12-O-TETRADECANOYL-PHORBOL-13-ACETATE (TPA) COUNTERACTS THE CAMP UP-REGULATION OF THE EXPRESSION OF THE STIMULATORY GUANINE-NUCLEOTIDE-BINDING PROTEIN (GS-ALPHA) AND GS-ALPHA MESSENGER-RNA IN CULTURED PIG THYROID-CELLS

In this study, we examined whether the protein kinase C (PKC) pathway could interfere with the regulation of Gs protein in porcine thyroid c ells. The two days culture of cells with 12-O-tetradecanoylphorbol 13- acetate (TPA) (0.1 mu M) alone neither affected adenylyl cyclase activ ity, nor the level of Gs alpha protein in membranes when compared with control cells. The co-addition of TPA with thyrotropin (TSH) (1 mU/ml ) or forskolin (fk) (10 mu M) in the culture medium, abolished the sti mulatory effects of either agonists on the activation of adenylyl cycl ase by fk or [AlF4](-) and on the increase of Gs alpha protein. By con trast, TPA had effects neither on the Gi-dependent inhibition of adeny lyl cyclase nor on Gi alpha proteins levels. The level of Gs alpha mRN A measured by Northern blot analysis was increased (200%) in TSH- or f k-treated cells and this increase was counteracted by TPA. The effects of TPA observed after 6-9 h of contact with cells were mimicked by me zerein, a non-phorbol protein kinase C activator and blocked by bisind olylmaleimide a specific protein kinase C inhibitor (GF 109203X). Thes e results suggest that the activation of the PKC pathway prevents the cAMP-dependent up-regulation of Gs alpha protein and Gsa mRNA expressi on.