Inhibitors of phosphodiesterase III block stimulation of xenopus laevis oocyte ribosomal S6 kinase activity by insulin-like growth factor-I

Abstract

Insulin-like growth factor-I (IGF-I) stimulated Xenopus laevis oocyte ribosomal S6 kinase activity 5- to 10-fold, with an apparent EC50 of 0.8 ± 0.1 nM after 90 min of hormone treatment. IGF-I-stimulated enzyme activity was inhibited by treatment of oocytes with nonselective phosphodiesterase (PDE) inhibitors, with apparent IC50 values of 2 ± 1 μM papaverine, 20 ± 2 μM isobutylmethylxanthine, and 128 ± 16 μM theophylline. Type III PDE inhibitors also inhibited IGF-I-stimulated S6 kinase activity with IC50 values of 9.7 μ 0.3 μM CI-930 and 84 μ 23 μM imazodan (Cl-914). These drugs apparently affected an intracellular molecular event leading to activation of S6 kinase, since Cl-930 prevented IGF-I-stimulation of S6 kinase, but had no direct inhibitory effect when added to the S6 kinase enzyme assay mixture. While hormone-stimulated S6 kinase activity was inhibited by isobutylmethylxanthine (nonselective PDE inhibitor) and Cl-930 (PDE III inhibitor), Ro 20, 1724 and rolipram (PDE IV inhibitors) and dipyridamole (PDE V inhibitor) had no significant effect on activated enzyme levels. The time course for IGF-I stimulation of oocyte S6 kinase displayed a small early peak of activity approximately 0.15-0.4 time required for 50% of cell population to display white spots (GVBD50) and a second major increase in activity at 0.6-0.7 GVBD50 that was sustained until meiotic maturation was complete. The second wave of enzyme activation was inhibited by Cl-930, but the early increase was not. These results further support the role for PDE III in the chronology of molecular events that trigger amphibian oocyte maturation and suggest that stimulation of PDE III is necessary for subsequent activation of S6 kinase by IGF-I.