Research reportThe influence of removing extracellular Ca2+ in the desensitization responses to capsaicin, zingerone and olvanil in rat trigeminal ganglion neurons
Introduction
Capsaicin, the pungent vanilloid in hot pepper, produces pain and inflammation when placed on skin or mucus membranes. These effects initially arise from capsaicin binding to channels [5]or G-protein coupled receptors [1]on the peripheral terminals of polymodal nociceptors that cause them to release peptides and other transmitters from their peripheral and central terminals [24]. Capsaicin's selectivity for polymodal nociceptors, coupled with the fact that after the initial excitatory phase the neurons become refractory to capsaicin (and to other irritants) [10]has made it clinically useful as a anti-nociceptive and anti-inflammatory compound [14]. This diminution of the response upon repeated applications is called desensitization. For capsaicin, desensitization has been shown to depend on a variety of factors including its concentration, the duration and time between applications, and most importantly for the purposes of this study, the presence of extracellular Ca2+[8]. Specifically, numerous studies have shown that removal of extracellular calcium diminished capsaicin-induced desensitization 5, 6, 7, 9, 12, 23, 27. This behavior was also found in the recently cloned capsaicin receptor/channel (VR1) [5]. Whole-cell patch clamp studies on cultured DRGs attributed desensitization to the action of calcium-dependent protein phosphatases that dephosphorylate capsaicin-sensitive channels [9]. In this study we investigate the effects of removing extracellular calcium on desensitization evoked by two capsaicin analogues, olvanil and zingerone.
Zingerone is a pungent pyrolytic product of ginger oleoresin found in ginger root [16], and olvanil is a synthetic non-pungent analogue that has a longer acyl chain than capsaicin [3]. Previously we found that in rat trigeminal neurons both the responses of zingerone and olvanil were inhibited by the competitive capsaicin receptor antagonist, capsazepine, suggesting that both these analogues bind to molecules in the family of vanilloid receptors [5]. Also, studies of rat spinal cord indicated that olvanil and capsaicin cross-desensitize, suggesting, but not proving, that they act at the same, or same class of, receptor(s) [11]. Regarding zingerone-induced desensitization, psychophysical and behavioral studies gave conflicting results. That is, in some studies zingerone did not exhibit desensitization 13, 26whereas other studies it did [22]. The differences appeared to be related to the times between applications. Whole-cell patch clamp studies using zingerone with rat TG neurons in a calcium-containing buffer showed that it exhibited complete desensitization [17].
Olvanil is similar to capsaicin in its anti-nociceptive and anti-inflammatory properties, but perceptually differs from capsaicin in that it is non-pungent 4, 11. Whole-cell patch clamp recordings using olvanil applied to rat TG showed the desensitization produced was markedly greater than it was for capsaicin [19]. The effect of calcium on this effect was not explored. In this study we have investigated and compared the effects of removing extracellular calcium on the currents evoked by capsaicin, zingerone, and olvanil using cultured rat trigeminal ganglion neurons.
Section snippets
Materials and methods
Salts were reagent grade. Olvanil was purchased from Tocris Cookson (Ballwin, MO) and zingerone from Pfaltz and Bauer (Waterbury, CT). Unless otherwise stated, all other drugs and enzymes were purchased from Sigma (St. Louis, MO).
Capsaicin
All investigators, including ourselves, found that removing extracellular calcium decreased capsaicin-induced desensitization 2, 6, 9, 18, 23. Here we report the effects on desensitization of removing calcium on two other capsaicin analogues, zingerone and olvanil. Fig. 1A shows representative data for experiments performed at −60 mV when seven 30-s applications of 1 μM capsaicin that was applied every 3 min. On the second and third applications the peak current decreased 23% and 38%,
Discussion
We, as well as others, have found that removing extracellular calcium (and replacing it with NaCl and EDTA) markedly decreased both the extent and rate of desensitization (Fig. 1, Fig. 4 and , ). In the elegant study using the cloned VR1 receptor it was found that the ratio of the peak currents (Ip) evoked from HEK293 cells in the first and third capsaicin applications were 95.3% and 13.0% in the absence and presence of calcium, respectively [5]. In these experiments, the HEK293 cells were held
Summary
The role of calcium in modulating the desensitizing responses of vanilloids, like capsaicin, zingerone and olvanil is, dependent on the particular agonist. With the VR1 receptor now cloned [5], and more subtypes and receptor types [1]likely to be found, one can now explore the kinetic steps underlying the differences in receptor activation and physiological responses between these three vanilloids.
Acknowledgements
We thank Dr. Robert Erickson for many criticisms. This work was supported in part from a grant from NIH DC-01065 and support from Phillip Morris.
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