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Northern blot analysis was used to investigate the effect of dexamethasone (Dex) or zinc on messenger RNA (mRNA) levels of metallothionein-IIa (MT-IIa) in fibroblasts from a patient with cortisol hyperreactive syndrome and from three normal subjects. Dex was seen to increase MT-IIa mRNA levels and brought them to a maximum after 12 h. Zinc also increased the levels of MT-IIa mRNA and brought them to a maximum at 8 h after the addition. Dex as well as zinc caused a dose-related increase in MT-IIa mRNA levels. Dex had a maximal inductive effect on MT-IIa at a concentration of 10(-6) mol/L and zinc at a concentration of 10(-4) mol/L. There was no significant difference in the levels of basal expression of the MT-IIa gene between the patient's and normal fibroblasts. But in three separate experiments induction of MT-IIa gene by Dex obtained for the patient's fibroblasts was almost twice as much as that for normal fibroblasts. On the other hand, there were no significant difference in induction by zinc between the patient's and normal fibroblasts. These data indicated that the patient's cells were hyperreactive to glucocorticoids as seen from the effect of Dex on the MT-IIa mRNA levels.

The ability of dexamethasone to induce aromatase activity was tested in fibroblasts from a patient with familial cortisol resistance, a patient with cortisol hyperreactive syndrome, and five normal subjects. Dexamethasone increased enzyme activity in all cases in a concentration-dependent manner (over a range of 1-1000 nmol/L). In fibroblasts from a patient with familial cortisol resistance, the response curve of dexamethasone-induced aromatase activity was shifted to the right compared to that of normal cells. However, the maximal induction of the enzyme at 1 mumol/L dexamethasone was unchanged in cortisol-resistant fibroblasts. On the other hand, in fibroblasts from the patient with the cortisol hyperreactive syndrome, the half-maximal effect of dexamethasone was similar to that in normal cells, but maximum induction of aromatase activity was 2 times greater than that in normal cells. The glucocorticoid antagonist RU 486 inhibited dexamethasone-induced aromatase activity in these patients' cells and in normal cells in a concentration-dependent manner, indicating that the altered effects of dexamethasone on aromatase induction observed in each cell type were mediated through glucocorticoid receptors.

One patient is reported who has the manifestations of Cushing's syndrome in spite of persistent hypocortisolemia. His serum levels of cortisol and free cortisol were below normal, and 24-h urinary excretion of 17-hydroxycorticosteroids and cortisol were decreased. There was a rapid and substantial increase in serum cortisol in response to synthetic ACTH-(1-24). Plasma levels of ACTH were marginally increased by successive administration of CRH and vasopressin, which were followed by substantial increases in serum cortisol. Glucocorticoid activity of the patient's serum, as measured by a RRA was low. There were no responses of urinary 17-hydroxycorticosteroids after metyrapone treatment. These laboratory examinations ruled out any known clinical conditions resulting in hypocortisolemia. The clinical condition could also be explained by cortisol hyperreactivity of the patient's cells. In vitro hyperreactivity to glucocorticoids was demonstrated in cultured skin fibroblasts whose aromatase activity was increased 1.5- to 1.8-fold above that of normal cells, and [3H]thymidine incorporation was inhibited more effectively by the addition of cortisol or dexamethasone. The mechanism by which the patient is hyperreactive to glucocorticoids remains unexplained.

The patient (age 46; female) had hypocalcemia without characteristic physical findings of Albright hereditary osteodystrophy in July, 1998. She had no evidence of tetany. Her blood test revealed elevated serum concentration of parathyroid hormone (PTH) in spite of hypocalcemia and hyperphosphatemia. Her Ellsworth-Howard test presented decreased excretion of both phosphate and cAMP in urine after PTH injection compared with normal range of response, indicating end-organ resistance to the actions of PTH. Finally, Ellsworth-Howard test, together with lack of clinical manifestations, resulted in diagnosis of pseudohypoparathyroidism type 1b.

Abnormalities of glucocorticoid receptors were assumed to cause resistance or hypersensitivity to glucocorticoid, while only glucocorticoid resistance has been described clinically. Patients with familial cortisol resistance have continuously elevated serum cortisol without any clinical manifestations of Cushing's syndrome due to hyposensitivity to cortisol in all tissues including the hypothalamus and the pituitary. Clinical symptoms of the disease are characterized by hypertension with hypokalemia and hyporeninemia, virilism in women, isosexual precocity in a boy and mild general fatigue. As the cause of the disease, a defect in glucocorticoid receptor affinity or binding capacity due to mutations in the glucocorticoid receptor gene has been reported. Another cause of the disease is the presence of heat labile glucocorticoid receptor. In 5 of 6 families with cortisol resistance reported so far, mutations of the glucocorticoid receptor gene have been demonstrated.

Tumors producing weak mineralocorticoids are exceedingly rare and most of them predominantly produce 11-deoxycorticosterone. We present a case of adrenocortical adenoma in which the overproduction of 18-hydroxydeoxycorticosterone exceeded that of 11-deoxycorticosterone.

An ELISA for cGMP in human plasma and urine using a monoclonal antibody is described. The monoclonal antibody was raised against succinyl cGMP conjugated to human serum albumin. The conjugate was adsorbed to the ELISA plate, giving an immobilized antigen approach which simplifies subsequent assay procedures. As low as 1.56 fmol/well of both plasma and urinary cGMP is measurable. Recoveries of added cGMP in plasma and urine were from 97% to 105%. Intra-assay coefficients of variation were less than 7.0% for plasma and 7.1% for urine samples. Inter-assay coefficients of variation for plasma and urine samples were less than 9.9% and 9.5%, respectively. The values obtained by ELISA correlated well with those by radioimmunoassay (RIA) (plasma: r = 0.96, n = 50; urine: r = 0.98, n = 60).

The effects of recombinant human somatomedin C/insulin-like growth factor (IGF-I) on the steroidogenic response of isolated rat adrenocortical cells to ACTH, forskolin, and (Bu)2cAMP were examined during short term incubations. The effect of IGF-I on cAMP production by cells stimulated with ACTH or forskolin was also examined. IGF-I inhibited ACTH-, forskolin-, and (Bu)2cAMP-induced corticosterone production in a concentration-dependent manner. IGF-I (30 ng/ml) also significantly inhibited ACTH-induced cAMP production. However, the peptide had no significant effect on forskolin-induced cAMP production. IGF-I suppressed ACTH-induced cAMP production both in the presence and absence of 3-isobutyl-1-methylxanthine, suggesting that IGF-I acts to inhibit the formation of cAMP rather than the stimulation of cAMP degradation. The observation that IGF-I inhibited steroidogenesis induced by (Bu)2cAMP strongly suggests that one site of inhibition is at some step(s) distal to cAMP formation. However, the inhibition of cAMP production after stimulation with ACTH also suggests a plasma membrane site of action for IGF-I in adrenocortical cells.

The action of the cyclic peptide polymyxin B (a well known inhibitor of protein kinase C) on adrenal steroid synthesis was examined with Y-1 adrenal tumor cells. Polymyxin B produces a biphasic effect on the stimulation of steroid synthesis by 2 nM ACTH in these cells, with inhibition at low concentrations (less than 10 microM) and a return to control levels at high concentrations (greater than 100 microM). Polymyxin B does not inhibit the stimulatory effect of Bu2cAMP on steroidogenesis. Inhibition of the steroidogenic response to ACTH by a fixed concentration (20 microM) of polymyxin B is overcome by high concentrations of ACTH. Polymyxin B causes a concentration-dependent stimulation of steroid synthesis by Y-1 cells and, over the same concentration range, increases the production of cAMP by these cells. Polymyxin B also partially inhibits the increased production of the cyclic nucleotide produced by ACTH. In addition, polymyxin B inhibits binding of [125I](Phe2,Nle4)ACTH-(1-38) to Y-1 cells. Polymyxin B, like ACTH, promotes rounding of Y-1 cells and partially inhibits rounding produced by ACTH. These effects of polymyxin B are specific to the extent that polymyxins E1 and E2 do not exert similar effects. The actions of polymyxin B are not confined to transformed cells, since responses similar to those seen with Y-1 cells were also observed with cultured rat fasciculata cells. On the other hand, the effect of polymyxin B is specific for adrenal cells, since the cyclic peptide does not influence the steroidogenic response of rat Leydig cells to LH. It is concluded that polymyxin B is a partial agonist of ACTH which is likely to prove useful in studying the molecular basis of the interaction between ACTH and its adrenal receptor.

The concentration of ACTH in extracts of rat anterior pituitary was measured by both radioimmunoassay and bioassay at different stages following adrenalectomy. Both types of ACTH activity decreased the day immediately following adrenalectomy but increased gradually afterwards. Immunological ACTH activity increased to 250% of the control value and biological ACTH activity increased to 490% of control value 3 weeks after adrenalectomy. The increase in biological ACTH activity occurred earlier, and the rate of increase was greater, than that of the immunological ACTH activity. The distributions of molecular weight forms of ACTH in extracts of anterior pituitary lobes was determined by gel filtration. Three molecular weight forms of immunoassayable ACTH were detected. Biological ACTH activity appeared in the 2nd and the 3rd peaks. A striking change was observed after adrenalectomy in the distribution of biologically active forms of ACTH. The ratio of biological ACTH activity to immunological ACTH activity in each peak changed at various stages after adrenalectomy. This indicated the heterogenous nature of the ACTH included in each peak. At 2 and again at 3 weeks, biological activity markedly increased until it exceeded the immunological ACTH activity in the 2nd peak. Dexamethasone had little influence on the elution profile of either immunoassayable and biologically active ACTH in gel filtration. Adrenalectomy may possibly have an effect on the intracellular posttranslational processing of ACTH precursors which leads to the development of biological ACTH activity.

Previous studies showed that Y-1 cells in which the concentration of cholesterol in plasma membranes (expressed relative to phospholipid as a molar ratio, C/P) was raised by incubation with cholesterol produced more 20 alpha-dihydroprogesterone than cells with normal or low plasma membrane C/P values. This difference was not associated with any difference in the production of cAMP. The cells respond to (Bu)2cAMP in the order high greater than normal greater than low C/P. High C/P is associated with higher levels of cholesterol in the cytosol and higher concentrations of this steroid in the inner mitochondrial membrane. The rate of transport of cholesterol to the inner membrane differed in the three groups of cells: high greater than normal greater than low C/P. Production of pregnenolone by mitoplasts was no different in the three groups, and the same applies to the production of pregnenolone by mitochondria from cells incubated with and those incubated without (Bu)2cAMP. 25-Hydroxycholesterol increased the production of 20 alpha-dihydroprogesterone by cells showing normal or low C/P values, but not those with high values. It is concluded that the transport of cholesterol to the inner mitochondrial membrane is regulated by at least two processes, namely the supply of substrate (cholesterol), which can saturate this transport mechanism, and some mechanism(s) that is stimulated by cAMP and is capable of stimulating transport above maximal levels produced by high concentrations of cholesterol alone. The second mechanism requires the synthesis of a new protein(s).

A 53-year-old woman with Nelson's syndrome was treated with 600 mg sodium valproate daily. Her plasma ACTH was effectively decreased, whilst the response of plasma ACTH to corticotrophin-releasing factor (CRF) was unaltered. The result of the CRF test suggested that sodium valproate, at least in the present patient, acted at the hypothalamic level.

An extract of porcine thyroid gland in 0.1 N acetic acid exerted dose-dependent potentiation of ACTH-induced corticosterone production in isolated rat adrenal cells. The extract by itself manifested no steroidogenic activity. Upon gel-filtration of the extract, potentiating activities were demonstrated in three main peaks with molecular weights of about 10,000, 5,000 and 2,000. These findings indicate the presence of heterogeneous forms of ACTH-potentiating factors in the thyroid. Significant enhancement of ACTH-induced steroidogenesis was readily apparent with three gel-filtration fractions at a lower concentration of ACTH (4.75 pM). At this concentration, dose-dependent potentiation was observed with these three fractions. Enhanced corticosterone production responses by cells preincubated with the thyroid extract were observed and the results indicated the existence of potentiating mechanisms other than inhibition of ACTH proteolysis. The lack of T4, T3 and thyroglobulin in this activity suggests that the activity resides in other constituents of the thyroid.

Growth hormone (GH)-producing pituitary adenoma from a 50-year-old acromegalic female was studied histochemically, immunohistochemically and electron microscopically. The adenoma was characterized by numerous crystal-like amyloid bodies of 5 to 40 micron in diameter. In the periphery of the crystal-like amyloid, bundles of amyloid fibrils were closely associated with deep invaginations of adenoma cells. The adenoma cells had numerous vesicles and vacuoles filled with amyloid fibrils, some of which were continuous with extracellular space. The crystal-like amyloids, as well as the adenoma cells, were immunohistochemically positive for GH. It might be possible that disorder of hydrolysis of "prohormone," from which GH is elaborated, is responsible for the amyloid production, and that amyloid discharge is accompanied with immunoreactive GH.

In an in vitro bioassay system for adrenocorticotropic hormone using isolated rat adrenal cells, kaurenol, a diterpene alcohol, stimulated corticosterone production and augmented the steroidogenic effect of adrenocorticotropin or forskolin, dose-dependently. Kaurenol had no effect on cyclic AMP production by the cells. The diterpene also had no stimulatory effect on the adrenal adenylate cyclase activity in a cell free system. The results suggest that this particular diterpene exerts a steroidogenic effect through a mechanism independent of cyclic AMP generation.

Studies made on cultured skin fibroblasts obtained from a patient with primary cortisol resistance are described. Whole cell in vitro assays, using the patient's fibroblasts revealed a reduction in the dexamethasone binding capacity (7.86 +/- 0.73 fmol/micrograms DNA, mean +/- SD, n = 3; normal: 15.2 +/- 1.90 fmol/micrograms DNA, n = 8) and an apparently normal dissociation constant (3.69 +/- 0.15 nM; normal: 3.74 +/- 0.40 nM). In addition, the effects of glucocorticoids on DNA synthesis in these cells were examined. DNA synthesis was inhibited by dexamethasone both in normal fibroblasts and in the patient's cells, but the patient's cells were less sensitive to this inhibition, indicating resistance of the cells to glucocorticoid in vitro. These results suggest that the resistance of target tissues to glucocorticoids is due to the reduction in receptor number and that this is the primary defect in this new type of primary cortisol resistance in man.

Three patients with renal hypouricemia in the same family are described. Serum urate levels in the mother were in the low normal range and were below normal in her 2 sons. In all 3 patients, the ratios of renal urate clearance to creatinine clearance were abnormally elevated. Clear responses to either pyrazinamide or probenecid administration were observed in these ratios. These results suggest that these 3 patients had renal hypouricemia with normal reabsorption of urate as judged by the criteria for differentiating abnormalities in renal urate handling. This corresponds to the previously postulated mechanism as renal urate hypersecretion. Possible limitations to the diagnostic use of probenecid and pyrazinamide are also discussed.

We have demonstrated the presence of ACTH-potentiating factors in porcine thymus which contained neither biologically active nor radioimmunoassayable ACTH. The factors were acidic and heat stable in nature, and were present in heterogeneous forms having various molecular weight. Purification of the factors with small molecular weights was performed using reversed-phase high-performance liquid chromatography following gel filtration and cation-exchange chromatography. A purified factor exerted dose-dependent ACTH-potentiating activity. Loss of the ACTH-potentiating activity of purified factors by treatment with carboxypeptidase Y indicated their peptidic nature. ACTH-potentiating activity was readily observable after preincubating the adrenal cells with the thymic extract. The enhancement of ACTH-induced steroidogenesis by the factors was observed in the presence of bacitracin at a concentration to prevent ACTH degradation. Therefore, the existence of potentiating mechanisms other than inhibition of ACTH proteolysis was indicated.

A calcitonin preparation derived from porcine thyroid glands exhibited potentiating activity on the steroidogenic action not only on ACTH-(1-24) but also of ACTH-(1-18) in isolated rat adrenal cells in vitro. This activity was demonstrated in various molecular weight fractions resulting from gel filtration of the preparation. This preparation also contained minute amount of immunoreactive and biologically active ACTH.

The concentration of cholesterol in plasma membranes of Y-l cells was altered between values of the molar ratio of cholesterol to phospholipids (C/P) of 0.6:1.6 to study the influence of plasma membrane cholesterol on the response to ACTH. Increasing concentrations of membrane cholesterol (C/P) were associated with an increase in the number of ACTH receptors and, hence, production of cAMP and steroids without affecting the nature of binding (Kd) of ACTH to its receptor. Binding studies revealed spare ACTH receptors at all concentrations of membrane cholesterol. However, production of cAMP and steroids by Y-l cells were tightly coupled. Studies with cholera toxin snowed that extreme changes in C/P were without influence on the activity of Ns or that of the cyclase itself. It was also shown that production of steroids in the absence of ACTH and that in response to cholera toxin involve some effect of membrane cholesterol not dependent on cAMP. (Endocrinology 120: 801–808, 1987)

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.

In a sensitive ACTH bioassay system using isolated rat adrenal cells, we tested the effect of gamma-MSH related peptides on ACTH-induced steroidogenesis. Peptides, including synthetic gamma1-, gamma2-, gamma3- and Lys-gamma3-MSH, exerted no effect in augmenting ACTH-induced steroidogenesis. None of the 16 kilodalton fragment of ACTH/beta-lipotropin precursor and its cleaved fragment had such an activity. The results are in contrast with previous reports concerning ACTH-potentiating activity of gamma-MSH related peptides and, therefore, indicate the necessity of further investigation of the principle involved in this unique biological activity.

High molecular weight ACTH fractions, obtained through gel filtration of boiled rat anterior pituitary extract, induced a marked increase in corticosterone production from isolated rat adrenal cells in the presence of low concentrations of ACTH-(1-24). This indicates the presence of heat-stable factors augmenting the steroidogenic action of ACTH in the rat anterior pituitary. We also noted that these factors potentiated the activity not only of ACTH-1(1-24) but also of ACTH-(1-8). The ACTH-potentiating factors in rat anterior pituitary extract are possibly present in heterogeneous forms according to their molecular weights (8,000, 10,000 and 15,000), their mobility in ion-exchange chromatography and their content in RIA-ACTH activity. Of these three forms, the former comigrated with biological ACTH activity. The remaining two forms were free of it. Since the effect of potentiating factors on modified ACTH-(1-9), shown to be less susceptible to proteolytic degradation from ACTH-(1-24), was similar to the effect on ACTH-(1-24), it is suggested that potentiation was not due to an inhibition of ACTH proteolysis.

Summary Studies on hypouricaemia observed in a patient with 48, XXYY syndrome revealed an abnormality in renal urate handling. His renal urate clearance was abnormally increased. Inosine administration and provocative tests using probenecid and pyrazinamide identified an isolated renal tubular abnormality with increased urate secretion. Since the serum urate in his brother with a normal sex chromosome constitution was also low, the association of renal hypouricaemia and 48, XXYY syndrome in this patient is probably coincidental. Although the brother was not investigated, these siblings may be a previously unreported case of familial hypouricaemia due to isolated renal hypersecretion.

This report describes studies of a man suspected of having primary cortisol resistance. This conclusion is based on his high plasma cortisol levels and high 24-h urinary 17-hydroxycorticosteroid and cortisol excretion, plus the fact that he had no manifestations of Cushing's syndrome. Among family members tested, his mother also had hypercortisolemia. Both mother and son had high levels of unbound plasma cortisol, but their plasma ACTH concentrations were within the normal range. Both were partially resistant to dexamethasone adrenal suppression, and both had mild hypertension without hypokalemia. To study this apparent end-organ resistance to cortisol, we examined the glucocorticoid receptors in peripheral mononuclear cells. Using whole cell assays, glucocorticoid receptors in both patients were found to have reduced total binding capacity. We conclude that these two patients, members of the same family, have primary cortisol resistance accompanied by a reduced number of glucocorticoid receptors.