N-n-Alkylpyridinium Analogs, a Novel Class of Nicotinic Receptor Antagonists: Selective Inhibition of Nicotine-Evoked [H]Dopamine Overflow from Superfused Rat Striatal Slices

Structural simplification of N-n-alkylnicotinium analogs, antagonists at neuronal nicotinic acetylcholine receptors (nAChRs), was achieved by removal of the N-methylpyrrolidino moiety affording N-n-alkylpyridinium analogs with carbon chain lengths of C1 to C20. N-n-Alkylpyridinium analog inhibition of [H]nicotine and [H]methyllycaconitine binding to rat brain membranes assessed interaction with 4 2* and 7* nAChRs, respectively, whereas inhibition of nicotine-evoked H overflow from [H]dopamine ([H]DA)-preloaded rat striatal slices assessed antagonist action at nAChR subtypes mediating nicotine-evoked DA release. No inhibition of [H]methyllycaconitine binding was observed, although N-n-alkylpyridinium analogs had low affinity for [H]nicotine binding sites, i.e., 1 to 3 orders of magnitude lower than that of the respective N-n-alkylnicotinium analogs. These results indicate that the N-methylpyrrolidino moiety in the N-n-alkylnicotinium analogs is a structural requirement for potent inhibition of 4 2* nAChRs. Importantly, N-n-alkylpyridinium analogs with n-alkyl chains C10 did not inhibit nicotine-evoked [H]DA overflow, whereas analogs with n-alkyl chains ranging from C10 to C20 potently and completely inhibited nicotine-evoked [H]DA overflow (IC50 0.12–0.49 M), with the exceptions of N-n-pentadecylpyridinium bromide (C15) and N-n-eicosylpyridinium bromide (C20), which exhibited maximal inhibition of 50%. The mechanism of inhibition of a representative analog of this structural series, N-n-dodecylpyridinium iodide, was determined by Schild analysis. Linear Schild regression with slope not different from unity indicated competitive antagonism at nAChRs mediating nicotine-evoked [H]DA overflow and a KB value of 0.17 M. Thus, the simplified N-n-alkylpyridinium analogs are potent, selective, and competitive antagonists of nAChRs mediating nicotine-evoked [H]DA overflow, indicating that the N-methylpyrrolidino moiety is not a structural requirement for interaction with nAChR subtypes mediating nicotine-evoked DA release. Nicotine (Fig. 1, structure 1) activates neuronal nicotinic acetylcholine receptors (nAChRs), which are members of a ligand-gated ion channel family, consisting of transmembrane pentameric proteins with diverse composition (Le Novère et al., 2002). Twelve genes encode 210 and 24 nAChR subunits, and in situ hybridization reveals their discrete, but overlapping, central nervous system distribution (Wada et al., 1989; Dineley-Miller and Patrick, 1992). Individual neurons elaborate multiple nAChR subtypes, and more than two different subunits can assemble forming functional nAChRs (Forsayeth and Kobrin, 1997; Zoli et al., 2002), greatly increasing the complexity of nAChR pharmacology. Thus, nAChR subtype diversity originates from differences in amino acid sequences of subunit proteins and from multiple combinations of subunit assemblies forming functional nAChRs. The exact subunit composition, stoichiometry, and arrangement of native nAChRs remain to be elucidated (Lukas et al., 1999). Although predominance does not necessarily reflect functional importance, the 4 2* subtype, probed by high-affinity [H]nicotine binding predominates in brain. Greater than 90% of [H]nicotine binding sites are immunoprecipitated with anti2 antibody (Whiting and Lindstrom, 1987; Flores et al., 1992), and 2-knockout mice do not exhibit highThis research was supported by National Institutes of Health Grants DA00399 and DA10934. 1 Current address: Targacept, Inc., 200 East First St., Suite 300, WinstonSalem, NC 27101-4165. 2 Current address: AstraZeneca, 1800 Concord Pike, P. O. Box 15437, Wilmington, DE 19850-5437. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.051789. ABBREVIATIONS: nAChR, neuronal nicotinic acetylcholine receptor; MLA, methyllycaconitine; DA, dopamine; NONI, N-n-octylnicotinium iodide; NDNI, N-n-decylnicotinium iodide; NMPI, N-methylpyridinium iodide; NEPI, N-ethylpyridinium iodide; NPrPI, N-n-propylpyridinium iodide; NBuPI, N-n-butylpyridinium iodide; NPPI, N-n-pentylpyridinium iodide; NHxPI, N-n-hexylpyridinium iodide; NHPI, N-n-heptylpyridinium iodide; NOPI, N-n-octylpyridinium iodide; NNPI, N-n-nonylpyridinium iodide; NDPI, N-n-decylpyridinium iodide; NUPI, N-n-undecylpyridinium iodide; NDDPI, N-n-dodecylpyridinium iodide; NPDPB, N-n-pentadecylpyridinium bromide; NEcPB, N-n-eicosylpyridinium bromide; ANOVA, analysis of variance. 0022-3565/03/3063-1011–1020$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 51789/1084233 JPET 306:1011–1020, 2003 Printed in U.S.A. 1011 at A PE T Jornals on A ril 0, 2017 jpet.asjournals.org D ow nladed from