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Summary

5. Summary

The consumption of the widespread addictive drug nicotine can cause severe diseases. The

nicotine-induced sensory effect is known to contribute to the addictive effect. Nicotine exists in

two stereoisomers, (+)- and (-)-nicotine, of which the (-)-enantiomer is much more abundant in

nature. The drug’s central nervous effect is mediated by nicotinic acetylcholine receptors. Both,

the olfactory system as well as trigeminal sensations like “burning” and “stinging” contribute to

the chemosensory impression induced by nicotine. The chemosensory impressions are

stereoselective. Subjects can discriminate the two isomers by olfactory sensation; the trigeminal

effects appear at different detection thresholds for each enantiomer. Besides nicotinic

acetylcholine receptors, the transient receptor potential channel A1, that mediates the “stinging”

mustard oil impression, is known to contribute to the nicotine impression. Psychophysical studies

indicate that nAChR and TRPA1 cannot fully explain the nicotine-induced chemosensory

impression.

In this study, we investigated the effect of nicotine on trigeminal expressed ionotropic receptors.

We used the electrophysiological two-electrode-voltage-clamp technique to characterize the

effect of (+)- and (-)-nicotine on heterologously expressed receptors. For homo- (α7) and

heteromeric (α2β4) human nAChRs the stereoselectivity for (-)-nicotine could be shown. For the

first time, (+)-nicotine could be shown to be a TRPA1-agonist. Additionally, no stereospecifity was

observed. Further, nicotine was identified as an antagonist for human 5-HT3-receptors with a

stereospecifity for the (-)-enantiomer. These effects were observed for homomeric 5-HT3A- as well

as heteromeric 5-HT3AB-receptors. Experiments analyzing ionotropic glutamate receptors revealed

a voltage-dependent block of NMDA-receptors (NR1-3a/NR2B) induced by (-)-nicotine (10 mM),

which looks similar to the known voltage-dependent Mg2+-block, but remains incomplete. No

effect was observed at GABAA-, Glycine-, AMPA- (GluR1(Q)flop), Kainat- (GluR6(Q)), P2X2-

receptors as well as TRPV1-3- and TRPM8-channels. In our experiments with human TRPV1,

(-)-nicotine could not evoke the positive modulation, that is known for TRPV1 from rat.

Further, in this study the effect of nicotine on murine trigeminal neurons was investigated in

calcium-imaging experiments. Under nAChR and TRPA1 blocking conditions a certain population

of neurons was found, showing a persisting (-)-nicotine response. With these results I could prove

the existence of an unknown (-)-nicotine receptor, expressed in trigeminal neurons, that may

contribute to the chemosensory impression of nicotine. Further, a population of neurons was

identified, responding specifically to (+)-nicotine. A specific receptor for this enantiomer is not

known to date. In further experiments, again under nAChR and TRPA1 blocking conditions, we

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Summary

could prove signals of specifically (+)-nicotine-responding neurons not to be mediated by both

known receptor families. Therefore, in this study the existence of a (+)-nicotine-specific receptor

expressed in trigeminal neurons was shown. As the unknown (+)- and (-)-nicotine-evoked signals

may be mediated by G-protein-coupled receptors, the involvement of metabotropic signal

pathways was investigated. Neither (+)- nor (-)-nicotine-induced signals were found under

Ca2+-free conditions, indicating that the observed nicotine-induced signals depend on extracellular

Ca2+-ions. Additionally, the blocking effect of nicotine on 5-HT3-receptors, for the first time, was

observed on native receptors in trigeminal neurons. 5-HT3-receptors were identified as the third

important target of nicotine in the trigeminal system with the capacity to influence nociception.

In a systematical approach, in this study, we analyzed the transcriptome of trigeminal and dorsal

root ganglia via next-generation-sequencing methods. We could quantify the trigeminal

expression of chemosensory relevant Cys-loop-, TRP- and KCNK-channels. In previous studies

these channels were detected by qualitative methods. Further, we tried to identify unknown

receptors of (+)- and (-)-nicotine by analyzing the amplicons of single cell transcriptomes, that

showed nicotine-signals independently from nAChRs and TRPA1. Transcriptome analysis of these

cells showed a clear expression of olfactory receptors in single neurons responsive under blocking

conditions. One neuron expressed Olfr293, the other Olfr1200. From the mass of known

transmembrane receptors, both cells shared just the expression of one single receptor, the

Latrophilin receptor Gpr137. Results gained from single-cell transcriptome analysis provide

further candidates of trigeminal expressed receptors that could be the unknown nicotine

receptors.