Gerd Kobal

Activation of Olfactory and Trigeminal Cortical Areas Following Stimulation of the Nasal Mucosa With Low Concentrations of S(-)-Nicotine Vapor-An fMRI Study on Chemosensory Perception

Applied to the nasal mucosa in low concentrations, nicotine vapor evokes odorous sensations (mediated by the olfactory system) whereas at higher concentrations nicotine vapor additionally produces burning and stinging sensations in the nose (mediated by the trigeminal system). The objective of this study was to determine whether intranasal stimulation with suprathreshold concentrations of S(−)‐nicotine vapor causes brain activation in olfactory cortical areas or if trigeminal cortical areas are also activated. Individual olfactory detection thresholds for S(−)‐nicotine were determined in 19 healthy occasional smokers using a computer‐controlled air‐dilution olfactometer. Functional magnetic resonance images were acquired using a 1.5T MR scanner with applications of nicotine in concentrations at or just above the individuals olfactory detection threshold. Subjects reliably perceived the stimuli as being odorous. Accordingly, activation of brain areas known to be involved in processing of olfactory stimuli was identified. Although most of the subjects never or only rarely observed a burning or painful sensation in the nose, brain areas associated with the processing of painful stimuli were activated in all subjects. This indicates that the olfactory and trigeminal systems are activated during perception of nicotine and it is not possible to completely separate olfactory from trigeminal effects by lowering the concentration of the applied nicotine. In conclusion, even at low concentrations that do not consistently lead to painful sensations, intranasally applied nicotine activates both the olfactory and the trigeminal system. Hum Brain Mapp, 2009.

Bimodal concentration-response of nicotine involves the nicotinic acetylcholine receptor, transient receptor potential vanilloid type 1, and transient receptor potential ankyrin 1 channels in mouse trachea and sensory neurons.

High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µM (−)-nicotine, a maximum at 100 µM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pHe-dependent, – five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

Cigarette smoke has sensory effects through nicotinic and TRPA1 but not TRPV1 receptors on the isolated mouse trachea and larynx.

Cigarette smoke (CS) exposes chemosensory nerves in the airways to a multitude of chemicals, some acting through the irritant receptors TRPV1 and TRPA1 but potentially also through nicotinic acetylcholine receptors (nAChR). Our aim was to characterize the differences in sensory neuronal effects of CS, gas phase, and particulate matter as well as of typical constituents, such as nicotine and reactive carbonyls. Isolated mouse trachea and larynx were employed to measure release of calcitonin gene-related peptide (CGRP) as an index of sensory neuron activation evoked by CS, by filtered CS gas phase essentially free of nicotine, and by dilute total particulate matter (TPM) containing defined nicotine concentrations. With CS stimulation of the superfused trachea, TRPV1 null mutants showed about the same large responses as wild-type mice, whereas both TRPA1−/− and double knockouts exhibited 80% reduction; the retained 20% response was abolished by mecamylamine (10 μM), indicating a distinct contribution of nAChRs. These phenotypes were accentuated by using TPM to stimulate the immersed trachea; 50% of response was retained in TRPA1−/− and abolished by mecamylamine. In contrast, the gas phase acted like a sheer TRPA1 agonist, consistent with its composition, among other compounds, of volatile reactive carbonyls like formaldehyde and acrolein. In the trachea, the gas phase and CS were equally effective in releasing CGRP, whereas the larynx showed much larger CS than gas phase responses. Thus nicotinic receptors contribute to the sensory effects of cigarette smoke on the trachea, which are dominated by TRPA1. How this translates to human perception affords future research.

Evaluation of the Effect of Ammonia on Nicotine Pharmacokinetics Using Rapid Arterial Sampling

INTRODUCTION The nicotine bolus theory states that the dependence-producing potential of cigarettes relates to a rapid increase in nicotine at brain receptor sites. It has been suggested that ammonia, a compound typically found in tobacco products, further increases the amount of nicotine absorbed and its absorption rate. The aim of this study was to determine whether different ammonia yields in cigarettes affected the rate or amount of nicotine absorption from the lungs to arterial circulation. METHODS 34 adult smokers received 3 separate puffs from each of 2 test cigarettes with different ammonia yields (ammonia in smoke: 10.1 μg per cigarette vs. 18.9 μg per cigarette), followed by rapid radial arterial blood sampling (maximum one sample per second) with 30 min between puffs. Arterial blood samples were assayed for nicotine by liquid chromatography tandem mass spectrometry. Pharmacokinetic modeling was performed and the two test cigarettes were assessed for bioequivalence. RESULTS No significant differences were found in area under the curve, C(max), or T((max)) and the 2 test cigarettes were found to be bioequivalent based on 2 one-sided tests at a significance level of 5%. In addition, the zero-order rate constant (k(0)) obtained from the initial slope of the curves and the model-dependent first-order rate constant (k(a)) were not significantly different. CONCLUSIONS This study provides strong evidence that the different ammonia yields of the test cigarettes had no impact on nicotine pharmacokinetics; thus, the ammonia did not increase the rate or amount of nicotine absorption from a puff of cigarette smoke.

Cigarettes With Different Nicotine Levels Affect Sensory Perception and Levels of Biomarkers of Exposure in Adult Smokers

INTRODUCTION Few clinical studies involving cigarettes have provided a comprehensive picture of smoke exposure, test article characterization, and insights into sensory properties combined. The purpose of these pilot studies was to determine whether cigarettes with different levels of nicotine but similar tar levels would affect sensory experience or smoking behavior so as to significantly alter levels of selected biomarkers of exposure (BOE). METHODS In 2 confined, double-blind studies, 120 adult smokers switched from Marlboro Gold cigarettes at baseline to either 1 of 2 lower nicotine cigarettes or 1 of 2 higher nicotine cigarettes and then to the other cigarette after 5 days. Urinary excretion of exposure biomarkers (nicotine equivalents [NE], total and free 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL], 1-hydroxypyrene, and 3-hydroxypropyl mercapturic acid) as well as carboxyhemoglobin and plasma cotinine were measured at baseline, Day 5, and Day 10. Daily cigarette consumption was monitored and sensory characteristics were rated for each cigarette. RESULTS With higher nicotine yield, urine NE, urine total NNAL, and plasma cotinine increased while nonnicotine BOE decreased without changes in cigarette consumption. In contrast, with lower nicotine yield, urine NE, urine total NNAL, and plasma cotinine dropped while nonnicotine BOE and cigarettes per day increased. Higher nicotine cigarettes were rated harsher and stronger than at baseline while lower nicotine cigarettes were less strong. All 4 test cigarettes were highly disliked. CONCLUSIONS These studies demonstrate that abrupt increases or decreases in nicotine and the resulting sensory changes impact BOE through changes in intensity or frequency of smoking.

Rapid automated blood sampling system for pharmacokinetics studies of cigarette smoking.

INTRODUCTION We developed an automated sampling system to allow multiple, discrete blood samples from a human participant to be collected rapidly and immediately following cigarette smoke exposure. We reported the details of the sampling system along with the results of a pilot study for evaluation of the system. METHODS Components of the system include silastic tubing, solenoid pinch valves, a peristaltic pump, and a fraction collector. This system incorporates a smoking machine that allows precise delivery of cigarette smoke through a mouthpiece and intricate timing to correlate blood samples with smoke inhalation. All components are controlled via integration from a user interface and are fully customizable. We performed several tests to evaluate the equipment, including tubing dead volume, leakage tests, and sample reproducibility. We also performed a pilot study with 6 adult smokers, who received 6 controlled puffs of a research test cigarette. Each inhalation was followed by radial arterial blood collection (1 sample per second tapered to 1 sample every 4 s) for 1 min. Samples were evaluated for nicotine via liquid chromatography-tandem mass spectrometric methods. RESULTS Sampling times and volumes were sufficient for nicotine analysis. No adverse effects were seen in the pilot study, and a 30-min washout period was deemed appropriate between puffs. A significant rise in plasma nicotine levels above baseline after inhalation of smoke was consistently detected in all participants. DISCUSSION The unique advantage of this system is to allow rapid blood sampling after a puff of cigarette smoke, with the benefits of reproducibility, reduction in labor intensity, and high temporal resolution.

The roles of TRPV1, TRPA1 and TRPM8 channels in chemical and thermal sensitivity of the mouse oral mucosa

Spices in food and beverages and compounds in tobacco smoke interact with sensory irritant receptors of the transient receptor potential (TRP) cation channel family. TRPV1 (vanilloid type 1), TRPA1 (ankyrin 1) and TRPM8 (melastatin 8) not only elicit action potential signaling through trigeminal nerves, eventually evoking pungent or cooling sensations, but by their calcium conductance they also stimulate the release of calcitonin gene‐related peptide (CGRP). This is measured as an index of neuronal activation to elucidate the chemo‐ and thermosensory transduction in the isolated mouse buccal mucosa of wild types and pertinent knockouts. We found that the lipophilic capsaicin, mustard oil and menthol effectively get access to the nerve endings below the multilayered squamous epithelium, while cigarette smoke and its gaseous phase were weakly effective releasing CGRP. The hydrophilic nicotine was ineffective unless applied unprotonated in alkaline (pH9) solution, activating TRPA1 and TRPV1. Also, mustard oil activated both these irritant receptors in millimolar but only TRPA1 in micromolar concentrations; in combination (1 mm) with heat (45 °C), it showed supraadditive, that is heat sensitizing, effects in TRPV1 and TRPA1 knockouts, suggesting action on an unknown heat‐activated channel and mustard oil receptor. Menthol caused little CGRP release by itself, but in subliminal concentration (2 mm), it enabled a robust cold response that was absent in TRPM8−/− but retained in TRPA1−/− and strongly reduced by TRPM8 inhibitors. In conclusion, all three relevant irritant receptors are functionally expressed in the oral mucosa and play their specific roles in inducing neurogenic inflammation and sensitization to heat and cold.