Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d-amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and compare its effects with those of d-amphetamine.[3H]-Dihydrotetrabenazine ([3H]DTBZ), used routinely to probe a high-affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a K 0 of 1.67 nM and Bmw, of 8.68 pmol/ mg of protein. Lobeline inhibited [ 3H]DTBZbinding with an IC 50 of 0.90 btM, consistent with its previously reported IC50 of 0.88~sM for inhibition of [ 3H]DAuptake into vesides. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [3H]DTBZbinding to vesicle membranes with an IC 50 of 39.4 1iM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d-amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [ 3H]DArelease from [3H]DA-preloaded synaptic vesicles in the absence of drug revealed at 112 of 2.12 mi Lobeline and d-amphetamine evoked [ 3HJDA release with EC 50 values of 25.3 and 2.22 heM, respectively. At a concentration 10 times the EC50, lobeline and d-amphetamine significantly decreased the t112 of [3H]DArelease to 1.58 and 1.48 mm, respectively. Thus, in contrast to d-amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28-fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.
A series of N-substituted nicotine analogues were synthesized and evaluated for their ability to inhibit nicotine-evoked l3H1dopamine ([3H]DA) release from rat striatal slices and for displacement of [3Hlnicotine binding from rat striatal membranes. O C the eleven compounds examined, the most efficacious analogues in the [3H]DA release assay were those that contained a pyridino N-alkyl substituent of three carbons or more in length. Structure-activity relationships indicate that potency in the L'HIDA release assay increases with increase in alkyl chain length. Introduction of an aromatic or unsaturated residue into the pyridino-N substituent also afforded compounds with significant antagonist activity. The most efficacious and potent compound in the series was S-N-octylnicotinium iodide (NONI). NONl had a potency approximately two-fold that of the classical nicotinic antagonists, mecamylamine (MEC) and dihydro-P-erythroidine (DHBE). In addition, over the concentration range examined, NONl completely blocked the effect of nicotine to evoke ["HIDA release, whereas the classical antagonists inhibited but did not completely block nicotine's effect. Furthermore, NONl did not possess agonist activity at concentrations which completely blocked nicotine-evoked 13H]DA release. Results from competition assays for ["Hlnicotine binding revealed that all the analogues were able to displace [3H]nicotine binding with a lower affinity as compared to the reference compound DHBE. A significant correlation between alkyl chain length and affinity for the [3H]nicotine binding site was observed. Moreover, there was a lack of correlation between displacement of [3H]nicotine binding and inhibition of nicotineevoked [3H]DA release, suggesting that different nicotinic receptor subtypes are responsible for modulation of DA release and [-'H]nicotine binding in striatum. The pKa values determined for S(-)nicotine and one of the active antagonists, S-N-allyl-nicotinium iodide (NANI), indicates that the quaternary ammonium analogues exist predominantly in their unprotonated forms at physiological pH. Molecular modeling studies suggest that these antagonists may interact with the nicotinic receptor in a novel binding mode which is different from the mode of interaction of nicotine with this receptor. This structure-activity data may provide useful information on the antagonist pharmacophore of the nicotinic receptor subtypes responsible for modulation of DA release and for nicotine binding in brain. Thus, it i s proposed that these antagonists bind to the receptor in their unprotonated forms and that the binding mode involves interaction of the quaternary pyridinium-N atom with the anionic site of the receptor. The unprotonated pyrrolidine-N atom serves as the hydrogen bond acceptor, which reverses the roles the nitrogens normally play in the binding of nicotine to the receptor. The N-alkyl substituent most likely binds to a site that extends beyond the normal agonist pharmacophore volume, which may prevent the receptor protein from ac...
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