Molecular and cellular pharmacology
Similar activity of mecamylamine stereoisomers in vitro and in vivo

https://doi.org/10.1016/j.ejphar.2013.10.018Get rights and content

Abstract

A previous characterization of mecamylamine stereoisomers using nicotinic acetylcholine receptors expressed in Xenopus oocytes revealed only small differences between the activity of the R and S forms of mecamylamine. However, that work was limited in the breadth of receptor subtypes tested, especially in regard to the discrimination of high and low sensitivity receptors, which differ in the ratios of alpha and beta subunits. We report new data using subunit concatamers, which produce uniform populations of high-sensitivity or low-sensitivity receptors, as well as alpha2, alpha5, and alpha6-containing receptors, which were not studied previously. Consistent with previous studies, we found that beta4-containing receptors were most sensitive to mecamylamine and that the IC50 values for the inhibition of net charge were lower than for inhibition of peak currents. No large differences were seen between the activities of the mecamylamine isomers. Additionally, a previously reported potentiation of high-sensitivity α4β2 receptors by S-mecamylamine could not be reproduced in the oocyte system, even with mutants that had greatly reduced sensitivity to mecamylamine inhibition or when the selective agonist TC-2559 was used. In vivo studies suggested that the R-isomer might be somewhat more potent than the S isomer at blocking CNS effects of nicotine. Although the potency difference was no more than a factor of two, it is consistent with lower LD50 estimates previously reported for the R isomer. Our results significantly extend knowledge of the nicotinic acetylcholine receptor activity profile of mecamylamine and support the hypothesis that these effects are not strongly stereoisomer selective.

Introduction

There are numerous subtypes of nicotinic acetylcholine receptors (nAChR) expressed in neurons which are pharmacologically distinct from the receptors of the neuromuscular junction (Millar and Gotti, 2009). The ganglionic blocker mecamylamine, a relatively nonselective noncompetitive antagonist for all the neuronal-type nAChR that does not block acetylcholine (ACh) binding, was among the very first drugs developed to target neuronal nAChR (Dennis et al., 1957) but has been long been abandoned for the treatment of hypertension, its original indication.

Normal brain function relies on a balance among neurochemical systems, which can be perturbed by disease, drug abuse, medications, or side effects of medications. It has been hypothesized that hypercholinergic function is an underlying feature of depression. Nicotine self-administration clearly disrupts normal cholinergic function. Certain antipsychotic mediations perturb the balance of dopaminergic and cholinergic function in the basal ganglia, resulting in dyskinesia. In order to treat these imbalances, mecamylamine has recently been considered for adjunct therapy to treat depression, nicotine dependence, and Tourette's syndrome. A prior history of relatively safe use for its original indication has allowed mecamylamine to be clinically tested for these indications, with mixed results (Rose et al., 1998, Silver et al., 2000, Singh et al., 2006).

Mecamylamine is a stereo-active compound, but the vast majority of experimental and therapeutic work has utilized racemic preparations. We previously tested whether the two stereoisomers of mecamylamine had equivalent selectivity and potency for the inhibition of nAChR subtypes expressed in Xenopus oocytes (Papke et al., 2001) but failed to find any striking differences in the activity profile of R(−)Mecamylamine (R-Mec) and S(+)Mecamylamine (S-Mec). However, a subsequent study (Fedorov et al., 2009) using nAChR expressed in cell lines reported a novel effect for S-Mec (TC-5214) on a specific α4-containing nAChR subtype with high sensitivity to agonists (HS α4β2 receptors, which have a subunit stoichiometry of two α4 and three β2 subunits). This effect that might have gone unnoticed in the earlier oocyte studies due to a limitation of the original approach.

Recently, various nAChR constructs have been developed which allow for the selective expression of receptors with specific subunit composition. We have used several constructs of linked nAChR subunits (concatamers) (Zhou et al., 2003) to extend our oocyte studies to test the activity of the mecamylamine stereoisomers on nAChR subtypes with defined subunit composition that could not previously be studied in isolation. While α4-containing receptors are the most abundant high-affinity nAChR in rodent brain, in primates there are additional high affinity-receptors containing α2 subunits (Han et al., 2000, Han et al., 2003). The degree to which α2 nAChR have a pharmacological profile similar to α4 receptors has not been well studied. Therefore we have also generated populations of α2 receptors with defined subunit composition and evaluated their responses to agonists, including TC-2559 and the mecamylamine stereoisomers. We also extended these studies to α6-containing receptors formed with a five-subunit concatamer.

We evaluated whether the previously reported selective potentiation of high-sensitivity α4β2 responses by S-Mec could be replicated in the oocyte expression system. Additionally, We have also conducted in vivo experiments to determine whether the mecamylamine stereoisomers given systemically differ in their potency for blocking some of CNS-dependent effects of nicotine in mice.

Section snippets

Chemicals

Fresh acetylcholine (Sigma; St. Louis, MO) stock solutions were made daily in Ringer's solution and diluted. Mecamylamine (N-2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine) stereoisomers were supplied by Layton Biosciences (Menlo Park, CA). TC-2559 was supplied by Targacept (Winston Salem, NC) or purchased from Tocris (c/o R&D Systems, Minneapolis MN). All other chemicals for electrophysiology were obtained from Sigma Chemical Co. (St. Louis, MO).

ACh receptor clones

Human nAChR clones, the β2-6-α4 and α4β2α6β2β3

Characterization of HS and LS α2β2 receptors

Additional experimental tools have become available in recent years that make it possible to study various nAChR subtypes with defined subunit composition. Receptors containing α4 and β2 subunits at a ratio of 2:3 (α4(2)β2(3)) are pharmacologically distinct from receptors with the subunit ratio 3:2 (α4(3)β2(2)). Receptors with the (α4(2)β2(3)) composition respond to relative low concentrations of ACh or nicotine and therefore have been identified as high sensitivity (HS) receptors, while

Discussion

The principal high-affinity nAChR subtype of mammalian brain is composed of five subunits of usually of two different types, α4 or α2 plus β2, in some cases co-assembled with alternative subunits such as α6, α5, or β3 (Gotti et al., 2009). While subunit composition may be differentially regulated in specific cell types or tissues in vivo, heterologous expression systems are permissive for forming mixed populations of receptors which require both α and β subunits. Since the original

Conclusions

In the present study, we have extended our knowledge of the in vitro activity (selectivity/potency) of mecamylamine for previously unstudied receptors. Our data suggest that mecamylamine will more effectively produce acute inhibition of LS forms of α4 and α2 receptors than the HS forms, which have been suggested to increase in abundance with chronic nicotine use. However, our analyses of net charge inhibition suggest that under chronic conditions there will be roughly equivalent inhibition of

Acknowledgments

We thank Targacept for supplying TC-2559 and Drs. R. Douglas Shytle and Ron Lukas for their comments. We also thank Drs. Jon Lindstrom and Edwin Johnson for supplying nAChR subunit DNA clones and concatamers. We thank Shehd A. A. Al Rubaiy, Lynda Cortes, Sara Copeland, Matthew R. Kimbrell, Matthew Isaacson, Lu W. Corrie, and Thomas F. Pack for conducting the OpusXpress experiments. We thank Dr. Lynn Wecker (University of South Florida) for the use of an additional OpusXpress 6000 and Tie-Shan

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