Related Experiment Videos
Src tyrosine kinase activity in rat thecal-interstitial cells and mouse TM3 Leydig cells is positively associated
C C Taylor1, D Limback, P F Terranova
1Department of Physiology, University of Kansas Medical Center, Kansas City 66160-7401, USA.
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 an in the mouse TM3 Leydig cell line. Treatment of TIC with herbimycin (1 microM) for 24 h inhibited basal and LH-stimulated PDE activity (approximately 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 (approximately 50%); however, protein kinase A activity was unaffected. TM3 cells stably transfected with a dominant negative Src tyrosine kinase (TM3Srck-) had lower PDE activity than cells transfected with a G418 resistance gene alone (TM3pSV2neo) 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 degrees C; the temperature at which the enzyme is active) than TM3pSV2neo 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.
Insights
Src tyrosine kinase activity regulates phosphodiesterase (PDE) activity in ovarian thecal-interstitial and Leydig cells. Inhibiting Src kinase reduces PDE activity, increasing cyclic adenosine monophosphate (cAMP) levels, which influences cellular differentiation.
Area of Science:
- Endocrinology
- Cell Biology
- Biochemistry
Background:
- Phosphodiesterases (PDEs) regulate intracellular cyclic nucleotide levels, impacting cellular differentiation.
- Src-selective tyrosine kinase inhibitor herbimycin A previously shown to potentiate LH-stimulated cAMP in ovarian thecal-interstitial cells (TIC).
Purpose of the Study:
- Investigate the effects of herbimycin and Src tyrosine kinase activity on PDE activity in rat TIC and mouse TM3 Leydig cells.
- Determine the role of Src family kinases in regulating PDE activity and cAMP levels in these cell types.
Main Methods:
- Treatment of TIC and TM3 cells with herbimycin A.
- Assessing PDE activity, cAMP and progesterone accumulation.
- Utilizing TM3 cells stably transfected with dominant-negative or temperature-sensitive Src tyrosine kinase.
Main Results:
- Herbimycin inhibited basal and LH-stimulated PDE activity in TIC and dose- and time-dependently in TM3 cells, increasing cAMP.
- Reduced Src tyrosine kinase activity correlated with lower PDE activity in TM3 cells.
- TM3 cells with dominant-negative Src had lower PDE activity, while those with active Src kinase had higher PDE activity.
- PDE activity was cAMP-specific and sensitive to PDE4 inhibitors, suggesting PDE4 family involvement.
Conclusions:
- Src family tyrosine kinase plays a significant role in regulating PDE activity in thecal and Leydig cells.
- This regulation impacts intracellular cAMP levels and consequently influences cellular differentiation.