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The dog thyrocyte I- trapping activity and the expression of the genes coding for dog thyrocyte thyroglobulin or thyroid peroxidase are enhanced by TSH through the cAMP cascade and reduced by mitogens such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol 13-acetate (TPA). In this work, we investigated whether H2O2 generation (a limiting step of thyroid hormone synthesis) is modulated by chronic treatment of the thyrocyte with TSH or mitogens such as EGF or TPA. We observed that both basal and carbachol- or ionomycin-stimulated H2O2 generation by the dog thyrocyte were concentration and time dependently enhanced by prolonged (12- to 72-h) exposure to TSH. This effect was reproduced by agents that increase the dog thyrocyte cAMP level or that mimic this increase. It was abolished when protein or RNA synthesis was inhibited. By contrast, EGF and TPA concentration and time dependently antagonized the effect of TSH. In addition, chronic exposure to EGF reduced both basal and carbachol- or ionomycin-stimulated H2O2 generation. The effect of TPA was reproduced by another protein kinase-C activating phorbol ester, phorbol dibutyrate, but not by beta-phorbol, an inactive phorbol ester. Modulation of dog thyrocyte H2O2 generation by chronic exposure to TSH or to the mitogens EGF and TPA was totally parallel to the modulation of their 125I- uptake. Taken together our results suggest that H2O2 generation (or at least one of its constituents) is a differentiation characteristic of the dog thyrocyte under tonic control of TSH through the cAMP cascade as iodide transport, thyroid peroxidase, and thyroglobulin.

Protein iodination by the dog thyrocyte (a marker of thyroid hormone synthesis) is stimulated by the Ca(2+)-phosphatidylinositol and cAMP cascades. We have shown previously that H2O2 generation, a limiting step of thyroid hormone synthesis, is modulated by these two cascades. In this work, we show that the I- release from preloaded thyrocytes is also activated by agents activating the Ca(2+)-phosphatidylinositol cascade and by Ca2+ ionophores, especially in synergy with 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase-C. The effect of carbachol is reduced when the extracellular Ca2+ is depleted. Thus, both arms of the Ca(2+)-phosphatidylinositol cascade, Ca2+ and diacylglycerol, acutely and synergistically activate dog thyrocyte I- release. This I- release was also accelerated by acute and chronic exposure to TSH, forskolin, or (BU)2cAMP. The chronic stimulation of I- release by TSH exposure was diminished by chronic epidermal growth factor treatment (which dedifferentiates the thyrocytes). In addition, the chronic stimulation of I- release by forskolin was not affected by withdrawal of the agent up to 4 h before the experiment, in contrast to the acute effect of forskolin, which vanished within 16 min after forskolin withdrawal. These results suggest that the chronic stimulation of I- release by TSH or forskolin involves a stable mechanism. The I- transport system causing the release of I- from the dog thyrocyte is almost insensitive to inhibition by NaClO4 and KSCN. Hence, the iodide release cannot be due to the action of the basolateral Na+/I- cotransporter. In addition, we show that I- release was less sensitive than I- uptake to the inhibition by dysidenin, a marine toxin isolated from the sponge, Dysidea herbacea, known to inhibit I- uptake by dog thyroid slices. In summary, this work suggests that in a well defined model of the thyroid, the dog thyrocyte in primary culture, an I- transport system distinct from the basolateral Na+/I- cotransporter, is responsible for the observed I- release. The complex modulation of this transport system, involving at least the Ca(2+)-phosphatidylinositol and cAMP cascades, parallels the regulation of protein iodination, which itself reflects thyroid hormone synthesis.

The effect of thyrotropin (TSH) on cyclic AMP accumulation, phosphatidylinositol bisphosphate (PIP2) hydrolysis and [Ca2+]i rise has been studied in CHO cells stably transfected with human TSH receptor (hTSHR) cDNA. In human thyroid slices, TSH activates these two intracellular cascades with a higher affinity for the adenylate cyclase activation (from 0.1 to 1 mU/ml TSH) than for phospholipase C activation (from 1 to 10 mU/ml TSH). The CHO cells transfected with the recently cloned cDNA of human TSH receptor respond in the same way to TSH. They respond between 0.1 and 1 mU/ml TSH for cyclic AMP accumulation and between 1 and 10 mU/ml TSH for inositol monophosphate (IP1) increase. In these same cells, TSH 10 mU/ml, but not forskolin (10 microM), or dibutyryl cyclic AMP (100 microM), clearly enhances intracellular calcium concentration [( Ca2+]i). Our results demonstrate unequivocally that a single transcription unit has the potential to encode receptor molecules coupled to both cascades.