A virilizing adrenocortical tumor in a female infant: in vivo and in vitro biochemical characteristics.

Normal adrenal and adrenal tumor cells from a female infant with a virilizing adrenal tumor were grown in tissue culture as monolayers for a period of 7 weeks. Half of the cultures were exposed to ACTH (0.1 U/ml). The cells grew well and continued to produce steroid hormones over the entire period in culture. Production of steroid hormones was measured by RIA of individual steroids in the culture medium. Unstimulated normal and tumor cells produced equivalent amounts of cortisol, 11 beta, 18, 21-trihydroxy-4-pregnene-3,20-dione, and 18,21-dihydroxy-4-pregnene-3,20-dione, but tumor cells produced lesser amounts of dehydroepiandrosterone (DHA), androstenedione, testosterone, and progesterone. Normal cells exposed to ACTH showed an increase in all steroids measured, whereas ACTH-exposed tumor cells showed an increase principally in DHA consistent with a deficiency in 3 beta-hydroxysteroid dehydrogenase activity. Circulating levels of DHA, androstenedione, and testosterone were elevated in the patient before removal of the adrenal tumor. The production of androgens by tumor cells in vitro resembled the pattern of circulating steroids in vivo. These studies demonstrate that tissue culture of human adrenal cells provides a means both to determine their biochemical characteristics and to investigate their responses to exogenous hormones.     

Comparative effects of prolactin versus ACTH, estradiol, progesterone, testosterone, and dihydrotestosterone on cortisol release and proliferation of the adrenocortical carcinoma cell line H295R

In this study, using the H295R cell line as a model system, we investigated the role of prolactin (PRL) and steroid hormones in the growth regulation and cortisol release of adrenocortical cells. H295R cells were treated with increasing doses (10(-13)-10(-6) M) of PRL, adrenocorticotropic hormone (ACTH), 17beta-estradiol (E(2)), progesterone (P(4)), testosterone (T), and dihydrotestosterone (DHT). As expected, ACTH raised cortisol secretion and increased the proliferation rate of cultured cells. Incubation with T, DHT, E(2), and P(4) for 24 h did not significantly increase cortisol release. Conversely, PRL concentrations of 10(-8)-10(-6) M caused a significant increase in the release of cortisol. Long-term (5 days) stimulation of H295R cells with E(2), P(4), and PRL was a trigger to increased cell proliferation, while T and DHT did not alter H295R cell proliferation. Taken together, these results indicate that steroid hormones exert differential effects on adrenocortical function. Additionally, the present study demonstrates that PRL had biphasic actions in regulating adrenocortical function. PRL may form a novel regulatory system for steroid hormone secretion and cell proliferation in the adrenal cortex.     

Prolactin has a direct effect on adrenal androgen secretion

The role of PRL in the secretion of androgens by the adrenal glands was investigated in vivo and in vitro. In women with hyperprolactinemia whose pituitary-adrenal function was normal, there was significant correlation between serum PRL and dehydroepiandrosterone sulfate [(DHEA-S) gamma = 0.48, P less than 0.05, n = 34] and DHEA (gamma = 0.50, P less than 0.05, n = 34), but not with androstenedione. Long term administration of sulpiride to normal women increased both serum PRL and DHEA-S, whereas acute elevation of PRL after a single iv dose of domperidone had no influence on the serum DHEA-S levels. Monolayer cultures of human adrenal cells were used in order to study the direct effect of PRL on adrenal androgen secretion. The daily secretion of DHEA-S, DHEA, androstenedione, and cortisol was determined. In the absence of ACTH, PRL had no effect on steroid secretion in a 7-day culture period. In the presence of ACTH, there was a daily increase in the secretion of steroids. PRL, when added in combination with ACTH, potentiated the effect of ACTH on DHEA-S and DHEA but not on androstenedione and cortisol secretion on the seventh day in culture. These results indicate that PRL has a direct synergistic effect with ACTH on adrenal cells to increase adrenal androgen release. Increases in DHEA-S and DHEA but not androstenedione in vitro and correlation between serum PRL and DHEA-S and DHEA but not androstenedione in women with hyperprolactinemia suggest that the synergistic effect of PRL on adrenal androgen secretion may result from partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase.     

Output of oestrogens, testosterone and their precursors by isolated human adrenal cells as compared with that of glucocorticosteroids.

The output of oestrogens, testosterone and their precursors was compared with that of glucocorticosteroids under standardized conditions, in a suspension of isolated human adrenal cells. Cortisol, corticosterone, androstenedione, dehydroepiandrosterone and its sulphate all increased in the same proportions after ACTH stimulation. The response to the logarithm of ACTH concentrations had a sigmoid shape but was fairly linear between 5 and 100 to 1000 muu./ml. The output of dehydroepiandrosterone plus that of its sulphate was of the same order of magnitude as the production of cortisol; the output of free dehydroepiandrosterone averaged half that of the sulphate indicating that the adrenal cortex is capable, under certain conditions, of producing large amounts of the free steroid. The output of androstenedione was very low, on average 35 times lower than that of cortisol, suggesting by extrapolation that the adrenal secretion may not be the main source of androstenedione in vivo or that ACTH is not the unique stimulus to adrenal androstenedione secretion. The output of testosterone was small to negligible and that of oestrogens was practically absent. In three additional experiments the influence of prolactin, prostaglandins, FSH and HCG was explored: no selective stimulation of androgen or oestrogen output was observed except in one experiment in which HCG stimulated adrenal testosterone production.     

Inhibition of Src tyrosine kinase stimulates adrenal androgen production

A unique characteristic of the primate adrenal is the ability to produce 19-carbon steroids, often called the adrenal androgens. Although it is clear that the major human adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), are produced almost solely in the adrenal reticularis, the mechanisms regulating production are poorly understood. Herein, we tested the hypothesis that the Src family of tyrosine kinases are involved in the regulation of adrenal androgen production. The NCI-H295R human adrenal cell line and primary human adrenal cells in culture were used to study adrenal androgen production and expression of enzymes involved in steroidogenesis. To examine the role of Src tyrosine kinase, cells were treated with PP2, a specific Src inhibitor. Alternatively, adrenal cells were transfected with an expression vector containing a dominant-negative form of Src. PP2 treatment inhibited basal cortisol production while significantly increasing the production of DHEA and DHEA-S (together referred to as DHEA(S)) in both adrenal cell models. The effect of PP2 on steroidogenesis occurred along with a rapid induction of steroidogenic acute regulatory (StAR) protein synthesis as revealed by Western analysis. Treatment with PP2 also increased mRNA levels for StAR, and cholesterol side-chain cleavage (CYP11A) and 17alpha-hydroxylase/17,20-lyase (CYP17) enzymes. Treatment of adrenal cells with the cAMP agonist dibutyryladenosine cyclic monophosphate (dbcAMP), stimulated the production of cortisol and DHEA(S). However, treatment of adrenal cells with a combination of PP2 and dbcAMP enhanced the production of DHEA(S) while inhibiting cortisol production. During dbcAMP treatment PP2 was able to augment the expression of CYP17 and to inhibit the induction of 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) levels. Increasing the CYP17 to HSD3B2 ratio is likely to promote the use of steroid precursors for the production of DHEA(S) and not for cortisol. Taken together these data suggest that the inhibition of Src tyrosine kinases causes adrenal cells to adopt a reticularis phenotype both by the production of DHEA(S) and by the steroidogenic enzymes expressed.     

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.     

Decreased adrenal androgen sensitivity to ACTH during aging

During the human aging process, basal plasma levels of cortisol and aldosterone demonstrate little change, while concentrations of adrenal androgens (AA) such as dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and androstenedione (A) decrease dramatically in men and women. There is no age-related change in ACTH concentrations to explain this observation. In this study, ACTH (cosyntropin, alpha1-24 corticotropin) stimulation tests were performed on elderly subjects and controls to test the hypothesis that analogous to the aging testicular. Leydig cell, AA-producing cells of the aging adrenal gland may become less sensitive to physiological levels of ACTH, but still retain their sensitivity to elevated ACTH levels. Results showed that in the elderly subjects, basal levels and ACTH-stimulatability of cortisol and aldosterone were unchanged. In agreement with earlier studies, basal levels of AA were found to be decreased in the elderly. However, ACTH stimulation demonstrated impaired reserve in DHA and A, and a total lack of stimulatability of DHAS. These findings could be explained by an age-related loss of adrenal enzymes or cell populations which produce AA, a loss or decrease in a subpopulation of ACTH receptors specific for AA production, or loss of a pituitary factor necessary for AA secretion.     

The NCI-H295 cell line: a pluripotent model for human adrenocortical studies

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones. Indeed, the lack of a human adrenocortical cell line retaining the ability to produce any of the major adrenal steroid products has slowed studies on normal and abnormal adrenal function. This obstacle has now been largely overcome with the availability of H295 cells, which represents the first adrenocortical cell line to maintain the ability, under specified conditions, to produce all the adrenocortical steroids (i.e., mineralocorticoids, glucocorticoids, and adrenal androgens). Thus, H295 cells appear to act as pluripotent adrenocortical cells capable of being directed to produce each of the zone-specific steroids. The H295 cell line should prove to be of value in studying the molecular and biochemical mechanisms controlling adrenal Steroidogenesis.     

The control of steroidogenesis by human fetal adrenal cells in tissue culture. I. Responses to adrenocorticotropin

A technique of monolayer tissue culture of human fetal adrenal cells was developed in order to study steroidogenic responses to factors such as ACTH. The daily production of 12 steroids [pregnenolone, 17-hydroxy pregnenolone, dehydroepiandrosterone (DHA), DHA sulfate, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, corticosterone, 11-desoxycortisol, cortisol, and aldosterone) was measured by RIA. Initially, fresh fetal adrenal cells produced DHA, DHA sulfate, 17-hydroxypregnenolone, and small amounts of cortisol, but in the absence of ACTH, the production of all steroids declined during culture to low levels. The addition of physiological amounts (1-10(4) pg/ml) of either alpha ACTH-1(1-24) or alpha ACTH-(1-39) or coculture with fetal pituitary cells elicited a progressive rise in steroid production during the first 4-6 days of incubation. The lowest ACTH doses elicited a proportionately greater adrenal androgen response (as reflected in the DHA to cortisol ratio), but with increasing ACTH dosage, there was greater stimulation of cortisol production, which equalled or exceeded that of DHA. The data demonstrate that fetal adrenal cells may be maintained in short term culture and can respond to physiological amounts of ACTH. The progressive increase in the production of cortisol and other delta 4, 3-ketosteroids in vitro suggests that the characteristic fetal pattern of steroidogenesis may result from the interaction of ACTH with some circulating inhibitor of adrenal 3 beta-hydroxysteroid dehydrogenase.     

Tumour-derived human adrenocortical cells express beta-adrenergic receptors: steroidogenic effects of beta-adrenergic input

It is well established that catecholamines have potent actions on adrenocortical function and steroidogenesis in different species. The effect of these substances on steroid production of the human adrenal cell line H295R is the subject of this study. H295R cells were cultured in the presence of the synthetic catecholamine, isoproterenol for four hours. Aldosterone, cortisol, and DHEA secretion was measured using direct radioimmunoassays. Administration of 10(-11)-10(-7) mol/L isoproterenol produced a dose-dependent increase in secretion of aldosterone, cortisol, and DHEA by H295R cells resulting in 3-fold, 2.5-fold, and 2-fold stimulation respectively, relative to basal values. Analysis of mRNA using nested PCR revealed the presence of all three types of beta-adrenergic receptors namely beta1, beta2, and beta3 in H295R cells. Isoproterenol had no effect on the proliferation rate of H295R cells as determined by 3H-incorporation assay and the colorimetric WST-1 cell proliferation assay.     

Control of adrenal androgen production

The major adrenal androgens are dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS) and androstenedione (delta 4). Studies by Cutler et al in 1978 demonstrated that these androgens are detectable in blood of all domestic and laboratory animals studied, but that only 4 species show increase in one or more with sexual maturation: rabbit, dog, chimpanzee and man. Studies by Grover and Odell in 1975 show these androgens do not bind to the androgen receptor obtained from rat prostate and thus probably are androgens only by conversion to an active androgen in vivo. Thomas and Oake in 1974 showed human skin converted DHEA to testosterone. The control of adrenal androgen secretion is in part modulated by ACTH. However, other factors or hormones must exist also, for a variety of clinical observations show dissociation in adrenal androgen versus cortisol secretion. Other substances that have been said to be controllers of adrenal androgen secretion include estrogens, prolactin, growth hormone, gonadotropins and lipotropin. None of these appear to be the usual physiological modulator, although under some circumstances each may increase androgen production. Studies from our laboratory using in vivo experiments in the castrate dog and published in 1979 indicated that crude extracts of bovine pituitary contained a substance that either modified ACTH stimulation of adrenal androgen secretion, or stimulated secretion itself - Cortisol Androgen Stimulating Hormone. Parker et al in 1983 showed a 60,000 MW glycoprotein was extractable from human pituitaries, which stimulated DHA secretion by dispersed canine adrenal cells in vitro, but did not stimulate cortisol secretion. This material contained no ACTH by radioimmunoassay. In 1982 Brubaker et al reported a substance was also present in human fetal pituitaries, which stimulated DHA secretion, but did not effect cortisol.     

Evidence for reduced 3 beta-ol-hydroxysteroid dehydrogenase activity in some hirsute women thought to have polycystic ovary syndrome

Congenital adrenal hyperplasia due to 3 beta-ol-hydroxysteroid dehydrogenase (3 beta-ol) deficiency is usually lethal. A partial deficiency in 3 beta-ol has been suggested in some women presenting with androgen excess. In this study, 24 women were investigated who had hirsutism and oligomenorrhea and high serum delta 5 androgens compared to delta 4 androgens. Of these women, 9 had significantly elevated 17-hydroxypregnenolone (17 Preg) to 17-hydroxyprogesterone (17 Prog) ratios when compared to controls under basal conditions. On further testing of 9 women with ACTH, 4 had significantly elevated 17 Preg to 17 Prog ratios. Eight women had elevated ratios of dehydroepiandrosterone sulfate to androstenedione, 4 had elevations of androstenediol (Adiol) to testosterone (T), and 4 had abnormal 17 Preg to cortisol ratios. Only 3 women out of the original 24 selected for study had elevated ratios for all 4 different steroid pairs measured. 17 Prog was normal in these women with 3 beta-ol deficiency in contradistinction to the high levels normally observed in women with congenital adrenal hyperplasia due to 21- or 11-hydroxylase deficiency. It is suggested that the cause of androgen excess in these women is high circulating levels of Adiol and, in part, the slightly elevated unbound T levels in these women. In conclusion, a subtle, incomplete form of 3 beta-ol deficiency may exist in adult women and is manifest by high delta 5 androgens such as dehydroepiandrosterone sulfate and Adiol and normal delta 4 androgens such as androstenedione and T.     

Differential production of adrenal steroids by purified cells of the human adrenal cortex is relative rather than absolute

OBJECTIVES: The adrenal cortex produces aldosterone, cortisol and androgens in response to ACTH and angiotensin II. To define the differential response of morphologically distinct cells of the adrenal cortex, we examined the phenotypical and functional characteristics of human adrenocortical cells. RESULTS: Tumour growth factor-beta receptor-1 (TGFbeta-R1) and CYP-11 were found to be expressed predominantly in the zona fasciculata, whereas human leukocyte antigen (HLA-DR) and CYP-17 were localised to the zona reticularis. The angiotensin II receptor, AT-1, was found to be predominantly expressed in the zona glomerulosa. Adrenocortical cells, separated by density, yielded two distinct fractions which displayed differential growth patterns. Lipid-rich cells of fraction I expressed TGFbeta-R1 and produced significantly more cortisol relative to androstenedione than unseparated or fraction II cells, whereas lipid-poor cells of fraction II expressed HLA-DR and produced more androstenedione relative to cortisol in the presence of ACTH. Aldosterone production by fraction II was significantly greater than fraction I or unseparated cells. TGFbeta-R1-positive fasciculata-type cells separated into fraction I and HLA-DR-positive cells consistent with reticularis cells separated into fraction II. Aldosterone-producing cells indicative of glomerulosa cells separated into fraction II. CONCLUSIONS: Our findings are consistent with the concept that all adrenocortical cells are capable of producing a range of steroids, but the relative production of cortisol, androgen and aldosterone differs.     

Effect of leptin on ACTH-stimulated secretion of cortisol in rhesus macaques and on human adrenal carcinoma cells

OBJECTIVE: Because glucocorticoids stimulate leptin release and, at least in vitro, leptin inhibits cortisol secretion, a feedback system between glucocorticoids and leptin has been proposed. However, in humans and non-human primates there are no in vivo studies to support any role for leptin in the control of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of leptin on (i) ACTH-stimulated secretion of cortisol in six male rhesus monkeys and (ii) basal and forskolin (FSK)-stimulated cortisol secretion by the human adrenal carcinoma cell H295R in vitro. DESIGN AND METHODS: In vivo studies: after suppression of endogenous ACTH with either dexamethasone (n=6) or a corticotropin-releasing factor (CRF) antagonist (d-Phe CRF(12-41)) (n=3), 1 microg bolus of human ACTH(1-24) was administered to stimulate adrenal cortisol release. Blood samples were collected every 15 min for 3 h. Leptin (1 mg) was infused over 4 h, starting 1 h before ACTH bolus. In vitro studies: NCI-H295R cells were incubated for 6, 12, 24 and 48 h in the absence or presence of 20 micromol/l FSK in combination with leptin (100 ng/ml medium). Cortisol levels in serum and medium were measured by solid phase radioimmunoassay. RESULTS: Acute leptin infusion to rhesus monkeys did not change basal cortisol levels, peak cortisol levels after ACTH(1-24) or the area under the curve when compared with studies in which leptin was not given. FSK increased cortisol levels in medium at 24 and 48 h, but leptin did not change cortisol release in either control or FSK-stimulated cells. CONCLUSIONS: Short-term leptin infusion affected neither the cortisol response to ACTH in non-human primates in vivo nor cortisol release (basal or FSK stimulated) by H295R cells, in vitro. These data suggest that leptin may not be an acute regulator of primate adrenal cortisol secretion.     

Adrenal androgen hyperresponsiveness to adrenocorticotropin in women with acne and/or hirsutism: adrenal enzyme defects and exaggerated adrenarche

To determine the adrenal contribution to elevated plasma androgens in 31 young hyperandrogenemic women with acne and/or hirsutism, we compared their responses to ACTH with those of 14 normal women. Each subject was given a low dose (10 micrograms/m2) of synthetic ACTH-(1-24) (Cortrosyn) after administration of 1.5 mg dexamethasone the night before the test. Thirty and 60 min responses of plasma 17 alpha-hydroxypregnenolone (17-Preg), 17 alpha-hydroxyprogesterone, (17-prog), dehydroepiandrosterone (DHEA), androstenedione, 11-deoxycortisol, and cortisol were measured. Eighteen (58%) patients had increased responses of at least one 17-ketosteroid or adrenal androgen precursor. All patients had cortisol responses within the range of those of the 14 normal subjects. Nine patients (29%) had evidence of steroid biosynthetic enzyme deficiencies, either mild congenital adrenal hyperplasia or the heterozygote state; after ACTH, 4 of these patients had elevated 17-prog in the range of values in heterozygote carriers of 21-hydroxylase deficiency, 2 had elevated levels of 11-deoxycortisol compatible with 11 beta-hydroxylase deficiency, and 3 had elevated levels of 17-Preg and DHEA, suggestive of 3 beta-hydroxysteroid dehydrogenase deficiency. Another 9 subjects (29%) had 17-ketosteroid (DHEA and/or androstenedione) hyperresponsiveness to ACTH with associated elevated 17-Preg responses. As a group, their patterns suggested relatively deficient 3 beta-hydroxysteroid dehydrogenase and relatively hyperactive C lyase without impairment of cortisol secretion. This pattern resembles exaggerated adrenarche, and we postulate that these 9 patients have hyperplasia of the zona reticularis. Neither basal levels of plasma androgens (free testosterone and DHEA sulfate) nor menstrual history predicted which patients would have abnormal ACTH responses. Although 5 of 11 (45%) patients with acne alone had abnormal responses to ACTH, 10 of 14 patients with acne and hirsutism (71%) had abnormal responses to ACTH. We conclude that an adrenal contribution is found in about half of hyperandrogenemic women with acne and/or hirsutism. This adrenal androgen hyperresponsiveness is heterogeneous. Some patients may have mild forms of congenital adrenal hyperplasia. However, functional androgenic hyperresponsiveness to ACTH, which resembles an exaggeration of adrenarche, is the most common abnormality found. Such findings may provide an explanation for the clinical observation of exacerbations of acne with stress.     

ACTH control of steroid secretion from adrenal cells of the developing rhesus monkey (Macaca mulatta)

Cells from adrenal glands of 140-160 days foetal, neonatal and infant Rhesus monkeys (Macaca mulatta) were prepared, by collagenase digestion and incubated with 10 pg-16 ng/ml ACTH. The production of cortisol, androstenedione, corticosterone, progesterone and prostaglandins was measured. The cortisol production in the 140 day and 160 day foetuses and in the neonatal adrenal cells was comparable. It was 2-fold higher in adrenal cells of 6 month infant monkeys. In all the groups there was an increasing production of cortisol with increasing ACTH concentration, and a response to low physiological concentrations of ACTH. The androstenedione production was significantly greater in the 160 day foetuses than in either those of 140 days or of the neonate which demonstrated a poor response to increasing ACTH concentrations. It responded well to increasing ACTH in adrenal cells from 6 month infant monkeys. Corticosterone output was 1/10th of cortisol with only the 140 day foetuses showing an increase in production with increasing ACTH concentrations. The results demonstrate that cells of the primate foetal adrenal gland are not inherently unresponsive to ACTH stimulation as regards cortisol production, which per/micrograms DNA does not appear to change over the last 25 days before term.     

Inadequately low serum levels of steroid hormones in relation to interleukin-6 and tumor necrosis factor in untreated patients with early rheumatoid arthritis and reactive arthritis

OBJECTIVE: To compare levels of steroid hormones in relation to cytokines and to study levels of cortisol or dehydroepiandrosterone (DHEA) in relation to other adrenal hormones in untreated patients with early rheumatoid arthritis (RA) and reactive arthritis (ReA) compared with healthy controls. METHODS: In a retrospective study with 34 RA patients, 46 ReA patients, and 112 healthy subjects, we measured serum levels of interleukin-6 (IL-6), tumor necrosis factor (TNF), adrenocorticotropic hormone (ACTH), cortisol, 17-hydroxyprogesterone (17-OH-progesterone), androstenedione (ASD), DHEA, and DHEA sulfate (DHEAS). RESULTS: RA patients had higher serum levels of IL-6, TNF, cortisol, and DHEA compared with ReA patients and healthy subjects, but no difference was noticed with respect to ACTH and DHEAS. However, in RA and ReA patients compared with healthy subjects, levels of ACTH, cortisol, ASD, DHEAS, and 17-OH-progesterone were markedly lower in relation to levels of IL-6 and TNF. Furthermore, the number of swollen joints correlated inversely with the ratio of serum cortisol to serum IL-6 in RA (R(Rank) = -0.582, P = 0.001) and, to a lesser extent, in ReA (R(Rank) = -0.417, P = 0.011). In RA patients, the mean grip strength of both hands was positively correlated with the ratio of serum cortisol to serum IL-6 (R(Rank) = 0.472, P = 0.010). Furthermore, in these untreated patients with RA and ReA, there was a relative decrease in the secretion of 17-OH-progesterone, ASD, and DHEAS in relation to DHEA and cortisol. This indicates a relative predominance of the nonsulfated DHEA and cortisol in relation to all other measured adrenal steroid hormones in the early stages of these inflammatory diseases. CONCLUSION: This study indicates that levels of ACTH and cortisol are relatively low in relation to levels of IL-6 and TNF in untreated patients with early RA and ReA compared with healthy subjects. The study further demonstrates that there is a relative increase of DHEA and cortisol in relation to other adrenal hormones, such as DHEAS. This study emphasizes that adrenal steroid secretion is inadequately low in relation to inflammation. Although changes in hormone levels are similar in RA and ReA, alteration of steroidogenesis is more pronounced in RA patients than in ReA patients.     

Lipoprotein requirements for secretion of ultraviolet-absorbing corticosteroids by guinea pig adrenocortical cells in vitro: inner versus outer cortices; zona glomerulosa versus zona fasciculata

Most studies of lipoprotein requirements for steroid secretion by the adrenal have examined the mixed cell population of the whole gland; none have examined lipoprotein requirements of guinea pig adrenocortical cells. In this study the effect of exogenous lipoprotein on the ability of cells from each of the different regions of the guinea pig adrenal cortex to synthesize and secrete steroids has been analyzed in vitro, under baseline and ACTH-stimulated conditions. Most studies have assessed the effects of lipoprotein on one or a few selected steroids. In this study the effects of lipoprotein and ACTH were examined both by an assay for fluorogenic steroids and by HPLC analysis of the spectrum of UV-absorbing steroids. Guinea pig outer adrenocortices, containing zona glomerulosa and zona fasciculata, maintained in vitro as fragments or as isolated cells, secreted at least 5 times more steroid than the inner cortex, predominantly zona reticularis, and were dependent upon lipoproteins for their secretion. Corticosterone (B) and cortisol (F) were the predominant products of both zones. Aldosterone (Aldo), 18-hydroxycorticosterone (18-OH B), deoxycorticosterone (DOC), 11 beta-hydroxyandrostenedione (11 beta-OH And), androstenedione (And), and deoxycortisol were less abundant products of the outer cortex, while the inner cortex secreted only very small amounts of these steroids. Each of the outer cortical cell types secreted a distinct spectrum of steroids. Aldo, 18-OH B, and DOC were characteristic of glomerulosa cells, but B was most prominent. Fasciculata cells secreted primarily F, with 11 beta-OH And as their next most prominent product. Low density lipoprotein (LDL) enhanced steroid secretion by glomerulosa cells to a greater extent than that by fasciculata cells, but the stimulation of LDL utilization by ACTH was greatest for fasciculata cells. LDL and ACTH also influenced the pattern of steroids secreted by each cell type. Addition of LDL to glomerulosa cells enhanced secretion of DOC and B, but not that of Aldo or 18-OH B. In fasciculata cell cultures, LDL enhanced secretion of both F and 11 beta-OH And. ACTH, particularly in the presence of LDL, stimulated secretion by glomerulosa cells of Aldo and 18-OH B, as well as that of F, And, and 11 beta-OH And. The combined presence of ACTH and LDL in fasciculata cell cultures preferentially stimulated secretion of F and B.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of adrenocorticotrophin on cortisol and androstenedione secretion from dispersed cells of guinea-pig adrenal zonae fasciculata and reticularis

We have studied cortisol and androstenedione secretion by dispersed cells of the outer zona fasciculata (ZF) plus zona glomerulosa, and the inner zona reticularis (ZR) plus medulla of the guinea-pig adrenal. The ZF and ZR were microdissected apart, the cells dispersed and incubated (200 000 cells/ml) for 90 min in the presence of adrenocorticotrophin (ACTH; 500 ng/l), dibutyryl cyclic AMP (dbcAMP; 1 mmol/l), pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol. The steroid concentrations were 5-25 mumol/l. Cortisol secretion was assayed by radioimmunoassay. There was no detectable cortisol secretion (less than 50 nmol/l) from the ZR in the controls (no additive) or after dbcAMP stimulation. Adrenocorticotrophin-stimulated cortisol secretion was also low (range less than 50-340 nmol/l). In contrast the ZF secreted 177-379 (control), 828-2052 (dbcAMP) and 2863-9735 (ACTH) nmol cortisol/l. There was no detectable (i.e. less than 2 nmol/l) cAMP production by ZR or ZF either basally (no ACTH) or after ACTH stimulation (500 ng/l). Challenge of the ZR cells with each cortisol precursor steroid (5 mumol/l) increased (P less than 0.05) cortisol secretion over that seen with the corresponding basal and ACTH-stimulated controls. Thus pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol (converted directly to cortisol by 21-hydroxylase) gave rise to (mean +/- S.D., n = 4) 406 +/- 86, 680 +/- 180, 1307 +/- 111, 1141 +/- 234 and 3160 +/- 419 nmol cortisol/l respectively.(ABSTRACT TRUNCATED AT 250 WORDS)