In Vivo Effects of Capsazepine on Trigeminal Nerve Sensitivity to Carbon Dioxide and

NicotineHessamedin Alimohammadi and Wayne L. Silver

Department of Biology, Wake Forest University, Winston-Salem, NC 27109alimhx00@wfu.edu, silver@wfu.edu

Methods ResultsIntroduction

Methods

Conclusions

Literature CitedFigure 4. Response to Nicotine Figure 6. Response 30 Minutes Post-

Treatment

Figure 5. Differential Effect of Capsazepine

Figure 3. Response to Carbon Dioxide

Mean percent response curves showing changes in the magnitude of the ethmoid nerve response to carbon dioxide (Figure 3) and nicotine (Figure 4) over a 10 minute time period (three stimulus presentations). Percent response values were calculated as a percentage of the first response magnitude, which was recorded 20 minutes after administration of either capsazepine or the control solution. Error bars represent one standard deviation unit. Administration of capsazepine resulted in a significant decrease in the ethmoid nerve response to carbon dioxide, whereas response to nicotine remained unchanged (n=5 for all groups).

Figure 5. Time course of relative capsazepine effect on carbon dioxide and nicotine. The relative strength of capsazepine effect was calculated as the quotient of the control and capsazepine treated response values at each stimulus presentation (capsazepine efficacy index). The effect of capsazepine on the nerve response to carbon dioxide increased over time. There was no change on the nerve response to nicotine vapor.Figure 6. Trigeminal nerve response to carbon dioxide and nicotine 30 minutes after administration of capsazepine. Response to carbon dioxide is ~50 of the original response, whereas response to nicotine is unchanged.

Figure 1. Experimental setup. Stimuli were presented via a computer-controlled air-dilution olfactometer. Data were recorded using an automated acquisition system controlled by the olfactometer. Respiration was monitored via a thermocouple inserted into the rat’s breathing tube.Figure 2. Representative data showing neural and integrated neural response of the ethmoid nerve to a 5 second presentation of carbon dioxide (50%) or nicotine vapor (12.5 ppm). Response magnitude was calculated as the amplitude difference between the maximum integrated response value and the mean baseline value 5-seconds prior to the onset of stimulus delivery.

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Figure 1. Experimental Setup

Figure 2. Representative Data

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Systemic administration of capsazepine specifically reduces nasal trigeminal nerve responses to carbon dioxide.

Nasal trigeminal nerve response to nicotine is not significantly affected by systemic administration of capsazepine.

Significant levels of reduction in the trigeminal nerve response to carbon dioxide are reached within 30 minutes of intraperitoneal injection of capsazepine.

Capsazepine treatment does not completely abolish trigeminal nerve sensitivity to carbon dioxide, suggesting the involvement of more than one receptor mechanism for carbon dioxide.

These results suggest that at the concentration tested, systemic CPZ does not interfere with nAChR function, and can be reliably used as a specific vanilloid receptor blocker in future in vivo studies.

1. Alimohammadi, H. & W. L. Silver. 2000. Evidence for nicotinic acetylcholine receptors on nasal trigeminal nerve endings of the rat. Chem. Senses. 25: 61-66.

2. Burnstock, G. & M. Williams. 2000. P2 Purinergic receptors: modulation of cell

function and therapeutic potential. J. Pharm. Exp. Therapeutics. 295: 862-869. 3. Caterina, M. J. & D. Julius. 2001. The vanilloid receptor: a molecular gateway to the

pain pathway. Annu. Rev. Neurosci. 24: 487-517.

4. Caterina, M. J., Leffler, A., Malmberg, A. B., Martin, W. J., Trafton, J., Peterson- Zeitz, K. R., Koltzenburg, M., Basbaum, A. I., & D. Julius. 2000. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 288: 306-313.

5. Finger, T. E., M. L. Getchell, T. V. Getchell & J. C. Kinnamon. 1990. Affector and effector functions of peptidergic innervation of the nasal cavity. In: Chemical Senses. Vol. 2: Irritation. B. G. Green, J. R. Mason & M. R. Kare, eds. Marcell Dekker, New York. pp. 1-20. 6. Ichikawa, H. & T. Sugimoto. 2000. Vanilloid receptor 1-like receptor-

immunoreactive primary sensory neurons in the rat trigeminal nervous system. Neuroscience. 101: 719-725.

7. Liu, L. & S. A. Simon. 1997. Capsazepine, a vanilloid receptor antagonist, inhibits

nicotinic acetylcholine receptors in rat trigeminal ganglia. Neurosci. Lett. 228(1): 29-32.

8. Liu, L. & S. A. Simon. 2000. Capsaicin, acid and heat-evoked currents in rat

trigeminal ganglion neurons: relationship to functional VR1 receptors. Phys. & Behav. 69: 363-378.

9. Lundblad, L., J. M. Lundberg, E. Brodin & A. Änggård. 1983. Origin and distribution of capsaicin-sensitive substance P-immunoreactive nerves in the nasal mucosa. Acta Otolaryngol. 96: 485-493. 10. Silver, W. L. & D. G. Moulton. 1982. Chemosensitivity of rat nasal trigeminal

receptors. Physiol. Behav. 28: 927-931. 11. Silver, W. L., D. B. Walker, M. W. Ogden & J. C. Walker. 1990. Nasal trigeminal responses to toluene presented by an automated delivery system. Chem. Senses. 15 (6): 701-712. 12. Walker, J. C., M. Kendal-Reed, C. J. Keiger, M. Bencherif & W. L. Silver. 1996. Olfactory and trigeminal responses to nicotine. Drug Dev. Res. 38: 160-168.

The peripheral receptors of the trigeminal system in the nasal

cavity are free nerve endings arising from the nasopalatine and

ethmoid branches of the trigeminal nerve. These chemosensitive A

and C fibers respond to a diverse list of chemicals and are scattered

throughout the respiratory epithelium.5,9 Previous and ongoing

research suggest that these fibers express a variety of receptor

molecules functioning as peripheral chemoreceptors, including the

nicotinic acetylcholine receptor (nAChR) and the capsaicin-, heat- and

acid-sensitive vanilloid receptor (VR-1).1,2,3,4,6,8 The functional

contribution of these receptor molecules to trigeminal

chemosensitivity can be characterized through the use of receptor

blocking compounds such as capsazepine (CPZ). CPZ, a competitive

inhibitor of VR-1, has previously been shown to also inhibit nAChRs

in cultured rat trigeminal ganglion neurons, calling into question its

reliability as a specific VR-1 blocking agent7.

To determine the reliability of using CPZ as a specific vanilloid

receptor blocker in an in vivo model, we examined the effects of

systemic capsazepine on nasal trigeminal nerve responses to nAChR

and VR-1 activating stimuli (nicotine and carbon dioxide,

respectively) in anesthetized Sprague-Dawley rats. Nicotine has been

shown to activate neuronal nAChRs present on trigeminal nerve

endings1, and carbon dioxide is believed to activate acid-sensitive

receptors, including VR-1, via a carbonic anhydrase mediated

intraepithelial acidification mechanism. Results suggest that at the

concentration tested, systemic CPZ does not interfere with nAChR

function, and can be reliably used as a specific vanilloid receptor

blocker.

A total of 10 male Sprague-Dawley rats weighing between 300

and 500 grams were used in this study. Rats were anesthetized with

urethane (ethyl carbamate: 1.0 g/kg injected i.p.). Two cannulae were

inserted into the trachea of each rat. One cannula, open to room air,

was inserted towards the rat’s lungs. A second cannula connected to a

vacuum line was inserted rostrally into the nasopharynx. Rats were

restrained in a head holder and the ethmoid nerve was exposed and

placed on a pair of platinum-iridium wire hook electrodes as

previously described.11

Rats in experimental groups received an intraperitoneal injection

of capsazepine (10-6 mol/kg). Control group rats were injected with

physiological saline (1.0 ml/kg, i.p.). Twenty minutes post-injection,

stimuli were delivered to the nares of the rats using a computer-

controlled air-dilution olfactometer.11 Rats were stimulated with

carbon dioxide and (-)-nicotine, at concentrations which have

previously been shown to stimulate the ethmoid nerve.10,12 Each rat

received a total of three presentations per stimulus, each lasting for 5

seconds. Rats were first stimulated with nicotine (12.5 ppm),

followed by 50% carbon dioxide 60 seconds later, with a 300 second

rest between each presentation set.

Multiunit neural activity (Neural Response) from the ethmoid

nerve was amplified and summated (Integrated Response) using an

averaging circuit. Respiration was recorded via a thermocouple

inserted into the rat’s breathing tube. Neural response, integrated

response, and respiration were recorded for a period of ~30 seconds

per stimulus presentation. The data were analyzed by MANOVA at

p < 0.05, using StatView Version 5.0.