The thyroid-stimulating hormone (TSH) binds to a receptor which activates adenylate cyclase and elevates cAMP concentration. In addition, effects of TSH on intracellular calcium and inositol phosphate accumulation have been reported. However, the mechanism of TSH-stimulated accumulation of inositol phosphates and elevation of calcium levels is unresolved. Previous work from this laboratory has shown TSH to cause acute transient increases in intracellular calcium in pig, human and FR TL-5 rat thyroid cells as well as in cell transfected with the human TSH receptor (JPO9 cells) in some (but not all) experiments. The aim of this study was to investigate the variability of the calcium response to TSH in JPO9 cells to learn more about the nature of this calcium signal induction. Calcium responses to TSH were determined using the fluorochrome fura-2 in both monolayers of adherent cells and adherent single cells. The responses to a single addition and to repetitive additions of TSH were compared. We also determined the cAMP response to TSH using these two protocols of TSH addition. Our data show that, whereas the cAMP response to TSH is highly predictable and consistent and does not require multiple exposures to TSH, cells were unlikely to respond to TSH with an increase in calcium unless they received multiple challenges with the hormone. A single addition of 10 mU/ml TSH failed to increase calcium in any of 40 single cells examined and in only 4 of 15 monolayers of cells (27%) examined; in contrast, 10 of 12 monolayers eventually responded with an increase in calcium after multiple exposure to TSH and 18 of 67 single cells. Similar data were obtained whether calcium was measured in single cells or in populations of cells. We also demonstrated cooperativity between an adenosine derivative, N6-(L-2-phenylisopropyl)adenosine, and TSH such that their co-administration resulted in a consistent and marked elevation in calcium levels not achieved with either agonist alone. In summary, we suggest that the coupling between the TSH receptor and the intracellular signalling system that leads to activation of intracellular calcium in JPO9 cells requires repetitive stimulation or the influence of other agonists, in contrast with the coupling between the TSH receptor and activation of the adenylate cyclase enzyme.

Abnormalities of the hypothalamus-pituitary-adrenal axis and hypersensitivity to corticosteroids have been suggested as major determinants of the development of visceral obesity. Since at the cellular level most effects of corticosteroids are mediated by specific receptors, we evaluated the number of type I and type II corticosteroid receptors in mononuclear leucocytes of 26 obese and 13 control subjects. We also studied the relationship between corticosteroid receptors, measured by radioreceptor assay, and abdominal visceral fat, evaluated by computed tomography scan, plasma and urine corticosteroid hormone concentrations and overall glucose metabolism, assessed by euglycaemic-hyperinsulinaemic clamp. We observed a decrease in type II receptors in the obese subjects (1746 +/- 160 vs 2829 +/- 201 per cell; P < 0.0001), with no change in type I receptors. Type II receptors decreased in relation to body mass index (r = -0.53; P < 0.005) and total glucose disposal (r = 0.51; P < 0.01). Abdominal visceral fat did not correlate with type II receptor number, but did correlate with total glucose disposal (r = -0.35; P < 0.05); the rate of glucose disposal was lower in obese subjects (3.3 +/- 0.3 vs 7.4 +/- 0.4 mg/kg per min; P < 0.001). Plasma and urine cortisol did not differ between the two groups. However, a direct correlation between type II receptor number and both plasma (r = 0.43; P < 0.02) and urine cortisol concentrations (r = 0.60; P < 0.05) was observed. In conclusion, the number of type II corticosteroid receptors in mononuclear leucocytes was found to be lower in obese subjects. This abnormality appears to be related to the degree of adiposity and to the main endocrine-metabolic features of the obesity syndrome, further supporting the hypothesis of involvement of hypothalamus-pituitary-adrenal axis hyperactivity in the pathophysiology of obesity.

ABSTRACT

In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band co-migrating with authentic calmodulin (M _r 17 000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of M _r 14 000–15 000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form.

Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0·03 nmol/l in urine to 18·6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0·79, P < 0·01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products.

J. Endocr. (1988) 118, 501–509