Cocaine‐amphetamine‐regulated transcript (CART) gene expression is regulated by intracellular calcium

Abstract

CART peptides are involved in numerous biochemical processes, including the reinforcement properties of psychostimulants. CART expression appears to be regulated via phosphorylated CREB (pCREB). Because, pCREB and the reinforcing actions of drugs of abuse are affected by Ca2+-mediated signaling, we hypothesized that increased intracellular Ca2+ levels may effect CART expression. GH3 cells, a rat pituitary cell line known to express endogenous CART, were treated with various concentrations of ionomycin, a Ca2+ ionophore. Several subsequent experiments support the involvement of Ca2+ in CART regulation. For instance, electrophoretic mobility shift assays demonstrated increased pCREB binding to the CRE oligo from the CART promoter after ionomycin treatment. Real-time PCR showed a time- and dose- dependent increase in CART mRNA levels. Finally, western blot analysis revealed elevated pCREB following ionomycin treatment. Furthermore, pretreatment with several inhibitors of Ca2+-mediated signalling, including calmidazolium, KN93, and BAPTA, reduced ionomycin-induced CART expression. In-vivo studies demonstrated that intra-accumbal injection of ionomycin increased CART mRNA levels in the rat nucleus accumbens as measured by in situ hybridization. Calmidazolium attenuated ionomycin–induced CART mRNA expression. These results suggest that the CART gene may be regulated by elevated Ca2+ via a calmodulin/pCREB signalling pathway in vitro in GH3 cells and, in vivo in rat nucleus accumbens. Supported by NIH grants RR00165, DA00418, DA15162. 1st and 2nd authors contributed equally to this work