The nonclassical binding kinetics of IGF-I and insulin to their respective receptors, suggestive of negative cooperativity, can be readily explained by our recently proposed novel binding mechanism whereby the bivalent ligand bridges the two receptor alpha-subunits alternatively at opposite sites in a symmetrical receptor structure. The bivalent binding mechanism also explains bell-shaped bioactivity curves. The possible role of different binding modes versus differences in downstream signaling by insulin and IGF-I in producing specific mitogenic or metabolic responses is discussed.

In most of the children with short stature, no organic basis can be found, which has led to the suggestion that some cases of growth failure may be due to an immunoreactive but bioinactive growth hormone (GH). In order to compare GH immunoreactivity and bioactivity (measured as receptor binding ability), a radioreceptor assay allowing the measurement of GH levels in human serum was developed using cultured human lymphocytes and 125I-labeled human GH purified by high-performance liquid chromatography. Serum samples were obtained after pharmacological stimulation with either insulin, glucagon or GRF from 19 healthy control subjects and 114 patients with various growth disturbances, aged 2.3-24.8 years. In general, there was a good correlation between the GH levels measured by the two methods, the RRA-GH levels being lower than the RIA-GH levels at all times irrespective of the stimulation test. In all the groups studied, most of the individual RRA/RIA ratios were within normal limits. It is concluded that the presence of an abnormal (bioinactive) GH molecule is extremely rare in patients with short stature.

To better understand the complex kinetics of human GH (hGH) binding to its receptors, we have further investigated, in IM-9 cultured human lymphocytes, the cellular locus corresponding to the slowly dissociating component of hormone binding, and to the homologous down-regulation of hGH receptors. First, we have detailed the biphasic kinetics of dissociation of bound hormone in control cells at 30 C. When the association at 30 C was extended from 0.5 to 3 h, the time required for half-dissociation of the fast component was slightly decreased (from 30 to 15 min) but that of the slow component increased considerably (from 6 h to 30 h). Concomitantly, the size of the slowly dissociating component increased from 50 to 80% of total. This indicates a maturation of bound hormone, from a rapidly to a slowly dissociating pool and, in the latter, an increase in the apparent affinity that may reflect a molecular rearrangement. Next, we have compared the effect of two procedures reported to inhibit receptor-mediated endocytosis at the level of coated pits. As previously reported, depletion of intracellular K+ abolished the slowly dissociating component and the down-regulation of hGH receptors. In contrast, upon incubation with 0.4 M sucrose, which like K+ depletion virtually abrogated hGH internalization, the dissociation kinetics remained non-first order, and the down-regulation of hGH-receptor was only slightly reduced. Thus, these procedures appear to block receptor-mediated endocytosis at two successive compartments of the cell surface. In conclusion, we propose that some conformational change of hGH-receptor at the cell surface (possibly associated with clustering) may considerably slow down their dissociation and may be sufficient for down-regulation.

Serum GH levels were measured by RIA and RRA in 133 subjects (19 healthy controls and 114 patients with various growth disturbances, aged 2.3-24.8 yr). Serum samples obtained from 147 stimulation tests representing a total of 1065 samples were analyzed by both methods, and the results compared. The data are expressed in absolute values and in RRA/RIA ratios. The area under the curve after a stimulation test (area GH) was calculated by planimetry. RIA was performed by the classical double antibody method using a polyclonal anti-serum. For the RRA, human cultured lymphocytes (IM-9 cells) were used, and 125I-labeled human GH was purified by high performance liquid chromatography. The same human GH standard was used in both assay systems. In control subjects a significant (P less than 0.0001) positive correlation was found at all ages between GH levels measured by RIA and RRA (r = 0.69 after insulin and r = 0.77 after glucagon). The RRA/RIA ratio (mean +/- SEM) for the peak GH level was 0.88 +/- 0.05, and the area under the GH curve was 0.85 +/- 0.05. The peak mean RRA/RIA ratios were significantly lower (P less than 0.05 and P = 0.03, respectively). No relationship was found with the absolute value of either peak or area GH. In patients with growth delay and Turner's syndrome, lower GH levels were found than in control subjects in both assay systems. The RRA/RIA ratios were also lower. In the other patients with some growth disorder, normal GH levels and ratios were found. In patients with renal failure, high levels of RIA-GH and RRA-GH were found, with a normal RRA/RIA ratio. In patients with documented pituitary GH deficiency, GH-releasing factor administration resulted in an increase in GH levels that was identical in both assays. The RRA/RIA ratio remained constant throughout the test. No correlation was found between the ratio and the absolute value of either RIA-GH or RRA-GH regardless of the stimulation test used. It is concluded that the presence of an abnormal GH molecule is extremely rare in patients with short stature. Thus, the presence of a bioinactive hormone is not a common cause of growth failure. During provocative testing some changes in the ratio may occur that do not appear after GH-releasing factor, further illustrating the different mechanisms involved in GH secretion.

We have investigated whether the slowly dissociating component of insulin and human growth hormone (hGH) binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell. To do so, we used intracellular K+ depletion, which has been shown to inhibit reversibly coated-pit formation and ligand internalization in some cell lines. IM-9 cells incubated in K+-free buffer after a hypotonic shock rapidly lost their K+, which was stabilized at +/- 50% of control by incubation in K+-free binding assay buffer. In K+-depleted cells, the hGH dissociation kinetics became monoexponential and, in contrast with control cells, compatible with the equilibrium constant derived from saturation and association data using a simple model. The loss of hGH receptors during competition studies was abolished. The down-regulation by unlabeled hGH was decreased by 80%. In contrast, insulin receptor kinetics remained unchanged (non-first-order) in the K+-depleted cells; the negative cooperativity and the down-regulation (60%) were identical to those of control cells. Quantitative electron microscopic autoradiography showed a decrease of +/- 50% in the fraction of 125I-labeled hGH internalized. The number of visible coated pits in the membrane was reduced by 80%. Thus, in IM-9 cells, association with coated pits and endocytosis appear to play a major role in the kinetics of hGH binding and in the down-regulation of its receptors, but not in insulin-receptor binding kinetics and down-regulation.

Human growth hormone (hGH) was labelled with 125Iodine to a specific activity of 50-80 μCi/μg by a stoichiometric modification of the chloramine-T method. After labelling, the crude reaction mixture containing 125I-hGH, unreacted iodide, chloramine-T, salts, etc. was purified by reverse-phase high performance liquid chromatography (HPLC) using a Synchropak RP-P column (Altech, USA) and a linear gradient. Biological activity of the purified material was measured by binding to IM-9 lymphocyte hGH receptors. The results were compared with those obtained after purification by gel filtration using a sephadex G-100 column. HPLC gave a much better separation than sephadex chromatography. The improved resolution was the result of an increase both in selectivity and efficiency. The analysis time was considerably reduced by HPLC (30 min vs 5 h). The recovery was comparable in both methods (95%). Specific binding of 125I-hGH to lymphocyte membrane receptors was higher by 20 % with the preparation purified by HPLC than with the sephadex material. The maximum bindability of the hormone by an excess of lymphocyte receptors was 86 % for the HPLC-purified 125I-hGH and 71 % for the sephadex-purified 125I-hGH. Both preparations behaved similarly in kinetic experiments. It is concluded that HPLC is a fast, convenient and reproducible method for purifying 125I-hGH for receptor studies.

Plasma levels of dehydroepiandrosterone-sulfate (DHEA-S), dehydroepiandrosterone (DHEA), delta 4-androstenedione (delta 4), testosterone and 17 alpha-OH-progesterone (17-OH-P) were studied in 58 samples collected in 18 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, during long-term ambulatory treatment with hydrocortisone. At each visit the patients were classified as being either in good control (GC) or in poor control (PC), based on well-defined clinical, auxological and biochemical criteria. The results were analyzed in relation to the degree of control and to chronological age (CA), bone age (BA), body surface (BS) and pubertal development. The most clear distinction between the children with GC and those with PC is found for DHEA-S (p less than 0.001 for BA). The majority of the DHEA-S values in the children with GC are closely grouped and significantly below the normal limits for CA, BA, BS and pubertal stage (p less than 0.001). In contrast, the PC children have wide-spread values, most of them being within or above the normal limits. The difference between GC and PC is also significant for testosterone (p less than 0.01) and delta 4 (p less than 0.05), but not for DHEA. Of the five steroids studied, DHEA-S is the most specific, whereas testosterone is the most sensitive and especially useful in girls and in prepubertal boys. delta 4 and 17-OH-P are almost as sensitive as DHEA-S, but they are less specific. DHEA is the less valid criterium.

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In this prospective longitudinal study, plasma androgen levels were determined during 1-7 years in 45 patients aged 5.6-23.8 years with either isolated growth hormone (GH) deficiency or multiple pituitary hormone deficiencies. Dehydroepiandrosterone sulfate, dehydroepiandrosterone, delta 4-androstenedione and testosterone were measured by RIA in 339 blood samples collected during the study period. Mean plasma androgen levels are normal in isolated GH deficiency. Patients with multiple pituitary hormone deficiencies, but normal ACTH reserve, have mean levels lower than normal. Patients with multiple deficiencies including ACTH deficiency have still lower plasma androgen levels. Longitudinal analysis of the data, however, shows that patients with either isolated GH deficiency or multiple pituitary deficiencies without ACTH deficiency constitute a heterogeneous population, with either normal or low to very low plasma androgen levels. Treatment with human GH as such does not have any effect on the adrenal androgen secretion. A dissociation is found in some patients between adrenarche and gonadarche, which indicates that the two events are not controlled by the same mechanisms. Our results support the existence of a specific hypothalamic-pituitary adrenal androgen-stimulating hormone (AASH). ACTH, although not identical to AASH, is essential for normal adrenarche. Induced puberty with estrogens in girls does not influence plasma androgen levels, and pubic and axillary hair growth is not achieved. It is suggested that replacement treatment with dehydroepiandrosterone sulfate should be administered to girls with hypopituitarism and very low plasma androgen levels at the time of the induction of puberty. Finally, it appears from this study that, to interpret the plasma androgens in hypopituitarism, body surface is as good as bone age.

A 7 5/12 year-old boy developed pseudoprecocious puberty. The diagnosis of Leydig cell tumour was suggested by clinical and hormonal findings and greatly facilitated by ultrasonographic investigation of the testes. Surgical exploration was in keeping with the diagnosis and the tumour was removed. Orchidectomy was not necessary. It appears that ultrasonography may be very useful in diagnosing a testicular tumour. It facilitates surgical intervention since it permits localization of the lesion which is usually very small and limited to a small part of the testis, as in this case.

Plasma levels of androstenedione,dehydroepiandrosterone, dehydroepiandrosterone sulphate (DHA-S), and testosterone (T), were measured (RIA after chromatographic separation) before and throughout puberty in 90 normal subjects and 235 children with various endocrine and growth disorders. In normal prepubertal boys, but not in girls, plasma levels of androgens rise significantly after the age of 6 years. The only sex differences are for DHA-S in the age range 1-6 and for T after 6 years. All values increase throughout puberty. In a longitudinal study (177 determinations in 39 hypopituitary patients), two distinct populations with either normal or low levels were found. In growth delay, levels are normal for bone age (BA), but not for chronological age (CA). In precocious puberty and simple obesity levels are high for CA, but normal for BA. It appears that plasma levels of adrenal androgens are a helpful tool in growth and puberty disorders.