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ABSTRACT
The effects of TSH and forskolin were examined on intracellular free calcium ([Ca2+]i) and calmodulin in normal pig thyroid cells in culture. TSH was found to produce acute increases in [Ca2+]i in pig cells. Responses were seen at concentrations of TSH between 0·01 and 10 mU/ml. Sensitivity to TSH was greater in adherent monolayers of cells than in cell suspensions and was also greater in subconfluent rather than confluent monolayers of cells. The increase in [Ca2+]i in response to TSH represented just over a doubling in [Ca2+]i whether examined at 22 °C or 37 °C. Forskolin failed to affect [Ca2+]i. TSH increased [Ca2+]i in the absence of extracellular calcium.
TSH, but not forskolin, produced a significant increase in intracellular calmodulin after 3 days of culture of cells with TSH. The increase in calmodulin was of the order of 60% and did not relate to any effect of TSH on thyroid cell number.
Journal of Endocrinology (1991) 129, 291–299
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ABSTRACT
The effect of α-melanocyte-stimulating hormone (α-MSH) on protein kinase C activity and distribution was investigated in murine B16 F1 melanoma cells, α-MSH was found to induce an increased association of protein kinase C (PKC) activity with the particulate fraction of the cells, with an associated loss of enzyme activity from the soluble fraction. The peak response to α-MSH occurred between 20 and 60 min of incubation time, and enzyme activities redistributed to those seen in the control cells over the following 12 to 24 h. The average response to α-MSH (1 nmol/l) was an approximate 2·5-fold increase in the percentage of enzyme activity associated with the membrane within 1 h of exposure to α-MSH; the particulate enzyme activity represented 19·2 ± 4·4% of total activity in the absence of α-MSH and 50·7 ± 4·7% (means ± s.e.m., n = 9, P < 0·005) in the presence of α-MSH (1 nmol/l). Cells which had a relatively small percentage of their PKC activity on the membrane initially were significantly (P < 0·01) more responsive to α-MSH stimulation than cells which initially had a relatively large percentage of PKC activity on the membrane. The association of PKC activity with the membrane showed some evidence of being dose-related to α-MSH. This is the first report, to the best of our knowledge, of α-MSH activating PKC.
Journal of Endocrinology (1992) 133, 333–340
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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.
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ABSTRACT
A bioassay for epidermal growth factor (EGF) is described using an eluted stain assay (ESTA) of dehydrogenase activity in pig thyrocytes. Optimal responsiveness to EGF was obtained in confluent cultures of primary pig thyrocytes cultured with EGF or biological samples containing EGF for either 24 or 48 h. Dehydrogenase activity was determined by measuring the production and then the release of a coloured formazan product produced by reduction of a 3-[4,5-dimethylthiazol-2 -yl]-2,5-diphenyltetrazolium bromide substrate added to the cells.
The assay responded equally to mouse, human and recombinant EGF and was suitable for measuring EGF activity in some but not all biological fluids. Specificity of detection of EGF activity was confirmed using antibody to EGF. The ESTA assay compared favourably with the radioreceptor assay for EGF in terms of sensitivity to EGF with half-maximal activation at 0·24 ±0·06 nmol/l (mean ± s.e.m., n= 22 experiments) for the ESTA assay and 0·60 ±0.13 nmol/l (n=7) experiments) for the radioreceptor assay.
Journal of Endocrinology (1992) 134, 449–457
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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
