5-HT2A receptors stimulate mitogen-activated protein kinase via H2O2 generation in rat renal mesangial cells

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells throug h a G protein-coupled 5-HT2A receptor. We tested the hypothesis that oxidan ts might be involved in the signal transduction pathway Linking the recepto r to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly incr eased the activity and phosphorylation of ERK. These effects were blocked b y the 5-HT2A receptor antagonist ketanserin. The peak effect was noted at 5 -10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemic al inhibitor and activator studies supported the involvement of phospholipa se CI protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H2O2 and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK acti vation cascade. Mapping studies supported a location for the NAD(P)H oxidas e enzyme and the ROS downstream from PKC. Our studies are most consistent w ith an ERK activation pathway as follows: 5-HT2A receptor --> G protein --> phospholipase C --> diacylglycerol - classical PKC - NAD(P)H oxidase - sup eroxide --> superoxide dismutase -->, H2O2 --> mitogen-activated extracellu lar signal-regulated kinase - ERK. These studies demonstrate a role for the 5-HT2A receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in cont eying the stimulatory signal from 5-HT. because I) chemical antioxidants attenuate th e 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar d egree, 3) 5-HT produces superoxide and H2O2 in these cells, and 4) a specif ic enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, w hich react selectively downstream of classical PKC.