Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells

Biochim Biophys Acta. 2005 Oct 30;1726(1):28-33. doi: 10.1016/j.bbagen.2005.08.008. Epub 2005 Sep 12.

Abstract

The small GTPase, RhoA, and its downstream effecter Rho-kinase (ROK) are reported to be involved in various cellular functions, such as myosin light chain phosphorylation during smooth muscle contraction and exocytosis. Indeed, growing evidence suggests that the RhoA/Rho-kinase pathway plays an important role in regulating exocytosis in these cells. However, it is not known whether the RhoA/Rho-kinase pathway has an effect on catecholamine synthesis. Using the rat pheochromocytoma cell line, PC12, we examined the effects of either Rho-kinase inhibitor (Y27632) or RhoA inhibitor (C3 toxin) on nicotine-induced catecholamine biosynthesis. We show that nicotine (10 microM) induces a significant, though transient, increase in RhoA activation in these cells. Treatment with either Y27632 (1 microM) or C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH) mRNA and the corresponding enzyme activity. TH catalyzes the rate-limiting step in the biosynthesis of catecholamine. Y27632 significantly inhibited nicotine-induced phosphorylation of TH at Ser40 as well as Ser19, which are known to be phosphorylated by Ca(2+)/calmodulin kinase II. Furthermore, Y27632 (10 microM) as well as C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of TH at the protein level. Thus, we propose that activation of RhoA, and its downstream effecter Rho-kinase, is a prerequisite for catecholamine biosynthesis in PC12 cells. At the concentrations used in our experiments, Y27632 does not affect cAMP/PKA activity or PKC activity, indicating that the inhibitory effect of Y27632 can be attributed to the inhibition of Rho-kinase activity as observed in chromaffin cells. In contrast, neither Y27632 (10 microM) nor C3 toxin (10 microg/ml) significantly altered catecholamine secretion in PC12 cells. In conclusion, we have demonstrated that inhibition of the Rho/Rho-kinase pathway in chromaffin cells lowers TH activity, probably through CaMKII inhibition. By contrast, neither Y27632 nor C3 toxin affect the secretion of catecholamine.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides / pharmacology
  • Analysis of Variance
  • Animals
  • Blotting, Western
  • Botulinum Toxins / pharmacology
  • Catecholamines / biosynthesis*
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • DNA Primers
  • Gene Expression Regulation, Enzymologic / drug effects*
  • Glutathione Transferase
  • Intracellular Signaling Peptides and Proteins
  • Nicotine / metabolism
  • PC12 Cells
  • Phosphorylation
  • Polymerase Chain Reaction
  • Protein Kinase C / metabolism
  • Protein Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein Serine-Threonine Kinases / metabolism
  • Pyridines / pharmacology
  • Rats
  • Signal Transduction / drug effects*
  • Tyrosine 3-Monooxygenase / metabolism
  • rho-Associated Kinases
  • rhoA GTP-Binding Protein / antagonists & inhibitors*
  • rhoA GTP-Binding Protein / metabolism

Substances

  • Amides
  • Catecholamines
  • DNA Primers
  • Intracellular Signaling Peptides and Proteins
  • Pyridines
  • Y 27632
  • Nicotine
  • Cyclic AMP
  • Tyrosine 3-Monooxygenase
  • Glutathione Transferase
  • Protein Serine-Threonine Kinases
  • rho-Associated Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • Botulinum Toxins
  • rhoA GTP-Binding Protein
  • botulinum toxin type C