Cracking the Betel Nut: Cholinergic Activity of Areca Alkaloids and Related Compounds

Abstract

Introduction: The use of betel quid is the most understudied major addiction in the world. The neuropsychological activity of betel quid has been attributed to alkaloids of Areca catechu. With the goal of developing novel addiction treatments, we evaluate the muscarinic and nicotinic activity of the four major Areca alkaloids: arecoline, arecaidine, guvacoline, and guvacine and four structurally related compounds.

Methods: Acetylcholine receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp.

Results: Both arecoline- and guvacoline-activated muscarinic acetylcholine receptors (mAChR), while only arecoline produced significant activation of nicotinic AChR (nAChR). We characterized four additional arecoline-related compounds, seeking an analog that would retain selective activity for a α4* nAChR, with diminished effects on mAChR and not be a desensitizer of α7 nAChR. We show that this profile is largely met by isoarecolone. Three additional arecoline analogs were characterized. While the quaternary dimethyl analog had a broad range of activities, including activation of mAChR and muscle-type nAChR, the methyl analog only activated a range of α4* nAChR, albeit with low potency. The ethyl analog had no detectable cholinergic activity.

Conclusions: Evidence indicates that α4* nAChR are at the root of nicotine addiction, and this may also be the case for betel addiction. Our characterization of isoarecolone and 1-(4-methylpiperazin-1-yl) ethanone as truly selective α4*nAChR selective partial agonists with low muscarinic activity may point toward a promising new direction for the development of drugs to treat both nicotine and betel addiction.

Implications: Nearly 600 million people use Areca nut, often with tobacco. Two of the Areca alkaloids are muscarinic acetylcholine receptor agonists, and one, arecoline, is a partial agonist for the α4* nicotinic acetylcholine receptors (nAChR) associated with tobacco addiction. The profile of arecoline activity suggested its potential to be used as a scaffold for developing new tobacco cessation drugs if analogs can be identified that retain the same nicotinic receptor selectivity without muscarinic activity. We report that isoarecolone is a selective partial agonist for α4* nAChR with minimal muscarinic activity and 1-(4-methylpiperazin-1-yl) ethanone has similar nAChR selectivity and no detectable muscarinic action.

Figure 1.

Nicotinic acetylcholine receptor (nAChR) responses to Areca alkaloids. Oocytes expressing either human α4 and β2 nAChR subunits or α7 subunits were first evaluated for their responses to two control applications to acetylcholine (Ach), 30 µM for the α4β2-expressing cells or 60 µM for the α7-expressing cells and then for their responses to the Areca alkaloids applied at 100 µM. Alkaloid responses were calculated relative to the average of the two initial ACh applications. Responses of the α4β2 receptors were calculated as peak currents and the α7 responses as net charge. Additionally, the α7 receptors were also tested with the alkaloids co-applied with 10 µM of the PAM PNU-120596, and those responses are scaled on the right-hand y-axis. In these experiments, the α4 and β2 subunit RNAs were co-injected at a 1:1 ratio.

Figure 2.

Characterization of Areca alkaloids analogs on M₁ and M₃ acetylcholine (AChR) expressed in Xenopus oocytes. The structures of the analogs, 1-(4-methylpiperazin-1-yl) ethanone (MPA), 4-acetyl-1,1-dimethylpiperazin-1-ium (DMPA), and 1-(4-ethylpiperazin-1-yl) ethanone (EPA) are shown accompanying the corresponding data. A two-application protocol as described for Supplementary Figure S1 was used to characterize these ligands for their effects on cells expressing M₁ and M₃ AChR. Test compounds were applied at 10 µM, and then after a 3-min wash period 10 µM acetylcholine was applied to determine whether the initial application was able to desensitize the receptor/channel system and decrease or eliminate further responses.

Figure 3.

Nicotinic acetylcholine receptor (nAChR) responses to isoarecolone. Oocytes expressing human α7, α3β4, α4β2 nAChR subunits, mouse muscle α1β1εδ subunits, or the human α4β2α4β2α3 concatamer were first evaluated for their responses to two control applications to ACh, and then for their responses to the analogs applied at 100 µM. Acetylcholine (ACh) controls were 60 µM for the α7-expressing cells, 100 µM for α3β4, and 30 µM for the other subtypes. Responses were normalized relative to the average of the two initial ACh applications from the same cells. Responses of the α7 receptors were calculated as net charge and as peak current for the other subtypes. Additionally, the α7 receptors were also tested with isoarecolone co-applied with 10 µM of the PAM PNU-120596, and those data are indicated by the asterisk. (Note that these are scaled on the same left-hand y-axis.)

Figure 4.

Nicotinic activity of the isoarecolone analogs (see Figure 3 for structures). After obtaining two acetylcholine control responses, 100 µM applications of 1-(4-methylpiperazin-1-yl) ethanone, 4-acetyl-1,1-dimethylpiperazin-1-ium, and 1-(4-ethylpiperazin-1-yl) ethanone were made to oocytes expressing the low sensitivity form of α4β2 (α4(3)β2(2)), the high sensitivity form of α4β2 (α4(2)β2(3)), or α7. Analog-evoked responses were calculated relative to the peak currents (α4β2 subtypes) or net charge (α7 receptors) of the ACh controls. Control ACh concentrations were 10 µM, 60 µM, and 100 µM for α4(2)β2(3), α7, and (α4(3)β2(2), respectively. An α4β2 dimer was co-expressed with monomeric α4 or β2 to yield the α4β2 subtypes indicated. Additionally, the α7-expressing cells were tested with co-applications of 10 µM PNU-120596 and 100 µM of the analogs. Those data, normalized to the responses of the same cells to 60 µM ACh alone, are plotted relative to y-axis on the right. All points are the averages of at least five oocytes (± SEM).