U18666A, a cholesterol-inhibition agent, modulates human neuronal nicotinic acetylcholine receptors heterologously expressed in SH-EP1 cell line

• Published in: Journal of neurochemistry 2009-03, Vol.108 (6), p.1526-1538
• Main Authors: Zheng, Chao, Wang, Meng-Ya, Liu, Qiang, Wakui, Makoto, Whiteaker, Paul, Lukas, Ronald J, Wu, Jie
• Format: Article
• Language: English
• Subjects: (3β)-3-[2-(diethylamino)ethoxy]androst-5-en-17-one dihydrochloride; Androstenes - chemistry; Androstenes - pharmacology; Anticholesteremic Agents - pharmacology; Biochemistry; Biological and medical sciences; Biophysical Phenomena - physiology; Cell Line, Transformed; Cell receptors; Cell structures and functions; Cellular biology; Dose-Response Relationship, Drug; Electric Stimulation - methods; Epithelial Cells - drug effects; Epithelial Cells - physiology; Fundamental and applied biological sciences. Psychology; Gene Expression - drug effects; Guanosine Diphosphate - analogs & derivatives; Guanosine Diphosphate - pharmacology; Humans; Medical sciences; Membrane Potentials - drug effects; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Neurology; Neurotransmitters; Nicotine - pharmacology; nicotinic acetylcholine receptor; patch clamp; Patch-Clamp Techniques; Pharmacology; Receptors, Nicotinic - drug effects; Receptors, Nicotinic - genetics; Receptors, Nicotinic - metabolism; SH-EP1 cell line; Thionucleotides - pharmacology; Tobacco, tobacco smoking; Toxicology; trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride - chemistry; trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride - pharmacology
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2009.05903.x

In this study, we evaluate the effects of (3β)-3-[2-(diethylamino)ethoxy]androst-5-en-17-one dihydrochloride (U18666A), a cholesterol synthesis/transporter inhibitor, on selected human neuronal nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the SH-EP1 cell line using whole-cell patch-clamp recordings. The results indicate that with 2-min pretreatment, U18666A inhibited different nAChR subtypes with a rank-order of potency (IC₅₀ of whole-cell peak current): α4β2 (8.0 ± 3.0 nM) > α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (> 100 μM), suggesting this compound is more selective to α4β2-nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on α4β2-nAChRs were investigated in detail. U18666A suppresses both peak and steady state components of whole-cell currents mediated by human α4β2-nAChRs in response to nicotine. In nicotine-induced concentration-response curves, U18666A reduces nicotine-induced current at maximally effective agonist concentrations without influencing nicotine's EC₅₀ value, suggesting a non-competitive inhibition. U18666A-induced inhibition of nAChR function is concentration-, voltage-, and use-dependent, suggesting an open channel block. Taken into consideration of ~10 000-fold enhancement of the potency of U18666A after 2-min pre-treatment, this compound also likely inhibits α4β2-nAChRs through a close channel block. In addition, the U18666A-induced inhibition in α4β2-nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data indicate that U18666A is a potent antagonist of α4β2-nAChRs and may be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as a potential candidate for smoking cessation.