Molecular pharmacology
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Molecular pharmacology · Dec 2003
Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.
Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). ⋯ H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.
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Molecular pharmacology · Dec 2003
Mu opioid transactivation and down-regulation of the epidermal growth factor receptor in astrocytes: implications for mitogen-activated protein kinase signaling.
Astroglia are a principal target of long-term mu antiproliferative actions. The mitogen-activated protein (MAP) kinase known as extracellular signal-regulated kinase (ERK), is a key mediator of cell proliferation. In studies on the mechanism of short- and long-term mu opioid regulation of the ERK signaling pathway, we show that the mu opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), acting via the endogenous mu opioid receptor (MOR), induced sequential epidermal growth factor receptor (EGF) receptor (EGFR) Tyr phosphorylation, Ser phosphorylation, and down-regulation in immortalized rat cortical astrocytes. ⋯ However, long-term application of U69,593 neither down-regulated EGFR nor inhibited EGF-induced ERK activation. Instead, it engendered a sustained activation of ERK. Collectively, our data suggest that long-term application of DAMGO initiates heterologous down-regulation of EGFR via a mechanism involving ERK in astrocytes.