LOBELINE DISPLACES [H-3]DIHYDROTETRABENAZINE BINDING AND RELEASES [H-3]DOPAMINE FROM RAT STRIATAL SYNAPTIC VESICLES - COMPARISON WITH D-AMPHETAMINE

Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classi fied as a nicotinic agonist and is currently used as a smoking cessati on agent. However, our previous in vitro studies demonstrate that lobe line does not act as a nicotinic agonist but alters presynaptic dopami ne (DA) storage by potently inhibiting DA uptake into synaptic vesicle s. Recently, d-amphetamine has been reported to act at the level of th e synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and comp are its effects with those of d-amphetamine. [H-3]- Dihydrotetrabenazi ne ([H-3]DTBZ), used routinely to probe a high-affinity binding site o n the vesicular monoamine transporter (VMAT2), bound to vesicle membra nes from rat striatum with a K-D of 1.67 nM and B-max of 8.68 pmol/mg of protein. Lobeline inhibited [H-3]DTBZ binding with an IC50 of 0.90 mu M, consistent with its previously reported IC50 Of 0.88 mu M for in hibition of [H-3]DA uptake into vesicles. These results suggest that l obeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [H-3]DTBZ binding to vesicle membranes with an IC50 of 39.4 mu M, a co ncentration 20 times greater than reported for inhibition of VMAT2 fun ction, suggesting that d-amphetamine interacts with a different site t han lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [H-3]DA release from [H-3]DA-preloaded synaptic vesicles in the absen ce of drug revealed a t(1/2), of 2.12 min. Lobeline and d-amphetamine evoked [H-3]DA release with ECS, values of 25.3 and 2.22 mu M, respect ively. At a concentration 10 times the EC50, lobeline and d-amphetamin e significantly decreased the t(1/2) of [H-3]DA release to 1.58 and 1. 48 min, respectively. Thus, in contrast to d-amphetamine, which is equ ipotent in inhibiting DA uptake and promoting release from the synapti c vesicles, lobeline more potently (28-fold) inhibits DA uptake (via a n interaction with the DTBZ site on VMAT2) than it evokes DA release t o redistribute presynaptic DA storage.