Teng L, Crooks P A, Dwoskin L P
College of Pharmacy and Graduate Center for Toxicology, University of Kentucky, Lexington 40536-0082, USA.
J Neurochem. 1998 Jul;71(1):258-65. doi: 10.1046/j.1471-4159.1998.71010258.x.
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 KD of 1.67 nM and Bmax of 8.68 pmol/mg of protein. Lobeline inhibited [3H]DTBZ binding with an IC50 of 0.90 microM, consistent with its previously reported IC50 of 0.88 microM for inhibition of [3H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [3H]DTBZ binding to vesicle membranes with an IC50 of 39.4 microM, 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]DA release from [3H]DA-preloaded synaptic vesicles in the absence of drug revealed a t1/2 of 2.12 min. Lobeline and d-amphetamine evoked [3H]DA release with EC50 values of 25.3 and 2.22 microM, respectively. At a concentration 10 times the EC50, lobeline and d-amphetamine significantly decreased the t1/2 of [3H]DA release to 1.58 and 1.48 min, 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.
洛贝林是一种从印度烟草（北美山梗菜）中提取的生物碱，被归类为烟碱样激动剂，目前用作戒烟药物。然而，我们之前的体外研究表明，洛贝林并非作为烟碱样激动剂发挥作用，而是通过有效抑制多巴胺（DA）摄取到突触小泡中来改变突触前DA储存。最近，有报道称右旋苯丙胺作用于突触小泡水平以改变突触前功能。目前的体外研究进一步阐明了洛贝林的作用机制，并将其作用效果与右旋苯丙胺进行比较。[3H]二氢丁苯那嗪（[3H]DTBZ）常用于探测囊泡单胺转运体（VMAT2）上的高亲和力结合位点，它与大鼠纹状体的囊泡膜结合，解离常数（KD）为1.67 nM，最大结合量（Bmax）为8.68 pmol/mg蛋白质。洛贝林抑制[3H]DTBZ结合的半数抑制浓度（IC50）为0.90 μM，与其之前报道的抑制[3H]DA摄取到囊泡中的IC50为0.88 μM一致。这些结果表明，洛贝林与VMAT2上的DTBZ位点特异性相互作用，以抑制DA摄取到突触小泡中。有趣的是，右旋苯丙胺抑制[3H]DTBZ与囊泡膜结合的IC50为39.4 μM，该浓度比报道的抑制VMAT2功能的浓度高20倍，这表明右旋苯丙胺与洛贝林在VMAT2上的作用位点不同，以抑制单胺摄取。在无药物情况下，对[3H]DA预装载的突触小泡中[3H]DA释放的动力学分析显示半衰期（t1/2）为2.12分钟。洛贝林和右旋苯丙胺诱发[3H]DA释放的半数有效浓度（EC50）分别为25.3和2.22 μM。在EC50的10倍浓度下，洛贝林和右旋苯丙胺分别将[3H]DA释放的t1/2显著降低至1.58和1.48分钟。因此，与右旋苯丙胺在抑制DA摄取和促进从突触小泡中释放方面具有同等效力不同，洛贝林抑制DA摄取（通过与VMAT2上的DTBZ位点相互作用）比诱发DA释放以重新分布突触前DA储存更为有效（28倍）。
