N-n-alkylnicotinium analogs, a novel class of antagonists at alpha 4 beta 2* nicotinic acetylcholine receptors: inhibition of S(-)-nicotine-evoked 86Rb+ efflux from rat thalamic synaptosomes.

Pyridine N-n-alkylation of S(-)-nicotine (NIC) affords N-n-alkylnicotinium analogs, previously shown to competitively inhibit [(3)H]NIC binding and interact with alpha4beta2* nicotinic receptors (nAChRs). The present study determined the ability of the analogs to inhibit NIC-evoked (86)Rb(+) efflux from rat thalamic synaptosomes to assess functional interaction with alpha4beta2* nAChRs. In a concentration-dependent manner, NIC evoked (86)Rb(+) efflux (EC(50) = 170 nmol/L). Analog-induced inhibition of NIC-evoked (86)Rb(+) efflux varied over a approximately 450-fold range. Analogs with long n-alkyl chain lengths (C(9)-C(12)) inhibited efflux in the low nmol/L range (IC(50) = 9-20 nmol/L), similar to dihydro-beta-erythroidine (IC(50) = 19 nmol/L). Compounds with shorter n-alkyl chain lengths (C(1)-C(8)) produced inhibition in the low micromol/L range (IC(50) = 3-12 micromol/L). C(10) and C(12) analogs completely inhibited NIC-evoked efflux, whereas C(1-9) analogs produced maximal inhibition of only 10% to 60%. While the C(10) analog N-n-decylnicotinium iodide (NDNI) did not produce significant inhibition of NIC-evoked dopamine release in previously reported studies, NDNI possesses high affinity for [(3)H]NIC binding sites (K(i) = 90 nmol/L) and is a potent and efficacious inhibitor of NIC-evoked (86)Rb(+) efflux as demonstrated in the current studies. Thus, NDNI is a competitive, selective antagonist at alpha4beta2* nAChRs.

Introduction of unsaturation into the N-n-alkyl chain of the nicotinic receptor antagonists, NONI and NDNI: effect on affinity and selectivity.

N-n-octylnicotinium iodide (NONI) and N-n-decylnicotinium iodide (NDNI) are selective nicotinic receptor (nAChR) antagonists mediating nicotine-evoked striatal dopamine (DA) release, and inhibiting [3H]nicotine binding, respectively. This study evaluated effects of introducing unsaturation into the N-n-alkyl chains of NONI and NDNI on inhibition of [3H]nicotine and [3H]methyllycaconitine binding (alpha4beta2* and alpha7* nAChRs, respectively), (86)Rb+ efflux and [3H]DA release (agonist or antagonist effects at alpha4beta2* and alpha6beta2*-containing nAChRs, respectively). In the NONI series, introduction of a C3-cis- (NONB3c), C3-trans- (NONB3t), C7-double-bond (NONB7e), or C3-triple-bond (NONB3y) afforded a 4-fold to 250-fold increased affinity for [3H]nicotine binding sites compared with NONI. NONB7e and NONB3y inhibited nicotine-evoked 86Rb+ efflux, indicating alpha4beta2* antagonism. NONI analogs exhibited a 3-fold to 8-fold greater potency inhibiting nicotine-evoked [3H]DA overflow compared with NONI (IC50 = 0.62 microM; Imax = 89%), with no change in Imax, except for NONB3y (Imax = 50%). In the NDNI series, introduction of a C4-cis- (NDNB4c), C4-trans-double-bond (NDNB4t), or C3-triple-bond (NDNB3y) afforded a 4-fold to 80-fold decreased affinity for [3H]nicotine binding sites compared with NDNI, whereas introduction of a C9 double-bond (NDNB9e) did not alter affinity. NDNB3y and NDNB4t inhibited nicotine-evoked 86Rb+ efflux, indicating antagonism at alpha4beta2* nAChRs. Although NDNI had no effect, NDNB4t and NDNB9e potently inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.02-0.14 microM, Imax = 90%), as did NDNB4c (IC50 = 0.08 microM; Imax = 50%), whereas NDNB3y showed no inhibition. None of the analogs had significant affinity for alpha7* nAChRs. Thus, unsaturated NONI analogs had enhanced affinity at alpha4beta2*- and alpha6beta2*-containing nAChRs, however a general reduction of affinity at alpha4beta2* and an uncovering of antagonist effects at alpha6beta2*-containing nAChRs were observed with unsaturated NDNI analogs.

Development of subtype-selective ligands as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release.

N-n-Alkylation of nicotine converts it from an agonist into an antagonist at neuronal nicotinic acetylcholine receptor subtypes mediating nicotine-evoked dopamine release. Conformationally restricted analogues exhibit both high affinity and selectivity at this site, and are able to access the brain due to their ability to act as substrates for the blood-brain barrier choline transporter.

Opposing effects of ethanol and nicotine on hippocampal calbindin-D28k expression.

Long-term ethanol exposure produces multiple neuroadaptations that likely contribute to dysregulation of Ca(2+) balance and neurotoxicity during ethanol withdrawal. Conversely, nicotine exposure may reduce the neurotoxic consequences of Ca(2+) dysregulation, putatively through up-regulation of the Ca(2+)-buffering protein calbindin-D(28k). The current studies were designed to examine the extent to which 10-day ethanol exposure and withdrawal altered calbindin-D(28k) expression in rat hippocampus. Further, in these studies, we examined the ability of nicotine, through action at alpha(7)(*)-bearing nicotinic acetylcholine receptors (nAChRs), to antagonize the effects of ethanol exposure on calbindin-D(28k) expression. Organotypic cultures of rat hippocampus were exposed to ethanol (50-100 mM) for 10 days. Additional cultures were exposed to 500 nM (-)-nicotine with or without the addition of 50 mM ethanol, 100 nM methyllycaconitine (an alpha(7)*-bearing nAChR antagonist), or both. Prolonged exposure to ethanol (>/=50 mM) produced significant reductions of calbindin-D(28k) immunolabeling in all regions of the hippocampal formation, even at nontoxic concentrations of ethanol. Calbindin-D(28k) expression levels returned to near-control levels after 72 h of withdrawal from 10-day ethanol exposure. Extended (-)-nicotine exposure produced significant elevations in calbindin-D(28k) expression levels that were prevented by methyllycaconitine co-exposure. Co-exposure of cultures to (-)-nicotine with ethanol resulted in an attenuation of ethanol-induced reductions in calbindin-D(28k) expression levels. These findings support the suggestion that long-term ethanol exposure reduces the neuronal capacity to buffer accumulated Ca(2+) in a reversible manner, an effect that likely contributes to withdrawal-induced neurotoxicity. Further, long-term exposure to (-)-nicotine enhances calbindin-D(28k) expression in an alpha(7)* nAChR-dependent manner and antagonizes the effects of ethanol on calbindin-D(28k) expression.

Effects of prolonged nicotinic ligand exposure on function of heterologously expressed, human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptors.

Effects of prolonged nicotinic ligand exposure on the function of human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EP1 human epithelial cells. Magnitudes of acute, nAChR-mediated, specific 86Rb+ efflux responses to 1 mM carbamylcholine were reduced after pretreatment with specific nAChR ligands in effects that depended on pretreatment drug dose, duration of drug pretreatment, and duration of drug-free recovery. Fifty percent inhibition of alpha4beta2-nAChR function following 5 min of recovery occurred after 1 min of pretreatment with 1 mM nicotine but also after 1-h pretreatment at 10 nM nicotine. Seventy-five percent loss in function persisted 1 h after drug removal following 15 min or more of exposure to 1 mM nicotine. However, functional recovery was nearly complete after 1 h in drug-free medium following 1 min to 24 h pretreatment with 0.1 to 1 microM nicotine, i.e., in the range of smoker plasma nicotine levels. alpha4beta4-nAChR was similarly sensitive to persistent inactivation by prolonged nicotine exposure. Carbamylcholine exhibited slightly lower persistent inactivation potency than nicotine at both alpha4beta2- and alpha4beta4-nAChR. The nAChR antagonist, mecamylamine, exhibited persistent inactivation potency and efficacy similar to nicotine at alpha4beta2-nAChR but had a reduced effect on alpha4beta4-nAChR. These studies illustrate persistent inactivation of human alpha4beta2- or alpha4beta4-nAChR induced by prolonged exposure to nicotine and show that other ligands induce nAChR persistent inactivation in a subtype-specific manner.

N-n-alkylnicotinium analogs, a novel class of nicotinic receptor antagonists: interaction with alpha4beta2* and alpha7* neuronal nicotinic receptors.

The current study demonstrates that N-n-alkylnicotinium analogs with increasing n-alkyl chain lengths from 1 to 12 carbons have varying affinity (Ki = 90 nM-20 microM) for S-(-)-[3H]nicotine binding sites in rat striatal membranes. A linear relationship was observed such that increasing n-alkyl chain length provided increased affinity for the alpha4beta2* nicotinic acetylcholine receptor (nAChR) subtype, with the exception of N-n-octylnicotinium iodide (NONI). The most potent analog was N-n-decylnicotinium iodide (NDNI; Ki = 90 nM). In contrast, none of the analogs in this series exhibited high affinity for the [3H]methyllycaconitine binding site, thus indicating low affinity for the alpha7* nAChR. The C8 analog, NONI, had low affinity for S-(-)-[3H]nicotine binding sites but was a potent inhibitor of S-(-)-nicotine-evoked [3H]dopamine (DA) overflow from superfused striatal slices (IC50 = 0.62 microM), thereby demonstrating selectivity for the nAChR subtype mediating S-(-)-nicotine-evoked [3H]DA overflow (alpha3alpha6beta2* nAChRs). Importantly, the N-n-alkylnicotinium analog with highest affinity for the alpha4beta2* subtype, NDNI, lacked the ability to inhibit S-(-)-nicotine-evoked [3H]DA overflow and, thus, appears to be selective for alpha4beta2* nAChRs. Furthermore, the present study demonstrates that the interaction of these analogs with the alpha4beta2* subtype is via a competitive mechanism. Thus, selectivity for the alpha4beta2* subtype combined with competitive interaction with the S-(-)-nicotine binding site indicates that NDNI is an excellent candidate for studying the structural topography of alpha4beta2* agonist recognition binding sites, for identifying the antagonist pharmacophore on the alpha4beta2* nAChR, and for defining the role of this subtype in physiological function and pathological disease states.

N-n-alkylnicotinium analogs, a novel class of nicotinic receptor antagonist: inhibition of S(-)-nicotine-evoked [(3)H]dopamine overflow from superfused rat striatal slices.

The structure of the S(-)-nicotine molecule was modified via N-n-alkylation of the pyridine-N atom to afford a series of N-n-alkylnicotinium iodide salts with carbon chain lengths varying between C(1) and C(12). The ability of these analogs to evoke [(3)H] overflow and inhibit S(-)-nicotine-evoked [(3)H] overflow from [(3)H]dopamine ([(3)H]DA)-preloaded rat striatal slices was determined. At high concentrations, analogs with chain lengths > or =C(6) evoked [(3)H] overflow. Specifically, N-n-decylnicotinium iodide (NDNI; C(10)) evoked significant [(3)H] overflow at 1 microM, and N-n-dodecylnicotinium iodide (NDDNI; C(12)) at 10 microM, whereas N-n-octylnicotinium iodide (NONI; C(8)), N-n-heptylnicotinium iodide (NHpNI; C(7)), and N-n-hexylnicotinium iodide (C(6)) evoked [(3)H] overflow at 100 microM. Thus, intrinsic activity at these concentrations prohibited assessment of inhibitory activity. The most potent N-n-alkylnicotinium analog to inhibit S(-)-nicotine-evoked [(3)H] overflow was NDDNI, with an IC(50) value of 9 nM. NHpNI, NONI, and N-n-nonylnicotinium iodide (C(9)) also inhibited S(-)-nicotine-evoked [(3)H] overflow with IC(50) values of 0.80, 0.62, and 0.21 microM, respectively. In comparison, the competitive neuronal nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-beta-erythroidine, had an IC(50) of 1.6 microM. A significant correlation of N-n-alkyl chain length with analog-induced inhibition was observed, with the exception of NDNI, which was devoid of inhibitory activity. The mechanism of N-n-alkylnicotinium-induced inhibition of the high-affinity, low-capacity component of S(-)-nicotine-evoked [(3)H] overflow was determined via Schild analysis, using the representative analog, NONI. Linear Schild regression and slope not different from unity suggested that NONI competitively interacts with a single nAChR subtype to inhibit S(-)-nicotine-evoked [(3)H]DA release (K(i) value = 80.2 nM). Thus, modification of the S(-)-nicotine molecule converts this agonist into an antagonist at nAChRs, mediating S(-)-nicotine-evoked DA release in striatum.