Adrenal Cortisol Production Research Articles

Glucocorticoid inhibition in the treatment of depression: can we think outside the endocrine hypothalamus?

Major depressive disorder affects a substantial percentage of the U.S. population, and can be highly debilitating. Selective serotonin reuptake inhibitors are commonly prescribed to treat depression, but may not be as effective for more severe or persistent depression. The authors review data concerning the effects of corticosteroid synthesis inhibitors (CSIs) in the management of depressive disorders, present a hypothesis as to their possible mechanisms of action based on recent data suggesting synergistic effects of glucocorticoids on extrahypothalamic corticotropin-releasing hormone (CRH), and consider alternative hypotheses. Published reports evaluating the efficacy of CSIs in treating depression are reviewed and presented in light of recent findings regarding actions of glucocorticoids on the central CRH system. Results from open label and double-blind studies by several groups have indicated that CSIs may be efficacious or of adjunctive value in some patients with depression, including those refractory to other agents; however, there is a need for more controlled studies. Several lines of data suggest that the mechanism of action of these agents may not be solely a function of inhibition of adrenal cortisol production. The authors propose that CSIs may be efficacious in part by reducing glucocorticoid enhancement of CRH action in neurons of the central nucleus of the amygdala and other structures outside the endocrine hypothalamus. Possible effects of systemically administered CSIs on glucocorticoid receptor regulation, neuroactive steroids, and classical monoamine systems are also discussed. We conclude that available clinical data suggest a potential role for CSIs in the management of depressive disorders, especially major depression with psychotic features.

Contrasting effects of eplerenone and spironolactone on adrenal cell steroidogenesis.

Spironolactone and eplerenone are widely used as mineralocorticoid antagonists. Spironolactone has several nonspecific actions including inhibition of androgen receptor and steroid hormone biosynthesis. While studies have shown that eplerenone does not exhibit nonspecific actions on androgen receptor, its effects on steroid hormone production have not been reported. Herein, the effects of eplerenone (0.1-30 microM) and spironolactone (0.1-30 microM) on steroid production were examined in human adrenocortical H295R cells. Spironolactone inhibited basal production of cortisol (91%) and aldosterone (53%). Treatment of H295R cells with angiotensin II (Ang II) for 24 h increased aldosterone production by 11-fold. Spironolactone inhibited Ang II stimulation of aldosterone production by 80%. Addition of pregnenolone increased aldosterone (9-fold) and cortisol (3-fold) production. Spironolactone inhibited pregnenolone metabolism to aldosterone (67%) and cortisol (74%). The inhibitory effects of spironolactone occurred at concentrations far higher than those needed to block mineralocorticoid receptor, suggesting an action directly on the enzymes involved in steroid production. In contrast, eplerenone did not inhibit basal, Ang II, forskolin, pregnenolone-stimulated cortisol, or aldosterone production. Together, these data demonstrate that opposed to spironolactone, pharmacologic concentrations of eplerenone do not inhibit adrenal cell aldosterone or cortisol production.

Evidence of a role for melatonin in fetal sheep physiology: direct actions of melatonin on fetal cerebral artery, brown adipose tissue and adrenal gland

Although the fetal pineal gland does not secrete melatonin, the fetus is exposed to melatonin of maternal origin. In the non-human primate fetus, melatonin acts as a trophic hormone for the adrenal gland, stimulating growth while restraining cortisol production. This latter physiological activity led us to hypothesize that melatonin may influence some fetal functions critical for neonatal adaptation to extrauterine life. To test this hypothesis we explored (i) the presence of G-protein-coupled melatonin binding sites and (ii) the direct modulatory effects of melatonin on noradrenaline (norepinephrine)-induced middle cerebral artery (MCA) contraction, brown adipose tissue (BAT) lypolysis and ACTH-induced adrenal cortisol production in fetal sheep. We found that melatonin directly inhibits the response to noradrenaline in the MCA and BAT, and also inhibits the response to ACTH in the adrenal gland. Melatonin inhibition was reversed by the melatonin antagonist luzindole only in the fetal adrenal. MCA, BAT and adrenal tissue displayed specific high-affinity melatonin binding sites coupled to G-protein (K(d) values: MCA 64 +/- 1 pm, BAT 98.44 +/- 2.12 pm and adrenal 4.123 +/- 3.22 pm). Melatonin binding was displaced by luzindole only in the adrenal gland, supporting the idea that action in the MCA and BAT is mediated by different melatonin receptors. These direct inhibitory responses to melatonin support a role for melatonin in fetal physiology, which we propose prevents major contraction of cerebral vessels, restrains cortisol release and restricts BAT lypolysis during fetal life.

Acute cold stress in rheumatoid arthritis inadequately activates stress responses and induces an increase of interleukin 6

Objective:Acute stress in patients with rheumatoid arthritis (RA) should stimulate a strong stress response. After cryotherapy, we expected to observe an increase of hormones of the adrenal gland and the...

Evaluation of effects of an oral contraceptive containing ethinylestradiol combined with drospirenone on adrenal steroidogenesis in hyperandrogenic women with polycystic ovary syndrome

To investigate whether the administration of an oral contraceptive containing the new antiandrogenic drospirenone is associated with reduced adrenal androgen synthesis in hyperandrogenic women with diagnosis of polycystic ovary syndrome. Drospirenone, an analogue of spironolactone and aldosterone antagonist, is a novel progestin under clinical development that is similar to the natural hormone progesterone, combining potent progestogenic with antimineralocorticoid and antiandrogenic activities. Prospective study. Healthy volunteers in University Department of Obstetrics and Gynecology. Fifteen women ages 18 to 28 years with the diagnosis of polycystic ovary syndrome. Three months of contraceptive use (30 mcg ethinylestradiol, 3 mg drospirenone). An adrenocorticotropic hormone test was performed before and after the study. Adrenal production of cortisol was unchanged after therapy with oral contraceptives. An interesting observation was reduced basal concentrations of androgens such as androstenedione, dehydroepiandrosterone sulfate, testosterone, and free testosterone during therapy. The ratios of the areas of substrates to products before and after oral contraceptive administration were compared for differences in 17alpha-hydroxylase (17-hydroxyprogesterone/progesterone) and 17,20-lyase (androstenedione/17-hydroxyprogesterone); activities were significantly reduced, indicating a reduction in the activities of these enzymes. The present results show for the first time that oral contraceptives containing drospirenone affect adrenal steroidogenesis by reducing synthesis and release of androgens in response to adrenocorticotropic hormone, leaving adrenal production of cortisol unchanged.

Estrogens and Glucocorticoid Hormones in Adipose Tissue Metabolism

Women have a higher percentage of body fat than men, and there is a gender-specific difference in fat distribution: Females tend to accumulate fat around the hips, buttocks, and thighs while men have a larger intra-abdominal (visceral) fat mass. After menopause, there is a redistribution of fat depots, and post-menopausal women develop increased amounts of visceral fat. The risk of developing obesity-related diseases is significantly lower in pre-menopausal women compared to men, a difference that is abolished after menopause, suggesting that the female sex steroid estrogen influences adipogenesis and adipose metabolism. Experimentally, estrogen increases the size and number of subcutaneous adipocytes and attenuates lipolysis. Post-menopausal women also develop a more atherogenic lipid pattern and decreased levels of the prothrombotic protein plasminogen activator inhibitor-1, which attenuates fibrinolysis. Pathologically increased circulating cortisol concentration is associated with dysmetabolic features e.g., central obesity, elevated blood pressure, insulin resistance, and dyslipidemia. In "simple obesity," glucocorticoid production is elevated. Peak levels of circulating cortisol are however low or normal, possibly because of increased clearance and/or tissue-specific changes in cortisol production. In addition to the adrenal production of cortisol, cortisol is also generated in adipose tissue by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) which converts inactive cortisone to active cortisol. The enzyme activity in subcutaneous fat increases with increasing body weight. Estrogen seems to have a tissue-specific influence on 11betaHSD1 enzyme activity, attenuating it in liver, kidney, and testis but upregulating 11betaHSD1 mRNA expression in preadipocytes from women. In the present review, we summarize and discuss the interaction between glucocorticoids and sex steroids and their influence on adipocyte metabolism.

The adrenal gland may be a target of LH action in postmenopausal women

LH receptor expression and function have been demonstrated in the human adrenal cortex, but their involvement in normal adrenal function remains elusive. Because cortisol levels have been reported to be higher in postmenopausal women than in age-matched men, the aim of the present study was to investigate a possible association of adrenal function with the elevated LH levels in postmenopausal women. A group of 112 endocrinologically normal postmenopausal women (mean age 67.6, range 50-88 years) was evaluated. A basal fasting morning sample of peripheral blood was taken for the determination of LH, cortisol, dehydroepiandrosterone-sulphate (DHEA-S), oestradiol (E2), testosterone, sex hormone-binding globulin (SHBG), insulin and glucose. Information about reproductive function, anthropometric parameters and arterial blood pressure was recorded. The correlation of LH and cortisol was bimodally distributed, with a significant linear correlation up to the LH level of 41 U/l (n = 78, P<0.01), after which the increase of cortisol levelled off. Significant associations were also found between serum DHEA-S and LH levels (P<0.05), as well as between cortisol and testosterone (P<0.0001), but not between E2 and LH. Multivariate analysis showed that the association of cortisol with LH was independent of age and testosterone; the association of DHEA-S with LH was independent of E2, cortisol and age. Significant associations were also found between E2, testosterone and DHEA-S levels (P<0.001). These results indicate that adrenal cortisol and DHEA-S production may be stimulated by the highly elevated postmenopausal levels of LH; the physiological significance of this association and plausible contribution to the metabolic syndrome observed after the menopause remain to be evaluated.

Potential Inflammatory biomarkers in Alzheimer's disease

The role of the brain's innate immune system in Alzheimer pathogenesis is now well established. Proinflammatory cytokines elaborated by this system, in particular activated microglia-derived interleukin-1 (IL-1), drive a cascade of neurotoxic changes that are important for the development and progression of both the neuritic plaques and neurofibrillary tangles characteristic of Alzheimer's disease. Cytokine expression may also be modulated by variants of genes. For instance, inheritance of certain IL-1 gene variants is associated with Alzheimer's disease. The potential for using blood levels of proinflammatory cytokines as biomarkers of disease progression, however, remains unrealized. The interpretation of cytokine levels in the blood is complicated by the fact, for example, that the overexpression of IL-1 in Alzheimer brain may act to increase adrenal cortisol production through the hypothalamic-pituitary-adrenal axis, which acts to limit macrophage activation and peripheral cytokine production.

Human hair follicles display a functional equivalent of the hypothalamic‐pituitary‐adrenal (HPA) axis and synthesize cortisol

The skin and its major appendages are prominent target organs and potent sources of key players along the classical hypothalamic-pituitary axis, such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and alpha melanocyte stimulating hormone (alpha-MSH), and even express key steroidogenic enzymes. Therefore, it may have established local stress response systems that resemble the hypothalamic-pituitary-adrenal (HPA) axis. However, functional evidence that this is indeed the case in normal human skin in situ has still been missing. We show that microdissected, organ-cultured human scalp hair follicles respond to CRH stimulation by up-regulating proopiomelanocortin (POMC) transcription and immunoreactivity (IR) for ACTH and alpha-MSH, which must have been processed from POMC. CRH, alpha-MSH, and ACTH also modulate expression of their cognate receptors (CRH-R1, MC1-R, MC2-R). In addition, the strongest stimulus for adrenal cortisol production, ACTH, also up-regulates cortisol-IR in the hair follicles. Isolated human hair follicles secrete substantial levels of cortisol into the culture medium, and this activity is further up-regulated by CRH. CRH also modulates important functional hair growth parameters in vitro (hair shaft elongation, catagen induction, hair keratinocyte proliferation, melanin production). Finally, human hair follicles display HPA axis-like regulatory feedback systems, since the glucocorticoid receptor agonist hydrocortisone down-regulates follicular CRH expression. Thus, even in the absence of endocrine, neural, or vascular systemic connections, normal human scalp hair follicles directly respond to CRH stimulation in a strikingly similar manner to what is seen in the classical HPA axis, including synthesis and secretion of cortisol and activation of prototypic neuroendocrine feedback loops.

Angiotensin II Dilates Bovine Adrenal Cortical Arterioles: Role of Endothelial Nitric Oxide

Adrenal steroidogenesis is modulated by humoral and neuronal factors and blood flow. Angiotensin II (AII) stimulates adrenal cortical aldosterone and cortisol production and medullary catecholamine release. However, AII regulation of adrenal vascular tone has not been characterized. We examined the effect of AII on diameters of cannulated bovine adrenal cortical arteries. Cortical arteries (average internal diameter = 230 microm) were constricted with U46619 and concentration-diameter responses to AII (10(-13) to 10(-8) mol/liter) were measured. In endothelium-intact arteries, AII induced dilations at low concentrations (maximum dilation = 25 +/- 6% at 10(-10) mol/liter) and constrictions at high concentrations (maximum constriction = 25 +/- 18% at 10(-8) mol/liter). AII constrictions were blocked by the angiotensin type 1 (AT1) receptor antagonist, losartan (10(-6) mol/liter). AII dilations were enhanced by losartan (maximal dilation = 48 +/- 8%), abolished by endothelial cell removal or N-nitro-L-arginine (L-NA, 3 x 10(-5) mol/liter) and inhibited by the angiotensin type 2 (AT2) receptor antagonist, PD123319 (10(-6) mol/liter, maximal dilation = 18 +/- 4%). In a 4,5-diaminofluorescein diacetate nitric oxide (NO) assay of isolated cortical arteries, AII stimulated NO production, which was abolished by PD123319, L-NA, or endothelial cell removal. Western immunoblot of arterial homogenates and endothelial and zona glomerulosa cell lysates revealed 48-kD and 50-kD bands corresponding to AT1 and AT2 receptors, respectively, in all three and a 140-kD band corresponding to endothelial NO synthase in endothelial cells and arteries. Our results demonstrate that AII stimulates adrenal cortical arterial dilation through endothelial cell AT2 receptor activation and NO release and AT1 receptor-dependent constriction.

Maternal melatonin selectively inhibits cortisol production in the primate fetal adrenal gland.

We tested the hypothesis that in primates, maternal melatonin restrains fetal and newborn adrenal cortisol production. A functional G-protein-coupled MT1 membrane-bound melatonin receptor was detected in 90% gestation capuchin monkey fetal adrenals by (a) 2-[(125)I] iodomelatonin binding (K(d), 75.7 +/- 6.9 pm; B(max), 2.6 +/- 0.4 fmol (mg protein)(-1)), (b) cDNA identification, and (c) melatonin inhibition of adrenocorticotrophic hormone (ACTH)- and corticotrophin-releasing hormone (CRH)-stimulated cortisol but not of dehydroepiandrosterone sulphate (DHAS) production in vitro. Melatonin also inhibited ACTH-induced 3beta-hydroxysteroid dehydrogenase mRNA expression. To assess the physiological relevance of these findings, we next studied the effect of chronic maternal melatonin suppression (induced by exposure to constant light during the last third of gestation) on maternal plasma oestradiol during gestation and on plasma cortisol concentration in the 4- to 6-day-old newborn. Constant light suppressed maternal melatonin without affecting maternal plasma oestradiol concentration, consistent with no effect on fetal DHAS, the precursor of maternal oestradiol. However, newborns from mothers under constant light condition had twice as much plasma cortisol as newborns from mothers maintained under a normal light-dark schedule. Newborns from mothers exposed to chronic constant light and daily melatonin replacement had normal plasma cortisol concentration. Our results support a role of maternal melatonin in fetal and neonatal primate cortisol regulation.

Chapter 16. The use of corticosteroids in the treatment of severe pediatric traumatic brain injury

With the lack of sufficient evidence for beneficial effect and the potential for increased complications and suppression of adrenal production of cortisol, the routine use of steroids is not recommended for children following severe traumatic brain injury.

Mt1 Melatonin receptor in the primate adrenal gland: inhibition of adrenocorticotropin-stimulated cortisol production by melatonin.

The pineal hormone melatonin participates in circadian, seasonal, and reproductive physiology. The presence of melatonin binding sites in human brain and peripheral tissues is well documented. However, in the mammalian adrenal gland, low-affinity melatonin binding sites have been detected only in the rat by some but not all authors. Conflicting evidence for a regulatory role of melatonin on adrenal cortisol production, prompted us to investigate this possibility in a New World primate, the capuchin monkey. Expression of melatonin receptors in the adrenal cortex was demonstrated through pharmacological characterization and autoradiographic localization of 2-[125I]iodomelatonin binding sites (dissociation constant = 96.9 +/- 15 pM; maximal binding capacity = 3.8 +/- 0.4 fmol/mg protein). The mt1 identity of these receptors was established by cDNA sequencing. Melatonin treatment of dispersed cells and explants from adrenal gland did not affect basal cortisol production. However, cortisol production stimulated by 100 nM ACTH was significantly inhibited by low melatonin concentrations (0.1-100 nM); this inhibitory effect was reversed by the mt1/MT2 melatonin antagonist luzindole. Melatonin also inhibited dibutyril-cAMP-stimulated cortisol production, suggesting that melatonin acts through a cAMP-independent signaling pathway. The present data demonstrate that the primate adrenal gland cortex expresses functional mt1 melatonin receptors and shows that melatonin inhibits ACTH-stimulated cortisol production.

CLINICAL AND ENDOCRINOLOGICAL FEATURES OF ADRENOCORTICOTROPIC HORMONE- INDEPENDENT BILATERAL MACRONODULAR ADRENOCORTICAL HYPERPLASIA

We report clinical findings in 5 patients with adrenocorticotropic hormone independent bilateral macronodular adrenocortical hyperplasia. In 4 males and 1 female 32 to 61 years old (median age 50) we evaluated clinical symptoms, endocrinological and radiological characteristics, treatment modality and postoperative clinical course. All cases presented with some features of Cushing's syndrome. Endocrinological examination revealed autonomous adrenal cortisol production with suppressed adrenocorticotropic hormone and a loss in the diurnal circadian rhythm of plasma cortisol. Abdominal computerized tomography showed bilateral enlargement of the adrenal glands with multiple nodules. 131Iodine labeled adosterol scintigraphy demonstrated remarkable bilateral uptake by the adrenal glands. The pituitary gland appeared normal on magnetic resonance imaging. Open unilateral complete adrenalectomy and contralateral partial adrenalectomy were performed in patient 1, open bilateral complete adrenalectomy was done in patients 2 and 3, and 2 and 1-stage laparoscopic bilateral complete adrenalectomy was performed in patients 4 and 5. Single removed adrenal glands weighed 32 to 108 gm. (median 60). The histological diagnosis was macronodular adrenocortical hyperplasia in all cases. Postoperative followup was 3 to 90 months. Clinical symptoms of Cushing's syndrome disappeared or improved after surgery in all cases. Although adrenocorticotropic hormone independent bilateral macronodular adrenocortical hyperplasia is a rare form of Cushing's syndrome, physicians are advised to consider it when diagnosing and treating cases of Cushing's syndrome with enlarged bilateral adrenal glands. Bilateral complete adrenalectomy is currently recommended as the treatment of choice.

Handling during Pregnancy in the Blue Fox (Alopex lagopus): The Influence on the Fetal Pituitary–Adrenal Axis

Previous studies revealed that handling is a stressor for farmed blue foxes. The present study was designed to examine the effects of a 1-min daily handling stress applied to pregnant blue fox vixens on the function of the fetal pituitary–adrenal system. Plasma concentrations of adrenocorticotropin hormone (ACTH), cortisol, and progesterone, adrenal content of cortisol and progesterone, in vitro adrenal production of these steroids and response to ACTH, and adrenal weights were measured in control (C; n = 73) and stressed (S; n = 58) fetuses. The ACTH levels were lower in stressed fetuses than in the controls (C: males, 128.6 ± 6.1 pg/ml; females, 165.9 ± 6.1 pg/ml; S: males, 122.3 ± 5.4 pg/ml; females, 145.0 ± 8.1 pg/ml; P < 0.05). In contrast, increased plasma cortisol concentrations in both sexes were demonstrated in stressed compared with control fetuses (C: males, 9.2 ± 0.4 ng/ml; females, 9.2 ± 0.4 ng/ml; S: males, 11.8 ± 0.7 ng/ml; females, 13.2 ± 0.7 ng/ml; P < 0.00001). The same difference was observed in plasma progesterone concentrations (C: males, 1.54 ± 0.07 ng/ml; females, 1.49 ± 0.10 ng/ml; S: males, 1.86 ± 0.11 ng/ml; females, 1.74 ± 0.10 ng/ml; P < 0.01). Prenatal stress did not change the baseline adrenal production of cortisol but prevented the cortisol response to ACTH in female fetuses and decreased the progesterone production in both sexes. Additionally, prenatally stressed fetuses of both sexes had significantly lower adrenal weights than controls (C: males, 9.4 ± 0.3 mg; females, 9.5 ± 0.4 mg; S: males, 8.1 ± 0.3 mg; females, 8.2 ± 0.4 mg; P < 0.001). These results indicate that prenatal handling stress induces a dysregulation of the pituitary–adrenal axis in the fetus and suggest that increased plasma glucocorticoids in the stressed dam can cross the placenta and influence the fetal hypothalamic–pituitary–adrenal axis.

Morphometric and hormonal changes following persistent handling in pregnant blue fox vixens (Alopex lagopus)

AbstractModern, intensive fox breeding implies a human-animal relationship but human contact causes stress in farm-bred blue foxes. Handling is a common part of routine farming practices, particularly during the breeding season. The purpose of this study was to determine how handling influences certain morphometric and hormonal parameters of adrenocortical and ovarian function and fertility in pregnant blue foxes and whether these effects are mediated through an increase in plasma concentrations of cortisol. Blue fox females were subjected to a 1-min daily-handling treatment in the last trimester of pregnancy (term = 52 days). Plasma concentrations of ACTH, cortisol, progesterone, oestradiol and testosterone, as well as thein vitroadrenal and gonadal production of steroids were measured by radio-immuno assay in control (C, no. = 6) and handled (H, no. = 7) vixens on days 47 to 48 of pregnancy. In addition, the number of viable and dead foetuses, body, adrenal and ovarian weights in pregnant foxes and body weights of their viable foetuses were recorded. Handling increased plasma concentration of cortisol (C: 6·8 (s.e. 1·6) ng/ml v. H: 22·8 (s.e. 5·9) ng/ml,P&lt; 0·05) as well asin vitroadrenal production of cortisol. In addition, handling decreased the body (C: 7·40 (s.e. 0·48) kg v. H: 6·24 (s.e. 0·13) kg,P&lt; 0·05) and ovarian (C: 3·45 (s.e. 0·26) g v. H: 2·73 (s.e. 0·17) g,P&lt; 0·05) weights of pregnant vixens and also the body weight of viable foetuses (C: 59·1 (s.e. 0·9) g, no. = 73 v. H: 50·6 (s.e. 1·2) g, no. = 58,P&lt; 0·01). Handling did not cause any changes in adrenal weight, plasma concentrations of progesterone and oestradiol orin vitroovarian steroid production. It is concluded that persistent handling of pregnant blue foxes is a mild stressor, which results in an increase in plasma concentration and adrenal production of cortisol and reduces maternal and foetal body weights.

Interactions between the hypothalamic-pituitary-adrenal axis and allergic inflammation

It is undeniable that glucocorticoids are remarkably effective in the therapeutic management of allergic diseases such as rhinitis, atopic dermatitis, and asthma. The potent synthetic drugs used clinically are analogues of the endogenous adre-nal hormone cortisol. A growing body of evidence now suggests that endogenous cortisol, which is produced in significant quantities by the body in a diurnal rhythm, is an important regulator of allergic disease expression and allergic inflammatory responses: lung function varies along with plasma cortisol levels; the number of circulating inflammatory cells varies with plasma cortisol levels; and low levels of endogenous cortisol may be associated with risk for asthma. Treatment studies suggest that the administration of inhaled or oral steroids is probably more effective when given in the evening when endogenous cortisol levels are low. Conversely, challenge studies show clearly that antigen-induced late-phase responses occur less readily if the challenge is performed in the morning at a time when endogenous cortisol levels are high. Finally, inflammatory responses may induce the production of cortisol by the hypothalamic-pituitary-adrenal axis as a feedback mechanism. Paradoxically, some inflammatory cytokines may induce resistance of inflammatory cells to glucocorticoids. The available information describes a complex yet balanced interplay between adrenal cortisol production and allergic inflammation. (J Allergy Clin Immunol 2000;106:S270-4.)

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C 18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.

Angiotensin II- and phorbol ester-induced steroidogenesis by ovine adrenocortical cells: effect of sex and the gonadal status of the donor animal.

1. Angiotensin II (AngII) is well recognized as a regulator of adrenal cortisol production in the sheep in vivo, but studies to date have failed to reveal this action on cultured ovine adrenocortical cells. Similarly, phorbol myristate acetate (PMA), an activator of protein kinase C, also has little effect on cortisol production from ovine adrenocortical cells in vitro. Previous studies have, however, only looked at the responses to single concentrations of AngII or PMA, but have led to the suggestion that important differences exist between adrenocortical cells of the sheep and other species. 2. We have extended previous studies by examining cortisol production by ovine adrenocortical cells over 3 h in response to a broad range of concentrations of either AngII (10(-10) to 10(-6) mol/L) or PMA (10(-10) to 10(-5) mol/L). In addition, we have investigated the possible role of gonadal factors in regulating these responses by comparing cultures derived from intact and gonadectomized male and female sheep. 3. Angiotensin II caused concentration-dependent increases (P < 0.05) in cortisol production in all cultures, with cortisol production in response to AngII being greater (P < 0.05) in cultures from intact male sheep than in the cultures from orchidectomized males or intact females. Cortisol production in response to AngII was modest in all groups (two- to 2.6-fold increases). 4. Phorbol myristate acetate elicited a concentration-dependent inhibition (P < 0.01) of basal cortisol production in adrenocortical cultures from all groups. Phorbol myristate acetate produced a greater inhibition (P < 0.05) of cortisol production in cultures from intact male sheep than either the orchidectomized males or intact females. 5. These findings demonstrate that ovine adrenocortical cells are responsive in a concentration-dependent fashion to AngII and PMA. In addition, sex and gonadal factors may play an important role in regulating the responsiveness of adrenocortical cells to these factors.

Effect of Estradiol on DHEAS Production in the Human Adrenocortical Cell Line, H295R

To determine if estradiol regulates DHEA and DHEAS production in a human adrenocortical (H295R) cell line and to determine if this effect is receptor mediated. NCI-H295 (H295R) cells were rinsed and placed in phenol red free Dulbecco's Modified Eagle's-F12 medium supplemented with 0.1% charcoal-stripped serum. After 24 hours, cells were rinsed and treated based on experimental design. The effects of estradiol were investigated by: 1) treatment of cells with increasing concentrations of estradiol (300-3000 nmol/L) with or without forskolin (10 mumol/L), 2) treatment of cells with the nonsteroidal synthetic estrogen diethylstilbestrol (DES) (300-3000 nmol/L) with or without forskolin (10 mumol/L), and 3) treatment of cells with an estradiol antagonist (ICI 182, 780) in the presence of estradiol. Estradiol alone increased the basal production of DHEAS in H295R cells in a concentration-dependent manner with a maximal effect at 1000 nmol/L. Forskolin treatment increased the basal production of DHEAS ten-fold. Estradiol also increased the forskolin stimulation of DHEAS production two-fold. In contrast, DES alone or DES in addition to forskolin did not stimulate DHEAS production. Estradiol, in contrast, inhibited H295R adrenal cell production of cortisol whereas DES exhibited a similar inhibition. The estrogen receptor antagonist ICI 182,780 was unable to inhibit the stimulatory effect of estradiol. Finally, estradiol in a concentration-dependent manner suppressed 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in H295R adrenal cells. These experiments support the role of estradiol in regulation DHEAS production by inhibiting 3 beta HSD activity; however, the mechanism appears to require high concentrations of estradiol and appears to be independent of the estrogen receptor.