12-O-tetradecanoyl-phorbol-13-acetate (TPA) counteracts the cAMP up-regulation of the expression of the stimulatory guanine nucleotide binding protein (Gsα) and Gsα 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 cells. The two days culture of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) (0.1 μM) alone neither affected adenylyl cyclase activity, nor the level of Gsα protein in membranes when compared with control cells. The co-addition of TPA with thyrotropin (TSH) (1 mU/ml) or forskolin (fk) (10 μM) in the culture medium, abolished the stimulatory effects of either agonists on the activation of adenylyl cyclase by fk or [AlF₄]^- and on the increase of Gsα protein. By contrast, TPA had effects neither on the Gi-dependent inhibition of adenylyl cyclase nor on Giα proteins levels. The level of Gsα mRNA measured by Northern blot analysis was increased (200%) in TSH- or fk-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 mezerein, a non-phorbol protein kinase C activator and blocked by bisindolylmaleimide a specific protein kinase C inhibitor (GF 109203X). These results suggest that the activation of the PKC pathway prevents the cAMP-dependent up-regulation of Gsα protein and Gsα mRNA expression.