Characterization of thyroid-stimulating immunoglobulin-induced cyclic AMP accumulation in the rat thyroid cell strain FRTL-5: potentiation by forskolin and calibration against reference preparations of thyrotrophin

Abstract

A clonal strain of rat thyroid cells (FRTL-5) has been used to investigate the biological activity of a Research Standard preparation of long-acting thyroid stimulator (LATS-B). Using the accumulation of intracellular cyclic AMP as a response parameter, significant stimulation was attained at a LATS-B dose of 0.75 mu./ml. The inter-bioassay coefficient of variation in response to a fixed dose of LATS-B (1.25 mu./ml) was 20.5%, as determined using eight sequential subcultures. Cells cultured directly from frozen stocks responded to both bovine TSH and LATS-B in a manner indistinguishable from cells subjected to regular subculturing. Cyclic AMP responses to incremental doses of LATS-B were potentiated after the inclusion of a low dose of forskolin (0.1 mumol/l). However, forskolin addition had no effect on the time-course of LATS-B-stimulated cyclic AMP accumulation, half-maximal responses being attained after 60 min in either the presence or absence of the diterpene. In the presence of 0.1 mumol forskolin/l, intracellular cyclic AMP responses to LATS-B were demonstrably parallel with those to human TSH (Second International Reference Preparation, 80/558), whilst parallel incremental cyclic AMP responses were also observed in respect of TSH and serial dilutions of a potent thyroid-stimulating immunoglobulin (TSIg) preparation, indicating that for this particular Graves' disease patient, TSIg bioactivity may be expressed in terms of a convenient and reproducible standard, as TSH microunit equivalents.