title: Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells.
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.
authors.affiliation: Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Fukuda
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Takekoshi
authors.forename: Toru
authors.initials: T
authors.lastname: Nanmoku
authors.forename: Kiyoaki
authors.initials: K
authors.lastname: Ishii
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Isobe
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawakami
mesh_terms: D000577:Amides / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000494:pharmacology; D002395:Catecholamines / Q000096:biosynthesis*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D009538:Nicotine / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000577:Amides; D002395:Catecholamines; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D009538:Nicotine; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein; C096324:botulinum toxin type C