Plasma corticosterone during postnatal ontogenesis in rats: comparison of protein-binding and fluorometric method.

Competitive protein-binding method was used for determination of plasma corticosterone levels in rat during postnatal ontogenesis until 600 days of age. The level of corticosterone was high after birth, decreased until 5th day of life and then again increased at the end of the second week. During adolescence, when the sexual differentiation begins the levels of plasma corticosterone in females become permanently higher than those of males. Moreover, the comparison of plasma corticosterone level as measured with the aid of competitive protein-binding method and fluorometric method was described in hypophysectomised, stressed and normal male rats. The correlation between both methods was satisfactory, but the results obtained with a competitive protein-binding method were, on an average, by 35% lower. The specificity, precision and recovery of competitive protein-binding assay were found to be satisfactory. This method was found to be of advantage for a determination of plasma corticosterone level in small laboratory animals because of a small volume of plasma necessary.     

Adrenal function in female rats before puberty: the effects of gonadectomy

The following study was designed to test whether the change in the amplitude of the adrenal rhythm and the response to stress seen at puberty in female rats is dependent upon stimulation of the adrenal system by gonadal steroids. Rats were gonadectomized either at two days of age or at 21 days of age and periodic blood samples were taken by cardiac puncture and assayed for corticosterone (B) by a fluorometric procedure. Ovariectomy at weaning age (21 days) had no effect on adrenal function until the time of normal puberty onset. In intact animals, a rise in resting levels of corticosterone and an increase in the incremental response to ether stress was noted at 35 days of age. (Puberty in females was 35.3 ± 1.2 days as indicated by vaginal opening). Ovulation occurred the following day in 9/10 rats. Gonadectomy at two days of age had essentially the same effect as later gonadectomy in females. By 70 days of age, resting corticosterone values and stress responses in gonadectomized females reached levels similar to intact females. It is concluded that an independent adrenarche can occur in females but that gonadal steroids present at the time of puberty modulate the timing of this process.     

Plasma corticosterone and restraint induced gastric pathology: age-related differences after administration of corticotropin releasing factor

Corticotropin releasing factor (CRF) or saline was administered i.p. to rats aged either 100 or 220 days, followed by either brief handling or water immersion restraint. Plasma corticosterone was measured 75 min. later. Age of the animals in itself was not a significant factor either for basal levels of plasma corticosterone or for extent of restraint induced gastric pathology. However after CRF administration, young but not older animals revealed a significant increase in plasma corticosterone levels, and post restraint gastric ulcerations were more severe in older than young animals. CRF significantly decreased the number of restraint induced ulcers in young rats, while the cumulative ulcer length was increased in older animals.     

Assessment of reduced glutathione: comparison of an optimized fluorometric assay with enzymatic recycling method

Glutathione is an important tripeptide involved in a variety of cellular processes. Thus, precise knowledge of its levels is essential. Glutathione exists in two free forms-reduced and oxidized-and a number of methods exist to measure its levels. The aim of our work was to optimize a spectrofluorometric assay for reduced glutathione based on the reaction between glutathione and o-phthalaldehyde. We found that a change of excitation wavelength to 340 nm and modification of pH to 6.0 enhance sensitivity and specificity of the method (intraassay coefficient of variation CV < 3%, interassay CV = 5.1%, recovery = 98-102%, linearity = 0-1000 μM GSH, calibration R2 = 1.00). We also anticipated possible effect of various amino acids on the fluorescence signal, but no interference was found. We compared the optimized fluorometric method with a popular enzymatic recycling glutathione assay and found very strong correlation of results (r = 0.99, n = 45). We introduce here an optimized fluorometric method possessing sufficient sensitivity and specificity that is comparable to the enzymatic glutathione assay. Because the fluorometric assay procedure is faster and lower in cost, it could be ideal for routine analysis of reduced glutathione levels in a large number of samples.     

Response of the adrenals to stress in animals from litters with different numbers of young.

Undernutrition and overnutrition during the suckling period -- achieved by adjusting the number of infant rats per litter to 4, 8 and 14 -- resulted in a decrease in the plasma corticosterone level and in corticosterone production by the adrenals in vitro in overfed animals at the age of 30, 180 and 300--360 days. An enhanced response to ether anaesthesia or immobilization stress was found in overfed animals from the smallest litters. The results show that a change in postnatal nutrition and in social relationships in the litter influences maturation of the hypoathalamo-hypophysio-adrenal regulatory system; in adulthood this is manifested in changes in production of the adrenal hormones and in the reaction of the adrenals to stress.     

Assessment of sperm viability in boar,rabbit and rooster: a modification of the fluorometric ethidium bromide exclusion procedure

The ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on boar, rabbit and rooster semen. Diluted semen was used for boars and roosters. Diluted rabbit semen did not allow for reliable fluorescence readings; the interference of granules characteristic of rabbit seminal plasma was suggested as its cause. Therefore, rabbit semen was washed on several Percoll and Optiprep density gradients, with the aim of removing the granules from the sperm suspension. The complete absence of granules was not obtained, however, the best result was provided by the 35/70% Percoll density gradient. Most spermatozoa formed a loose pellet with low contamination. Although the washing procedure resulted in a selective action, Percoll washed semen was used to assess the EtBr procedure. The fluorescence intensities of stained fresh and stained digitonin-permeabilized samples were corrected, respectively, for the nonspecific fluorescence measures of fresh and digitonin-permeabilized samples both unstained. The contribution of the dye was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for boars and roosters (8-32 x 10(6) cell/ml) and Percoll washed semen for rabbits (4-16 x 10(6) cell/ml). The reliability of the fluorometric method was compared with the traditional nigrosin-eosin (NE) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges. For boars and roosters the values determined by fluorometry agreed closely with those determined using the NE method.     

Influence of stress and phenobarbital on corticosterone metabolism during early postnatal life of rat

Postnatal changes in liver corticosterone metabolism in vitro were investigated in male rats pretreated for three days twice daily by physiological saline i.p./stress/ or by phenobarbital /20 mg/kg/. Perinatally, both stress and phenobarbital decrease corticosterone side chain metabolism while no change was observed in A ring reduction rate. In older animals no effect of the stress on corticosterone metabolism was observed. The inhibitory influence of phenobarbital on the side chain metabolism was still apparent at age of 14 days, but not in adult animals. The A ring reduction rate was increased by phenobarbital at age of 14 days and in adult animals.Measurements of serum corticosterone and corticosterone production by adrenal glands in vitro confirmed earlier reports showing that during perinatal period increased level of circulating corticosterone can be associated with minor or transient changes in adrenal cortex activity.It is concluded that changes in liver corticosterone metabolism are likely to play an important role in regulation of glucocorticoid activity perinatally when the responsiveness of pituitary-adrenal system to environmental stimuli is decreased.     

Low doses of L-tryptophan are lethal in rats with adrenal insufficiency

Adrenalectomy decreased the LD50 value for L-tryptophan from greater than 1000 mg/kg in normal rats to 11.6 mg/kg. The LD50 in adrenalectomized rats was restored to normal by corticosterone replacement therapy. Administration of metyrapone, which blocks the synthesis of adrenal steroids, to normal rats decreased plasma corticosterone levels by approximately 50% and decreased the LD50 from greater than 1000 mg/kg to 24.9 mg/kg. Neurochemical analysis revealed a large increase in tissue tryptamine levels following administration of L-tryptophan in rats with adrenal insufficiency. Therefore, it appears that death due to L-tryptophan in animals with adrenal insufficiency is due to formation of excess tryptamine and, consequently, an elevation in blood pressure and other cardiovascular dysfunctions.     

The effect of recaging into groups or into isolation on the pituitary adrenal response to immobilization of different age groups of C57BL/6J mice

Although there is evidence which suggests that age and social environment are significant variables in all experiments dealing with stress in intact animals, there is relatively little information available on the manner in which these variables interact to influence the pituitary adrenal response to stress. Inbred mice of strain, C57BL/6J, of 3 different age groups (49, 255 and 720 days) were subjected to a brief immobilization stress 24 hours subsequent to regrouping them 1, 2 or 4 per cage. Plasma corticosterone concentration was measured by radioimmunoassay prior to and 15 minutes after immobilization or 60 minutes after treatment with ACTH. It was found that preimmobilization levels of corticosterone and increments in corticosterone in response to ACTH treatment were smaller in 255 day than in 49 or 720 day old mice and that preimmobilization corticosterone levels of control and recaged 49 and 255 day old mice were similar. In 49 day old mice, recaging increased the immobilization evoked increment in corticosterone, but in 255 and 720 day old mice recaging in groups or pairs did not change the immobilization evoked response. However, recaging of 720 day old mice in isolation resulted in a decrease in the immobilization evoked increment. Therefore, it appears that the act of transfer itself increased the pituitary adrenal function in the 49 day old mice while, in the oldest mice, isolation itself reduced pituitary adrenal activity. Finally, it appears that in the 255 day old mice, recaging is only a minor stress since after 24 hours′ there is no evidence of elevated steroid levels.     

Analysis of ecdysteroids by fluorometry

All ecdysteroids are fluorescent when treated with sulphuric acid. The fluorescence can be used for the detection on thin-layer plates as well as for the quantitation of ecdysteroids in ethanolic solution. A standard procedure for the quantitative analysis of ecdysteroids by fluorometry is given. The assay is linear over two orders of magnitude of steroid concentration and is sensitive down to the range of 10⁻¹¹ mol ecdysteroid. The assay is group specific for ecdysteroids. Ecdysone gives the highest fluorescence and is therefore used as the standard. Other ecdysteroids exhibit a fluorescence value which is about 50% relative to ecdysone. Any interference in the assay by other steroids including cholesterol and 7-dehydrocholesterol is low. The fluorometric assay may be used to detect and quantify ecdysteroids in extracts of biological material after a chromatographic step. This is shown by the analysis of ecdysteroids from the blue blowfly, Calliphora vicina.     

Japanese quail selected for high plasma corticosterone response deposit high levels of corticosterone in their eggs

Poor habitat quality or body condition often correlates with high responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis rather than with elevated baseline levels of glucocorticoids. We hypothesized that, for egg-laying vertebrates, high responsiveness of the HPA axis would correspond to high concentrations of corticosterone in yolk. We tested the prediction that Japanese quail (Coturnix coturnix japonica) selected for high plasma corticosterone response to brief immobilization (HS quail) would lay eggs with higher yolk corticosterone concentrations than birds selected for low response (LS quail). Quail from both lines were left undisturbed, outside of the stressors associated with daily management, before a first round of egg collection. In a second experiment, quail of both lines were experimentally stressed during the week before egg collection. In both cases we found quail from the HS line to lay eggs with significantly higher yolk corticosterone concentrations than quail of the LS line. After exposure to added experimental stressors, the line difference was more pronounced (increasing from 62% to 96%). There was no line difference in concentrations of yolk testosterone. Our results suggest that (1) genetic differences underly differences in the transfer of maternal corticosterone to yolk and (2) females may be able to control deposition of corticosterone into yolk through a mechanism independent of baseline corticosterone titers.     

Adrenocorticotropin and corticosterone levels in pre-weanling spontaneously hypertensive rats

Circulating plasma ACTH and corticosterone levels were measured in spontaneously hypertensive rats (SHR) and the corresponding, normotensive Wistar-Kyoto (WKY) strain at 10 and 20 days of age. In addition, stored levels of ACTH were measured in the pituitary glands of these animals. Circulating corticosterone levels were significantly lower, in both strains, at 10 days than at 20 days. Although the glucocorticoid was undetectable in WKY animals at 10 days, significant levels were observed in age-matched SHR. No difference in corticosterone concentrations was observed between the two strains at 20 days. Circulating ACTH levels did not reflect the values for circulating glucocorticoids. There were no significant differences in the levels of ACTH between strains or between age groups. Moreover, pituitary stores of ACTH between animals of different strains and ages were not found to be significantly different among any of the groups tested. These results demonstrate that there is a difference in circulating corticosterone levels between spontaneously hypertensive and Wistar-Kyoto rats at 10 days postnatally which is not evident just prior to weaning (20 days). This difference is not due to variations in stored or circulating ACTH. Indeed, ACTH levels are high at a time (10 days) when corticosterone is low - thus suggesting that the difference may reside within the responsiveness of the adrenal cortex.     

Fluorometric determination of urinary kynurenic acid by flow injection analysis equipped with a "bypass line"

A flow injection analysis involving a photochemical reaction and fluorometric detection has been developed for the determination of urinary kynurenic acid. Kynurenic acid was found to fluoresce on irradiation with ultraviolet light at pH 7.2 in the presence of hydrogen peroxide. This method was applied to flow injection analysis using a new procedure involving a "bypass line" for the simultaneous determination of urinary kynurenic acid and background fluorescence. The calibration graph showed linearity over the range of 0.20 to 120 pmol. For pretreatment of urinary kynurenic acid, a PRE-SEP C18 cartridge was used. The mean recovery of kynurenic acid from urine was 94.5%. The content of urinary kynurenic acid was 13.0 +/- 2.68 mumol/day. There was good correlation (r = 0.9729) between values determined by flow injection analysis and high-performance liquid chromatography.     

Effect of Escapable and Inescapable Stress on Levels of Catecholamines in Adrenals and Corticosterone in Blood Plasma in Young and Aged Rats

Effect of escapable and inescapable electrical stress (ES, IS) on the catecholaminergic system was studied in young (3 months) and aged (25 months) male Wistar rats on the day 3 after stress, using radioimmune analysis and high-pressure liquid chromatography. Catecholamine concentration in adrenals and corticosterone level in blood of control aged rats was lower than in young animals. On the third day after the electrical stimulation in cages with current-conducting floor, production of hormones of adrenal cortical and medullar layers rose significantly in aged rats, with a more pronounced increase of noradrenaline after IS, while of blood adrenaline and corticosterone, after ES. In young rats no significant changes in catecholamines were revealed, whereas the blood corticosterone level was increased after IS. Thus, in aged rats, a low basal level of catecholamines and corticosterone and a delayed stress response can be established. In old animals after ES, a long post-action was observed, which was quite comparable with the results obtained after IS in the both age groups.     

Inhibition of the ACTH adrenal response to stress by treatment with hydrocortisone, prednisolone and dexamethasone in the rat

16- and 4-week-old intact and adrenalectomized rats have been treated with different doses of the three glucocorticoids hydrocortisone, prednisolone and dexamethasone by gavage. The delayed feedback effect on plasma ACTH and corticosterone response to an ether stress have been assessed. Almost complete suppression of corticosterone response 20 min after an ether stress and an ACTH suppression to 20% of control values 5 min after an ether stress were observed with 25 micrograms of dexamethasone, 10 mg of prednisolone and 20 mg of hydrocortisone. Although the percent inhibition of corticosterone and ACTH response to stress was comparable, a striking dissociation of the ACTH and corticosterone release was observed in terms of absolute concentrations. A mean ACTH concentration of 462 ng/l after 25 micrograms of dexamethasone was measured together with a barely measurable corticosterone concentration of 3 micrograms%. Similarly, after 10 mg of prednisolone, the mean ACTH concentration was 404 ng/l, whilst the mean corticosterone concentration was 3 micrograms%. This dissociation demonstrates that the corticosterone concentration on its own does not necessarily reflect the ACTH release. At 4 weeks of age, the ACTH response to stress is more difficult to suppress than in adult animals. This is more obvious after adrenalectomy, where the excessive ACTH secretion was less inhibited by all glucocorticoids used. The time between the last steroid gavage and stress must be considered. In 4-week-old animals the ACTH response 16 h after 12.5 micrograms of dexamethasone was inhibited by 22%, whereas 4 h after the same dexamethasone dose the inhibition was 85%.(ABSTRACT TRUNCATED AT 250 WORDS)     

The release of corticosterone and a corticosterone-binding protein by incubated rat adrenal slices

Stimulation of incubated rat adrenal slices with ACTH(1-24) resulted in an increase in the release of both corticosterone and specific corticosterone-binding protein into the incubation medium. The release of corticosterone and binding protein was dose and calcium dependent with adrenals from animals pretreated with betamethasone. While the secretion of corticosterone was continuous throughout the incubation period, there appeared to be a limit to the increase in binding capacity. The specificity of steroid binding to the adrenal protein showed a similar profile to that of corticosteroid-binding globulin (CBG) in rat serum. A Western blot analysis using anti-rat CBG as the primary antiserum, showed that the adrenal protein was not CBG. [3H]corticosterone binding with disc electrophoresis, run at 2 degrees C, gave a single peak with approximately the same Rf value for rat serum, purified CBG, and adrenal incubate; at 22 degrees C peaks were only seen for rat serum or purified CBG. The data presented provides further evidence for the existence of a specific corticosterone-binding protein of adrenal origin released in conjunction with corticosterone. The adrenal protein would appear to have a lower affinity for corticosterone than does CBG, and to be functionally more labile. It is possible that the adrenal protein may be CBG that has been internalized, modified and released with corticosterone.     

Proliferative and cytotoxic immune functions in aging mice. IV. Effects of suppressor cell populations from aged and young mice

The changes with age in three splenic suppressor cell populations were studied in C57BL/6 mice. Allospecific Ts cells and nonspecific non-T suppressor cells were both generated in vitro in allogeneic MLC. The presence of "pre-existing" suppressor cells in fresh spleen cells from normal mice was examined. Suppressor cell activities were assayed for their ability to suppress proliferation in a fresh allogeneic MLC after treatment to prevent their own proliferation. The ability to generate both specific and nonspecific suppressor cells decreased with age, whereas pre-existing suppressor cells were detected in spleens from the majority of the aged animals but not in spleens from young animals. The decrease in suppressor cell activity was not due to any requirement for age matching between donors of suppressor and target cells. The specific and nonspecific MLC-generated suppressor cells inhibited both the proliferative response in the assay MLC and the generation of cytotoxic cells. The pre-existing suppressor cells only suppressed the proliferative response and not the generation of cytotoxic cells. The changes seen with age in these suppressor cell populations suggest that the ability to generate suppression (both allospecific and nonspecific) to newly encountered Ag declines with age, whereas a resident splenic suppressor cell population accumulates over the lifetime of the animals.     

The influence of oral corticosterone replacement on plasma prolactin levels of adrenalectomized female rats

The administration of 80 μg of corticosterone/ml of drinking solution to adrenalectomized (ADX) rats resulted in a 24 hour serum corticosterone pattern similar to that of intact animals except that the magnitude of the afternoon-nocturnal surge was one third. Basal plasma prolactin levels and the estrogen-induced afternoon prolactin surge were similar for intact and for ADX animals receiving corticosterone in the drinking solution. Adrenalectomized animals receiving 0.9% NaCl to drink, however, had an afternoon prolactin surge that was significantly lower than that of intact animals while basal levels were similar.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8–10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.