Mechanisms by Which Low Salt Condition Increases Sensitivity of Thyroid Stimulating Antibody Assay*

Abstract

Mechanisms for high sensitivity in the assay of thyroid stimulating antibody under low salt (NaCl-deprived) hypotonic condition were analyzed using FRTL-5 rat thyroid cells. First, intracellular and extracellular cAMP contents after stimulations with TSH and Graves' immunoglobulin (Ig) were measured under both low salt (hypotonic) and high salt (isotonic) conditions. Higher total and much higher extracellular cAMP production were observed under the low salt condition. Under the high salt condition, the intracellular cAMP content reached a plateau after 10 min, whereas it increased progressively up to 120 min under the low salt condition. Thus, other cellular mechanisms, as well as increase in membrane permeability, seem to be involved in the enhanced response under the low salt condition. On the other hand, pretreatment of the cells with low salt solution without stimulator resulted in a decrease in subsequent cAMP response to TSH or Graves' Ig. Furthermore, [125I]bovine TSH binding to the cells under low salt condition also decreased after the cells were pretreated with the low salt solution. Scatchard analysis revealed that this decrease was due to a decrease in the Cmax of the low affinity binding site of the TSH receptor. Presence of stimulatory ligands to the TSH receptor from the beginning of exposure of the cells to low salt conditions was considered necessary. When the cells had been pretreated at 4 C with stimulator preparations in low salt condition, subsequent incubation at 37 C without stimulator resulted in more cAMP production than the case of high salt pretreatment. Moreover, low salt ligand-free incubation after the high salt binding resulted in higher cAMP production than high salt ligand-free incubation after the low salt binding. Thus, both initial binding and incubation under the low salt condition appear capable of augmenting cAMP production. The fact that the sensitivity of the low salt assay to Graves' Ig was higher than that to bovine TSH was found to relate closely to the increase in initial binding. In conclusion, augmentation of cAMP production under low salt conditions is considered to be related not only to increased ligand binding and increased membrane permeability, but also to some alterations of postreceptor mechanisms during incubation. The proposed postreceptor mechanisms seem to be induced by stimulator binding, which prevents the TSH receptor from damage and promotes intracellular cAMP production progressively. Details of this remain to be elucidated.