Relationships between the GTP content and the TSH-stimulated adenylate cyclase activity of cultured thyroid cells

The adenylate cyclase activity of a crude membrane fraction derived from cells cultured for 4 days in the presence of TSH (0.1 mU/ml), when acutely stimulated with 25 mU/ml, is 5-8 times higher than that derived from control cells. It has been suggested that changes in the intracellular content of GTP resulting from TSH chronic treatment were the cause of the modified responsiveness of the cyclase. To investigate this hypothesis, a method for GTP determination was developed. The steady-state concentration of GTP in 4-day TSH-treated cells is 2-3 times higher than in 4-day control cells. The increase in GTP content is concentration dependent between 5 and 500 microU/ml TSH in the culture medium. It presents a maximum on day 4 of culture, but remains elevated up to day 5. Nevertheless the GTP content is not the only factor controlling the cyclase activity, indeed the addition of 0.1 mM GTP to membranes from control cells does not increase the response up to the level reached by membranes from TSH-treated cells. Treatment of the cells with virazole, a drug inhibiting the biosynthesis of guanyl nucleotides, greatly decreases the GTP level, but is unable to suppress the positive effect of the TSH chronic treatment on adenylate cyclase activity. These results show that the increase in GTP level resulting from culture of the cells in the presence of minute amounts of TSH is not exclusively linked to adenylate cyclase responsiveness to TSH.