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PAIN MECHANISMS

A High Concentration of Resiniferatoxin Inhibits Ion Channel Function in Clonal Neuroendocrine Cells

From the Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts.

Address correspondence and reprint requests to G. Strichartz, PhD, MRB 613/BWH, 75 Francis St., Boston, MA 02115-6110. Address e-mail to gstrichz{at}zeus.bwh.harvard.edu.

Abstract

BACKGROUND: Resiniferatoxin (RTX) is a potent agonist of the transient receptor potential vanilloid 1 channel (TRPV1) found in peripheral nociceptors. RTX causes cellular excitation first, followed by a long-lasting refractory state, which has suggested its therapeutic use for pain control. RTX's effect could result from specific actions on TRPV1 channels, but might also arise from previously reported TRPV1-independent effects. We have tested whether exposure to RTX compromises ion channels in a TRPV1-independent manner.

METHODS: Clonal rat anterior pituitary (GH₃) cells, loaded with the Ca⁺²-sensitive fluorescent dye (fluo-4), were stimulated with the Na⁺ channel activator veratridine (VTD) or directly depolarized by 60 mM K⁺ solution. The physiological effects of exposure to RTX were evaluated by stimulated increases of fluorescence from raised intracellular [Ca²⁺].

RESULTS: The presence of 10 µM RTX acutely reduced the median fluorescence changes by VTD and 60 mM K⁺ to 45% and 50%, respectively (P = 0.018 and 0.043). Prolonged exposure (24 h) of cells to 10 µM RTX, followed by a 2 h washout, reduced the median fluorescence changes by VTD and 60 mM K⁺ to 5.6% and 42% of control changes, respectively (P = 0.027 and 0.011). Cell responses to VTD partially recovered, to 42% of control, after incubation in RTX-free medium for 24 h.

CONCLUSION: RTX at 10 µM directly and acutely inhibited voltage-dependent Ca²⁺ channels, in a TRPV1-independent manner. Prolonged exposure (24 h) to 10 µM RTX inhibited voltage-dependent Na⁺ channels in addition to the Ca²⁺ channels, in at least a partially reversible manner.

The transient receptor potential vanilloid 1 channel (TRPV1) is a nonselective cation channel that is recognized as a molecular integrator of various noxious stimuli such as noxious heat and protons and the vanilloid capsaicin, the pungent ingredient of hot peppers.1 Capsaicin has a biphasic action through TRPV1, initial excitation is followed by a long-lasting refractory state during which capsaicin-stimulated neurons are insensitive not only to subsequent capsaicin application but also to other noxious stimuli. Based on this effect, capsaicin and its analogs have been studied as potential therapeutic agents for pain control. Resiniferatoxin (RTX) is one of the most potent vanilloid agonists of TRPV12 and, importantly for analgesia, is even more effective in the desensitization action than capsaicin. In animal experiments, RTX shows a far more favorable ratio of desensitization to irritation than capsaicin. In previous reports, either intraganglionic or intrathecal administration of RTX was shown to be effective in reducing experimental inflammatory hyperalgesia and cancer-related pain, without affecting locomotor function.3,4 Perineural application of RTX (0.0003%–0.001%, equivalent to 4.8–15.9 µM) causes several days worth of functional neural blockade in both inflammatory and neuropathic pain models.5,6 Interestingly, RTX at high concentrations seems to have an effect beyond TRPV1-dependent actions, such as transient analgesic effects on mechanical nociception.5 Since TRPV1-independent effects of capsaicin on diverse ion channels have been reported7–9 and long-lasting behavioral actions have been characterized at these high concentrations,2 we tested whether RTX compromises the functions of ion channels in a TRPV1-independent, reversible manner.

METHODS

Cell Culture
Clonal rat anterior pituitary (GH₃) cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco's modified Eagle medium (DMEM, Gibco/BRL, Rockville, MD; supplemented with 10% fetal bovine serum, penicillin, and streptomycin) under 95% O₂ and 5% CO₂ at 37°C.

Reagents and Solutions
RTX, veratridine (VTD), and tetrodotoxin (TTX) were obtained from Sigma-Aldrich (St. Louis, MO). RTX and VTD were dissolved in pure dimethylsulfoxide (DMSO) to make stock solutions of 1 and 25 mM, respectively, and TTX (as a citrate-containing powder) was dissolved in high-purity deionized water. All reagents were kept at –20°C before use. The imaging buffer (IB) for most physiological assays contained: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH adjusted to 7.4. High K⁺ solution contained: 85 mM NaCl, 60 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH adjusted to 7.4. For the experiments with zero extracellular Ca²⁺, the IB was modified by replacing all the added Ca²⁺ by Mg²⁺, thus increasing the latter ion's concentration to 4 mM; all other ingredients were the same.

Fluorescence Measurements
Changes in intracellular [Ca²⁺] in GH₃ cells were measured as described previously.10 Briefly, cells were loaded with 5 µM Ca²⁺-sensitive fluorescent dye, Fluo 4-AM, dissolved in 0.005% Pluronic F-127 (Molecular Probes, Eugene, OR). After two brief, 5-min washes with IB to remove any extracellular dye, GH₃ cells were superfused with IB for 30 s, exposed to RTX or to IB containing vehicle for 10 min (for acute exposure experiments) and then stimulated for 45 s with 50 µM VTD, a selective chemical "activator" of voltage-gated Na⁺ channels,8,9 or high K⁺ (60 mM), which depolarizes the cells and opens voltage-gated calcium channels, and superfused again with IB for 75 s, all in the continued presence of RTX. All procedures were conducted at room temperature (20°C–21°C). The cells were imaged at 490–500 nm using a DG4 multiwavelength light source with a Stanford Photonics 12 bit digital intensified CCD and the data displayed and analyzed using QED imaging software (QED Software, Pittsburgh, PA). The maximal fluorescence changes that occurred during any stimulation by VTD or high K⁺ (60 mM) solution were collected for all identical experiments in one recording session, and processed for the analysis of cellular responses.

For the experiments on the acute effects of RTX, dye-loaded cells were exposed to RTX for 10 min before stimulation by VTD or high K⁺, in the continued presence of RTX. In experiments to measure the effects of prolonged exposure to RTX and its reversal after RTX washout, we waited until 24 h of RTX exposure, or until 24 h of washout after a 24 h exposure to RTX, and then loaded the cells with dye in RTX-free medium, followed by the usual brief washes to remove extracellular dye before measurement of fluorescence changes. The cells under these conditions were nominally "RTX-free."

Statistics
Statistical analysis was performed using Statview 5.0.1 (Abacus Concepts, Berkeley, CA). Data were analyzed using Mann–Whitney U-test or Wilcoxon's signed rank test for nonparametric comparisons. Results were expressed as median and ranges. For calculation of inhibition intensity, the median value of the RTX-treated group was divided by the median value of the control, vehicle-treated group. Inhibition was expressed as mean ± sem of this ratio and its statistical significance was assessed by comparing the ratios of control and treatment groups using ANOVA. A P value <0.05 was considered statistically significant.

RESULTS

Na⁺ Influx Induced by VTD Triggers an Increase in Intracellular Ca²⁺
To assess the effects of acute and chronic exposure of RTX on neuronal activity, we adopted an optical assay to quantify Na⁺ channel activity. In this assay, GH₃ cells preloaded with a Ca²⁺ sensitive dye (Fluo-4) were stimulated with VTD, a specific activator of voltage-gated Na⁺ channels. VTD binds to the open conformation of Na⁺ channels, resisting their normal inactivation to a nonconducting state and resulting in persistently open channels.11,12 By this effect, VTD evokes membrane depolarization that opens voltage-gated Ca²⁺ channels, permitting the rapid entry of Ca²⁺ into the cell. This increased Ca²⁺ concentration is detected by the Fluo-4 fluorescence intensity. Application of 50 µM VTD for 45 s caused a fluorescence increase in Fluo-4 loaded cells (Fig. 1A). This is consistent with the previous reports that GH₃ cells express both several isoforms of Na⁺ channels (all of the TTX-sensitive type) and also voltage-gated L-type Ca²⁺ channels13–15; accordingly, opening of the Na⁺ channels allows an inward current that depolarizes the cells and thereby opens voltage-gated Ca²⁺ channels. Since each individual cell varies in baseline fluorescence intensity, and the kinetics and latency of fluorescence intensity changes also differ among individual cells, we quantified the data as follows. For each cell image, F₀ is the mean fluorescence intensity in a single cell before stimulation, and F is the increase in fluorescence intensity of that cell over the period of acute exposure to VTD or to high K⁺ (Fig. 1B). The relative change in fluorescence intensity of each cell is expressed as F/F₀ and F_max is the maximum increase in fluorescence intensity of that cell over the entire stimulation period.

View larger version (5K): [in this window] [in a new window]   Figure 1. Na+ influx induced by veratridine (VTD) triggers Ca2+ increase in clonal anterior pituitary cells. A, A video image of a fluorescence increase induced by 50 µM VTD in GH3 cells loaded with the Ca2+-sensitive dye Fluo-4. B, Corresponding fluorescence trace (in arbitrary units, a.u.) shows the VTD-stimulated change, F, of that cell measured over the observation period. Relative changes in fluorescence intensity of this and other cells are expressed as F/F0, where F0 is the mean baseline fluorescence intensity in a single cell before stimulation. C, The maximum Ca2+-induced fluorescence increase is indicated by (Fmax/F0) for cells stimulated by VTD while exposed to vehicle (control), 1 µM tetrodotoxin (TTX), or 0 mM Ca2+ external solution (0 Ca). Data are expressed as medians (horizontal lines) with 1st and 3rd quartiles (boxes) and 10th and 90th percentiles (vertical lines). **P < 0.01 for the difference in distributions of these Fmax/F0 values between control and either TTX- or 0 Ca treated cells (Mann–Whitney U-test).

The variance over time in F₀, which is the noise in this baseline fluorescence measurement, was on average 0.02 of the mean F₀; a true response was therefore set at F/F₀ >0.1, i.e., at a signal:noise ratio of 5. Changes in normalized fluorescence less than this value were considered "nonresponses." Histograms of F_max/F₀ for all the individual cells in a plated population show a non-normal distribution (Fig. 2B). We used two parameters to assess the sensitivity of responses to VTD: one is the median value of the population of maximum cellular responses and the other is the fraction of nonresponding cells. Neither of these is dependent on interpretations of a particular amplitude of response and so is independent of the kinetics of rise or decay of intracellular [Ca²⁺].

View larger version (9K): [in this window] [in a new window]   Figure 2. Resiniferatoxin (RTX) inhibits the veratridine (VTD)-induced Ca2+ increase. A, GH3 cells were stimulated with VTD in the presence of different concentrations of RTX. Percentage of the median value of maximum fluorescence increase (Fmax/F0) divided by the control (RTX = 0) increase is expressed as mean ± sem. *P < 0.05 for the comparison of the distributions of Fmax/F0 between control and RTX-treated cells (Mann–Whitney U-test). B, A histogram shows the distribution of values of the maximum VTD-stimulated increases (Fmax/F0) in GH3 cells acutely exposed to 10 µM RTX (see Methods). The number of cells with fractional increases in fluorescence ranging from 0 to 0.1 (scored as nonresponders, see Results) up to 0.9–1.0 are listed within the ranges noted on the horizontal axis. The absence of a column means that no cells had a signal with amplitudes in this range.

In contrast to VTD, the acute application of RTX (1–10 µM) did not produce any detectable Ca²⁺ increase in GH₃ cells (data not shown), indicating that functional TRPV1 receptors are not expressed, and that other modes for changing [Ca²⁺]_in are not affected by RTX.

Exposure of cells to IB containing the vehicle alone, 1% (v:v) DMSO for RTX at 10 µM or 0.2% for VTD at 50 µM, caused no acute changes in fluorescence nor did 1% DMSO have any long-term effect on GH3 cell responses (see below).

We next analyzed the role of Na⁺ channels and the requirement of external calcium for the VTD-induced intracellular [Ca²⁺] increase. Incubation of GH₃ cells with the Na⁺ channel-specific inhibitor TTX (1 µM) abolished the VTD-induced intracellular [Ca²⁺] increase, demonstrating that this signal is dependent on Na⁺ influx through voltage-dependent Na⁺ channels (Fig. 1C). The direct binding of VTD to the Na⁺ channel is unaltered by the presence of TTX, which binds at a different location.12 Removal of Ca²⁺ from the external solution also prevented the VTD-induced intracellular [Ca²⁺] 'increase (Fig. 1C), demonstrating the requirement for Ca²⁺ entry from the bathing solution, consistent with the role of voltage-gated Ca²⁺ channels.

RTX Acutely Inhibits the VTD-Induced Ca²⁺ Increase
Besides the receptor-specific effects produced through TRPV1, capsaicin and its homologues are reported to have some direct inhibitory effect on Na⁺ channels.7,8 To test this possibility, we stimulated cells with VTD after brief incubation and then in the continued presence of different concentrations of RTX. In GH₃ cells that were exposed to 10 µM RTX, median values of the VTD-induced intracellular [Ca²⁺] increase were reduced to 45% of median control values in GH₃ cells exposed to vehicle (P = 0.018), whereas acute exposure to 3 µM RTX caused a modest (28%), but statistically insignificant, reduction (Fig. 2A). Examination of Figure 2B shows that the median value in relative fluorescence decreased because of 2 factors: (1) the fluorescence intensity of the maximally responding cells was reduced (from 0.9 to 1.0 for control to 0.7 to 0.8 for RTX-treated), and (2) the fraction of "nonresponding" cells (F_max/F₀ <0.1) was increased by 75%, due to acute RTX exposure.

RTX Inhibits Ca²⁺ Increase Evoked by Elevated K⁺
Because the VTD-evoked increase in intracellular [Ca²⁺] requires ion flux through both Na⁺ channels and Ca²⁺ channels, inhibition by RTX might occur through a direct effect on Ca²⁺ channels. To test this, cells were stimulated with a 60 mM KCl solution (high K⁺) that depolarizes cells in a Na⁺ channel-independent manner. Exposure to such high K⁺ solution caused rapid, large and sustained intracellular [Ca²⁺] increases (Fig. 3A). In contrast to the increase from VTD, the one induced by high K⁺ was insensitive to 1 µM TTX, confirming that this intracellular [Ca²⁺] increase is Na⁺ channel-independent (Fig. 3B). No change in dye signal was detected when cells were depolarized by high K⁺ in Ca²⁺-free solution, showing that Ca²⁺ entry was essential for this response, consistent with an entry through the (L-type) voltage-gated calcium channels known to carry virtually all of this inward current in GH₃ cells.13–15

View larger version (5K): [in this window] [in a new window]   Figure 3. Resiniferatoxin (RTX) inhibits Ca2+ increase evoked by high potassium chloride. A, Example of the time-course of a fluorescence increase induced by 60 mM KCl solution (high K+) in a Fluo-4 loaded GH3 cell. B, High K+-induced maximum fluorescence increase (Fmax/F0) under vehicle (control) and 1 µM tetrodotoxin (TTX). Data are expressed as medians (horizontal lines) with 1st and 3rd quartiles (boxes) and 10th and 90th percentiles (vertical lines). P > 0.05 between these two distributions (Mann–Whitney U-test). C, The average of the ratio of median values of high K+-induced maximum fluorescence increase (Fmax/F0) in RTX (10 µM)-treated cells to those in vehicle-treated cells, from three separate experiments, shows a significant reduction of the high K+ response due to acute exposure to RTX (P < 0.05, ANOVA).

When GH₃ cells were acutely exposed to 10 µM RTX, the median value of the high K⁺-induced intracellular [Ca²⁺]-related fluorescence increase was reduced to 50% of the median control value (P = 0.043, Fig. 3C), essentially the same as the reduction of the VTD-induced change caused by this RTX treatment. These data demonstrate that 10 µM RTX has a direct inhibitory effect on Ca²⁺ channels that is sufficient to account for the acute suppression of the VTD-induced signal, which also relies on Ca⁺² entry during cell depolarization.

Long-Term Exposure to RTX Results in a Long-Lasting Inhibition of Na⁺ Channel Activity
Previous electrophysiological reports observed that capsaicin and its chemical analogues have residual effects that can persist after 5–10 min of washout, so we tested whether the more potent vanilloid RTX also has effects after washout. GH₃ cells were incubated with 10 µM RTX for 1 h, loaded with Fluo-4 in RTX-free conditions, a procedure that takes 2 h., and then stimulated with VTD in the absence of RTX. After such an RTX treatment, the median value of the VTD-induced fluorescence signal was significantly reduced to 60.2% of the median control value in GH₃ cells incubated with vehicle for the same time (Fig. 4A).

View larger version (6K): [in this window] [in a new window]   Figure 4. Long-term exposure of cells to resiniferatoxin (RTX) results in a residual inhibitory effect on the veratridine (VTD)-stimulated Ca2+ increase. A, GH3 cells were incubated with vehicle (control) or 10 µM RTX for 1 or 24 h, then loaded with Fluo-4 in RTX-free medium (for 2 h) and then stimulated with VTD in the absence of RTX in the bathing medium. Percentages of the median value of maximum Ca2+ increase (Fmax/F0) against control for three experiments are expressed as mean ± sem. *P < 0.05 for responses of RTX-treated cells versus controls (ANOVA). B, GH3 cells were incubated with RTX (10 µM) for 24 h and then returned to RTX-free medium for 0 or 24 h. Those cells were then loaded with Fluo-4 in RTX-free condition and stimulated with VTD in the absence of RTX. Percentage of median value of maximum fluorescence increase (Fmax/F0) against control is expressed as mean ± sem. *P < 0.05 for response after 24 h washout compared to response of cells before washout (ANOVA). C, GH3 cells were incubated with vehicle (control) or RTX (10 µM) for 24 h, loaded with Fluo-4 in RTX-free condition and stimulated with high K+-in the absence of RTX. Percentage of median value of (Fmax/F0) against control are expressed as mean ± sem. *P < 0.05 for mean of the ratio responses of RTX-treated cells versus control (vehicle-treated) cells.

When the period of GH₃ cell incubation in RTX was lengthened to 24 h, the VTD-induced fluorescence increase was nearly abolished, with its median value reduced to 5.6% of median control values in GH₃ cells incubated with vehicle (P = 0.027, Fig. 4A). After such treatment, almost no Ca²⁺ enters the cell when exposed to VTD.

The reversibility of effects from the 24 h exposure to RTX was examined by returning the cells to RTX-free medium for 24 h, then loading them with dye (2 h) and stimulating with VTD. Median values of the VTD-induced fluorescence change were restored to 42% of control values by this washout procedure, showing about a seven-fold return of activity over this 24 h period (P = 0.010, 0 h rest vs 24 h rest, Fig. 4B).

When GH₃ cells exposed to 10 µM RTX for 24 h were stimulated with high K⁺, the median value of the fluorescence increase was marginally, albeit significantly, reduced to 42% of the median control value, considerably less than the approximately 95% inhibition of the VTD-induced change (P = 0.011 for inhibition of VTD response vs inhibition of K⁺ response, Fig. 4C). Therefore, reduction of the VTD-induced response by long-term exposure to RTX cannot be accounted for by the decrease in entry through voltage-gated Ca²⁺ channels.

DISCUSSION

The present study demonstrates that relatively high concentrations (1–10 µM range) of RTX have inhibitory effects on voltage-dependent Na⁺ and Ca²⁺ channels in a TRPV1-independent manner. Behavioral studies show that RTX applied perineurally at similar concentrations, 5–16 µM,5,6 results in an antihyperalgesic action that reverses after several days, with no motor effects and no accompanying neurohistological changes in the peripheral axons.16 Applications of RTX, by intrathecal delivery for the treatment of experimental canine bone cancer,4 or by intraganglionic microinjection for long-term prevention of nocifensive responses in rats,3 delivered initial concentrations equivalent to 0.3 µM and 16–160 µM, respectively, yielding behavioral deficits that lasted for at least several weeks and were accompanied by the ablation of TRPV1-expressing sensory neurons. By comparison, exposure of isolated cells to concentrations of RTX as low as 1–10 nM activated TRPV-1 receptors/ channels, induced Ca²⁺ entry, and altered mitochondrial appearance through TRPV1-involving mechanisms, sparing cells that do not express the vanilloid receptor.17,18 These studies report a variety of histological consequences in tissues where different administration locations and concentrations of RTX have effects lasting from days to months, implying that different cellular mechanisms may be engaged by different routes and different doses of RTX.

Preliminary results on the antinociceptive actions of perineurally applied RTX in the rat show that the increase in thermal latency caused by an even lower [RTX], 0.16 µM (0.00001%), can be only partially prevented by very high concentrations of the TRPV-1 antagonist capsazepine (at 80 µM, 0.003%; unpublished observation, I. Kissin, Pain Research Center, Brigham & Women's Hospital, Boston, MA), suggesting that some of these behavioral actions of RTX in vivo do not involve TRPV-1 receptors. In summary, in the present study, we have used doses and conditions that are relevant to many of the reported therapeutic actions of RTX.

Other investigators have reported that high doses of capsaicin inhibit several types of ion channels, including voltage-dependent Na⁺ and Ca²⁺ channels.7–9 Two groups report that 30 µM capsaicin blocked Na⁺ channels in a TRPV1-independent manner; in one case, the channels were assayed using patch clamp recordings of heterologously expressed channels, from the rat skeletal muscle -subunit, in cells lacking TRPV1,7 and in the other, the channels were assayed in sensory neurons from TRPV1-knockout mice.19 In the absence of TRPV1, capsaicin elicited a time-dependent block of inactivation-deficient Na⁺ currents, with the 50% inhibitory concentration (IC₅₀) of 6.8 µM for open Na⁺ channels.8 In addition, capsaicin produced use-dependent block of Na⁺ currents indicating that open Na⁺ channels have higher affinity for this vanilloid than Na⁺ channels in the resting states.8,19 These data support the hypothesis of the existence of a vanilloid binding site on voltage-dependent Na⁺ channels, totally independent of TRPV1.

Interestingly, this inhibitory effect was absent when batrachotoxin (BTX) was used to keep Na⁺ channels open. This observation is consistent with a competition between BTX and vanilloids, raising the possibility that the capsaicin binding site sterically overlaps or is located adjacent to the BTX receptor (site 2). Regarding this issue, Duan et al.9 have reported that TRPV1 modulatory drugs, such as capsaicin and capsazepine, inhibited the binding of BTX to voltage-dependent Na⁺ channels. Since in their experiments the initial association rate of BTX was not changed by these TRPV1 ligands, there is no direct competition with BTX for the site 2-binding domain but rather an allosteric action. VTD has been shown to share binding domains on the Na⁺ channel with BTX (site 2), so the acute inhibitory effect of RTX on Na⁺ channels might come from reduced VTD binding to that site. However, we found that the acute inhibitory effect of 10 µM RTX was effectively the same for intracellular [Ca²⁺] increases caused by VTD and high K⁺, the latter mediated by voltage-gated calcium channels. Thus, there is evidence for a direct effect of RTX on calcium channels, and an acute inhibition of sodium channels is not necessary to explain these acute results.

Since such high concentrations of vanilloids inhibit several types of voltage-gated cation channels, it is possible that general physicochemical actions, such as stiffening of the lipid bilayer, might account for these inhibitory effects,7 although the structural similarities of the channels could account for a common binding motif for these agents, as is the case for local anesthetics.20

These acute inhibitory effects of capsaicin on Na⁺ channels were fully reversed after several minutes of wash in vanilloid-free solution, which is distinctly different from the slow recovery of Na⁺ channel function that we observed after long-term exposure to RTX, implying that a simple reversible binding to Na⁺ channels does not explain the actions of 24 h exposure.

Twenty-four hours of exposure to 10 µM RTX caused a very strong inhibition of the VTD-evoked intracellular [Ca²⁺] increase, even after 2 h of washout of RTX. Since 1 h exposure to10 µM RTX causes less inhibition, duration of exposure is an important determinant of RTX's residual effectiveness in suppressing the VTD-induced response. More importantly, 24 h exposure to RTX had a much weaker effect on high K⁺-induced intracellular [Ca²⁺] increases (Fig. 4C). Therefore, unlike the situation with equally inhibited responses to acute RTX exposure, the differential effect of 24 h exposure to 10 µM RTX means that the relatively weak inhibition of Ca²⁺ channels (stimulated by high K⁺) cannot account for the very strong inhibition of Na⁺ channels (stimulated by VTD).

The precise mechanism of this long-term effect of RTX is unknown, although a decrease in the density of channels on the plasma membrane is a serious candidate. The number of functional sodium channels is determined by several factors, including cytosolic [Ca²⁺] and protein kinase C (PKC; Refs. 21 and 22). Since RTX at 10 µM has an inhibitory effect on Ca²⁺ channels, decreased Ca²⁺ channel activity and a consequent decreasing of cytoplasmic free [Ca²⁺] might play a role in a reduced expression of Na⁺ channels. In fact, prolonged exposure to antagonists of L-type calcium channels decreases the expression of Na⁺ channels on the surface in GH₃ cells.23 Furthermore, RTX itself contains a phorbol ester-like structure and at high concentration has a weak but direct effect on PKC.24,25 In support of this hypothesis, activation of PKC has been shown to down-regulate Na⁺ channel expression in cells homologous to sympathetic ganglion neurons.22

In conclusion, RTX at 10 µM directly and acutely inhibited voltage-dependent Ca²⁺, and possibly Na⁺, channels in clonal neuroendocrine cells in a TRPV1-independent manner. Prolonged exposure (24 h) to RTX at 10 µM inhibited voltage-dependent Ca²⁺ channels and, to a much greater degree, voltage-dependent Na⁺ channels in a TRPV1-independent, partially reversible manner. Since RTX at similar concentrations has been shown to induce a prolonged but reversible analgesia in vivo, these cellular responses might contribute to the behavioral pharmacology of RTX.

Footnotes

Accepted for publication January 25, 2008.

Supported by NIH-GM065834.

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