Biochem. J. (2000) 347
(61–67) (Printed in Great Britain)
5-Hydroxytryptamine1A receptor/Gibg stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of Src in Chinese hamster ovary fibroblasts
Yurii V. MUKHIN*†, Maria N. GARNOVSKAYA*†, Georgiann COLLINSWORTH*†, Jasjit S. GREWAL*†, DeKisha PENDERGRASS*†, Toshio NAGAI*†, Stephen PINCKNEY*†, Eddie L. GREENE*†, and John R. RAYMOND*†1
*Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, U.S.A., and †Ralph H. Johnson Veterans' Affairs Medical Center, 109 Bee Street, Charleston, SC, 29401, U.S.A.
The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)1A receptor, which activates the extracellular signal-regulated kinase (ERK) through a Gibg-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT1A receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT1A receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT1A receptor in Chinese hamster ovary (CHO) cells downstream of Gibg subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT1A receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide.
Abbreviations used: AEBSF, 4-(2-aminoethyl)-benzenesulphonyl fluoride; AEBSAC, 4-(2-aminoethyl)-benzenesulphonic acid; AEBSNH
2, 4-(2-aminoethyl)-benzenesulphonamide;
a-LA,
a-lipoic acid; BAL, British anti-Lewisite, 2,3-dimercaptopropanol; BSO, buthionine sulphoximine; CHO, Chinese hamster ovary; DCF-DA, 2´,7´-dichlorofluorescin diacetate; diamide, azodicarboxylic acid bis(dimethylamide); DPI, diphenylene iodinium; 5-HT, 5-hydroxytryptamine or serotonin; ERK, extracellular signal-regulated kinase; HMAP, 4´-hydroxy-3´-methoxyacetophenone; MAEBSF, 4-(2-
N-methylaminoethyl)-benzenesulphonyl fluoride; MAPK, mitogen-activated protein kinase; MEK, mitogen and extracellular signal-regulated kinase kinase; NAC,
N-acetylcysteine; PAO, phenylarsine oxide; PKC, protein kinase(s) C, calcium and phospholipid-dependent protein kinases; PP1, 4-amino-5-(4-methylphenyl)-7-(
t-butyl)pyrazolo(3,4-
d)pyrimidine; ROS, reactive oxygen species; SOD, superoxide dismutase; TBH,
tert-butylhydroperoxide; TLCK;
Na-
p-tosyl-
L-lysine chloromethylketone; VSMC, vascular smooth muscle cells.
1 To whom correspondence should be addressed (e-mail raymondj@musc.edu).
Key words: G protein, phosphorylation, proliferation, serotonin receptor, tyrosine kinase.
Received 12 July 1999/3 November 1999; accepted 4 January 2000
The Biochemical Society, London © 2000
Full Editorial Board
RSS feeds
Table of contents by email
My BJ toolbox
