Lobeline effects on tonic and methamphetamine-induced dopamine release

doi:10.1016/j.bcp.2007.11.019

Biochemical Pharmacology

Volume 75, Issue 6, 15 March 2008, Pages 1411-1415

https://doi.org/10.1016/j.bcp.2007.11.019 Get rights and content

Abstract

The mechanisms of interaction between lobeline and the dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT-2) are not clear. The goal of this study was to elucidate the effects of lobeline on these transporters in a cell system co-expressing the DAT and VMAT-2. Lobeline caused release of [³H]dopamine to a similar extent as reserpine (VMAT-2 inhibitor), but was less efficacious than methamphetamine or dopamine. Additionally, lobeline decreased the [³H]dopamine-releasing effects of methamphetamine, unlike reserpine which increased release by methamphetamine. These results suggest that lobeline has unique properties at the DAT and VMAT-2 which may make it useful as a pharmacotherapeutic to treat methamphetamine abuse.

Introduction

Lobeline has been proposed as a potential treatment for psychostimulant abuse [1], and may be a good treatment for methamphetamine abuse for a number of reasons. First, lobeline interacts with both the vesicular monoamine transporter (VMAT-2) [2] and the cell surface dopamine transporter (DAT) [3]. These proteins are key sites of action for methamphetamine and amphetamine [4], [5]. Thus, lobeline may be able to directly inhibit the effects of methamphetamine. Secondly, in self-administration paradigms, lobeline is only a weak reinforcer in mice [6], and does not support self-administration in rats [7]. Lobeline also has a long history of use in people (see [1] for review), thus lobeline appears to have a low abuse potential. Further, lobeline decreases self-administration of methamphetamine in rats [8]. Lobeline interacts with nicotinic acetylcholine receptors, and has been used as a smoking cessation aid with mixed results (see [1]). Additionally, lobeline evokes calcium-independent (nonvesicular) release of [³H]dopamine [3], [9], [10]. Previous studies suggest that lobeline is able to inhibit amphetamine-induced dopamine release [11], however the precise mechanism for this effect is unclear. The purpose of this study was to determine the mechanism(s) of lobeline's action at the DAT and VMAT-2, and its effects on methamphetamine-induced changes in [³H]dopamine release by comparing the effects of lobeline to those of drugs with known activity at the transporters. Therefore, we used HEK-293 cells stably transfected with the DAT and VMAT-2 to minimize the potential confounds of more complicated systems. Primary cultures and synaptosomes contain numerous receptors, and endogenously synthesize and metabolize dopamine, which could interfere with data interpretation.

Section snippets

Materials

[³H]dopamine (3,4-[7-³H]dihydroxyphenylethylamine, 5.8–9.7 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ, U.S.A.). RTI-55 was a generous gift from Dr. F. Ivy Carroll at the Research Triangle Institute (Research Triangle Park, NC, U.S.A.), supplied by the National Institute on Drug Abuse drug supply program. Eco-Lume scintillation fluid was purchased from ICN biochemicals, Inc. (Aurora, OH, U.S.A.). All water used in these experiments was purified by a Milli-Q system (Millipore

Results

To examine the specific mechanistic interactions of lobeline, we used DAT antagonists (nomifensine and RTI-55), a VMAT-2 antagonist (reserpine) and a DAT and VMAT-2 substrate (dopamine). Characterization of these cells, as determined by radioligand binding, suggests that the ratio of DAT to VMAT-2 (DAT:VMAT-2; 1.35 – 5.22) is comparable to that found in human brain (DAT:VMAT-2; 0.76 – 2.31) [12], [13], [14]. The ability of the drugs to induce [³H]dopamine release was compared. In addition, the

Discussion

Lobeline inhibits binding of [³H]dihydrotetrabenazine (a VMAT-2 antagonist) to the VMAT-2 with relatively high affinity (900 nM) [2], and also causes release of [³H]dopamine from rat striatal slices [2], [3]. Our results demonstrate that lobeline induces some release of [³H]dopamine from mammalian cells expressing the DAT and VMAT-2. These results are corroborated by the observation that lobeline induces much greater [³H]dopamine release than the DAT antagonists nomifensine and RTI-55 (Fig. 1).

Acknowledgements

This work was supported by NIH grants DA017541-02, AA007468-19 (CJW), by NIH/VA interagency agreement Y1-DA5007, by grant P50DA018165, and the VA Merit Review and Career Scientist Programs (A.J.).

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Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures
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These metabolites have previously been reported in other Lobelia species, including the Chinese medicinal plant L. chinensis (Mfuh and Larionov, 2015), but appear to have never been tested for DAT activity. Lobeline itself inhibits the DAT and VMAT2 (Wilhelm et al., 2008) but was not found in mutant culture extracts or extracts of L. cardinalis (Brown et al., 2016b) (or in L. chinensis Yang et al., 2014). In addition to these Lobelia alkaloid–like metabolites, many other metabolites were increased in one or more mutants containing increased DAT inhibitory activity.
The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and "non-classical”" DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, "target-directed evolution" was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are "mined" for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC₅₀ similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.
Modulation of aggressive behavior in mice by nicotinic receptor subtypes
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Previous studies of the effect of nicotine on aggression in mice have shown that nicotine can reduce offensive aggression measures in socially-isolated Swiss Webster mice in home cage resident–intruder encounters [13], however this study did not evaluate the contribution of locomotor or sociability changes due to nicotine. In neutral cage encounters of socially-isolated OF1 mice with anosmic opponents, nicotine administered either acutely or chronically did not reduce aggression, whereas lobeline, a less specific nAChR agonist that also interacts with the dopamine transporter and vesicular monoamine transporter [27] reduced aggression even at doses that did not affect locomotion [28,29]. Acute nicotine administration also did not influence sociability significantly in socially isolated OF1 mice [30].
Aggression is frequently comorbid with neuropsychiatric conditions and is a predictor of worse outcomes, yet current pharmacotherapies are insufficient and have debilitating side effects, precluding broad use. Multiple models of aggression across species suggest that the nicotinic acetylcholine receptor (nAChR) agonist nicotine has anti-aggressive (serenic) properties. Here we demonstrate dose-dependent serenic effects of acute nicotine administration in three distinct mouse strains: C57BL/6, BALB/c, and CD1. While acute nicotine administration (0.25 mg/kg) modestly reduced solitary homecage locomotion, this could not account for nicotine’s serenic effects since social encounters eliminated the hypolocomotor effect, and nicotine did not alter social interaction times. Pretreatment with the homomeric (α7 subunit) nAChR antagonist methyllycaconitine (5 mg/kg), but not the heteromeric (β2 or β4 subunit-containing) nAChR antagonist dihydro-β-erythroidine (DHβE, 3 mg/kg), blocked the serenic effects of nicotine. By contrast, pretreatment with DHβE blocked the effect of acute nicotine administration on locomotion, uncoupling nicotine’s serenic and hypolocomotor effects. Finally, the α7 nAChR partial agonist GTS-21 reduced aggression in C57BL/6 mice. These results support the idea that acute nicotine administration has serenic effects and provide evidence for specificity of this effect distinct from effects on locomotion. Furthermore, pharmacological studies suggest that activation of α7 nAChRs underlies the serenic effects of nicotine. Further studies of nAChRs could enhance understanding of the neurobiology of aggression and may lead to the development of novel, more specific treatments for pathological aggression.
The vesicular monoamine transporter-2: An important pharmacological target for the discovery of novel therapeutics to treat methamphetamine abuse
2014, Advances in Pharmacology
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Moreover, lobeline is more potent in inhibiting DA uptake at VMAT2 than it is in releasing DA from the vesicles, whereas amphetamine is more potent in releasing DA from the vesicles than it is in inhibiting DA uptake into the vesicles. Importantly, lobeline reduces METH-evoked DA release in vitro while concurrently increasing extracellular dihydroxyphenylacetic acid (DOPAC) concentrations (Miller et al., 2001; Wilhelm, Johnson, Eshleman, & Janowsky, 2008). Collectively, these results suggest that the decreased reinforcing effects of METH are the result of lobeline decreasing the cytosolic pool of DA available for METH-induced reverse transport by DAT (Dwoskin & Crooks, 2002).
Methamphetamine abuse escalates, but no approved therapeutics are available to treat addicted individuals. Methamphetamine increases extracellular dopamine in reward-relevant pathways by interacting at vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine uptake and promote dopamine release from synaptic vesicles, increasing cytosolic dopamine available for reverse transport by the dopamine transporter (DAT). VMAT2 is the target of our iterative drug discovery efforts to identify pharmacotherapeutics for methamphetamine addiction. Lobeline, the major alkaloid in Lobelia inflata, potently inhibited VMAT2, methamphetamine-evoked striatal dopamine release, and methamphetamine self-administration in rats but exhibited high affinity for nicotinic acetylcholine receptors (nAChRs). Defunctionalized, unsaturated lobeline analog, meso-transdiene (MTD), exhibited lobeline-like in vitro pharmacology, lacked nAChR affinity, but exhibited high affinity for DAT, suggesting potential abuse liability. The 2,4-dicholorophenyl MTD analog, UKMH-106, exhibited selectivity for VMAT2 over DAT, inhibited methamphetamine-evoked dopamine release, but required a difficult synthetic approach. Lobelane, a saturated, defunctionalized lobeline analog, inhibited the neurochemical and behavioral effects of methamphetamine; tolerance developed to the lobelane-induced decrease in methamphetamine self-administration. Improved drug-likeness was afforded by the incorporation of a chiral N-1,2-dihydroxypropyl moiety into lobelane to afford GZ-793A, which inhibited the neurochemical and behavioral effects of methamphetamine, without tolerance. From a series of 2,5-disubstituted pyrrolidine analogs, AV-2-192 emerged as a lead, exhibiting high affinity for VMAT2 and inhibiting methamphetamine-evoked dopamine release. Current results support the hypothesis that potent, selective VMAT2 inhibitors provide the requisite preclinical behavioral profile for evaluation as pharmacotherapeutics for methamphetamine abuse and emphasize selectivity for VMAT2 relative to DAT as a criterion for reducing abuse liability of the therapeutic.
High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding
2012, Journal of Biological Chemistry
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Thus, the acetylcholine receptor does not appear to be required for the effect of lobeline on muscle cell expression of WFA-binding glycans. Lobeline is also known to act through alternative receptors, including the dopamine transporter and the vesicular monoamine transporter (30–32). A role for lobeline as a protein folding chaperone has been reported, suggesting that lobeline may increase protein abundance by reducing protein misfolding and degradation (33).
Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.
Background: Genetic alteration of muscle cell glycosylation in muscular dystrophy models has ameliorated disease.
Results: A high throughput screen identified a small molecule, lobeline, which altered muscle cell glycosylation and improved laminin binding.
Conclusion: Lobeline increased abundance of sarcolemmal glycoproteins and increased laminin binding in an N-glycan-dependent manner.
Significance: A novel approach revealed an unexpected role for N-glycans in muscle cell function.
Tetrabenazine inhibition of monoamine uptake and methamphetamine behavioral effects: Locomotor activity, drug discrimination and self-administration
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The VMAT2 inhibitor, lobeline, decreases both methamphetamine-evoked DA release and methamphetamine self-administration in outbred rats (Harrod et al., 2001, 2003; Miller et al., 2001). However, lobeline also acts as a potent antagonist at nicotinic receptors, and less potently inhibits DAT function (Miller et al., 2000; Zheng et al., 2005; Wilhelm et al., 2008). Lobelane, a des-oxy lobeline analog, inhibits VMAT2 more potently and selectively than its parent compound (Miller et al., 2004; Zheng et al., 2005), and similarly decreases methamphetamine-evoked DA release and methamphetamine self-administration (Neugebauer et al., 2007; Nickell et al., 2010).
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Lobeline effects on tonic and methamphetamine-induced dopamine release

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgements

Biochem Pharmacol

Prog Neurobiol

Pharmacol Biochem Behav

Brain Res

Brain Res

Neuroscience

Lobeline displaces [3H]dihydrotetrabenazine binding and releases [3H]dopamine from rat striatal synaptic vesicles: comparison with d-amphetamine

J Neurochem

Lobeline and nicotine evoke [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake

J Pharmacol Exp Ther

Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport

J Neurosci

Lobeline and nicotine evoke [³H]overflow from rat striatal slices preloaded with [³H]dopamine: differential inhibition of synaptosomal and vesicular [³H]dopamine uptake