RESUMO
The desensitization of follicle-stimulating hormone (FSH)-evoked cAMP synthesis occurs upon continuous or repeated hormonal stimulation, and it involves the hormone-receptor interaction and post-receptor events. These mechanisms were studied in a murine granulosa cell line (KK-1) stably transfected with the human FSH receptor (hFSHR) complementary deoxyribonucleic acid (cDNA) under a powerful viral promoter. Hence, the FSHR transcriptional regulation was eliminated from the experimental model. Stimulation of the cells with recombinant human FSH (rhFSH) or a phorbol ester, 12-O-tetradecanoylphorbol-13 acetate (TPA), resulted in clear desensitization, i.e. subsequent rhFSH-stimulated cAMP formation was 73.4 +/-2.2%, (P < 0.001) and 66.3 +/-3.4%, (P < 0.0001), respectively, of that of cells preincubated in medium. TPA prestimulation evoked also clear inhibition (65-74% of control) of rhFSH or forskolin (a non-specific activator of adenylate cyclase) induced progesterone production. The suppression by TPA preincubation of the rhFSH-induced cAMP synthesis was completely abolished by the protein kinase C (PKC) inhibitor staurosporine (STR). Preincubation with STR exhibited a significant (P < 0.0001) increasing effect on the rhFSH-stimulated cAMP accumulation. The specific involvement of PKC was further evidenced by other inhibitors, all of them exerted significant elevation of cAMP synthesis following rhFSH restimulation. Furthermore, only the PKC beta isoform appeared to be constitutively expressed in these cells during desensitization. Prestimulation of the G-protein activity by sodium fluoride (NaF) or cholera toxin (CT), followed by rhFSH challenge, accounted for a decrease in the cAMP-mediated responsiveness, down to 69.4 +/- 2.8 or 74.2 +/- 1.9%, of control (P < 0.001), respectively, indicating that the post-receptor events are critical for desensitization. [125I]iodo-rhFSH binding to the cells did not change significantly during desensitization and the different stimulations. In contrast, approximately 50% increase (P < 0.001) occurred in the steady-state levels of FSHR mRNA in the cells stimulated with FSH. This was apparently due to prolonged half-time of mRNA, and not to altered transcription, since the FSHR cDNA was driven by a powerful viral promoter. In accordance, the cells transfected with Simian Virus (SV40) promoter-driven luciferase gene did not display alterations in luciferase activity following stimulatory treatments. The effects of the post-receptor stimulations (NaF or CT) on [125I]iodo-rhFSH binding were minor (8-12% reduction). Taken together, these data provide evidence that the agonist-responsive hFSHR desensitization appears through a PKC-beta isoform-mediated modulation of cAMP production. The desensitization of FSH action involves modifications of functional properties of the existing components of the FSH signal transduction complex, and does not require concomitant suppression of transcription or translation of the FSHR gene.
