Different responses to repeated applications of zingerone in behavioral studies, recordings from intact and cultured TG neurons, and from VR1 receptors

Abstract

When applied repetitively to the cornea, capsaicin, the pungent compound in hot pepper, causes an initial eye-wiping response that diminishes upon repeated exposure (tachyphylaxis). This diminution, however, is not observed upon repetitive application of its pungent analogue, zingerone, to the cornea or tongue. In addition, compared with capsaicin, the lingual application of zingerone produces a gustatory response with a shorter latency and duration. Because both the tongue and the cornea are innervated by the trigeminal nerve, and because zingerone and capsaicin are structurally related, it is not evident why the responses to these compounds should give such different behavioral and psychophysical endpoints. We have addressed this issue by measuring the neural responses from rat trigeminal ganglion neurons (TG) to repeated applications of zingerone applied to the cornea, from cultured rat TG neurons, and from cloned capsaicin receptors (VR1) expressed in Xenopus oocytes and then comparing these effects to those evoked by capsaicin. Extracellular recordings from the trigeminal ganglion revealed that the responses to repeated corneal applications of 30 mM zingerone show desensitization. Cultured TG neurons, and oocytes expressing VR1 receptors, were also desensitized by repeated applications of zingerone. Electrophysiological recordings revealed that these two vanilloids could activate the same receptor (VR1), currents in the same neuron, and crossdesensitize. The more rapid onset and shorter duration responses seen with zingerone (compared with capsaicin) provides a rationalization for its more rapid onset and shorter duration gustatory response. We attribute the different behavioral responses to periodic applications of these two agonists to two competing effects: one leading to sensitization, and the other to tachyphylaxis. Which of these dominates depends on the concentration, exposure time, and interstimulus interval. Consequently, whether or not zingerone will exhibit tachyphylaxis depends critically on the experimental conditions.

Introduction

Capsaicin (Fig. 1) is a pungent vanilloid found in hot peppers that is eaten daily by over a third of the world's population [35]. When placed on the human tongue, it induces a warm sensation, a burning and sometimes a stinging/pricking sensation, and at increasing concentrations a painful sensation 11, 13, 14, 15, 18, 19, 32, 33. The capsaicin analogue, zingerone, is a pungent pyrolytic product of ginger oleoresin responsible for the piquancy of ginger. When placed on human tongues, zingerone produces stinging, tingling, numbing, burning, and warm taste sensations 30, 31, 32. In this article, the responses of capsaicin and zingerone will be compared at behavioral, psychophysical, cellular, and molecular levels [6].

Recently, the capsaicin-sensitive VR1 receptor was cloned and expressed in Xenopus oocytes and HEK 293 cells 1, 40. Using immunocytochemistry and in situ hybridization methods it has been found that VR1 subunits are present in rat TG and cornea 6, 16. VR1 channels have been found to be cation selective, and exhibit desensitization in the presence of extracellular calcium 6, 40. When currents evoked from individual cells to repetitive agonist applications are decreased, this process will be referred to as desensitization. When the currents are decreased in the presence of an agonist, this process will be referred to as acute desensitization. In behavioral or psychophysical experiments, the diminution of a response to repetitive applications will be referred to as tachyphylaxis. These characteristics are found in primary TG 24, 25, 26, DRG 9, 12, 21, 23, 27 neurons, and also in rat subnucleus caudalis neurons when capsaicin is placed on the rat cornea-conjunctiva or tongue [5]. Another characteristic of the VR1 receptor and of capsaicin receptors in TG and DRG neurons that may be important in rationalizing its behavioral effects is that when the pH is lowered, such as occurs during inflammation or ischemia, the sensitivity of these receptors to capsaicin increases 29, 34, 41.

There is some evidence that capsaicin and zingerone can activate the same receptor(s), and/or a common pathway. That is, capsaicin and zingerone both induce pain and/or irritation when placed onto rat corneas 2, 5, 39, contractions of isolated urinary bladder tissue [28], and hypothermia, vasodilatation, bradycardia, and apnoea [38]. Also, after capsaicin-induced desensitization, the application of zingerone does not produce hypothermia, vasodilatation, bradycardia, or apnoea, suggesting that they crossdesensitize 24, 38, 39. Finally, patch-clamp measurements obtained from rat TG neurons showed that capsazepine, a competitive inhibitor of capsaicin-activated currents [4], inhibits the inward currents evoked by both capsaicin and zingerone [24].

In other experiments, however, zingerone (ED₅₀ = 30 mM) was shown not to simply behave as a less potent analogue of capsaicin (ED₅₀ = 0.7 μM) [24]. One of the most striking differences in the responses evoked by these two vanilloids is that zingerone behaves differently from capsaicin in its ability to produce complete tachyphylaxis. Specifically, Szolcsanyi and Jancho–Gabor [39] found that corneal applications of capsaicin (every 3 min) initially evoked painful/irritating sensations (as evidenced by the frequency in which rats wiped their eyes), but as the number of capsaicin applications increased, the frequency of eye wiping monotonically decreased—until it completely disappeared (17, 39, and Fig. 2). In contrast, in rat corneas that were not previously exposed to capsaicin, it was found that with repeated applications of 1% zingerone, the eye-wiping responses remained approximately constant (Fig. 2), indicating that under these conditions, zingerone does not exhibit marked tachyphylaxis.

In rat corneas desensitized by capsaicin, 1% zingerone placed onto the same cornea 2 h later evoked an eye-wiping response, albeit smaller, than that of the initial capsaicin application in the naı̈ve cornea, or in the cornea not previously exposed to capsaicin (Fig. 2). This smaller response to zingerone shows that the effects of capsaicin were still present, and subsequent zingerone applications did not evoke eye wiping (Fig. 2). Thus, if capsaicin and zingerone activate the same receptors in the same neurons, the question arises as to why, upon repeated applications, do they elicit such different behavioral responses? To address this question we performed measurements designed to determine whether these two vanilloids activate the same neurons. In addition, we measured TG neuron responses produced by repeated applications of zingerone onto rat corneas to determine whether, at this level of the trigeminal axis, zingerone induces desensitization.

One behavioral gustatory difference between capsaicin and zingerone is that the peak of irritation for zingerone is reached earlier, and the duration is much briefer than it is for capsaicin 30, 31, 32. As an initial step in rationalizing the different behavioral and psychophysical responses of these compounds, we will determine whether capsaicin and zingerone can activate the same receptor and measure the differences in the kinetics of the currents activated by these agonists.

Another aspect of the responses evoked by lingual applications of zingerone or capsaicin that will be addressed is sensitization (the intensity of the capsaicin response increases upon repeated applications) 11, 13 (Fig. 3A). When zingerone is applied immediately after capsaicin-induced sensitization, it initially produces an even more irritating sensation (hypersensitizes), but subsequent zingerone applications induce tachyphylaxis (Fig. 3B). In the psychophysical literature there is some disagreement as to whether zingerone application to human tongues produces tachyphylaxis (compare Fig. 3C and 3D). For example, Green found that 1% zingerone, applied using filter paper to the surface of the tongue, produced neither sensitization nor tachyphylaxis ([13], Fig 3C), whereas Prescott and Stevenson [31] found that whole mouth rinses of 1% zingerone produced marked tachyphylaxis (Fig. 3D). The data obtained here is used to elucidate and eliminate some of the mechanisms underlying the differences between capsaicin and zingerone 13, 33.