Adrenocortical Cell Proliferation Research Articles

Adrenocorticotropin Induction of Stress-activated Protein Kinase in the Adrenal Cortex in Vivo

A broad array of stressors induce ACTH release from the anterior pituitary, with consequent stimulation of the adrenal cortex and release of glucocorticoids critical for survival of the animal. ACTH stimulates adrenocortical gene expression in vivo and inhibits adrenocortical cell proliferation. Binding of ACTH to its G-protein-coupled receptor stimulates the production of cAMP and activation of the protein kinase A pathway. The stress-activated protein kinases (SAPKs) (or c-Jun N-terminal kinases) and the extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase family of serine/threonine kinases, which have recently been implicated in G-protein-coupled receptor intracellular signaling. The SAPKs are preferentially induced by osmotic stress and UV light, whereas the ERKs are preferentially induced by growth factors and proliferative signals in cultured cells. In these studies, ACTH stimulated SAPK activity 3-4-fold both in the adrenal cortex in vivo and in the Y1 adrenocortical cell line. 12-O-Tetradecanoylphorbol-13-acetate but not cAMP induced SAPK activity in Y1 cells. The isoquinolinesulfonamide inhibitors H-8 and H-89 blocked ACTH induction of SAPK activity at protein kinase C inhibitory doses but not at protein kinase A inhibitory doses. The calcium chelating agent EGTA inhibited ACTH-induced SAPK activity and the calcium ionophore A23187 induced SAPK activity 3-fold. In contrast with the induction of SAPK by ACTH, ERK activity was inhibited in the adrenal cortex in vivo and in Y1 adrenal cells. Together these findings suggest that ACTH induces SAPK activity through a PKC and Ca+2-dependent pathway. The induction of SAPK and inhibition of ERK by ACTH in vivo may preferentially regulate target genes involved in the adrenocortical stress responses in the whole animal.

Sympathetic effects on the steroidogenesis and proliferation of adrenocortical cellsin vitro.

Recent evidence supports the hypothesis that sympathetic neurons play a significant role in the regulation of adrenocortical cell proliferation and steroidogenesis. Co-cultures of rat adrenocortical and sympathetic ganglion cells have been established to study sympatho-adrenal interactions. In these studies we have compared differentiation, growth and secretion of adrenocortical cells grown in co-culture with those grown alone. Adrenocortical cells were identified using 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) histochemistry or immunocytochemistry; sympathetic neurons were identified using immunocytochemical localization of tyrosine hydroxylase. The sympathetic neurons usually form clusters of 3-10 cells, and extend neurites to adrenocortical cells. Adrenocortical cells continue to proliferate, express 3 beta HSD and sequester lipid droplets. In the co-cultures, the adrenocortical cells are smaller, form larger clusters and show denser 3 beta HSD staining than the adrenocortical cells alone. The presence of the sympathetic neurons enhances adrenocortical cell proliferation as shown by a 2 fold increase in the number of 3 beta HSD(+) cells as well as increased BrdU incorporation after 48 hrs. Steroidogenesis appeared to be enhanced in the presence of sympathetic neurons as demonstrated by 3 beta HSD(+) staining and a 2-fold greater corticosterone and aldosterone secretion. However, when secretion is expressed per number of adrenocortical cells, the rates are comparable, indicating that secretion rate per cell remains unaltered by the presence of neurons. The effects of sympathetic neuron activation on adrenocortical cells remain to be determined.

Effects of TRH and TRH-like peptide pGLU-HIS-GLY-NH2 on adrenocortical cell proliferation in rats

The effects of three peptides-colon mitosis inhibitor (CMI), its amide, and thyroliberin (TRH)-on the adrenocortical cell proliferation were studied. As an index of cell proliferation, the incorporation of bromodeoxyuridine (BrDU) into adrenocortical cell nuclei was used. It was found that: Twelve hours after the injection, only CMI-amide significantly increased the total number of BrDU-immunopositive cell nuclei per equatorial section of the adrenal cortex. This mitogenic effect was observed separately for the entire section, zona glomerulosa, and zona fasciculata. TRH stimulated the cell proliferation of the adrenal cortex 24 h after the injection. Its mitogenic effect was observed for entire section and zona glomerulosa. These findings suggest that these two related peptides, TRH and CMI-amide, should be considered as growth factors for the adrenal cortex.

Evidence for fibroblast growth factor mediation of compensatory adrenocortical proliferation.

The role of basic fibroblast growth factor (bFGF) in the neurally mediated control of compensatory adrenocortical cell proliferation which occurs in response to unilateral adrenalectomy has been investigated. Three isoforms of bFGF have been identified in the rat adrenal with Western blots and bFGF immunoreactivity is most concentrated in the glomerulosa cells. A high affinity binding site (Kd = 10 pM) was identified in primary cultures of rat glomerulosa cells. Using autoradiography of 125I-bFGF binding, in vivo bFGF binding sites were found concentrated in the glomerulosa as well as the capsule cells. The compensatory adrenocortical proliferation was blocked by suramin and bFGF receptor density appeared to be regulated during this proliferation. These results support a role for bFGF in autocrine and paracrine stimulation of proliferation in the adrenal cortex and capsule. To specifically block the receptor-mediated effect of bFGF in this response, we have developed an antisense strategy. Antisense oligodeoxynucleotide targeted against bFGF-receptor mRNA blocks the proliferative effect of bFGF in primary glomerulosa cell cultures by approximately 50%. These results indicate that this antisense strategy interferes with the expression of bFGF-receptors and is an effective technique to reduce the proliferative effect of bFGF via the effect on its receptor.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals.

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.

Proliferogenic effect of neurotensin (NT) and neuromedin-N (NMN) on the rat adrenal cortex: Evidence that angiotensin-II mediates the effect of NMN, but not of NT

NT and NMN, two biologically active peptides acting via the same specific receptor, are both able to stimulate in vivo the proliferative activity of rat adrenocortical cells. The present study aimed to investigate whether the mechanism underlying this effect of NT and NMN may involve an enhanced production of angiotensin-II (ANG-II), a potent adrenocortical proliferogenic factor. Metaphases per adrenal section were counted 120 min after vincristin injection. A bolus administration of ANG-II resulted in a marked increase in the number of metaphase-arrested cells 12, 24 and 48 h after the beginning of the experiment; the concomitant administration of saralasin (SAR), a competitive antagonist of ANG-II, completely blocked the proliferogenic efect of ANG-II. NT-evoked rise in the number of metaphases occurred 48 h after administration and was not influenced by the simultaneous SAR injection. On the contrary, NMN injection induced a burst of adrenocortical cell proliferation within 12 h, and this effect was prevented by SAR. These data suggest that ANG-II mediates the proliferogenic effect of NMN, but not that of NT.

Basic fibroblast growth factor and its receptor messenger ribonucleic acids are expressed in human ovarian epithelial neoplasms

Our purpose was to determine whether basic fibroblast growth factor is present in, and synthesized by, human ovarian epithelial neoplasms and to evaluate the expression of gene for the basic fibroblast growth factor receptor. The synthesis of basic fibroblast growth factor and its receptor was investigated in seven primary human ovarian epithelial neoplasms. Neoplastic tissues were homogenized and the cytoplasmic extracts purified by heparin-sepharose chromatography with a linear salt gradient of 0.6 to 3 mol/L sodium chloride in Tris-hydrochloric acid. The in situ synthesis of basic fibroblast growth factor and its receptor was demonstrated by polymerase chain reaction. Total ribonucleic acid was reverse transcribed and then amplified with two oligonucleotide primers specific for the bovine and human basic fibroblast growth factor gene and its human receptor gene. As assessed by both bioassay and radioimmunoassay a peak of basic fibroblast growth factor-like activity was present in all tumors in the chromatographic fractions eluted with 3 mol/L sodium chloride. The mitogenic effect on bovine adrenocortical endothelial cell proliferation varied from 35% to 153% above control cultures. Levels of basic fibroblast growth factor-like immunoreactivity were between 4 and 33 ng/ml. Qualitatively similar results were obtained after purifying the cytoplasmic extract of dispersed human ovarian tumor cells. The mitogenic effect was completely abolished by a specific neutralizing anti-basic fibroblast growth factor antibody. Single major deoxyribonucleic acid bands of the expected size (354 and 661 bp) were detected in all tumors studied. The identify of this material with the human basic fibroblast growth factor sequence was confirmed by restriction enzyme analysis. These data demonstrate that both basic fibroblast growth factor and its receptor are present in and synthesized by human ovarian tumor cells. Thus basic fibroblast growth factor might stimulate their abnormal proliferation through an autocrine mechanism.

Effect of nilvadipine on adrenocortical cell proliferation and steroidogenesis in early stage of adrenal regeneration.

The influence of nilvadipine, a novel calcium channel antagonist on adrenal regeneration was investigated in male Wistar rats. The estimation of the mitotic index after administration of colchicine was supplemented by an immunoperoxidase technique with monoclonal antibodies to bromodeoxyuridine. Plasma corticosterone was determined by a radioimmunological assay. Nilvadipine was given subcutaneously in two doses (0.2 and 1 mg/kg) to animals subjected to adrenal enucleation combined with contralateral adrenalectomy. It was found that nilvadipine inhibited the proliferation ratio of adrenocortical cells on the 4th day after surgery in a dose-dependent manner. The inhibitory effect of nilvadipine became less significant by 8 days after operation. However, no changes in corticosterone secretion were observed.

Oxygen availability as a regulatory factor of androgen synthesis by adrenocortical cells.

Molecular oxygen is an obligatory substrate of all cytochrome P-450 (cyt P-450) hydroxylases involved in the steroid biosynthetic pathways. However, oxygen-derived free radicals are highly destructive species resulting from cyt P-450-catalyzed steroid hydroxylation reactions. Cells in culture are usually exposed to an atmospheric pO2 that is well above the estimated in situ pO2 in vivo. It has been suggested that lowering pO2 might prevent the loss of biosynthetic enzymatic activities in bovine adrenocortical or Leydig cells in culture. The present study was performed to examine the effect of low pO2 pressure (1% oxygen in the gas phase) compared to routinely employed conditions (19% oxygen) on bovine adrenocortical cell steroidogenic activities under both basal and stimulated conditions. Lowering the pO2 showed no significant effect on the cultured adrenocortical cell proliferation rate or their ability to produce cortisol. By contrast, it resulted in a dramatic drop in androgen secretion and a slight increase in corticosterone synthesis. The mechanism involved in the qualitative modulation of steroid biosynthesis by oxygen availability was examined in some detail using purified steroid hydroxylase components. We found that cyt P-450(17 alpha, which can catalyze both the steroid 17 alpha-hydroxylation and the 17-20-lyase reaction is probably the major target explaining the oxygen effect. Indeed, cyt P-450(17 alpha) hydroxylase activity exhibits a clearly higher affinity for oxygen (Km, 22 microM) than its lyase activity (Km, 66 microM). These observations suggest that 1) oxygen availability is able to modulate the balance between androgen and corticosteroid pathways in bovine adrenocortical cell; and 2) adrenocortical cell functions studied in vitro under relatively high pO2 do not exactly reflect the in vivo situation.

Oxygen availability as a regulatory factor of androgen synthesis by adrenocortical cells

Molecular oxygen is an obligatory substrate of all cytochrome P-450 (cyt P-450) hydroxylases involved in the steroid biosynthetic pathways. However, oxygen-derived free radicals are highly destructive species resulting from cyt P-450-catalyzed steroid hydroxylation reactions. Cells in culture are usually exposed to an atmospheric pO2 that is well above the estimated in situ pO2 in vivo. It has been suggested that lowering pO2 might prevent the loss of biosynthetic enzymatic activities in bovine adrenocortical or Leydig cells in culture. The present study was performed to examine the effect of low pO2 pressure (1% oxygen in the gas phase) compared to routinely employed conditions (19% oxygen) on bovine adrenocortical cell steroidogenic activities under both basal and stimulated conditions. Lowering the pO2 showed no significant effect on the cultured adrenocortical cell proliferation rate or their ability to produce cortisol. By contrast, it resulted in a dramatic drop in androgen secretion and a slight increase in corticosterone synthesis. The mechanism involved in the qualitative modulation of steroid biosynthesis by oxygen availability was examined in some detail using purified steroid hydroxylase components. We found that cyt P-450(17 alpha, which can catalyze both the steroid 17 alpha-hydroxylation and the 17-20-lyase reaction is probably the major target explaining the oxygen effect. Indeed, cyt P-450(17 alpha) hydroxylase activity exhibits a clearly higher affinity for oxygen (Km, 22 microM) than its lyase activity (Km, 66 microM). These observations suggest that 1) oxygen availability is able to modulate the balance between androgen and corticosteroid pathways in bovine adrenocortical cell; and 2) adrenocortical cell functions studied in vitro under relatively high pO2 do not exactly reflect the in vivo situation.

The effects of suramin on human adrenocortical cells in vitro: Suramin inhibits cortisol secretion and adrenocortical cell growth

Suramin, a polycyclic and polyanionic drug, has been successfully used in the therapy of inoperable adrenocortical cancer. The present study was undertaken to investigate the effects of suramin on normal human adrenocortical cells in primary monolayer cultures. The proliferation and the basal, as well as the adrenocorticotropin (ACTH)-stimulated, cortisol secretion of these cells were studied. The data show that suramin decreases basal, as well as ACTH-stimulated, cortisol secretion in a dose-dependent manner ( P < .05 from 300 μmol/L upward). At a suramin concentration of 3 mmol/L, cortisol secretion was inhibited by 70% ± 4% in ACTH-stimulated cells and by 42% ± 6% in unstimulated cells. The proliferation of adrenocortical cells in response to fetal calf serum was also inhibited by suramin at concentrations from 300 μmol/L upward, maximal suppression (71% ± 6%, P < .01) being observed at a concentration of 10 mmol/L. Both inhibition of cortisol secretion and inhibition of adrenocortical cell proliferation were not due to toxicity of the compound, as could be shown by restimulation of cortisol secretion in suramin-treated cells with ACTH. Our results indicate that suramin exerts an inhibitory influence on the cortisol secretion and on the proliferation of normal human adrenocortical cells. Suramin may not only be useful in the treatment of adrenocortical cancer, but may also have an ameliorative effect on other malignant conditions with augmented steroid hormone production, resistant to conventional forms of therapy.

Indomethacin inhibits the basal and angiotensin II-stimulated mitotic activity of adrenocortical cells in vivo and in organ culture.

The aim of the experiment in vivo was to examine the effect of a 3-day peroral administration of indomethacin (IND: 4.0-4.7 mg/kg b.wt./24 h) on the adrenocortical cell proliferation in rats in basal conditions and after a single i.p. injection of angiotensin II (ANG: 100 micrograms per rat). The in vitro study was to test the effect of IND on the mitotic incidence in adrenocortical cells or organ-cultured rat adrenal explants during a 24-h incubation with or without ANG. It was shown that IND significantly decreased the mean mitotic activity rate (MMAR) of adrenocortical cells in vivo as well as in organ culture. That decrease concerned all the three cortical zones vs respective controls. In turn, both in vivo and in vitro ANG markedly increased the MMAR of the zona glomerulosa cells as compared to the values recorded in controls, while there were no changes of the MMAR of the zona glomerulosa after ANG in the groups receiving IND. The results obtained indicate that IND exhibits an inhibitory effect on the adrenocortical cell proliferation which may suggest that prostaglandins (PGs) play an important role in the process of adrenocortical hyperplasia, their participation in the ANG-induced zona glomerulosa cell proliferation being strongly assumed.

Melatonin inhibits mitotic activity of adrenocortical cells in vivo and in organ culture.

The goal of this study was to examine the effects of melatonin, as well as those of melatonin and corticotropin (1-24 adrenocorticotrophic hormone (ACTH); Synacthen Depot) administered together, on the mitotic activity of adrenocortical cells in male and female mice. Melatonin was given subcutaneously once daily, in late-afternoon injections (between 16:00 and 18:00) in doses of 1 microgram, 10 micrograms, and 100 micrograms, and ACTH in a dose of 0.1 mg (10 U) daily for 10 days. Additionally, the highest dose of melatonin (100 micrograms daily) was administered together with ACTH. The metaphase-arrest technique using colchicine as a stathmokinetic agent was employed in the study. Melatonin, in all the examined doses, significantly decreased mean mitotic activity rate (MMAR) of the adrenal cortex in both male and female mice. Moreover, in a dose of 100 micrograms, melatonin suppressed the mitogenic effect of ACTH on the adrenal cortex. Furthermore, the present study investigated the effects of melatonin (5 x 10(-7)M), N-acetylserotonin (NAc-5HT) (5 x 10(-7)M), and ACTH (250 mU/ml or 1,000 mU/ml) alone as well as the effect of ACTH (250 mU/ml) applied jointly with melatonin on the mitotic activity of adrenocortical cells in rat adrenal explants incubated in vitro. Both pineal indoleamines (melatonin and NAc-5HT) significantly decreased the MMARs of adrenocortical cells. Corticotropin, as well as ACTH and melatonin applied together, also reduced the MMAR of adrenocortical cells. The present data suggest that melatonin may be directly involved in the inhibitory control of adrenocortical cell proliferation.

Intraventricular 6-hydroxydopamine increases the adrenal cortex mitotic activity in rats.

Effects of intraventricular injections of 6-hydroxydopamine (6-OHDA) on adrenocortical cell proliferation in rats have been investigated by means of the colchicine metaphase-arrest technique. In the group of animals receiving 6-OHDA alone, an increase of mean mitotic activity rate (MMAR) was observed at 96 h and 144 h after injection. This rise of MMAR was completely inhibited by pretreatment of animals with desmethylimipramine (DMI) - a blocker of norepinephrine uptake. It is concluded that enhancement of ACTH secretion is responsible for the above mentioned increase of mitotic activity after 6-OHDA administration. This phenomenon is related to abolition of the inhibitory noradrenergic effect on ACTH secretion by 6-OHDA treatment. Additionally, the results suggest little or no involvement of dopaminergic neurons in early changes of ACTH secretion after intraventricular 6-OHDA.

Hormonal Control of Adrenocortical Cell Proliferation

A primary bovine adrenocortical cell culture system responsive to physiological concentrations of ACTH has been established. When added to cultures, ACTH inhibited DNA synthesis and cell division over the same concentration range required for stimulation of fluorogenic steroid production (0.01-10 nM). With chronic exposure to ACTH, cells became desensitized to the growth inhibitory effects of ACTH. Though cell growth was initially completely inhibited by ACTH, cells ultimately began to grow in its continued presence. The lag time to initiation of cell growth, the rate of growth, and the final density achieved depended on the ACTH concentration. Desensitization to ACTH(1-39) was not induced by monobutyryl cyclic AMP nor by ACTH(11-24). Specificity of desensitization was apparent because cells which had become desensitized to ACTH(1-39) remained fully responsive to monobutyryl cyclic AMP, prostaglandin E(1), and cholera toxin. Though the effects of ACTH on cell growth were readily reversible upon hormone removal, the desensitized response to readdition of ACTH persisted for at least 8 h. Fibroblast growth factor (FGF) stimulated both the growth rate and saturation density achieved. FGF did not alter the growth inhibitory effects of ACTH nor the reduced growth rate observed in desensitized cells maintained in ACTH. However, FGF greatly increased the saturation density achieved by cultures maintained with ACTH. Through the process of desensitization, adrenocortical cells are able to grow in the presence of high concentrations of ACTH and to respond to the effects of a growth factor by increasing the cell density achieved. This pattern of response may be a general one for growth control under the combined effects of antimitotic and mitotic factors.

Effect of dexamethasone on cell population kinetics in the adrenal cortex of the prepubertal male rat.

SUMMARY The effect of a single injection of dexamethasone on adrenocortical cell proliferation was studied in prepubertal male rats using tritiated thymidine. After a short latent period, all zones of the adrenal cortex showed a rapid decrease in both labelling and mitotic indices. After a prolonged period when very low indices were apparent, there was a rapid rise in both proliferative indices with most zones showing a considerable increase above control values. A more detailed study of the initial depression showed that after a latent period of about 5 h the labelling index fell approximately 8 h before the mitotic index. This differential response in the labelling and mitotic indices was consistent with a block in the cell cycle late in the pre-DNA synthetic interval of the cell cycle (G1), with cells being prevented from entering DNA synthesis. This hypothesis was also supported by an experiment involving continuous labelling of control and dexamethasone-treated animals; again after a latent period of 5–6 h, the rate of increase of the continuous labelling index fell as cells became blocked in late G1. By analogy with other tissues, results are interpreted in terms of a direct action of dexamethasone on adrenocortical cells; this steroid-sensitive step in the cell cycle may be important in the control of growth in the adrenal cortex.

A dexamethasone sensitive step in the cell cycle of adrenocortical cells.

Corticotrophin (ACTH) has been shown to increase adrenocortical DNA content (Bransome, 1968) and to stimulate adrenal DNA polymerase and thymidine kinase activity (Masui & Garren, 1970). Bransome (1968) has shown that dexamethasone also produces a significant decrease in adrenocortical DNA. It is therefore surprising that a recent report by Ueberberg, Stöcker & Städtler (1970) concluded that cell proliferation is not influenced by dexamethasone treatment. The present results indicate that in prepubertal rats dexamethasone has a profound inhibitory effect or adrenocortical cell proliferation by acting on a particular point in the cell cycle. Male Wistar rats aged 14 days were given a single i.p. injection of 3 μg dexamethasone phosphate (Merck, Sharp and Dohme)/g body weight. Controls received normal saline. Animals were killed serially thereafter. Tritiated thymidine ([3H]Tdr) was administered 1 h before death, and autoradiographs of the adrenal glands prepared as previously described (Wright, 1971). The adrenal cortex was