Lieju Liu

Capsazepine, a vanilloid receptor antagonist, inhibits nicotinic acetylcholine receptors in rat trigeminal ganglia

Vanilloid receptors are activated by capsaicin, the pungent ingredient in hot pepper. They are also specifically and competitively inhibited by capsazepine (CPZ). To determine whether CPZ is specific to vanilloid receptors, its effects were tested on the currents evoked by nicotine in rat trigeminal ganglia. We found that 10 microM CPZ, a concentration frequently used to inhibit capsaicins physiological responses attributed to capsaicin, reversibly inhibits (40%) the magnitude of the currents activated by 100 microM nicotine. We conclude that 10 microM capsazepine can alter the effects of channels other than those activated by capsaicin, and thus caution must be used in attributing all the CPZ-sensitive physiological effects to those only produced by blocking of vanilloid receptors.

Capsaicin, acid and heat-evoked currents in rat trigeminal ganglion neurons: relationship to functional VR1 receptors

Activation of primary trigeminal (TG) neurons by protons, capsaicin, or heat can evoke a variety of sensations, including tingling, stinging, warmth, and burning. Capsaicin and acid are trigeminal stimulants that are important in gustatory physiology. These stimuli can activate H(+)-gated ion channels and heterologously expressed VR1 receptors (vanilloid receptor 1). We have obtained evidence by using electrophysiological and pharmacological measurements on TG neurons that these three stimuli can activate many receptors, and we have determined the extent they behave similarly to VR1 receptors and H(+)-gated channels from the DEGenerin/ENaC superfamily. Whole-cell recordings from rat TG neurons revealed that protons evoked transient (Tp), sustained (Sp), and biphasic (TSp) currents. Tp currents had reversal potentials (Vr) of 24-45 mV, a pH(0.5) range from 5.5 to 6.5, and were inhibited by amiloride, suggesting the presence of functional H(+)-gated channels. Sp currents were inhibited by the VR1 antagonist capsazepine, had Vrs approximately 0 mV, and had pH(0.5) = 6.4. Capsaicin also activated transient (Tc), sustained (Sc), and biphasic (TSc) currents. At pH 5.9, the sensitivity of the Sc currents increased by about a factor of 10, which may partially account for the synergistic responses of acid in foods containing capsaicin. Heating TG neurons evoked a thermally active, capsazepine-inhibitable current with threshold temperature of 43 degrees C and Vr = 5 mV that is also present in neurons activated by and protons (Sp) and capsaicin (Sc). These data suggest that TG neurons have functional receptors that behave similarly to VR1. Activation of such receptors should result in a burning sensation, whereas activation of the transient and biphasic currents should result in other taste descriptors.

Acidic stimuli activates two distinct pathways in taste receptor cells from rat fungiform papillae

A sour taste sensation may be produced when acidic stimuli interact with taste receptor cells (TRCs) on the dorsal surface of the tongue. We have searched for pathways in TRCs that may be activated by acidic stimuli using RT-PCR and changes in intracellular calcium (Ca(2+)(I)) induced by acidic stimuli in rat fungiform papillae. RT-PCR revealed the presence of proton-gated subunits ASIC-beta and VR1. Ca(2+) imaging measurements of the TRCs revealed two distinct responses to acidic stimuli: Ca(2+)(i) was increased in 9% (28/308; Type I) and was decreased in 39% (121/308; Type II). Neither of these responses was affected by the removal of extracellular Ca(2+), indicating that the changes arise from the release and sequestration of Ca(2+) from intracellular stores. These responses were also not inhibited by the vanilloid receptor antagonist, capsazepine, suggesting they do not arise from the activation of vanilloid receptors. The Type I, but not the Type II response was inhibited by amiloride. Dose-response measurements for Types I and II responses yielded pH(50%) of 4.8 and 4.9, respectively. Type II responses were inhibited by pertussis toxin, suggesting G-protein involvement. TRCs that exhibit Type II responses could also be activated by quinine (which increased Ca(2+)(I)) thus suggesting a mechanism by which the addition of acid may be suppressive to other chemical stimuli.

Neuronal nicotinic acetylcholine receptors in rat trigeminal ganglia.

The application of nicotine to the various epithelia served by the trigeminal nerve produces irritation and/or pain by activating neuronal nicotinic acetylcholine receptors (NnAChRS) in sensory neurons. In this study the NnAChRs were identified in rat trigeminal ganglia (TG) using RT-PCR and immunocytochemistry. With RT-PCR the subunits of NnAChRs in rat TG were determined, and with immunocytochemistry the localization of three prominent subunits (alpha 7, alpha 4 and beta 2) were localized in intact TG neurons. The relative abundance of the alpha and beta subunits were: alpha 7 approximately alpha 3 > alpha 6 > alpha 4 approximately alpha 5 > alpha 9 > or = alpha 2, and beta 2 approximately beta 3 > beta 4. This is the first report of the alpha 9 subunit in TG. Immunohistochemical studies revealed that almost all TG neurons contained alpha 7-LI and alpha 4-LI, and that 85% had beta 2-LI. For these three subunits much of the label was internalized. Immunocytochemical studies using antibodies raised against chick alpha 8 subunits did not specifically label rat TG. These data reveal that rat TG neurons contain the entire spectrum of mammalian NnAChR subunits.

A non-pungent resiniferatoxin analogue, phorbol 12-phenylacetate 13 acetate 20-homovanillate, reveals vanilloid receptor subtypes on rat trigeminal ganglion neurons.

Capsaicin, the vanilloid responsible for the pungent taste of hot peppers, binds to receptors found primarily in polymodal nociceptors. Capsaicin initially stimulates polymodal nociceptors and subsequently inhibits them from responding to a variety of stimuli. This property makes it useful clinically as an analgesic and anti-inflammatory compound. There is mounting, albeit indirect, evidence for the existence of several subtypes of vanilloid receptors. One such piece of evidence comes from studying analogues of capsaicin, such as phorbol 12-phenylacetate 13 acetate 20-homovanillate. This compound binds to (capsaicin) vanilloid receptors on sensory neurons, but unlike capsaicin it is non-pungent and does not produce hypothermia. To determine how sensory neurons respond to phorbol 12-phenylacetate 13 acetate 20-homovanillate, and to compare these responses with those evoked by capsaicin, whole-cell patch-clamp measurements were performed on cultured rat trigeminal ganglion neurons. It was found that 63% of the neurons held at -60 mV were activated by 3 microM, phorbol 12-phenylacetate 13 acetate 20-homovanillate, and 87% of these were also activated by 1 microM capsaicin. In a given neuron, phorbol 12-phenylacetate 13 acetate 20-homovanillate, like capsaicin, could activate kinetically distinct inward currents. The current-voltage curves characterizing phorbol 12-phenylacetate 13 acetate 20-homovanillate responses were asymmetric and had reversal potentials at -5.8 +/- 6.0 mV and 10.4 +/- 4 mV. The averaged dose-response curves for phorbol 12-phenylacetate 13 acetate 20-homovanillate were fit to the Hill equation and had binding constants (K(1/2)s) of 2.73 microM and 0.96 microM and Hill coefficients (ns) of approximately 1 for a rapidly- and slowly-activating current, respectively. These parameters are consistent with those obtained from binding experiments and calcium-influx experiments on sensory nerves. Repeated applications of phorbol 12-phenylacetate 13 acetate 20-homovanillate every 3 min caused a complete reduction in the rapidly-activating currents leaving only a reduced slowly-activating current. This provides strong evidence for the independence of these currents and the existence of subtypes of vanilloid receptors. Additional evidence for the existence of receptor subtypes is that 10 microM capsazepine, a specific and competitive inhibitor of capsaicin-evoked responses, did not inhibit the phorbol 12-phenylacetate 13 acetate 20-homovanillate-induced currents in some neurons and partially inhibited them in other neurons. Thus, there are capsazepine-sensitive and capsazepine-insensitive subtypes of vanilloid receptors. In summary, we have obtained electrophysiological and pharmacological evidence for distinct subtypes of vanilloid receptors.

Identification of acetylcholine receptors in adult rat trigeminal ganglion neurons

Nicotinic acetylcholine receptors (nAChRs) were identified in a subpopulation of cultured adult rat trigeminal ganglia (TG) neurons by whole-cell patch-clamp recordings. Dimethylphenylpiperazinium (DMPP), a nAChR agonist, induced inward currents in 21/68 of TG neurons having soma diameters greater than 28 microns. These currents were inhibited by hexamethonium, mecamylamine and atropine, indicating the presence of neuronal ganglionic-type nAChRs. This interpretation is consistent with the finding that the nicotine- or DMPP-induced currents were not inhibited by alpha-bungarotoxin (alpha-Bng) in 5 of the 9 cells tested with this compound. However, in 2 of the 9 cells tested, the DMPP-induced currents were completely inhibited by alpha-Bng, and in the remaining two cells tested, the currents were partially inhibited by alpha-Bng. About 22% of the cells having diameters > or = 28 microns were specifically labeled with FITC-labeled alpha-Bng, whereas only 2% of the cells with soma diameters < 28 microns were labeled. These data taken together suggest that more than one subtype of nAChR is present in TG.

The influence of removing extracellular Ca2+ in the desensitization responses to capsaicin, zingerone and olvanil in rat trigeminal ganglion neurons.

Desensitization is a process that describes the diminishing effect of a drug upon repeated applications. In regard to capsaicin, the pungent compound in hot pepper, it is well established that removal of extracellular calcium markedly diminishes desensitization. To explore whether this behavior extends to other analogues of capsaicin, we have determined the effect of removing extracellular calcium with capsaicin analogues, zingerone and olvanil, by whole-cell patch clamping cultured rat trigeminal ganglion neurons. Zingerone, like capsaicin, is pungent but has a shorter acyl chain, whereas olvanil is non-pungent and has a longer acyl chain. The currents evoked by 30-s applications of 30 mM zingerone or 1 microM olvanil repeated every 3 min differ in two important ways from the responses evoked by 1 microM capsaicin under these same conditions. In the presence of extracellular calcium, repeated applications of zingerone and olvanil produce nearly complete desensitization. Also in contrast to capsaicin, removing extracellular calcium for these two agonists does not diminish desensitization. These data analyses suggest the existence of calcium-independent pathways that can result in desensitization, and that pungency is not related to the phenomenon of desensitization.

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

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.

Capsaicin activated currents in rat dorsal root ganglion cells

&NA; Capsaicin is a pungent‐tasting compound produced by plants in the Capsium family that activates a subset of primary afferent neurons associated with pain and thermoreception. Previous studies from dorsal root ganglion (DRGs) neurons suggest that many of capsaicins physiological responses are a consequence of its activating a cation‐selective current. To further characterize the responses to capsaicin whole‐cell patch‐clamp measurements were performed on rat DRGs to which 0.1–10 &mgr;M capsaicin was continuously applied. The capsaicin‐activated currents exhibited marked variability in their thresholds, amplitude (to 15 nA), rates of desensitization, and the number of distinct maxima in the evoked current. Similar responses were found in rat trigeminal ganglion cells. The heterogeneity in the magnitude of the currents evoked by 0.1 &mgr;M capsaicin likely reflects different types of capsaicin‐sensitive neurons; a result consistent with in vitro extracellular recordings from capsaicin‐sensitive sensory afferents (Seno and Dray 1993).

Changes in osmolality modulate voltage-gated calcium channels in trigeminal ganglion neurons.

Voltage-gated calcium channels (VGCCs) participate in many important physiological functions. However whether VGCCs are modulated by changes of osmolarity and involved in anisotonicity-induced nociception is still unknown. For this reason by using whole-cell patch clamp techniques in rat and mouse trigeminal ganglion (TG) neurons we tested the effects of hypo- and hypertonicity on VGCCs. We found that high-voltage-gated calcium current (I(HVA)) was inhibited by both hypo- and hypertonicity. In rat TG neurons, the inhibition by hypotonicity was mimicked by Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator but hypotonicity did not exhibit inhibition in TRPV4(-/-) mice TG neurons. Concerning the downstream signaling pathways, antagonism of PKG pathway selectively reduced the hypotonicity-induced inhibition, whereas inhibition of PLC- and PI3K-mediated pathways selectively reduced the inhibition produced by hypertonicity. In summary, although the effects of hypo- and hypertonicity show similar phenotype, receptor and intracellular signaling pathways were selective for hypo- versus hypertonicity-induced inhibition of I(HVA).