Tissue differences in the up-regulation of glucocorticoid-binding proteins in the rat.

The magnitude of increase in glucocorticoid receptor concentration and transcortin-like binding was examined in a variety of peripheral tissues and brain structures after adrenalectomy. Glucocorticoid binding was assayed in liver, heart, kidney, pituitary, hippocampus, cerebral cortex, amygdala-entorhinal area, and hypothalamus. Glucocorticoid receptor concentration, measured using [3H]dexamethasone as ligand, increased in all eight tissues, but the magnitude of this increase varied 30-fold among tissues. The largest increase was shown by kidney cytosol, followed by amygdala-entorhinal cortex, hippocampus, liver, cerebral cortex, hypothalamus, pituitary, and heart. These increases were not due to a selective enhancement of mineralocorticoid receptors. The increase in transcortin in peripheral tissues was variable and exceeded the increase in plasma transcortin by an order of magnitude. It was concluded that up-regulation of the glucocorticoid receptor after adrenalectomy is a response common to most, if not all, glucocorticoid target tissues. However, the magnitude of this response was tissue specific and was not directly related to initial receptor density. The marked increase in tissue transcortin ([3H]corticosterone binding in the presence of excess dexamethasone) suggested that plasma transcortin is sequestered by peripheral tissues in substantial amounts in the acutely adrenalectomized rat. The increase in transcortin uptake by tissues and the increases in cytosolic receptor number are apparently subject to different regulatory control.

Persistent alteration of pituitary-adrenal function in the rat by prepuberal corticosterone treatment.

The possible existence of periods of sensitivity to elevated levels of corticosterone in the postnatal development of the pituitary-adrenal axis was investigated. Long-Evans rats were implanted ip with corticosterone (25 mg/100 g body wt) mixed with an equal amount of cholesterol on postnatal days 3, 6, 12 or 18. Eight blood samples were obtained from each animal at various ages by cardiac puncture. Steroid treatment during the first postnatal week resulted in lowered adult basal levels of plasma corticosterone without affecting the diurnal rhythm. Males treated on day 3 had lowered peak (PM) and trough (AM) levels of the steroid as adults, while males treated on day 6 showed only a lowered PM level. A similar reduction in basal levels of plasma corticosterone was observed in immature females, but did not persist into adulthood. Steroid treatment at day 12 had no effect on basal or diurnal pituitary-adrenal activity. Treatment at day 18 reduced the amplitude of the adult diurnal rhythm in both sexes and delayed the onset of the rhythm in males. No differences in stress responsiveness or adrenal weights were detected. Thus, two distinct periods exist in the postnatal development of the rat during which basal and diurnal pituitary-adrenal activity can be differentially and persistently altered by treatment with the naturally occurring glucocorticoid.

Sex differences in the binding of type I and type II corticosteroid receptors in rat hippocampus.

Binding parameters of soluble Type I and Type II receptors were assessed in hippocampus of adult, adrenalectomized, male and female rats. No sex differences in the number of either Type I or Type II receptors could be demonstrated between gonadally intact animals. When females treated with 17 beta-estradiol benzoate (10 micrograms/day) were compared with males, a statistically significant reduction in Type II receptors was observed in the females; progesterone produced no further decrease in receptor numbers. The amount of tissue-associated corticosteroid-binding globulin in gonadally intact animals (perfused with dextran-saline) was twice as great in females as males. Sex-dependent differences in these gonadally intact rats were found in the affinity, measured as the dissociation constant (Kd), of both the Type I and Type II receptors. For both receptors, affinity in cytosols from females was reduced. The difference for the Type II receptor was slight, but the Kd value of the Type I receptor was several-fold higher in females. The difference in affinity was evident with both natural and synthetic steroid ligands. There appears to be little, if any, difference in affinity between the hippocampal Type I and the Type II receptors in females. This suggests that the occupancy of Type I receptors in females is substantially less than that of males at low circulating concentrations of corticosteroids.

[3H]corticosterone binding in the caudate-putamen.

Autoradiographic data indicate little specific binding of corticosterone in the caudate-putamen. However, chronic glucocorticoid treatment has been reported to alter certain biochemical parameters within the caudate-putamen, including high-affinity choline uptake. We asked whether glucocorticoid receptors in the caudate-putamen of adrenalectomized rats could be detected by a biochemical approach. We determined the amount of specific [3H]corticosterone binding in cytosols from the caudate-putamen and the ability of nuclei in this brain region to retain [3H]corticosterone in vivo. Results indicated that 72 h post-adrenalectomy, the caudate-putamen has 56% of the maximal binding capacity of the hippocampus (205 +/- 11 vs 361 +/- 19 fmol/mg protein). The apparent Kd of [3H]corticosterone in the two tissues did not differ substantially. The cytosolic binding capacity of the caudate-putamen was similar to that of the cerebellum, a brain region which also fails to demonstrate marked retention of glucocorticoids in autoradiographic studies. When cytosol binding was measured at a shorter time period after adrenalectomy (12 h), no differences were observed between the binding capacities of the hippocampus and the caudate-putamen. In competition experiments using [3H]corticosterone as ligand, no differences between the caudate-putamen and the hippocampus were found in the relative affinities of any of 8 steroids; the order of relative affinities obtained was in good agreement with those reported for the classical glucocorticoid receptor in other tissues. Rats were injected via the tail vein with [3H]corticosterone, 36 nmol/kg, an amount calculated to saturate more than half the nuclear sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Hippocampal cytosol binding capacity of corticosterone: no depletion with nuclear loading.

The recurrence every 24 h of glucocorticoid elevation led us to investigate the temporal relationship between glucocorticoid receptor occupation in brain cell nuclei and the availability of cytosol sites. Adrenalectomized rats were injected i.v. with [3H]corticosterone in doses ranging from 15 to 106 nmol/kg. Peak nuclear binding occurred 1-2 h after [3H]corticosterone injection and was preceded by a peak of cytosol receptor labeling at 15-30 min. Yet 1 h after 55 or 105 nmol/kg [3H]corticosterone, no depletion in the total in vitro binding capacity of the cytosol could be detected even though estimated depletion should have been approximately 30% had it occurred. Injection of 80 nmol/kg of corticosterone per rat plus 40 nmol/kg dexamethasone also failed to reduce total cytosol binding capacity, even though estimated depletion should have been approximately 40%. No change in binding affinity of cytosol sites was observed in the injected animals compared to uninjected controls. The in vivo nuclear binding capacity of hippocampus for [3H]corticosterone (fmol/hippocampus) is about 40% of the cytosol binding capacity measured in vitro. Moreover, no more than 40% of total cytosol sites are occupied in vivo as a result of [3H]corticosterone injections which occupy nuclear sites to 80% of estimated capacity. Yet, even with the larger in vitro cytosol binding capacity, a depletion approaching 40% of cytosol binding sites would have been seen, had it occurred as a result of nuclear translocation. The apparent lack of depletion of cytosol receptors is supported by experiments which showed that two injections of [3H]corticosterone 2 h apart fail to fatigue the nuclear uptake mechanism. The present results suggest (1) that in the hippocampus an excess of extranuclear glucocorticoid binding proteins exists, and (2) that the availability of functional cytosol receptors may be regulated to maintain a relatively constant cellular level.

Sexual dimorphism of glucocorticoid binding in rat brain.

Glucocorticoids bind with high affinity to intracellular receptors located in high density within discrete regions of the rodent and primate brain. The binding of [3H]corticosterone was compared in the brains of male vs female rats. The number and affinity of cytosol receptors in the hippocampus and hypothalamus were examined in vitro. The cytosolic binding capacity of the hippocampus is greater in the female than in the male. This difference in binding capacity is not dependent on the presence of gonadal steroids: the effect of gonadectomy was not significant for either sex. The difference is not due to transcortin since the binding capacity of [3H]dexamethasone is also greater in the female hippocampus. Receptor affinity in the female hippocampus is half that of the male value. In the hypothalamus, the dimorphism is in the opposite direction: the number of [3H]corticosterone cytosolic binding sites was found to be greater in the male. The male hypothalamus also showed a greater affinity for [3H]corticosterone than did the female. Ovariectomy increased the number of binding sites in the female hypothalamus. In vivo nuclear uptake of a tracer dose of [3H]corticosterone was determined in animals having intact gonads. The percent of tissue [3H]corticosterone present in cell nuclei from 4 brain regions, including the hippocampus and hypothalamus, was calculated per unit DNA. The concentrations of [3H]corticosterone in nuclei relative to tissue homogenates were higher in females than males for the 4 brain regions, but not for the pituitary or liver. The data are interpreted as suggesting that glucocorticoid secretion under basal conditions and during stress may differentially effect specific brain structures in male vs female rats.

Photoperiod regulation of mineralocorticoid receptor mRNA expression in hamster hippocampus.

Hippocampal mineralocorticoid receptor mRNA expression was increased in male hamsters exposed to 18 days of short photoperiod relative to animals maintained under long day illumination (p < 0.05). Short day hamsters were also characterized by increased weight gain, and heavier adrenal glands (p < 0.05). The larger adrenals showed selective increases in the widths of the zonae reticularis and glomerulosa (p < 0.001). Incidences of torpor and reduced body temperature were observed in the short day animals. No changes were found in reproductive organ weights, systolic blood pressure, open-field behavior, or stress levels of plasma corticosteroids. We conclude that the hamster brain-adrenal axis responds rapidly to changes in photoperiod, raising the possibility that this axis is a primary mediator of shortened photoperiod responses.

Sex difference in glucocorticoid binding in rat pituitary is estrogen dependent.

Sex-dependent differences in corticosteroid binding were assessed in individual pituitaries from adult male and female rats that had been adrenalectomized 12 h before sacrifice. Soluble binding was assayed in duplicate on LH-20 columns. Gonadally intact females showed significantly less 3H-dexamethasone binding than did intact males (p less than 0.01). This difference was confirmed in a second study (p less than .001). However, when ovariectomized females were compared with gondadectomized males, there was no difference in receptor concentration. Estrogen was able to reverse the effect of ovariectomy: ovariectomized females receiving estrogen (10 micrograms/rat/day) had significantly fewer receptors than intact males; p less than 0.01). Progesterone (500 micrograms/rat/day) did not antagonize the effect of estrogen in the pituitary. A sex difference was also found in the Type I (mineralocorticoid) receptor subpopulation which comprised approximately 10% of the total receptors, with females having fewer receptors than males. These results demonstrate that in the pituitary, the level of functional corticosteroid receptors is subject to a 20% down-regulation by circulating levels of estrogen. This raises the possibility that the lower number of receptors in females may act to reduce their sensitivity to the negative feedback effects of glucocorticoids at the level of the pituitary.

"Isolation stress" revisited: isolation-rearing effects depend on animal care methods.

Early reports of enhanced behavioral reactivity in isolation-reared rats attributed this syndrome to "isolation stress." In the studies reported here, this "isolation stress syndrome" was reliably obtained in adult rats reared from weaning in individual hanging metal cages. Such isolates showed behavioral and adrenocortical symptoms of profound fear during open-field testing, unlike group-housed controls or littermate isolates reared singly in plastic cages. Animals in hanging metal cages are never touched by human caretakers, whereas rats reared in plastic cages are picked up and put in clean cages twice weekly. Handling hanging-cage isolates twice weekly to model the handling associated with cage changes completely protected against this syndrome. Further, there was no hormonal, neurochemical or anatomical evidence of chronic stress even in hanging-cage isolates. Littermates housed in social groupings (three rats per plastic cage) also froze and defecated in the open field at rates comparable to hanging-cage isolates if they were the first animals to be tested from their social group cage. It is probable that odor cues from familiar cagemates in the open field protected socially reared animals tested subsequently from the same cage from this syndrome. It is concluded that isolates are not chronically stressed, and that rearing effects are the result of a complex interaction between prior handling, social experience and test conditions.

Adrenergic control of motor activity: effects of PNMT inhibition upon open field behavior in the rat.

Two inhibitors of brain PNMT were given to rats in doses which caused equivalent reductions in enzyme activity. Each drug produced similar and dose-related decreases in ambulation, rearing and defecation during open field behavioral testing. Central adrenergic (epinephrine containing) system may be necessary for normal open field behavior. This may reflect an interaction with other monoamines, particularly dopamine.

Influence of gonadal steroids on brain corticosteroid receptors: a minireview.

Sex differences exist in the functioning of the two brain corticosteroid receptor systems. Ovarian steroid replacement alters receptor mRNA expression, receptor binding capacities, and receptor affinity. The abundance of both mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) message can be reduced by estrogen. Progesterone is able to partially antagonize the action of estrogen and to induce MR transcription. The effect of estrogen on receptor binding capacity is more modest than its transcriptional actions. Estrogen decreases MR binding more reliably than it does GR. Progesterone has high affinity for the MR and can substantially reduce MR affinity for corticoids. Androgen apparently regulates corticoid receptor transcription but may not affect binding capacity. Estrogen and androgen are both more potent in regulating pituitary-adrenal function than would be suggested by their actions on receptor binding parameters.

Sex differences in the distribution and regulation of glucocorticoid receptors in cardiac tissues of rats.

We compared the binding of [3H]-dexamethasone in cytosols prepared from atria and ventricles. The effects of steroid treatment and adrenalectomy on receptor concentration were measured in both male and female rats. In male rats the distribution of receptors was similar in atria and ventricles. In contrast, the atria of female rats in all treatment groups had twice the number of receptors as did the ventricles. Adrenalectomy in females resulted in receptor up-regulation, but dexamethasone treatment, which was effective in reducing binding in males and in the ventricles of females, failed to alter atrial binding in females. These results suggest that the atria of female rats may be more responsive than ventricles to the effects of circulating glucocorticoids.

Effects of postnatal corticosterone treatment on reproductive development in the rat.

Reproductive development was studied in rats in which corticosterone (25 mg/100 g body wt) mixed with an equal amount of cholesterol was implanted i.p. at Days 3, 6, 12 or 18 after birth. Vaginal perforation in rats treated on Days 12 or 18 was advanced by 10 days but reproductive function in the treated animals was generally disrupted. A variable incidence of persistent vaginal oestrus was observed in adult females given implants on Days 3 and 6, and of prolonged vaginal dioestrus in those treated on Days 12 or 18. All treated females had lordosis quotients lower than those of untreated controls, the effect being most pronounced at the earlier ages of treatment. The results are believed to indicate two periods in reproductive development which are differentially affected by corticosterone.

Serotonergic and cholinergic interaction in the regulation of pituitary-adrenal function in rats.

It has been proposed that the central serotonergic inputs which modulate pituitary-adrenal secretion are mediated by cholinergic neurons. We have tested this hypothesis in intact rats. Male Sprague-Dawley rats were injected with cholinergic and serotonergic agents which enhanced transmitter function and with receptor blocking agents. Agents were injected, singly and in combination, into both unstressed and stressed animals. Since the response to cholinergic agents might be due to changes to vasopressin release, Brattleboro (vasopressin deficient) rats were also injected with cholinergic agents. The level of plasma corticosterone at 1-h post-injection was determined. Results indicate that the serotonin receptor blockade decreased the stimulatory, cholinergic effect of physostigmine. Cholinergic receptor blockers did not significantly reduce the corticosterone rise induced by 5-hydroxytryptophan. These results do not support the hypothesis of cholinergic mediation of serotonergic input. Nicotinic and muscarinic receptors appeared to exert opposing influences on the system. The nicotinic receptor antagonist was able to block the stimulatory effect of physostigmine. The muscarinic receptor antagonist significantly elevated plasma corticosterone levels. No differences were found in the effect of physostigmine on Brattleboro rats as compared to controls. These data are interpreted as suggesting that 1) the acetylcholine-induced stimulation of pituitary-adrenal function is mediated, in part, by serotonergic neurons; and 2) stimulation of nicotinic receptors is facilitatory whereas stimulation of muscarinic receptors is inhibitory to pituitary-adrenal function.

Effects of psychological stress on joint inflammation and adrenal function during induction of arthritis in the Lewis rat.

Glucocorticoids are effective immunosuppressive and anti-inflammatory agents, but some aspects of stress appear to be proinflammatory. This study investigates this apparent paradox as it applies to stress exposure and the development of arthritis in a rat strain that has subnormal hypothalamic-pituitary-adrenal (HPA) responsiveness. Female Lewis rats were subjected to 1 week of rotating, psychological stressors for 5 h daily, beginning 7 days following inoculation with type II collagen. The collagen-induced arthritis (CIA) group exposed to stress showed reduced ankle width increase (p < 0.001) and decreased hindlimb severity scores (p < 0.001). At sacrifice, 2 days following stress termination, no differences in either measure remained and there was no difference in hind paw volume. However, the area of the tibia invaded by stroma, as quantitated by image analysis, was reduced in the stressed rats (p < 0.05). In animals exposed to stress, adrenal weights were increased (p < 0.005) and plasma corticosterone levels were elevated at sacrifice (p < 0.02). Both injected groups had significantly larger adrenal (p < 0.005) and lower thymus weights (p < 0.05) than did uninjected controls. Likewise, both CIA groups had reduced glucocorticoid receptor immunoreactivity in synovial membranes compared to controls (p < 0.001), suggesting that the Lewis rat's HPA deficiency may be intensified by glucocorticoid receptor downregulation during the induction of CIA. These data indicate that the responsiveness of the HPA axis to psychological stress in this strain is sufficient to alter disease progression.

Comparison of brainstem and adrenal circadian patterns of epinephrine synthesis.

The enzyme which converts to norepinephrine to epinephrine, phenylethanolamine N-methyltransferase (PNMT), is found in brain as well as in the adrenal medulla. PNMT activity is subject to regulation by glucocorticoids, hormones which have a diurnal rhythm. We asked (1) whether a diurnal fluctuation exists in adrenal and brain PNMT activity and (2) what relationship this fluctuation might have to the diurnal rhythm in circulating glucocorticoids. Rats were sacrificed at 4-hour intervals over a 24-hour period. The PNMT activity in the adrenals and brainstems of these animals was determined by radioenzymatic assay, and the plasma levels of corticosterone were measured by competitive protein binding. No significant temporal variation was found in adrenal PNMT activity. Brainstem PNMT activity, however, showed a distinct diurnal fluctuation in activity, with a nadir at 7 a.m. and a peak at 3 p.m. The rise in brainstems PNMT clearly preceded by several hours the circadian rise in plasma corticosterone. We conclude that the circadian rhythm in circulating corticosterone does not drive the diurnal variation in brain PNMT activity. In unstressed animals, injection of exogenous corticosterone failed to elevate brainstem PNMT activity, whereas injection of specific inhibitors of PNMT activity significantly elevated plasma corticosterone levels. These data raise the possibility that the converse is true: changes in epinephrine synthesis may modulate the diurnal rhythm in pituitary-adrenal activity.