Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.

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.