Human thyroid adenylate cyclase in non-toxic goitre: sensitivity to TSH, fluoride and thyroid stimulating immunoglobulins.

SUMMARY Homogenates prepared from human thyroid tissue obtained at operation for non-toxic goitre were separated by differential centrifugation into 1000 g, 5000 g, 10,000 g and 37,000 g fractions. The fractions showed varying amounts of TSH (12.5 mu/ml) and fluoride (10 mmol/1) sensitive adenylate cyclase activity. The 5000 g pellet contained the highest amount of TSH-sensitive adenylate cyclase activity calculated per mg protein. Of other potential activators tested only fluoride, immunoglobulins from patients with Graves' disease and adrenaline (10−6 mol/l) caused significant stimulation in this fraction, which was selected for further studies. Maximal activation of adenylate cyclase was obtained with 75 mu TSH/ml incubate, half maximal stimulation with 2.9 mu TSH/ml and detectable stimulation was found with 0.0125 mu TSH/ml. Supramaximal concentrations (1.25 u/ml) gave a decreased response. Stimulation with TSH resulted in a linear production of cyclic AMP for 40–60 min dependent on the TSH concentration. 30 min pre-incubation of homogenate with TSH at 4°C did not influence the response after addition of ATP-buffer and subsequent incubation at 37°C, suggesting a very rapid equilibrium for binding of TSH to its receptor after addition of ATP-buffer to the system. In the 5000 g fraction stimulation of adenylate cyclase with TSH 12.5–75 mu/ml incubate exceeded that obtained with fluoride, and when combined in maximal concentrations an additive effect was observed after incubation for 40 and 60 min, but not after 10 and 20 min, and fluoride caused a linearization of the cAMP response to maximally stimulatory concentrations of TSH. Thus, fluoride appears to maintain the TSH stimulated adenylate cyclases in a maximally activated state. The 5000 g fraction was sensitive to stimulation with immunoglobulins (TSI) from ten of eleven randomly selected patients with untreated Graves' disease. TSI stimulated adenylate cyclase activity could exhibit a lag-phase of at least 20 min, and stimulation was most readily detected after 60 min incubation at 37°C. The TSI preparations from patients with Graves' disease exhibit varying degrees of lag-phase indicating that TSI is a heterologous class of immunoglobulins with activation kinetics distinctly different from those of TSH.