Src tyrosine kinase activity in rat thecal–interstitial cells and mouse TM3 Leydig cells is positively associated with cAMP-specific phosphodiesterase activity

Abstract

Phosphodiesterases (PDEs) play a critical role in the regulation of intracellular cyclic nucleotide concentration and, consequently, regulate the state of cellular differentiation. We have reported that the Src-selective tyrosine kinase inhibitor, herbimycin A, potentiates luteinizing hormone (LH)-stimulated cAMP accumulation in culture media by ovarian thecal–interstitial cells (TIC; see Taylor, C. and Terranova, P.F. (1995) Lipopolysaccharide inhibits rat ovarian thecal–interstitial cell steroid secretion in vitro. Endocrinology 136, 5527–5532). The present study was conducted to investigate the effects of herbimycin, and changes in Src tyrosine kinase activity, on PDE activity in rat TIC and in the mouse TM3 Leydig cell line. Treatment of TIC with herbimycin (1 μM) for 24 h inhibited basal and LH-stimulated PDE activity (≈50 and 70%, respectively) and was associated with an increase in cAMP and progesterone accumulation in culture media. Treatment of TM3 cells with herbimycin inhibited PDE activity and increased cAMP accumulation in a dose- and time-dependent manner. TM3 cell cultures challenged with herbimycin had lower Src tyrosine kinase activity than controls (≈50%); however, protein kinase A activity was unaffected. TM3 cells stably transfected with a dominant negative Src tyrosine kinase (TM3 Srck − ) had lower PDE activity than cells transfected with a G418 resistance gene alone (TM3 pSV2neo) which served as control cells. Conversely, TM3 cells expressing a temperature-sensitive Src kinase had significantly greater PDE activity at the Src active temperature (35°C; the temperature at which the enzyme is active) than TM3 pSV2neo control cells grown at the same temperature. TM3 cell lysates hydrolyzed minimal amounts of cGMP, indicating a cAMP-specific PDE. Phosphodiesterase activity in both TM3 and rat TIC was sensitive to the PDE4-selective inhibitor RO20-1724, indicating the predominant active enzyme is probably a member of the cAMP-specific PDE4 family. From the present data, we conclude that a tyrosine kinase of the Src family may play an important role in regulating phosphodiesterase activity in thecal and Leydig cells, and thus regulate intracellular cAMP and the state of cellular differentiation.