ECE2006 Poster Presentations Diabetes, metabolism and cardiovascular (174 abstracts)
Biological Sciences, University of Warwick, Coventry, United Kingdom.
Orexin-A (OR-A) and orexin-B (OR-B) play an important role in the regulation of energy balance and the control of sleep-wake cycle. They act via G-protein coupled receptors, namely orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). OX2R has equal affinity for both OR-A and OR-B, whilst OX1R has a 10-fold greater affinity for OR-A. Orexin-mediated functions have been extensively explored, however, the intracellular signalling pathways remain poorly understood. Using HEK-293 cells, we investigated the signalling pathways involved in extracellular-regulated-kinase 1 and 2 (ERK1/2) were mediated by OX1R and OX2R activation.
Full-length human OX1R and OX2R were amplified from human brain by RT-PCR and subsequently cloned and transfected into HEK-293 cells. Using OR-A and OR-B (0.01, 0.1, 1, 10 and 100 nM), phosphorylation of ERK1/2 were examined time course (5, 10, 15, 20 and 30 min). These effects were further dissected by the use of specific inhibitors for PKA and PKC and Gi/o-protein blocker (pertussis toxin, PTX).
The results demonstrated a dose-dependent phosphorylation and activation of ERK1/2 with a maximal activation by OR-A and OR-B (5 and 15 min). Activation of ERK1/2 by OR-A was greater than OR-B in HEK293-OX1R transfected cells, whereas a similar level of ERK1/2 activation by OR-A or OR-B was observed in HEK293-OX2R transfected cells. In HEK293-OX1R and OX2R transfected cells, OR-A- and OR-B-induced ERK1/2 activation was abolished by PKC inhibition. Inhibition of PKA also attenuated OR-A- and OR-B-induced ERK1/2 activation. However, the effect was weaker than that observed for PKC. This suggests that PKC is mainly involved in OX1R- and OX2Rmediated ERK1/2 activation. In addition, orexin-activated phosphorylation of ERK1/2 was inhibited with pre-treatment of PTX in HEK293-OX2R transfected cells, but not in HEK293-OX1R transfected cells.
In conclusion, activation of ERK1/2 by OR-A an OR-B via both receptors resulted in a dosedependent response with a maximal activation between 5 and 15 min. These data demonstrated that PKC and PKA signalling pathways are involved in the phosphorylation of ERK1/2 via OX1R and OX2R. Furthermore OX2R activation to ERK1/2 is associated with PTX-sensitive G-protein.