is in a state of hypersecretion of A C T H secretagogues 17. Therefore, one could postulate a shortloop negative feedback effect of hypothalamic CRF to down-regulate its receptors. Alternatively, a hypersecre-
A
mm 4month HYPO
lib
PM
SC
HIPPO
STR
•
12 month
•
18 month
r,
z
~ 24mo.th
om l mL
E
AP
CB
FC
PC
OB
Fig. 1. Effect of age on []2SI]ovine CRF binding sites in discrete regions of rat brain and in anterior pituitary. Values represent the mean +_ S.E.M.; n = 6 rats/group. HYPO, Hypothalamus; MB, midbrain; PM, pons medulla; SC, spinal cord; HIPPO, hippocampus; STR, striatum; AP, anterior pituitary; CB, cerebellum; FC, frontal cerebral cortex; PC, parietal cerebral cortex; OB, olfactory bulb. *, Significantly different at P < 0.01 from corresponding values in 4-month-old rats; t, significantly different at P < 0.05 and P < 0.01 from corresponding values in 12- and 18-month-old rats, resp. Data were analysed for differences using one-way ANOVA and Duncan's multiple range test.
ted hypothalamic factor and/or glucocorticoid could cause the death of cells in the hypothalamus containing CRF receptors. Although not reaching statistical significance, several of the other brain regions such as the striatum and the hippocampus showed an overall trend toward receptor decline as a consequence of aging. The relevance of the decreases in CRF receptors in these brain areas is unclear. The loss of CRF receptors in the hippocampus may be secondary to the cell loss that has been reported in this brain region as a consequence of aging 15 and may contribute to age-related cognitive deficits. CRF affects a variety of behaviors including locomotor activityl°; the decrease in CRF receptors in the striatum may relate to alterations in behavioral activity )°. CRF receptors were not significantly altered in the cerebral cortex of aged rats confirming data in postmortem human samples demonstrating no significant relationship between age and the density of CRF receptors in a variety of cerebral cortical areas 4. The most profound age-related change was observed in the anterior pituitary gland demonstrating approx, a 60% overall reduction in CRF binding from 4- to 24-month-old-rats. The rate of receptor decline was also seen to increase with age. There was an approximate 13% decline in CRF binding from 4-month- to 12month-old rats and about a 30% decline between groups thereafter. To determine if this decrease in CRF binding observed in the anterior pituitary was due to a reduction in the number of CRF receptors (Bmax) o r a change in the affinity of the receptor for CRF (Ko), saturation analysis was performed using anterior pituitary tissue isolated from 4- and 24-month-old rats. As shown in Fig. 2, a 63% reduction in the number of CRF binding sites was observed with respect to increasing age with no apparent change in the K o. These results indicate that CRF receptors in the anterior pituitary are highly susceptible to loss during the aging process. In what may be a physiological consequence of this age-related CRF receptor decrease, the aged pituitary has been shown to have a dampened sensitivity to CRF 9. Following a single injection of CRF, aged rats have a diminished ability to secrete ACTH. Several mechanisms may be involved in the age-related loss of CRF receptors in the anterior pituitary. Hypersecretion of CRF from the hypothalamus may down-regulate CRF receptors at the level of the anterior pituitary. In support of this hypothesis, the basal and stimulated release of a corticotropin-releasing factor(s) from the hypothalamus has been reported to be increased in aged rats tT. The elevated levels of circulating corticosteroids that have been reported to occur in the course of aging ~3 may also contribute to the loss of CRF receptors in the anterior lobe of the pituitary; glucocorticoids have been reported to decrease CRF receptors in
157 the anterior pituitary 3. Alternatively, the reduction of CRF receptors in this tissue may simply reflect a loss of corticotropes. To indirectly assess if the number of corticotropes were reduced as a result of the aging process, anterior pituitary glands from young and aged rats were analyzed for their content of proopiomelanocortin (POMC) messenger RNA (mRNA). Total RNA was isolated from tissue using the guanidinium isothiocyanate/cesium chloride density gradient method ~6, subjected to formaldehyde gel electrophoresis and capillary blotted overnight onto Nytran membranes (Scheicher & Schuell, Inc., Keene, NH). The membranes were prehybridized in 5 x SSC, 50 mM sodium phosphate (pH 6.5), 1 mM EDTA, 1% sodium dodecyl sulfate (SDS), 2.5 x Denhardt's solution, and 200 /~g/ml sheared single-stranded herring sperm DNA for 4.5 h at 66 °C. The membrane was initially probed with a 32p-end-labeled POMC oligonucleotide (Du Pont-NEN) overnight at the same temperature. The blots were washed for 5 min in 1 x SSC and 0.5% SDS at 34 °C and then for 20 min in the same solution at room temperature. Blots were covered in plastic wrap and placed in X-ray cassettes apposed to Kodak X-AR film overnight at -70 °C. Blots were subsequently stripped of probe by incubating for 30 min at 65 °C in 20 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0), and 0.1% SDS and reprobed with a 32p-end-labeled 18S ribosomal RNA (rRNA) oligonucleotide using the same protocol described above except that exposure time to X-AR film was decreased to 30 min. Autoradiographic images were evaluated using a PC-based computerized image analysis system (Loats Associates, Westminster, 4.5
_ 31
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p,,,
o
S
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o.1
,,
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0.2
,,
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o.s
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o.5
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0.6
~, i,,,,
o.7
o.a
12S I-oCRF (nM) Fig. 2. Saturation profile of [125I]ovine CRF binding sites in rat anterior pituitary membranes. Using the non-linear least squares curve-fitting program LIGAND 11. The receptor densities (//max) were 3.29 and 1.44 fmol/mg protein for 4- and 24-month-old rats resp. with K a values of 155 and 126 pM, resp.
1.8
B
1.6 z
1.4 1.2
•-
1
~0.8
18S D.-
0.6 0.4 0.2 0
/
ooO" ///
Fig. 3. Blot hybridization analysis of POMC mRNA in 4-month- and 24-month-old rats. A: the relative amount of POMC mRNA was calculated as a ratio of the quantified POMC and 18S rRNA images. • , Indicates level of POMC mRNA in liver (control). B: total RNA was isolated from the anterior pituitary tissue of 4-month- and 24-month-old rats and hybridized as described above. Liver was used as a negative control for POMC mRNA. The total amount of sample applied in each lane was 2 ~g.
MD). Hybridization of the blots using an 18S rRNA probe was performed to normalize for the amount of total RNA applied to each lane. The relative amount of POMC mRNA was calculated as a ratio of the quantified POMC and 18S rRNA images. As shown in Fig. 3, there were no differences observed in the levels of POMC mRNA between 4-month- and 24-month-old rats. This observation is supported by others who report no difference in the basal or stimulated release of ACTH from anterior pituitary gland preparations from young and old rats 17. If the assumption that an equal amount of POMC mRNA is transcribed per corticotrope is true, one could conclude that there are an equivalent number of corticotropes within anterior pituitary glands of young and old rats. Overall, the data would suggest that CRF receptors located in the anterior pituitary gland are down-regulated, possibly as a consequence of CRF and/or glucocorticoid hypersecretion in the aged rat. Glucocorticoid receptors located in the hippocampus have been shown to decline in an age related manner in the rat 12. Because the hippocampus is a primary site of negative feedback in the HPA axis for CRF TM, decline of glucocorticoid receptors in this brain area could lead to a state of CRF hypersecretion by the hypothalamus with a subsequent down-regulation of CRF receptors in the anterior pituitary. In support of this hypothesis, recent in vitro studies have indicated that the hypothalamus is in a state of hyperactivity in the aged rat 17. Alternatively, since there is a hypersecretion of CRF and possibly other POMC-stimulating factors in the aged rat, total POMC mRNA may remain unchanged even if there is an age-related loss of corticotropes in the anterior pituitary. Further investigation is necessary to
158 distinguish b e t w e e n these possibilities a n d to elucidate the exact m e c h a n i s m of C R F r e c e p t o r decline seen in the a n t e r i o r pituitary of the aged rat.
1 Bissette, G., Reynolds, G.P., Kilts, C.D., Widerlov, W. and Nemeroff, C.B., Corticotropin-releasing factor-like immunoreactivity in senile dementia of the Alzheimer type, J. Am. Med. Assoc., 254 (1985) 3067-3069. 2 Brodish, A., Control of AC~H secretion by corticotropinreleasing factor(s), Vitamin. Horm., 37 (1979) 111-153. 3 Childs, G.V., Morell, J.L., Niendorf, A. and Aguilera, G., Cytochemical studies of corticotropin-releasing factor (CRF) receptors in anterior lobe corticotropes: binding, glucocorticoid regulation and endocytosis of [biotinyl-ser1] CRF, Endocrinology, 119 (1986) 2129-2142. 4 De Souza, E.B., Whitehouse, P.J., Kuhar, M.J., Price, D.L. and Vale, W.W., Reciprocal changes in corticotropin-releasing factor (CRF)-like immunoreactivity and CRF receptors in cerebral cortex of Alzheimer's disease, Nature, 319 (1986) 593-595. 5 De Souza, E.B., Whitehouse, P.J., Folstein, S.E., Price, D.L. and Vale, W.W., Corticotropin-releasing hormone (CRH) is decreased in the basal ganglia in Huntington's disease, Brain Research, 437 (1987) 355-359. 6 De Souza, E.B., Corticotropin-releasing factor receptors in the rat central nervous system: characterization and regional distribution, J. Neurosci., 7(1) (1987) 88-100. 7 De Souza, E.B., CRH defects in Alzheimer's and other neurologic diseases, Hosp. Practice, 23 (1988) 59-71. 8 Hess, G. and Riegle, G., Adrenocortical responsiveness to stress and ACTH in aging rats, J. Gerontol., 25 (1970) 354-360. 9 Hylka, V.W., Sonntag, W.E. and Meites, J., Reduced ability of old male rats to release ACTH and corticosterone in response to CRF administration, Proc. Soc. Exp. Biol. Med., 175 (1984) 1-4. 10 Koob, G.F., Corticotropin-releasing factor and behavior, Perspect. Behav. Med., 2 (1985) 39-52. 11 Munson, P.J. and Rodbard, D., LIGAND: a versatile comput-
The authors wish to thank Dr. Nathan Appel for his generous help with the project, Win. Mark Mithchell for his assistance in quantitating the northern blot autoradiographic images and Denise Pearsall for help with the CRF receptor assays.
erized approach for characterization of ligand binding systems, Anal. Biochem., 297 (1980) 220-228. 12 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., Corticosterone receptors decline in a site-specific manner in the aged rat, Brain Research, 289 (1983) 235-240. 13 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., The adrenocortical stress-response in the aged male rat: impairment of recovery from stress, Exp. Gerontol., 18 (1983) 55-64. 14 Sapolsky, R.M., Krey, L.C., and McEwen, B.S., Glucocorticoid-sensitive hippocampal neurons are involved in terminating the adrenocortical stress response, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6174-6177. 15 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis, Endocr. Rev., 7 (1986) 284-301. 16 Sambrook, J., Fritsch, E.E and Maniatis, T., Molecular Cloning, 2nd edn., Cold Spring Harbor Laboratory Press, 1989, pp. 7.19-7.22. 17 Scaccianoce, S., Di Sciullo, A. and Angelucci, L., Age-related changes in hypothalamo-pituitary-adrenocorticai axis activity in the rat, Neuroendocrinology, 52 (1990) 150-155. 18 Tang, G. and Phillips, R., Some age-related changes in pituitaryadrenal function in the male laboratory rat, J. Gerontol., 33 (1978) 377-382. 19 Vale, W., Rivier, C., Brown, M.R., Spiess, J., Koob, G., Swanson, L., Bilezikjian, L., Bloom, F. and Rivier, J., Chemical and biological characterization of corticotropin releasing factor, Recent Prog. Horm. Res., 39 (1981) 245-270. 20 Whitehouse, P.J., Vale, W.W., Zweig, R.M., Singer, H.S., Mayeux, R., Price, D.L. and De Souza, E.B., Reductions in corticotropin-releasing factor-like immunoreactivity in cerebral cortex in Alzheimer's disease, Neurology, 37 (1987) 905-909.
155
BRES 24544
Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland Jeffrey A. Heroux, Dimitri E. Grigoriadis and Errol B. De Souza Laboratory of Neurobiology, Neuroscience Branch, Addiction Research Center, National Institute on Drug Abuse, Baltimore, MD 21224 (U.S.A.)
(Accepted 13 November 1990) Key words: Aging; Adrenocorticotropic hormone; Corticotropin-releasing factor; Corticosterone; Hypothalamic-pituitary-adrenocortical axis; Hypothalamus; Proopiomelanocortin; Receptor down-regulation
Corticotropin-releasing factor (CRF) receptors were measured in discrete areas of brain and in anterior pituitary of 4-, 12-, 18-, and 24-month-old male Fischer rats. No significant age-related alterations in [~25I]ovineCRF binding were observed in the olfactory bulb, cerebral cortex, hippocampus, brainstem, and cerebellum; there was a trend for CRF binding to decrease in the striatum as a consequence of aging. Significant age-related decreases were observed in 125I-ovineCRF binding in the anterior pituitary and hypothalamus with maximal reductions of 60 and 27%, respectively. Saturation analysis in the anterior pituitary indicated an age-related reduction in the density of CRF receptors (i.e. Bmax)without an alteration in the affinity (i.e. Kd) of CRF for its binding site. Northern analysis of proopiomelanocortin (POMC) mRNA in the anterior pituitary indicated no significant differences in the levels of POMC mRNA between 4- and 24-month-old rats. These and other data suggest that the age-related decrease in anterior pituitary CRF receptors may be due to hypersecretion of hypothalamic CRF rather than a loss of corticotropes in the anterior pituitary.
The aging process has been previously shown to have a profound impact on the normal functioning of the hypothalamic-pituitary-adrenocortical ( H P A ) axis in the rat 15. Aged rats have been reported to have increased basal levels of circulating corticosterone as well as an impaired ability to recover from their adrenocortical stress response 13. Following exposure to a stressor, aged rats possess a diminished ability to terminate corticosterone secretion 13. In addition, the aged pituitary and adrenal gland have a dampened response to corticotropin-releasing factor (CRF) 9 and adrenocorticotropic hormone ( A C T H ) a,ls, respectively. In addition to the key role of C R F in the physiologic regulation of the H P A axis 2'19, C R F has been strongly implicated as a neurotransmitter in the central nervous system (CNS) as evidenced by its ability to produce a broad spectrum of autonomic and behavioral effects 1°,19. Recent studies have suggested that C R F may be important in the manifestation of certain dementias associated with several neurodegenerative disorders 7. For example, decreases in CRF-like immunoreactivity accompanied by reciprocal increases in C R F receptor binding have been documented in postmortem cerebral cortex samples of Alzheimer's patients L4,7,2°. In addition, alterations in the levels of C R F in other brain regions have been reported
in Parkinson's disease, Huntington's chorea, and progressive supranuclear palsy 5'7'2°. In view of the alterations in the H P A axis as a consequence of aging and changes in C R F accompanying several neurodegenerative disorders which occur to a greater degree in the aged, the present study was undertaken to determine the effect of the aging process on levels of C R F receptors in the rat CNS and pituitary gland. Male Fischer rats (Harlan Industries, Indianapolis, IN) were sacrificed by decapitation, and brain and pituitary regions of interest were dissected on dry ice and immediately frozen in liquid nitrogen. Samples were stored at -70 °C until assayed. On the day of assay, brain tissues were weighed and homogenized in 10 vols. of ice-cold 50 m M "Iris containing 10 m M MgCI2, 2 mM E G T A , 0.1% bovine serum albumin, 0.15 m M bacitracin, and 1.0 mM phenylmethylsulfonylfluoride, p H 7.2, at 22 °C using a Polytron (setting 6.0) for 20 s. Membranes were washed twice by centrifugation (40,000 g for 12 min at 4 °C) in the same buffer and resuspended for assay at a concentration of 40 mg original wet wt. tissue/ml buffer. Anterior pituitary tissue was homogenized in 1.5 ml microfuge tubes by 10 passes of a plastic pestle homogenizer in a volume of 1.0 ml buffer. Membranes were washed twice and resuspended at a
Correspondence: E.B. De Souza, The Du Pont Merck Pharmaceutical Co., Experimental Station, E400/4352, P.O. Box 80400, Wilmington, DE 19880-0400, U.S.A.
0006-8993/91/$03.50 t~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
156 tissue concentration of 10 mg/ml of buffer. Determination of CRF binding to membranes was performed as previously described 6 using a final concentration of [125I]Tyr°-ovine CRF ([]zSI]oCRF) of 100 pM (Du PontNew England Nuclear, Boston, MA; specific activity, 2200 Ci/mmol) and 900 pM unlabeled ovine CRF (oCRF; Peninsula Laboratories, Inc, Belmont, CA) for single point binding assays. For saturation studies, [125I]oCRF was used at various concentrations as indicated. Nonspecific binding was determined by the addition of 1 pM unlabeled rat/human CRF (Peninsula). The regional levels of CRF receptor binding as a function of age are shown in Fig. 1. Of the CNS areas assayed, only the hypothalamus showed a significant change with a 27% reduction in CRF binding from the youngest to the oldest rats. The mechanism of this specific regional decrease of CRF receptors is unknown, however previous studies have indicated that the aged hypothalamus is in a state of hypersecretion of A C T H secretagogues 17. Therefore, one could postulate a shortloop negative feedback effect of hypothalamic CRF to down-regulate its receptors. Alternatively, a hypersecre-
A
mm 4month HYPO
lib
PM
SC
HIPPO
STR
•
12 month
•
18 month
r,
z
~ 24mo.th
om l mL
E
AP
CB
FC
PC
OB
Fig. 1. Effect of age on []2SI]ovine CRF binding sites in discrete regions of rat brain and in anterior pituitary. Values represent the mean +_ S.E.M.; n = 6 rats/group. HYPO, Hypothalamus; MB, midbrain; PM, pons medulla; SC, spinal cord; HIPPO, hippocampus; STR, striatum; AP, anterior pituitary; CB, cerebellum; FC, frontal cerebral cortex; PC, parietal cerebral cortex; OB, olfactory bulb. *, Significantly different at P < 0.01 from corresponding values in 4-month-old rats; t, significantly different at P < 0.05 and P < 0.01 from corresponding values in 12- and 18-month-old rats, resp. Data were analysed for differences using one-way ANOVA and Duncan's multiple range test.
ted hypothalamic factor and/or glucocorticoid could cause the death of cells in the hypothalamus containing CRF receptors. Although not reaching statistical significance, several of the other brain regions such as the striatum and the hippocampus showed an overall trend toward receptor decline as a consequence of aging. The relevance of the decreases in CRF receptors in these brain areas is unclear. The loss of CRF receptors in the hippocampus may be secondary to the cell loss that has been reported in this brain region as a consequence of aging 15 and may contribute to age-related cognitive deficits. CRF affects a variety of behaviors including locomotor activityl°; the decrease in CRF receptors in the striatum may relate to alterations in behavioral activity )°. CRF receptors were not significantly altered in the cerebral cortex of aged rats confirming data in postmortem human samples demonstrating no significant relationship between age and the density of CRF receptors in a variety of cerebral cortical areas 4. The most profound age-related change was observed in the anterior pituitary gland demonstrating approx, a 60% overall reduction in CRF binding from 4- to 24-month-old-rats. The rate of receptor decline was also seen to increase with age. There was an approximate 13% decline in CRF binding from 4-month- to 12month-old rats and about a 30% decline between groups thereafter. To determine if this decrease in CRF binding observed in the anterior pituitary was due to a reduction in the number of CRF receptors (Bmax) o r a change in the affinity of the receptor for CRF (Ko), saturation analysis was performed using anterior pituitary tissue isolated from 4- and 24-month-old rats. As shown in Fig. 2, a 63% reduction in the number of CRF binding sites was observed with respect to increasing age with no apparent change in the K o. These results indicate that CRF receptors in the anterior pituitary are highly susceptible to loss during the aging process. In what may be a physiological consequence of this age-related CRF receptor decrease, the aged pituitary has been shown to have a dampened sensitivity to CRF 9. Following a single injection of CRF, aged rats have a diminished ability to secrete ACTH. Several mechanisms may be involved in the age-related loss of CRF receptors in the anterior pituitary. Hypersecretion of CRF from the hypothalamus may down-regulate CRF receptors at the level of the anterior pituitary. In support of this hypothesis, the basal and stimulated release of a corticotropin-releasing factor(s) from the hypothalamus has been reported to be increased in aged rats tT. The elevated levels of circulating corticosteroids that have been reported to occur in the course of aging ~3 may also contribute to the loss of CRF receptors in the anterior lobe of the pituitary; glucocorticoids have been reported to decrease CRF receptors in
157 the anterior pituitary 3. Alternatively, the reduction of CRF receptors in this tissue may simply reflect a loss of corticotropes. To indirectly assess if the number of corticotropes were reduced as a result of the aging process, anterior pituitary glands from young and aged rats were analyzed for their content of proopiomelanocortin (POMC) messenger RNA (mRNA). Total RNA was isolated from tissue using the guanidinium isothiocyanate/cesium chloride density gradient method ~6, subjected to formaldehyde gel electrophoresis and capillary blotted overnight onto Nytran membranes (Scheicher & Schuell, Inc., Keene, NH). The membranes were prehybridized in 5 x SSC, 50 mM sodium phosphate (pH 6.5), 1 mM EDTA, 1% sodium dodecyl sulfate (SDS), 2.5 x Denhardt's solution, and 200 /~g/ml sheared single-stranded herring sperm DNA for 4.5 h at 66 °C. The membrane was initially probed with a 32p-end-labeled POMC oligonucleotide (Du Pont-NEN) overnight at the same temperature. The blots were washed for 5 min in 1 x SSC and 0.5% SDS at 34 °C and then for 20 min in the same solution at room temperature. Blots were covered in plastic wrap and placed in X-ray cassettes apposed to Kodak X-AR film overnight at -70 °C. Blots were subsequently stripped of probe by incubating for 30 min at 65 °C in 20 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0), and 0.1% SDS and reprobed with a 32p-end-labeled 18S ribosomal RNA (rRNA) oligonucleotide using the same protocol described above except that exposure time to X-AR film was decreased to 30 min. Autoradiographic images were evaluated using a PC-based computerized image analysis system (Loats Associates, Westminster, 4.5
_ 31
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p,,,
o
S
i,,
o.1
,,
i0,
0.2
,,
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o.s
i,,
0.4
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o.5
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0.6
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o.7
o.a
12S I-oCRF (nM) Fig. 2. Saturation profile of [125I]ovine CRF binding sites in rat anterior pituitary membranes. Using the non-linear least squares curve-fitting program LIGAND 11. The receptor densities (//max) were 3.29 and 1.44 fmol/mg protein for 4- and 24-month-old rats resp. with K a values of 155 and 126 pM, resp.
1.8
B
1.6 z
1.4 1.2
•-
1
~0.8
18S D.-
0.6 0.4 0.2 0
/
ooO" ///
Fig. 3. Blot hybridization analysis of POMC mRNA in 4-month- and 24-month-old rats. A: the relative amount of POMC mRNA was calculated as a ratio of the quantified POMC and 18S rRNA images. • , Indicates level of POMC mRNA in liver (control). B: total RNA was isolated from the anterior pituitary tissue of 4-month- and 24-month-old rats and hybridized as described above. Liver was used as a negative control for POMC mRNA. The total amount of sample applied in each lane was 2 ~g.
MD). Hybridization of the blots using an 18S rRNA probe was performed to normalize for the amount of total RNA applied to each lane. The relative amount of POMC mRNA was calculated as a ratio of the quantified POMC and 18S rRNA images. As shown in Fig. 3, there were no differences observed in the levels of POMC mRNA between 4-month- and 24-month-old rats. This observation is supported by others who report no difference in the basal or stimulated release of ACTH from anterior pituitary gland preparations from young and old rats 17. If the assumption that an equal amount of POMC mRNA is transcribed per corticotrope is true, one could conclude that there are an equivalent number of corticotropes within anterior pituitary glands of young and old rats. Overall, the data would suggest that CRF receptors located in the anterior pituitary gland are down-regulated, possibly as a consequence of CRF and/or glucocorticoid hypersecretion in the aged rat. Glucocorticoid receptors located in the hippocampus have been shown to decline in an age related manner in the rat 12. Because the hippocampus is a primary site of negative feedback in the HPA axis for CRF TM, decline of glucocorticoid receptors in this brain area could lead to a state of CRF hypersecretion by the hypothalamus with a subsequent down-regulation of CRF receptors in the anterior pituitary. In support of this hypothesis, recent in vitro studies have indicated that the hypothalamus is in a state of hyperactivity in the aged rat 17. Alternatively, since there is a hypersecretion of CRF and possibly other POMC-stimulating factors in the aged rat, total POMC mRNA may remain unchanged even if there is an age-related loss of corticotropes in the anterior pituitary. Further investigation is necessary to
158 distinguish b e t w e e n these possibilities a n d to elucidate the exact m e c h a n i s m of C R F r e c e p t o r decline seen in the a n t e r i o r pituitary of the aged rat.
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The authors wish to thank Dr. Nathan Appel for his generous help with the project, Win. Mark Mithchell for his assistance in quantitating the northern blot autoradiographic images and Denise Pearsall for help with the CRF receptor assays.
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