Effects of unilateral adrenalectomy on the remaining adrenal cortex of adrenocorticotropic hormone-treated male and female hamsters

Sham-operated and unilaterally adrenalectomized male and female hamsters were administered 25 micrograms adrenocorticotropic hormone (ACTH) for 5 days after the operation in order to examine the effects of ACTH on compensatory adrenal growth. In ACTH-treated male and female hamsters, unilateral adrenalectomy did not change the relative weight of the remaining adrenal. There were no significant differences in the volumes of the adrenocortical zones and their parenchymal cells, as well as in the number of adrenocortical cells per gland if compared with unilaterally adrenalectomized and sham-operated ACTH-treated male hamsters, while 3H-thymidine incorporation per gland was lower in monoadrenalectomized animals. On the contrary, in ACTH-treated females, unilateral adrenalectomy resulted in a significant hypertrophy of zona fasciculata cells and in an enhanced 3H-thymidine uptake by the remaining gland. These findings stress the existence of notable sex-related differences in the compensatory adrenal growth in hamsters.     

IGF1 and 2 in two models of adrenal growth

Insulin-like growth factors (IGFs) 1 and 2 were measured in the adrenal glands of rats undergoing either compensatory growth following left unilateral adrenalectomy or adrenal regeneration following bilateral adrenal enucleation. In normal rat adrenal gland, the tissue concentration of IGF2 (7.45 +/- 0.99 pg/micrograms protein) wa higher than IGF1 (1.26 +/- 0.23 pg/micrograms protein), both peptides being more abundant in the inner zones of the adrenal gland compared to the capsule-glomerulosa. During compensatory growth of the right adrenal gland, IGF1 and 2 increased significantly compared with control right adrenal glands at 24 h following left unilateral adrenalectomy (P less than 0.001). At 68 h, the increase remained significant for IGF1 (P = 0.012). The two peptides were measured in the regenerating adrenal gland at 7, 14 and 21 days following bilateral enucleation. Whilst there was a trend towards an increase in the IGF1 and 2 content of regenerating adrenal glands, the increase was significant only for IGF2 in the left adrenal gland at 21 days following enucleation. Plasma IGF1 and 2 did not increase compared to controls during the experiments (110.97 +/- 1.95 and 46.33 ng/ml, respectively), suggesting that the changes in tissue IGF reflect increased local production during rapid growth of the adrenal gland.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.     

Influences of the adrenal gland and photoperiod on the hamster oestrus cycle

The influence of the adrenal gland and photoperiod on the adult female hamster estrous cycle was investigated. Hamsters were exposed to 14 hours of light per day and estrous cycles were monitored. Normal cycles were characterized by a copious discharge every 4th day. Hamsters were then adrenalectomized, adrenalectomized and blinded, or blinded only. Adrenalectomized/blinded hamsters were given a .2% sodium saccharine-1%(w/v) sodium chloride solution to drink which promotes long-term survival in most adrenalectomized hamsters. With blinding alone, it took from 18 to 62 days for cessation of estrous cycles. Cycles ceased in a mean of 36.2 days in blinded/adrenalectomized hamsters. After adrenalectomy only, 3 hamsters continued to show 4-day estrous cycles for at least 9 weeks in response to the long days. 6 adrenalectomized animals died due to adrenal insufficiency but displayed several 4-day estrous cycles. It is suggested that in the hamster, adrenal hormones are nonessential for the long-term expression of estrous cyclicity. However, further research is needed to determine whether adrenal hormones are capable of modulating photoperiodic time measurement or ovarian regression.     

Rat insulin-like growth factor-I and -II mRNAs are unchanged during compensatory adrenal growth but decrease during ACTH-induced adrenal growth

The insulin-like growth factors (IGFs) may be important autocrine and paracrine mediators of organ growth. We used solution-hybridization/ribonuclease protection assays to examine IGF-I and IGF-II mRNA abundance during hypertrophy or the rat adrenal gland induced by unilateral adrenalectomy or by adrenocorticotropic hormone (ACTH) infusion. Adrenal IGF-I mRNA did not change during the period of rapid organ growth at 18 or 66 h after unilateral adrenalectomy. ACTH infusion induced a time- and dose-dependent decrease in adrenal IGF-I mRNA despite significant increases in gland size. IGF-II mRNA also remained unchanged after unilateral adrenalectomy and decreased after ACTH infusion, to a greater extent than IGF-I mRNA. Liver IGF-I mRNA did not change with ACTH exposure, indicating an effect specific to the adrenal. We also measured adrenal P450scc mRNA as a marker of steroidogenic capacity. P450scc mRNA was unchanged after unilateral adrenalectomy and increased with ACTH infusion. Thus IGF-I and IGF-II mRNAs respond in parallel, but in different fashions with different stimuli for adrenal growth. The decrease in IGF mRNA after exposure to ACTH may be a factor in the ACTH-induced inhibition of compensatory hypertrophy after unilateral adrenalectomy.     

Sex differences in adrenocortical structure and function

Summary Adrenal glands of adult male hamsters are larger and secrete more cortisol than those of females. Stereology was therefore used to study zonal and cellular aspects of development of the adrenal cortex of male and female hamsters. Adrenal glands were studied at weekly intervals from day 21 to day 77 of postnatal ontogenesis. Within this period, body weight did not differ significantly between the sexes. During development, absolute and relative adrenal weights were higher in males; their zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR) become markedly larger than those in females. No marked changes in the volume of individual ZG cells occurred although ZF cells and ZR cells become larger in male than female animals. The total number of adrenocortical cells increased within the period studied, a greater increase being observed in ZG and ZF in males. No distinct sex difference was observed in the number of ZR cells throughout development. From day 56 of postnatal life the adrenal cortex of male hamster contained more parenchymal cells than the female gland. These results thus indicate that sex differences in hamster adrenal cortex depend upon changes in number and size of parenchymal cells.Supported in part by a grant from the Committee of Zoology, Polish Academy of Sciences     

Further data on the existence of a neural pathway from the adrenal gland to the hypothalamus

Summary The effect of unilateral adrenalectomy on the incorporation of tritiated leucine into the ventromedial nucleus of both sides of the rat hypothalamus was studied by light- and electron-microscopical autoradiography. The left adrenal had been removed and 14 days later labeled amino acid was given intravenously. Rats were killed 5, 30, 60 and 120 min after isotope administration. Following unilateral adrenalectomy there is a marked difference in the autoradiographic reaction of both sides of the hypothalamic ventromedial nucleus. On the right side the number of autoradiographic silver grains is much higher than on the left side, the difference being statistically significant for each animal and for each time interval. In accordance with previous findings the data suggest the existence of a neural pathway from the adrenal gland to the hypothalamus.     

ACTH-induced ultrastructural changes in the zona fasciculata of the hamster adrenal cortex. Are intraadrenal thrombi regulators of corticosteroid secretion?

Correlated stereological and functional studies were performed on the effect of massive ACTH doses on adrenal cortex of the female hamster. ACTH resulted in a marked increase in adrenal gland weight at day 6 of treatment followed by a drop at day 9. Stereology showed significant enlargement of the zona fasciculata (ZF) cells with the highest value at day 6 and subsequent drop at day 9 of treatment. This hypertrophy was due to a notable increase in the volume of mitochondrial, SER, Golgi apparatus and lipid droplet compartments. Cortisol secretion by adrenal slices and homogenates was also highest at day 6 of ACTH administration and notably lower at day 9. At day 6 of ACTH treatment in outer ZF thrombi were seen. In their vicinity the subendothelial space was dilated and endothelial cells dissociated from the basal lamina. Numerous erythrocytes were also visible among dissociated ZF cells. At day 9 of experiment in outer part of ZF numerous spaces devoid of parenchymal cells appeared. The earlier authors considered the "empty spaces" or "holes" in hyperstimulated adrenal cortex as a sign of holocrine secretion of steroid hormones. The present findings enable us to introduce a new hypothesis on the development of these spaces. In our opinion in hyperstimulated adrenal cortex numerous thrombi may be formed leading thus to the degeneration of adrenocortical cells. Thus, the appearance of the "empty spaces" or "holes" in the gland is not connected with the holocrine secretion but with the regulation of the number of secretory cells in adrenal cortex by the thrombi-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compensatory adrenal growth in aldosterone-treated male and female hamsters

The aim of the study was to investigate the compensatory adrenal growth in aldosterone-treated male and female hamsters. Hemiadrenalectomised and sham-operated animals were treated for 5 days with a daily d-aldosterone dose of 25 micrograms/animal. In both male and female aldosterone-treated hamsters monoadrenalectomy did not change the relative adrenal weight if compared with sham-operated groups. The fasciculata zonae of monoadrenalectomised aldosterone-treated males was larger and contained more parenchymal cells than in appropriate control group. There was no difference in the volume of adrenocortical zones, average cell volume and in cell number between sham-operated and unilaterally adrenalectomised females. In vitro 3H-thymidine incorporation per adrenal was markedly higher in monoadrenalectomised than in sham-operated aldosterone-treated males while the opposite was true for female hamsters. Thus, the action of aldosterone on CAG in the hamster seems to depend on sex, with no effect in males and inhibitory action in females.     

Effects of adrenalectomy and nephrectomy on adrenal regeneration hypertension in the rat.

The involvement of the regenerating adrenal gland and kidney, and the contribution of deoxycorticosterone (DOC) and prostaglandin E2 (PGE2), in the development of adrenal regeneration hypertension (ARH) was evaluated in young female Sprague-Dawley rats. Based on tail-cuff plethysmographic measurement, animals subjected to nephrectomy and adrenalectomy on the right side and adrenal enucleation (removal of the adrenal cortex) on the left side developed significant (P less than 0.05, n = 12) hypertension within 6 weeks following operation. Subsequent left nephrectomy in these ARH rats produced a further elevation, whereas a secondary adrenalectomy resulted in an acute and discernible reduction in blood pressure within 24-36 hours. It is interesting to note that the progressive increase in blood pressure following left nephrectomy was significantly reversed by PGE2 (10 or 20 micrograms/kg, i.p.). At the same time, the reduction in blood pressure after secondary adrenalectomy was significantly retarded by deoxycorticosterone trimethylacetate (2 mg/kg, i.p.). These data demonstrated that both the kidney and the regenerating adrenal cortex are involved in the pathogenesis of ARH. Furthermore, it is probable that the secretion of DOC by the regenerating adrenal cortex is responsible for the elevation in blood pressure, in a process that is balanced by PGE2, possibly secreted by the kidney.     

The involvement of cyclic AMP-dependent protein kinase(s) in the induction of ornithine decarboxylase in the regenerating rat liver and in the adrenal gland after unilateral adrenalectomy

We investigated the temporal relationship between the level of cyclic AMP, the activation of cyclic AMP-dependent protein kinase(s), and the induction of ornithine decarboxylase in two important rapid growth systems: the regenerating rat liver and the remaining adrenal gland following unilateral adrenalectomy. There was a biphasic increase in the aactivity of ornithine decarboxylase at 4 and 14 h following partial hepatectomy. The concentration of cyclic AMP increased 2-fold compared to sham-operated animals within 2–3 h, returned to baseline by 8 h, and was elevated again 3-fold by 12 hours. The activation of cyclic AMP-dependent protein kinase(s) occured in a similar biphasic manner. From a control activity ratio (?cAMP/+cAMP) of 0.4, values for total soluble kinase activation reached 0.75 at both 2 and 14 h. After a delay of 2 h following unilateral adrenalectomy, ornithine decarboxylase activity in the remaining adrenal gland increased 15–20-fold of control level by 8 h. Cyclic AMP concentrations in the adrenal were elevated 3.5-fold within 30 min. The protein kinase activation increased from 0.25 to nearly a totally activated state of 1.0 within 1 h, decline to 0.4 by 2 h, and returned to the level of the sham-operated controls at 8 h. In both the rat liver in response to partial hepatectomy and the adrenal gland undergoing hypertrophy, cyclic AMP-dependent protein kinase(s) was markedly activated prior to increases in ornithine decarboxylase activity.     

Zone-specific cell proliferation during compensatory adrenal growth in rats

Compensatory adrenal growth after unilateral adrenalectomy (ULA) leads to adrenocortical hyperplasia. Because zonal growth contributions are not clear, we characterized the phenotype of cortical cells that proliferate using immunofluorescence histochemistry and zone-specific cell counting. Rats underwent ULA, sham adrenalectomy (sham), or no surgery and were killed at 2 or 5 days. Adrenals were weighed and sections immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Adrenal weight increased after ULA compared with sham and no surgery at both time points, and there was no difference between sham and no surgery. However, either ULA or sham increased Ki67-positive cells in the outer fasciculata at both time points compared with no surgery. Outer fasciculata-restricted proliferation is thus associated with adrenal weight gain in ULA but not sham. Experiment repetition using proliferating cell nuclear antigen and bromodeoxyuridine showed similar results. After ULA, adrenal DNA, RNA, and protein increased at both time points, whereas after sham, only adrenal DNA increased at 2 days. Compensatory growth thus results from hyperplasia and hypertrophy, whereas sham induces only a transient adrenal hyperplasia. Dexamethasone pretreatment prevented the increase in adrenal weight after ULA and blocked Ki67 labeling in the outer fasciculata but not zona glomerulosa in all groups. These results clearly show that the outer fasciculata is the primary adrenal zone responsible for compensatory growth, responding to steroid-suppressible stress signals that alone are ineffective in increasing adrenal mass.     

The differential effect of atrazine on luteinizing hormone release in adrenalectomized adult female Wistar rats

High doses of atrazine (ATR), administered for 4 days, suppress luteinizing hormone (LH) release and increase adrenal hormones levels. Considering the known inhibitory effects of adrenal hormones on the hypothalamo-pituitary-gonadal axis, we investigated the possible role the adrenal gland has in mediating ATR inhibition of LH release. To determine the extant and duration of adrenal activation, ovariectomized Wistar rats were given a single dose of ATR (0, 50, or 200 mg/kg), and corticosterone (CORT) levels were assayed at multiple time points posttreatment. CORT levels were increased within 20 min and remained elevated over 12 h postgavage in 200-mg/kg animals. To determine the effects of adrenalectomy on ATR inhibition of the LH surge and pulsatile LH release, adrenalectomized (ADX) or sham-operated ovariectomized rats were treated for 4 days with ATR (0, 10, 100, or 200 mg/kg), and an LH surge was induced with hormone priming. In the afternoon following the last dose of ATR, blood was sampled hourly for 9 h. Another cohort of ovariectomized rats was examined for pulsatile patterns of LH secretion after ATR (0, 50, or 200 mg/kg) and sampled every 5 min for 3 h. ADX had no effect on ATR inhibition of the LH surge but prevented the ATR disruption of pulsatile LH release. These data indicate that ATR selectively affects the LH pulse generator through alterations in adrenal hormone secretion. Adrenal activation does not play a role in ATR's suppression of the LH surge, and therefore ATR may work centrally to alter the preovulatory LH surge in female rats.     

Capsaicin-sensitive adrenal sensory fibers participate in compensatory adrenal growth in rats

Compensatory adrenal growth, in which one gland undergoes hyperplasia after removal of the other, is mediated by a neural reflex. In the present studies, a method employing capsaicin to selectively remove adrenal sensory fibers was developed and applied to determine whether adrenal capsaicin-sensitive fibers participate in compensatory adrenal growth. The splanchnic nerves of anesthetized male rats were treated with capsaicin or vehicle. Capsaicin treatment selectively removed adrenal calcitonin gene-related peptide-positive fibers. One week after drug treatment, rats underwent left adrenalectomy or sham surgery and recovered for 5 days. Capsaicin treatment bilaterally or to the left splanchnic nerve alone (i.e., the afferent nerve in the reflex) impaired compensatory adrenal growth at 5 days compared with vehicle controls, whereas capsaicin treatment to the right splanchnic nerve alone did not affect growth. Moreover, left adrenalectomy induced c-Fos immunolabeling in ipsilateral dorsal spinal cord that was prevented by capsaicin treatment. These data suggest that adrenal capsaicin-sensitive afferent nerves participate in compensatory adrenal growth and that this effect is primarily on the afferent limb of the reflex.     

Sex differences in adrenocortical structure and function

Summary Adrenal glands of adult female rats are heavier than the glands of corresponding male rats. Postpubertal orchiectomy increases the adrenal weight, an effect restored by testosterone replacement. Under the same conditions ovariectomy of 8 weeks duration does not change the adrenal weight while estradiol replacement enhances the relative adrenal weight.Karyometric studies have shown that nuclei in the female zona fasciculata cells are larger (app. 18%) than those of the male. Similar but only slight differences (2%) were observed in the zona reticularis. Orchiectomy results in enlargement of cell nuclei within all zones of the adrenal cortex; testosterone replacement has the opposite effect. Ovariectomy of 8 weeks duration slightly enhances the volume of nuclei of the zona glomerulosa cells, has no effect on the nuclei of the zona fasciculata and reduces the volume of nuclei in the zona reticularis. Estradiol replacement reduces the volume of nuclei of the zona glomerulosa cells but increases nuclear volume in the zona fasciculata and in the zona reticularis.Thus testosterone has an inhibitory effect on the adrenal cortex of the rat while the physiologic effect of estradiol on the morphology of this gland, particularly on the zona fasciculata cells is rather dubious.The author wishes to thank Mrs. B. Westerska and Miss K. Siejak for excellent technical assistance.This research was supported in part by a grant from the Zoological Committee, 2nd Department, Polish Academy of Sciences.This paper is dedicated to the memory of late Kazimierz Mietkiewski, M.D., Ph.D. whose encouragements and suggestions were most stimulating for my work.     

Immunoreactive delta sleep-inducing peptide in the rat hypothalamus, pituitary and adrenal gland: effects of adrenalectomy

Immunoreactive (IR) delta sleep-inducing peptide (DSIP) was examined by immunocytochemistry in the rat pituitary and adrenal gland and found to be colocalized with IR thyroid-stimulating hormone in the pituitary and with noradrenaline in the adrenal medulla. IR-DSIP was also detectable in nerve fibers in the posterior pituitary. By radioimmunoassay, IR-DSIP was quantified in plasma and tissue extracts after uni- or bilateral adrenalectomy. Significantly elevated plasma levels of IR-DSIP were measured 5 days after bilateral adrenalectomy (p less than 0.001). IR-DSIP was increased (p less than 0.05) in pituitary extracts from bilaterally adrenalectomized rats after 5 days, but not after 14 or 28 days. Sham- and unoperated animals did not significantly differ in plasma or tissue concentration of IR-DSIP. High-performance liquid chromatography of C18 SEP-PAK purified hypothalamus extracts revealed a single peak of IR-DSIP material of lower hydrophobicity than synthetic DSIP. The elevated concentration of IR-DSIP in the rat pituitary and plasma after bilateral adrenalectomy is consistent with the previously suggested role of DSIP to influence the activity of the hypothalamic-pituitary-adrenal axis.     

Age-related differences in skeletal muscle protein synthesis: relation to markers of immune activation

A quantitative analysis of zone-specific proliferation was done to determine the recovery of adrenal cortical zonation during regeneration after enucleation. Adult male rats underwent adrenal enucleation [unilateral enucleation (ULE)] or sham surgery, both accompanied by contralateral adrenalectomy. At 2, 5, 10, and 28 days, blood and adrenals were collected to assess functional recovery. Adrenal sections were immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P-450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P-45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Recovery of fasciculata secretory function occurred by 28 days as reflected by plasma ACTH and corticosterone, whereas glomerulosa function reflected by plasma aldosterone remained low at 28 days. At 5 days, ULE adrenals showed increased Ki67+ cells in the glomerulosa and inner fasciculata, whereas at 10 and 28 days increased proliferation was restricted to the outer fasciculata. These data show that enucleation results in transient elevations in glomerulosa and inner fasciculata cell proliferation followed by a delayed increase in the outer fasciculata. To assess adrenal growth in enucleated adrenals previously suppressed by the presence of an intact adrenal, rats underwent ULE and sham surgery; after 4 wk, the intact adrenal was removed and enucleated adrenals were collected at 2, 5, and 10 days. Overall, proliferation was delayed in this model, but at 5 days, Ki67+ cells increased in the outer fasciculata, whereas by 10 days, increased proliferation occurred in the outer and inner fasciculata. The key novel finding of increased proliferation in the inner fasciculata suggests that the delayed growth of the enucleated adrenal results in part from a regenerative response.     

Effects of the hypocholesterolemic drug, 4-aminopyrazolo (3,4-d) pyrimidine (4-APP), on the hamster adrenal cortex. An ultrastructural and functional study.

The aim of this study was to gain insight into the effects of 4-aminopyrazolo(3,4-d)pyrimidine (4-APP), a hypocholesterolemic drug, on the adrenal cortex of the hamster, representing an animal species in which steroidogenesis primarily relies on utilization of cholesterol synthesized de novo in the gland. 4-APP administration (1.5 mg/animal day for 3 days) to intact or dexamethasone-suppressed hamsters resulted in a marked proliferation of adrenocortical cells. However, the volume of parenchymal cells was unchanged in intact animals and lowered in the zona glomerulosa (ZG) and zona reticularis (ZR) of dexamethasone-administered hamsters. In both groups of animals, 4-APP strikingly increased the volume of the lipid-droplet compartment and markedly reduced the surface area of smooth endoplasmic reticulum in ZF cells, without significantly affecting the volume of the mitochondrial compartment and the surface area of mitochondrial cristae. These morphologic changes displayed no evident correlation with adrenal cortisol content and secretion. Since most of the 4-APP-induced changes were not prevented by dexamethasone, it seems legitimate to suggest that they could mainly depend on a direct effect of 4-APP on the hamster adrenocortical cells.     

Compensatory hypertrophy of the adrenal glands following unilateral adrenalectomy in juvenile rats

Left-handed adrenalectomy in rats aged 1, 5, 10, 15, 20 and 30 days produces compensatory hypertrophy of the remaining gland. Hypertrophy of the right adrenal is unmarked and variable depending on the time elapsed after operation (1, 3, 5, 7, 14, 21 and 30 days). The index of hypertrophy as regards one right control adrenal does not exceed 133.6%, whereas that as regards both control adrenals 66.2%. Zona fasciculata reticularis plays the main role in the increase of the dimension and weight of the hypertrophied gland. The body weight of operated rats is lower as compared to control, the weight lag being inversely proportional to the animal's age at the moment of operation.     

Effect of striatum destruction on the hypothalamo-pituitary-adrenal axis function in adrenalectomized rats

The influence of the bilateral striatectomy on activation of the hypothalamus-pituitary-adrenal axis after unilateral adrenalectomy was investigated. Increasing of the intact adrenal gland's mass in striatectomized rats was less than in the control group. It was combined with decreasing of the production of corticosteroids by intact gland. Such disturbances were more obvious in rats with destruction of the dorsal striatum than the ventral one. These facts suggest the striatum participation, its dorsal zone in particular, in the processes of regulation of the hypothalamus-pituitary-adrenal axis.