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A procedure for the continuous flow incubation of isolated adrenal cells is described. In this way the advantages of continuous flow incubations of adrenal tissue are combined with those of isolated adrenal cells. Suspensions of isolated adrenal cells were prepared by a modification of the collagenase method. A sigmoid dose-response curve was obtained when these cells were incubated with ACTH in batch incubations. Under these conditions (in the presence of 1 mU ACTH/ml) the corticosterone production rate remained constant during at least 240 min. This production rate was linearly related to the number of cells. Pre-incubation of the cells during 3 h resulted in an increased response to ACTH. In continuous flow incubations without ACTH the corticosterone production was negligible. With 100 μU ACTH/ml corticosterone production increased sharply after a short lag period. A maximum was reached after 60–75 min followed by a slow decrease. Cells pre-incubated in the continuous flow apparatus had a slightly diminished ACTH response without loss of affinity to ACTH. The continuous flow incubation of isolated adrenal cells offers new possibilities for the dynamic study of steroid biosynthesis in vitro. The method may also be valuable to study processes in a wide variety of other tissues.

The present study shows that in a group of 6 euadrenal patients, previously treated by complete adrenalectomy for pituitary dependent Cushing's syndrome, the stress stimulus of insulin induced hypoglycaemia is followed by a plasma ACTH response which is of similar magnitude as the response obtained with lysin-vasopressin. Both observations indicate that the central nervous system-pituitary axis is basically normal in pituitary dependent Cushing's syndrome as assessed by insulin induced hypoglycaemia. It is concluded that non-responsiveness of the pituitary-adrenocortical system to insulin induced hypoglycaemia in untreated patients with pituitary dependent Cushing's syndrome does not represent a fundamental defect of the stress mechanism, but is due to hypercorticism per se.

The change in the plasma concentration of cortisol after the administration of thyrotrophin releasing hormone (TRH) and LH releasing hormone (LH-RH) was studied in normal dogs and in dogs with pituitary-dependent hyperadrenocorticism (PDH). The normal dogs showed a small but significant increase in the plasma concentration of cortisol 15 min after intravenous injection of TRH and LH-RH. In ten of the dogs with PDH the response to TRH was not significantly different from that in the normal dogs, but in 13 the response was significantly greater. In 15 of the dogs with PDH the response to LH-RH administration was within or below the range of responses in the normal dogs and in only one dog was the response to LH-RH greater than that in the normal dogs. These findings are discussed in relation to the pathogenesis of PDH.

The response has been studied in nine dogs with hyperadrenocorticism due to adrenocortical tumours to the administration of dexamethasone, insulin, lysine-vasopressin and tetracosactide by measuring the changes in plasma cortisol concentration. Administration of dexamethasone did not produce a decrease in the plasma concentration of cortisol in any of these dogs. Administration of insulin caused slight increases in the plasma concentration of cortisol in four out of eight dogs. Lysine-vasopressin increased the plasma concentration of cortisol in eight out of nine dogs, three responded supranormally. Eight out of the nine dogs responded to tetracosactide administration, three responded supranormally. It is concluded that in the dog, in contrast to man, the lysine-vasopressin test cannot be used to differentiate between pituitary-dependent hyperadrenocorticism and hyperadrenocorticism due to an adrenocortical tumour. Apparently pituitary ACTH is not completely depleted in dogs with hyperfunctioning adrenocortical tumours.

Abstract. In 34 acromegalic patients either untreated or inadequately treated by surgery and/or radiotherapy the plasma GH levels and plasma Prl levels were determined during 8 h after the acute administration of the dopamine agonist bromocriptine and during 150 min after the administration of the dopamine antagonist pimozide.A 50% or more suppression of the basal GH levels was arbitrarily defined as a positive response. Seventeen patients displayed a positive response to bromocriptine, 17 patients displayed a negative response to bromocriptine. Plasma Prl levels were elevated in 6 of 17 bromocriptine responders and in 2 of 17 bromocriptine nonresponders. There was no difference in the plasma GH response to pimozide administration in the patients with a positive or a negative GH response to bromocriptine.With one exception there was in no patient a positive GH response to pimozide. In further experiments the plasma GH response to pimozide was measured during 11 h in 6 bromocriptine sensitive and 6 bromocriptine insensitive patients. Again no difference was found between bromocriptine responders and non-responders. There was also no difference in the Prl response to bromocriptine or pimozide between GH responders and GH non-responders. The Prl response to pimozide was blunted in comparison to a control group.The present study does not lend support to the idea that there is a fundamental difference in the degree of hypothalamic dopaminergic control of GH or Prl secretion between bromocriptine sensitive and bromocriptine insensitive patients with acromegaly.

Hypothalamic corticotrophin releasing factor (CRF) activity was determined in five dogs with spontaneous hyperadrenocorticism and in three control animals (one untreated, one treated with high doses of ACTH for 2 months and one treated with high doses of cortisone for 2 months). Hypothalamic CRF activity was low or undetectable in four dogs with spontaneous pituitary-dependent hyperadrenocorticism and was reduced in one dog with Cushing's syndrome due to an adrenocortical tumour. The results are compatible with a pituitary origin for pituitary-dependent hyperadrenocorticism in the dog but are not conclusive; direct information about the rates of hypothalamic CRF secretion is required.

The biochemical characterization of 22 cases of pituitary-dependent hyperadrenocorticism in the dog, is reported. The principal characteristics of the disease include excessive and non-rhythmic production of cortisol, decreased sensitivity of the hypothalamic-pituitary system to the suppressive effects of dexamethasone, decreased responsiveness of the pituitary-adrenocortical system to the stimulus of insulin-induced hypoglycaemia and increased responsiveness of the system to stimulation with lysine-vasopressin. From these observations it is concluded that pituitary-dependent hyperadrenocorticism in the dog is a valid model for study of the pathogenesis of the disease in man.For the diagnosis of hyperadrenocorticism itself, the measurement of the concentration of corticosteroids in a single sample of plasma obtained 8 h after intravenous injection of 0·01 mg dexamethasone/kg was sufficient. The level of 11-hydroxycorticosteroids was less than 140 nmol/l plasma in normal dogs, whereas higher values were found in dogs with hyperadrenocorticism. For purposes of differential diagnosis, measurement of the level of corticosteroids in the plasma both before and 4h after intravenous injection of 0·05 mg dexamethasone/kg is adequate: suppression is obtained only in cases of pituitary-dependent hyperadrenocorticism.

Administration of cyproheptadine for 2 months to five dogs with pituitary-dependent hyperadrenocorticism (PDH) at a dose rate of 0·3 mg/kg per 24 h (group 1) and to four dogs with PDH at a dose rate of 1 mg/kg per 24 h (group 2) did not result in any clinical improvement. The hyperadrenocorticoid state, as indicated by the circulating cortisol levels, the urinary corticosteroid excretion and the response of the hypothalamo-pituitary-adrenal axis to lysine-vasopressin, thyrotrophin releasing hormone and dexamethasone did not change consistently, although there was a tendency to normalization of some parameters in the dogs of group 2. However, these changes were not found to be consistent for each individual dog but were limited to one parameter per dog. It is concluded that cyproheptadine is not suitable for the treatment of PDH in the dog.J. Endocr.(1984) 101, 311–314

Abstract. Twenty-seven patients with active acromegaly despite previous treatment by surgery and/or radiotherapy received bromocriptine in a dose of 10–20 mg daily for a period of 6–9 months. The results of chronic bromocriptine treatment were evaluated by measurement of plasma growth hormone (GH) levels during the day and by subjective and objective criteria of clinical activity. The results of chronic bromocriptine treatment were also compared with four biochemical criteria obtained before treatment e.g. basal plasma prolactin (Prl) levels and the plasma GH response to oral administration of 2.5 mg bromocriptine respectively iv administration of 200 μg TRH and 500 μg somatostatin. The main observations may be summarized as follows: 1) The mean pre-treatment GH levels during the day ranged from 6–207 mU/l. Hyperprolactinaemia was present in 6 patients. 2) During bromocriptine treatment mean plasma GH levels decreased to less than 50% in 11 patients (GH responders) whereas in 19 patients changes of mean plasma GH and of subjective criteria of clinical activity were concordant. 3) Glucose tolerance improved significantly (P< 0.01) in 10 GH-responders and the urinary hydroxyproline/creatinine ratio decreased significantly (P< 0.05) in 8 GH-responders. 4) Five out of 6 patients with hyperprolactinaemia belonged to the group of GHresponders. 5) A single dose of 2.5 mg bromocriptine induced a more than 50% decrease of plasma GH in 8 of 11 GH-responders and in 5 of 16 GH non-responders. 6) The iv injection of 200 μg TRH was followed by a rise of plasma GH of more than 100% in 9 of 11 GHresponders and in 6 of 16 non-responders. 7) Responsiveness to both a single dose of bromocriptine and TRH was found in 8 of 11 GH-responders and in 3 of 16 GH non-responders.

Dogs with spontaneous pituitary-dependent hyperadrenocorticism were divided into two groups, one with normal plasma concentrations of α-MSH (normal α-MSH dogs, n= 26) and the other with high plasma concentrations of α-MSH (high α-MSH dogs, n= 14), on the presumption that high α-MSH concentrations indicated a parent cell of pars intermedia origin.The urinary corticoid/creatinine ratios of the high α-MSH dogs were significantly higher than those of the normal α-MSH dogs. The percentage decrease of the corticoid/creatinine ratios following dexamethasone administration was significantly higher in the normal α-MSH dogs than in the high α-MSH dogs. Dexamethasone resistance occurred in both the normal α-MSH dogs (4 out of 26) and the high α-MSH dogs (7 out of 14), indicating a relative rather than an absolute difference.The short-term effect of orally administered bromocriptine, at a dose (10 μg/kg body weight) known to be effective in lowering prolactin concentrations in dogs, was investigated by measuring concentrations of cortisol, ACTH and α-MSH in plasma at 4, 6 and 8 h after administration. Significant decreases were observed for cortisol in both groups and for α-MSH only in the high α-MSH dogs.The effect of 5 days of bromocriptine administration (10 μg at 12-h intervals) was assessed by measurements of urinary corticoid/creatinine ratios. Considering both groups as a whole, only the corticoid/creatinine ratios of the high α-MSH dogs decreased significantly on the first day of treatment. In each group there was one dog in which bromocriptine administration lowered the corticoid/creatinine ratios for several days, but the values stayed above the reference range and the animals did not recover.It is concluded that high α-MSH concentrations are not unambiguously associated with the theoretically expected characteristics of pars intermedia disease. The results indicate that the pituitary lesions causing hyperadrenocorticism may not maintain the regulation characteristics of the lobe of origin.J. Endocr. (1988) 118,271–277

Abstract. Eleven patients with active acromegaly were treated with 10–20 mg bromocriptine daily for a period of 6–9 months. The clinical response was evaluated by a 'clinical and metabolic improvement score'. The biochemical response was evaluated by measurement of both the mean plasma growth hormone (GH) level during the day and the somatomedin-C (Sm-C) concentration.Before and at the end of the treatment period plasma samples were fractionated by Sephadex G-100 chromatography in order to study the effects of chronic bromocriptine treatment on the concentrations of total GH and its different molecular forms.The main observations may be summarized as follows:Three immunoreactive components were observed on Sephadex chromatography corresponding to molecular weight above 100 000 (big-big GH), 40000–60000 (big GH) and 20000–22000 (little GH).Bromocriptine treatment induced preferentially a reduction of little GH. There was a very good correlation between the decrease of little GH and total GH, and both were significantly correlated with the clinical response.The correlation between the decrease of Sm-C values and that of little and total GH as well as between the decrease of Sm-C and the clinical response was poor.It is concluded that a) measurement of little GH is not superior to the determination of total GH in the assessment of disease activity of bromocriptine treated acromegalic patients; b) both methods are superior to the measurement of plasma Sm-C levels; c) clinical response out of proportion ot the fall of total GH which can be explained by a preferential reduction of little GH, has not been observed in our investigations.

Insulin-131I metabolism was studied in 10 patients receiving insulin therapy and in 12 untreated subjects (7 normal controls, 4 diabetics treated exclusively with oral hypoglycaemic agents and 1 untreated diabetic).In the subjects not receiving insulin therapy, insulin-131I was rapidly accumulated in the liver and kidneys, and degradation was followed by rapid urinary excretion; in cases in which insulin therapy had induced antibody formation, degradation was inhibited.In tracer studies using different animal insulins and human insulin, species-specific differences in combining with antibodies could be demonstrated both in vitroand in vivo.

In seven cases of proven Cushing's syndrome, the intravenous infusion of dexamethasone at a rate of 1 mg/h for a period of 5 hours readily distinguished pituitary dependent Cushing's syndrome from other causes of Cushing's syndrome. Thus the 5 h – iv dexamethasone test may give the same differential diagnostic information as the classical oral dexamethasone suppression test of Liddle. The test is quick to perform, obviates the need for accurate urine collections and is not influenced by variations in the intestinal absorption of the glucocorticoid.

The plasma cortisol response following intramuscular administration of synthetic lysin-8-vasopressin was measured in nine patients suffering from various forms of Cushing's syndrome. A significant response was obtained in six patients with pituitary dependent Cushing's syndrome, whereas no response was observed in two patients with Cushing's syndrome due to an adrenal tumour and in one patient with an ectopic ACTH syndrome. The possible technical errors which are inherent to this test are discussed in relation to a few contradicting observations from the literature.In three patients with pituitary dependent Cushing's syndrome the use of lysin-8-vasopressin and metyrapone is compared in the evaluation of pituitary-adrenal reserve capacity during various stages of treatment by unilateral adrenalectomy and external pituitary irradiation.

The present study describes a radioimmunoassay for plasma adrenocorticotrophin (ACTH) in man with a sensitivity of at least 15 pg/ml. In-vitro studies using various fragments of ACTH showed immunobiological dissociation. However, this phenomenon does not appear to be of major importance in vivo, since stimulation and suppression tests showed overall parallel changes of immunoreactive plasma ACTH and plasma cortisol. The results obtained from patients with various forms of Cushing's syndrome are presented. It is concluded that basal plasma ACTH determinations are useful in the differential diagnosis of Cushing's syndrome and may help in predicting the development of a pituitary tumour after adrenalectomy.The supranormal plasma cortisol response to i.m. injection of lysinevasopressin in subjects with pituitary-dependent Cushing's syndrome is attributed both to an increased ACTH release by the pituitary and to an excessive response of the hyperplastic adrenal cortex. The i.v. infusion of dexamethasone at a rate of 1 mg/h for 5 h in subjects with pituitarydependent Cushing's syndrome was followed by significant and parallel decreases of both immunoreactive plasma ACTH and plasma cortisol. No rise of plasma ACTH levels was found with insulin-induced hypoglycaemia in subjects with untreated pituitary-dependent Cushing's syndrome.