Control of the intracellular Ca2+-concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: Evidence for different kinetics of Ca2+-phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production

Abstract

Carbachol, through a muscarinic receptor, thyrotropin‐releasing hormone (TRH), prostaglandin F_2α (PGF_2α), bradykinin, and adenosine triphosphate (ATP) in creased the apparent [Ca²⁺], (intracellular free Ca²⁺ concentration) of dog thyrocytes in primary culture. The [Ca²⁺], measured by the Quin‐2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca²⁺], declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF_2α and in the case of bradykinin, TRH, and ATP, the [Ca²⁺], returned to the basal value. If the extracellular Ca²⁺ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca²⁺], followed by a decline of the [Ca²⁺], often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca²⁺ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca²⁺ influx into the thyrocytes. Carbachol, bradykinin, TRH, PGF_2α, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF_2α, or ATP. All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H₂O₂ generation. This enhancement was severely reduced in the absence of extracellular Ca²⁺ and was mimicked by Ca²⁺ ionophores in the presence of extracellular Ca²⁺ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H₂ O₂ generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF_2α, bradykinin, and ATP through their action on the Ca²⁺‐phosphatidylinositol cascade.