h-89 and erbstatin

Epidermal growth factor (EGF) has been shown to influence FSH-stimulated estradiol (E2) and progesterone (P4) production from granulosa cells. RG 50810, a tyrosine kinase inhibitor (TKI), has previously been shown to inhibit the EGF-receptor tyrosine kinase. RG 50810 has also been shown to inhibit FSH-stimulated increases in mRNA for steroidogenic enzymes, implying a functional role of tyrosine kinases in FSH action in granulosa cells. However, inhibition of FSH-stimulated steroidogenesis by TKIs has not been evaluated in connection with the effects of EGF in granulosa cells. In the present studies, FSH-stimulated E2 production was inhibited similarly by inhibitors of protein kinase A (H-89) and protein kinase C (calphostin C) and by TKIs, and none of the inhibitors were capable of reversing the EGF-induced inhibition of FSH-stimulated E2 production. FSH-stimulated P4 production was enhanced dramatically in serum-containing medium with concentrations of TKI that were near previously reported IC50s. The enhancing effect of TKIs was less evident in serum-free medium. Addition of EGF to serum-free medium enhanced FSH-stimulated P4 production, and the TKIs reversed EGF-enhanced P4 production, but in a manner similar to that of protein kinase A inhibitor H-89. Compared to results in serum-free medium, the potency of RG 50810 and genistein to inhibit the effects of EGF on P4 production was 3- to 8-fold greater relative to H-89. These studies have demonstrated that TKIs RG 50810 and genistein selectively inhibit the effects of EGF on FSH-stimulated P4 production in granulosa cell cultures. In contrast, these studies have demonstrated nonselective inhibition of FSH-stimulated E2 and P4 production by TKIs in serum-free medium, in which it is not clear which enzyme system is affected by the compounds tested.

Topics: Animals; Cells, Cultured; Culture Media, Serum-Free; Enzyme Inhibitors; Epidermal Growth Factor; Estradiol; Female; Follicle Stimulating Hormone; Genistein; Granulosa Cells; Hydroquinones; Isoquinolines; Naphthalenes; Progesterone; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Steroids; Sulfonamides; Tyrphostins

1. The differential effects of inhibitors of protein kinase (PK) or tyrosine kinase (TK) on phosphatidylcholine (PC) biosynthesis in monocyte-like U937 cells were compared in pulse-chase-studies in which the cells prelabelled with [3H]choline for 30 min were chased in the absence or presence of kinase inhibitors. 2. PKA inhibitor (H-89) decreased the label incorporation into PC, while PKA activator (8-BrcAMP) had no effect. 3. PKC inhibitors (chelerythrine and hypericin) inhibited PC biosynthesis; on the other hand, PKC activator (SC-10) was stimulatory. 4. The inhibition of PC biosynthesis by H-89 and chelerythrine was accompanied by the inactivation of CTP: cholinephosphate cytidylyltransferase (CT). 5. In contrast, TK inhibitor (genistein) markedly stimulated CT and PC biosynthesis, while erbstatin and tyrphostin No. 25 showed no effect.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Catechols; Cyclic AMP-Dependent Protein Kinases; Genistein; Humans; Hydroquinones; Isoflavones; Isoquinolines; Monocytes; Nitriles; Phosphatidylcholines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Sulfonamides; Tumor Cells, Cultured; Tyrphostins