Abstract
The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity.
Highlights
Tobacco use is recognized as a significant, but avoidable health hazard
The Tobacco Control Act gave the Food and Drug Administration (FDA) the ability to extend the regulations—the so-called deeming rule—to cover all other categories of products that meet the Tobacco Control Act definition of ‘‘tobacco product.’’ These products include electronic cigarettes (e-cigarettes), cigars, pipe tobacco, waterpipe tobacco, and novel products like nicotine gels, tobacco sticks, pellets and strips that dissolve in the mouth, and other dissolvable products not currently under FDA’s regulatory authority as smokeless tobacco products
Multiple nicotinic acetylcholine receptors (nAChRs) subtypes are expressed in these circuits and different subunits have been associated with different aspects of nicotine addiction
Summary
Tobacco use is recognized as a significant, but avoidable health hazard. The Family Smoking Prevention and Tobacco Control Act, commonly referred to as the Tobacco Control Act, signed into law in 2009, gave the US Food and Drug Administration (FDA) immediate responsibility for regulating the manufacture, distribution, and marketing of tobacco products, cigarettes, cigarette tobacco, roll-your-own tobacco, and smokeless tobacco products. Nicotine is the primary addictive component of tobacco products and additional ingredients may modulate nicotine’s action at the receptor level.[1] Nicotine acts by mimicking the endogenous neurotransmitter acetylcholine that activates ionotropic cholinergic receptors to depolarize neuronal membranes. Functional brain imaging in humans and laboratory animal studies have demonstrated nicotine-induced activation of the prefrontal cortex, thalamus, and visual system, indicative of activation of cortico-basal ganglia-thalamic brain circuits, and increased DA concentration in the ventral striatum/nucleus acumbens.[3] Multiple nAChR subtypes are expressed in these circuits and different subunits have been associated with different aspects of nicotine addiction. This study describes the development and validation of highthroughput electrophysiology-based assays in four recombinant cell lines, each expressing a different subtype of human nicotinic receptor: a3b4, a3b4a5, a4b2, and a7/RIC-3. Assays for each subtype were optimized and pharmacologically validated with reference compounds in an automated electrophysiology system (IonWorks BarracudaÒ [IWB]; Molecular Devices, LLC, Sunnyvale, CA)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
