Introduction

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Capsaicin is one of the flavoring ingredients present in the hot pepper Capsicum family. It has been studied for its reactivity with the nociceptor, the "pain" mediator in the neuronal system (for reviews, see Bevan and Szolcsanyi, 1990; Dray, 1992). Capsaicin acts on pain-sensing neurons, including dorsal root ganglion cells (Wood et al., 1988), vagal sensory C-type neurons (Marsh et al., 1987), and trigeminal neurons (Liu and Simon, 1994, 1996). In these cells, capsaicin mediates membrane depolarization and the opening of cation-selective ion channels. By this pathway, capsaicin transfers the pain signal in sensory neurons. Furthermore, the capsaicin-induced large Ca²⁺ entry induces cell injury and desensitization to most other signals in the neuron (Marsh et al., 1987). Thus, the desensitization of signals caused by capsaicin has been thought to be a promising therapeutic tool to mitigate neuropathic pain and pathological conditions in which neuropeptides released from primary sensory neurons play a major role (Dray, 1992; Szallasi and Blumberg, 1996). With systemic or topical administration, capsaicin and its structural analogs produce a reversible antinociceptive and anti-inflammatory effect after an initial undesirable effect (Janusz et al., 1993).

It has been generally accepted that many of the capsaicin effects are mediated by vanilloid receptors. Vanilloid receptors are expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflammation (for a review, see Szallasi and Blumberg, 1996). The VR1 receptor, the first cloned vanilloid receptor, reveals the same distribution (Caterina et al., 1997). VR1 forms a nonselective cation channel that recognizes not only capsaicin as a ligand of the vanilloid receptor but also heat of which the threshold is decreased by H⁺ (Tominaga et al., 1998). Until now, at least two different receptors (C- and R-type) were known for capsaicin, and they were detected in rat dorsal root ganglion cells (Acs et al., 1997) and murine mast cells (Biro et al., 1998). The receptors show similar characteristics with regard to antagonists and agonists but different binding properties. On the other hand, it has been suggested that there are different subtypes of vanilloid receptors or non-VR1-mediated capsaicin effects. This hypothesis is due to the observation that some capsaicin-mediated effects did not follow the typical features of vanilloid receptors, such as effective concentration (Nakazawa et al., 1994; Zhu et al., 1997) and unusual effects of vanilloid antagonists (Docherty et al., 1997; Liu and Simon, 1997).

Various capsaicin effects have been studied in neuronal cells, and the importance of capsaicin studies with regard to the neuronal system has been recognized. We therefore studied the effect of capsaicin on receptor-mediated phospholipase C (PLC) activation and intracellular Ca²⁺ elevation in rat pheochromocytoma PC12 cells. PC12 cells have been used as a good model system in which to study the neuroendocrine system because the receptors and their signaling are well characterized. It is therefore possible to study the regulation of receptor-induced norepinephrine secretion in these cells. PC12 cells express the purinoceptors P2X₂, which is a nonselective cation channel, and P2Y₂, which is coupled to G protein and PLC (Park et al., 1997). Bradykinin receptors are also coupled to PLC in PC12 cells (Suh et al., 1995). Here we report that capsaicin inhibits receptor-mediated Ca²⁺ increase by blocking the store-operated Ca²⁺ entry (SOCE; formerly referred to as capacitative Ca²⁺ entry) that occurs subsequent to PLC activation.

	Experimental Procedures

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Materials. Capsaicin, ATP, bradykinin, and sulfinpyrazone were purchased from Sigma Chemical Co. (St. Louis, MO). Thapsigargin was purchased from Alomone Labs (Jerusalem, Israel). Fura-2/pentaacetoxymethyl ester (fura-2/AM) was obtained from Molecular Probes (Eugene, OR). [³H]Norepinephrine and [³H]inositol-1,4,5-trisphosphate (InsP₃) were purchased from New England Nuclear (Boston, MA). RPMI 1640 and penicillin/streptomycin were purchased from Gibco (Grand Island, NY). Bovine calf serum and horse serum were obtained from Hyclone (Logan, UT).

Cell Culture. PC12 cells were grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated bovine calf serum, 5% (v/v) heat-inactivated horse serum, and 1% (v/v) penicillin/streptomycin. The concentrations of penicillin and streptomycin were 5 U/ml and 50 µg/ml, respectively. The culture medium was changed every day, and the cells were subcultured weekly. Jurkat T cells were maintained at 37°C in RPMI 1640 supplemented with 10% (v/v) heat-inactivated bovine calf serum and 1% (v/v) penicillin/streptomycin. The culture medium was changed every day. All cells were cultured in a humidified atmosphere of 95% air/5% CO₂.

Measurement of [³H]Norepinephrine Secretion. Catecholamine secretion by PC12 cells was measured by the method reported by Suh and Kim (1994). In brief, cells were loaded with [³H]norepinephrine (1 µCi/ml) during incubation in RPMI 1640 for 1 h at 37°C. The cells were then washed twice and incubated in Locke's solution (154 mM NaCl, 5.6 mM KCl, 5.6 mM d-glucose, 1 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES buffer adjusted to pH 7.4) for 15 min for stabilization. Then, the cells were reincubated in fresh Locke's solution for 15 min to measure basal secretion. The cells were subsequently stimulated with the drugs under study for 15 min. The medium was removed from each well, and residual catecholamine was extracted from the cells through the addition of 10% trichloroacetic acid. The radioactivity was measured with a scintillation counter. The amount of [³H]norepinephrine secreted was calculated as the percentage of total [³H]norepinephrine content.

Measurement of [Ca²⁺]_i. [Ca²⁺]_i was determined using the fluorescent Ca²⁺ indicator fura-2 as reported previously (Suh et al., 1995). Briefly, the cell suspension was incubated in Locke's solution with 3 µM fura-2/AM for 50 min at 37°C under continuous stirring. The loaded cells were then washed twice with Locke's solution. Sulfinpyrazone (250 µM) was added to all solutions to prevent dye leakage. For the fluorometric measurement of the [Ca²⁺]_i, 1 × 10⁶ cells/ml was placed into a quartz cuvette in a thermostatically controlled cell holder at 37°C and continuously stirred. Fluorescence ratios were monitored with dual excitation at 340 and 380 nm and emission at 500 nm. Calibration of the fluorescent signal in terms of [Ca²⁺]_i was performed as described by Grynkiewicz et al. (1985) using the following equation: [Ca²⁺]_i = K_D[(R  R_min)/(R_max  R)](S_f2/S_b2), where R is the ratio of fluorescence emitted after excitation at 340 and 380 nm, and S_f2 and S_b2 are the proportionality coefficients at 380 nm excitation of Ca²⁺-free fura-2 and Ca²⁺-saturated fura-2, respectively. To obtain R_min, the fluorescence ratios of the cell suspension were measured successively at a final concentration of 4 mM EGTA, 30 mM Trizma base, and 0.1% Triton X-100. Then, the cell suspension was treated with CaCl₂ to a final concentration of 4 mM Ca²⁺, which is sufficient to saturate fura-2 with Ca²⁺ under these conditions, and the fluorescence ratios were measured to obtain R_max.

Mn²⁺ Quenching of fura-2 Fluorescence. The Mn²⁺ quench assay was performed as described by Lee et al. (1997) to measure the influx of Ca²⁺ from the extracellular space. Briefly, as described above; fura-2-loaded cells (5 × 10⁶ cells/ml) were placed into a quartz cuvette in a thermostatically controlled cell holder at 37°C and continuously stirred. Fluorescence was excited at 360 nm (i.e., the isosbestic wavelength at which Ca²⁺ does not affect fura-2 fluorescence and at which, therefore, any changes are caused by Mn²⁺ quenching). Emission was recorded at 500 nm. The potency and slope of the change in the fluorescence intensity were recorded after the application of 1 mM MnCl₂ and the drugs to be tested.

Measurement of InsP₃ Production. InsP₃ mobilization was determined by competition assay using [³H]InsP₃ as described previously (Lee et al., 1997). To determine InsP₃ production, confluent cells on six-well plates were stimulated with the drugs to be tested. The reactions were terminated by the addition of ice-cold 5% trichloroacetic acid containing 10 mM EGTA. The supernatant of the lysate was saved, and trichloroacetic acid was extracted with diethylether. The aqueous fraction after the final extraction was neutralized with 200 mM Trizma base and adjusted to pH 7.4. Then, 20 µl of the extract was added to 20 µl of assay buffer (0.1 M Tris buffer containing 4 mM EDTA) and 20 µl of [³H]InsP₃ (100 nCi/ml). The mixture was incubated for 15 min on ice and then centrifuged at 2000g for 10 min. Next, 100 µl of water and 1 ml of liquid scintillation cocktail were added to the pellet to measure the radioactivity. The InsP₃ concentration in the samples was determined by comparison with a standard curve and expressed as picomoles per milligram of protein. Total cellular protein concentration was determined with the Bradford method after sonication of cells.

Analysis of Data. All quantitative data are expressed as mean ± S.E. We calculated EC₅₀ and IC₅₀ values with the Microcal Origin for Windows program. Differences were determined by one-way ANOVA and considered to be significant only for P < .05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

We studied the effect of capsaicin on PLC-mediated [Ca²⁺]_i increase and norepinephrine secretion in PC12 cells. Figure 1A shows that 50 µM capsaicin markedly reduced the sustained [Ca²⁺]_i level elevated by extracellular ATP, whereas it had less of an effect on the peak [Ca²⁺]_i level. We had reason to assume that the difference in the inhibitory effect of capsaicin on the peak and the sustained [Ca²⁺]_i level might be due to differences in [Ca²⁺]_i-elevating pathways. Cytosolic Ca²⁺ increase triggered by the PLC pathway could be distinguished as Ca²⁺ release from the intracellular Ca²⁺ reservoir and Ca²⁺ influx from the extracellular space. In the absence of extracellular Ca²⁺, the ATP-induced [Ca²⁺]_i rise was not inhibited by capsaicin (data not shown), suggesting that capsaicin does not exert an inhibitory effect on the ATP-induced Ca²⁺ release from Ca²⁺ stores in the cell. The data also suggest that capsaicin has an inhibitory effect on the ATP-induced Ca²⁺ influx from the extracellular space. There is a possibility that 1 to 10 mM streptomycin could inhibit PLC directly (Schwertz et al., 1984; Bian et al., 1998; Gergawy et al., 1998) and thus affect the capsaicin effect on PLC-mediated signaling, because the PC12 cells were cultured in a medium containing streptomycin (~34 µM). We tested capsaicin on cells cultured in antibiotic-free medium and found no detectable difference. To confirm that the inhibitory effect of capsaicin is on responses elicited by the PLC-coupled receptor, we tested whether capsaicin had an effect on responses to bradykinin, which is known to activate PLC in PC12 cells (Fig. 1B). Capsaicin inhibited the bradykinin-induced Ca²⁺ elevation without significant attenuation of the peak level, but capsaicin did not inhibit the bradykinin-induced [Ca²⁺]_i rise in the absence of extracellular Ca²⁺, as was the case for the extracellular ATP stimulation. Furthermore, we tested the effect of capsaicin on the production of InsP₃ to determine whether capsaicin directly inhibited PLC. As seen in Fig. 1C, there were no statistically significant differences in the extracellular ATP- or bradykinin-induced InsP₃ production in the presence or absence of capsaicin (P > .05). The result shows that capsaicin inhibited neither extracellular ATP- nor bradykinin-induced InsP₃ production. We then looked for a capsaicin effect on norepinephrine secretion in PC12 cells. In the [³H]norepinephrine-loaded PC12 cells, PLC-activating ligands (e.g., ATP or bradykinin) induced the secretion of norepinephrine with an increase in [Ca²⁺]_i. Figure 2 shows that capsaicin inhibited the extracellular ATP-induced norepinephrine secretion. The inhibition shows a concentration-dependent manner with an IC₅₀ value of 42.9 ± 7.7 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of capsaicin on ATP- and bradykinin-induced [Ca2+]i rise and InsP3 production in PC12 cells. Fura-2-loaded cells were challenged with 300 µM ATP (A) or 3 µM bradykinin (BK; B) in the presence (b) or absence (a) of 50 µM capsaicin (Cap). Typical Ca2+ traces from more than three separate experiments are presented. The results were reproducible. C, capsaicin effect on InsP3 production was monitored in PC12 cells. Cells were preincubated with (hatched column) or without (open column) 50 µM capsaicin for 3 min and then treated with 300 µM ATP or 3 µM bradykinin for 15 s. The production of InsP3 was measured as described in Experimental Procedures. Each result is the mean ± S.E. of triplicate assays. The experiments were performed three times independently. and the results were reproducible.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of capsaicin on [3H]norepinephrine secretion and its inhibitory effect on extracellular ATP-induced [3H]norepinephrine secretion by PC12 cells. [3H]Norepinephrine-loaded PC12 cells were treated with 300 µM extracellular ATP in the presence of the indicated concentrations of capsaicin. The secreted [3H]norepinephrine was measured as described in Experimental Procedures and is expressed as the percentage of total [3H]norepinephrine. Three separate experiments were done, and each point is the mean ± S.E. The results were reproducible.

PLC activation can cause [Ca²⁺]_i elevation by InsP₃-dependent Ca²⁺ release from internal stores and subsequent SOCE from the extracellular space. We tested whether capsaicin inhibits SOCE, which is responsible for the sustained level of the Ca²⁺ increase after PLC-coupled receptor activation. We triggered SOCE by treatment with 1 µM thapsigargin, which inhibits microsomal Ca²⁺-ATPase. Figure 3 demonstrates that 10 to 100 µM capsaicin markedly inhibited the thapsigargin-induced Ca²⁺ level when added during the sustained Ca²⁺ elevation. The effect was concentration-dependent, and the IC₅₀ value was 24.8 ± 2.4 µM. The thapsigargin-induced Ca²⁺ influx was confirmed by influx of Ba²⁺ and Mn²⁺ added to the extracellular space to monitor the influx of Ca²⁺ separate from the release of Ca²⁺. Capsaicin inhibited fluorescence changes induced by the influx of Ba²⁺ (Fig. 4A). Capsaicin also decreased the rate of fluorescence quenching, which indicates binding of fura-2 and Mn²⁺ (Fig. 4B). The results suggest that the target of the inhibitory action of capsaicin is Ca²⁺ influx through calcium release-activated channels. Next, we confirmed the inhibition of the thapsigargin-induced SOCE by capsaicin with SK&F96365 (1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), an antagonist of SOCE (Merritt et al., 1990). As shown in Fig. 5, 20 µM SK&F96365 decreased the thapsigargin-induced sustained Ca²⁺ elevation as did capsaicin; however, the subsequent addition of capsaicin did not change the inhibition of the sustained Ca²⁺ elevation, and vice versa.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of capsaicin on thapsigargin-induced SOCE in PC12 cells. A, fura-2-loaded cells were treated with indicated concentrations of capsaicin (Cap) after incubation with 1 µM thapsigargin (TG). Stimuli were vehicle (a), 10 µM capsaicin (b), 50 µM capsaicin (c), and 100 µM capsaicin (d). B, concentration-dependent effect of capsaicin on thapsigargin-induced SOCE. Cells were treated with various concentrations of capsaicin after incubation with 1 µM thapsigargin. The net decreases in [Ca2+]i are expressed as the percentage of control (thapsigargin-induced Ca2+ level without capsaicin treatment). Each point was obtained from triplicate experiments and is the mean ± S.E. The results were reproducible.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of capsaicin on thapsigargin-induced Ba2+ and Mn2+ influx in PC12 cells. A, fura-2-loaded cells were stimulated with 1 µM thapsigargin with or without capsaicin (Cap) in Ca2+-free medium and then 5 mM Ba2+ was added: vehicle (a), 10 µM capsaicin (b), 50 µM capsaicin (c), and 100 µM capsaicin (d). The experiments were independently carried out more than three times. The results were reproducible. B, Mn2+-induced fura-2 fluorescence quenching was recorded in fura-2/AM-loaded cells incubated with 1 mM Mn2+ and drug addition at the indicated time (arrow). Stimuli were vehicle (a), 1 µM thapsigargin with 100 µM capsaicin (b), and 1 µM thapsigargin (c). The influx of Mn2+ was measured as described in Experimental Procedures. The data are depicted as fluorescence intensity at 360 nm (F360). The presented data are representative of four independent experiments. The results were reproducible.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of SK&F96365 on inhibition of the thapsigargin-induced SOCE by capsaicin. A, fura-2-loaded PC12 cells were treated with 1 µM thapsigargin (TG) and then challenged with 100 µM capsaicin (Cap) in the presence of 20 µM SK&F96365 (SKF). B, cells were treated with 1 µM thapsigargin (TG) and then challenged with 20 µM SK&F96365 (SKF) in the presence of 100 µM capsaicin (Cap). The data are representative of more than four independent experiments. The results were reproducible.

SOCE has been well studied, and it is prominent in Jurkat-T cells, human T cell leukemia cells (Randriamampita and Tsien, 1993; Berridge, 1995). If the capsaicin-induced inhibition occurred only in PC12 cells, the inhibitory effect would not be a general phenomenon. We therefore tested the capsaicin effect on thapsigargin-sensitive SOCE in Jurkat-T cells. Figure 6 shows that capsaicin inhibited the thapsigargin-induced sustained Ca²⁺ elevation in Jurkat-T cells within a similar range of concentrations as in PC12 cells. The IC₅₀ value for Jurkat-T cells was 21.5 ± 6.9 µM, which compares well with the IC₅₀ value for PC12 cells. The results suggest that the capsaicin-mediated inhibition of thapsigargin-sensitive SOCE may be a general phenomenon and not specific to neuronal cell type.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of capsaicin on the thapsigargin-induced SOCE in Jurkat-T cells. A, fura-2-loaded Jurkat-T cells were treated with indicated concentrations of capsaicin (Cap) after incubation with 1 µM thapsigargin (TG). Stimuli were vehicle (a), 10 µM capsaicin (b), 50 µM capsaicin (c), and 100 µM capsaicin (d). B, concentration-dependent effects of capsaicin on the thapsigargin-induced SOCE in Jurkat-T cells. Cells were treated with various concentrations of capsaicin after incubation with 1 µM thapsigargin. The net decrease in [Ca2+]i is depicted as percent of the control (thapsigargin-induced Ca2+ level without capsaicin treatment). Each point was obtained from triplicate experiments and is the mean ± S.E. The results were reproducible.

We tested the capsaicin effect with regard to agonists and antagonists for vanilloid receptors. Resiniferatoxin is known as a potent vanilloid agonist from Euphorbia that is 100 times more potent than capsaicin (Winter et al., 1990). Resiniferatoxin (1 µM), which is a concentration 100 times higher than that generally used, did not exhibit any inhibitory effect on the thapsigargin-induced SOCE (Fig. 7A). It has been reported that ruthenium red and capsazepine act on the vanilloid receptor in an inhibitory manner (Amann and Maggi, 1991). Still, 10 µM ruthenium red and 30 µM capsazepine did not reverse the capsaicin-induced inhibition of the thapsigargin-induced SOCE (Fig. 7, B and C). Furthermore, the antagonist capsazepine revealed an effect similar to that of capsaicin on the thapsigargin-induced SOCE (Fig. 7C). Capsazepine inhibited SOCE induced by thapsigargin as did capsaicin. The inhibitory effect of capsazepine was concentration-dependent (IC₅₀ = 9.6 ± 0.3 µM) and about 2.5 times more potent than that of capsaicin (Fig. 8).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effects of resiniferatoxin, ruthenium red, and capsazepine on the capsaicin-evoked inhibition of thapsigargin-induced [Ca2+]i rise in PC12 cells. A, fura-2-loaded cells were treated with 1 µM resiniferatoxin (Res) after incubation with 1 µM thapsigargin (TG). B, cells were treated with 1 µM thapsigargin and then challenged with 100 µM capsaicin (Cap) in the presence of 10 µM ruthenium red (RR). C, after the pretreatment with 1 µM thapsigargin, the cells were challenged with 100 µM capsaicin in the presence (dashed trace) or absence (dotted trace) of 30 µM capsazepine (Capz). All data presented are typical Ca2+ traces of more than five separate experiments. The results were reproducible.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Capsazepine-induced inhibition of the thapsigargin-induced [Ca2+]i rise in PC12 cells. Fura-2-loaded cells were treated with indicated concentrations of capsazepine after incubation with 1 µM thapsigargin. The net decrease in [Ca2+]i is expressed as a percentage of the control (thapsigargin-induced Ca2+ level without capsazepine treatment). Each point was obtained from triplicate experiments and is the mean ± S.E. The results were reproducible.

It has been reported that phorbol ester activates protein kinase C and subsequently inhibits the SOCE (Montero et al., 1993, 1994; Petersen and Berridge, 1994; Song et al., 1998). We tested the possibility of the involvement of protein kinase C with phorbol-12-myristate-13-acetate (PMA) and GF109203X, an activator and inhibitor of protein kinase C, respectively. As shown in Fig. 9A, 3 µM PMA reduced the sustained Ca²⁺ elevation caused by thapsigargin. However, a subsequent capsaicin treatment added to the reduction in Ca²⁺, although 3 µM PMA is the maximal concentration for inhibition of thapsigargin-induced SOCE. The result suggests that the inhibitory mechanisms of PMA and capsaicin are independent of each other. In addition, pretreatment with 10 µM GF109203X did not reverse the capsaicin effect, whereas it completely blocked the inhibitory effect of PMA (Fig. 9B). Thus, we conclude that capsaicin inhibits thapsigargin-sensitive SOCE via a mechanism separate from the activation of protein kinase C. 

View larger version (16K): [in this window] [in a new window]   Fig. 9.   GF109203X effect on PMA and capsaicin-evoked inhibition of the thapsigargin-induced [Ca2+]i rise. A, fura-2-loaded PC12 cells were treated with 1 µM thapsigargin (TG) and then challenged sequentially with 3 µM PMA and 100 µM capsaicin (Cap). The trace of the effect by capsaicin only (without PMA treatment) is the negative control (dotted trace). B, after treatment with 10 µM GF109203X for 10 min, the cells were treated with 1 µM thapsigargin and then challenged with 3 µM PMA and 100 µM capsaicin. The trace of the treatment with capsaicin only (without PMA treatment) is the negative control (dotted trace). All presented data are typical Ca2+ traces of more than four separate experiments. The results were reproducible.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In this study, we demonstrate that capsaicin inhibits thapsigargin-sensitive SOCE. We detected that capsaicin inhibited extracellular ATP-induced [Ca²⁺]_i increase and norepinephrine secretion, even though capsaicin itself did not trigger any significant effects, but capsaicin slightly inhibited the peak level in the [Ca²⁺]_i rise. In addition, the same was observed in the bradykinin response. This confirms the hypothesis and suggests that capsaicin does not inhibit Ca²⁺ signaling in a nonspecific manner.

PLC-mediated signaling plays an important role in the modulation of neurotransmitter release in PC12 cells. Many neurotransmitters in neuronal cells perform their signaling via a PLC-mediated pathway. The regulation of PLC-mediated signaling is therefore very important to the understanding of neurotransmission by neuronal cells. The activation of PLC results in the production of InsP₃, the opening of the InsP₃ receptors of intracellular Ca²⁺ pools, and the subsequent Ca²⁺ release. The depletion of the Ca²⁺ stores induces Ca²⁺ influx from the extracellular space to refill the emptied Ca²⁺ stores (Putney and Bird, 1993; Fasolato et al., 1994; Berridge, 1995). It has been suggested that the depletion of the intracellular Ca²⁺ pools produces a diffusible messenger, the Ca²⁺-influx factor CIF (Randriamampita and Tsien, 1993; Parekh et al., 1995), although the nature of CIF is still obscure. Thapsigargin, an inhibitor of Ca²⁺-ATPase in the Ca²⁺ stores, is generally used to inhibit the pumping of cytosolic Ca²⁺ into the stores in various cells, including PC12 cells and Jurkat-T cells. Although the opinion that every InsP₃-sensitive store can be depleted by thapsigargin is controversial, the depletion of a thapsigargin-sensitive Ca²⁺ store is sufficient to induce SOCE.

We demonstrated that capsaicin inhibits SOCE that is activated subsequent to PLC activation and depletion of the thapsigargin-sensitive Ca²⁺ stores. The evidence is as follows: 1) capsaicin does not inhibit calcium release from internal stores in the absence of external calcium, (2) capsaicin does not inhibit extracellular ATP- or bradykinin-induced InsP₃ production, (3) capsaicin inhibits thapsigargin-induced sustained Ca²⁺ elevation, (4) capsaicin also inhibits thapsigargin-induced Ba²⁺ influx and fluorescence quenching with Mn²⁺ influx, and (5) capsaicin does not inhibit thapsigargin-sensitive SOCE in SK&F96365-treated cells.

The effect of capsaicin can be classified into two categories: vanilloid receptor-dependent and -independent effects. Generally, the effects mediated by vanilloid receptors have typical characteristics. Capsaicin works at nanomolar concentrations and opens the cation channels dominantly. These effects are inhibited by vanilloid receptor antagonists (ruthenium red, capsazepine) and activated with agonists (resiniferatoxin). Vanilloid receptors are exclusively localized in sensory neuronal cells as exemplified in the vanilloid receptor VR1, which has been cloned and exists only on dorsal root ganglia and trigeminal neurons (Caterina et al., 1997).

However, our results strongly suggest that capsaicin acts through a non-VR1-mediated pathway in PC12 cells. The suggestion is supported by the following results: 1) capsaicin shows its inhibitory effects in micromolar concentrations, which are relatively high compared with those required for the VR1-mediated responses; 2) the VR1 antagonist capsazepine exhibited a partial agonistic effect instead of an antagonistic effect; 3) another VR1 antagonist, ruthenium red, did not inhibit the capsaicin effect; and 4) the VR1 agonist resiniferatoxin did not mimic the capsaicin effect. It has been reported that capsaicin inhibits the acetylcholine- or 80 mM KCl-induced [Ca²⁺]_i rise at micromolar concentrations in PC12 cells (Nakazawa et al., 1994). In equine tracheal smooth muscle, capsaicin induced relaxation via the activation of Ca²⁺-sensitive K⁺ channels at 100 µM, which is a very high concentration compared with the EC₅₀ value of VR1 (Zhu et al., 1997). It has been reported that capsaicin in micromolar concentrations inhibits K⁺ and Ca²⁺ currents in Xenopus laevis embryo spinal neurons (Kuenzi and Dale, 1996). The major characteristics of the non-VR1-mediated responses are their high effective concentration ranges compared with the VR1-mediated response. It is unclear why such a relatively high concentration of capsaicin is required; however, it is possible that capsaicin acts on a receptor distinct from the vanilloid receptors or directly on unique, unknown target sites.

In addition, it is noteworthy that a typical antagonist for the vanilloid receptor, capsazepine, which shares structural similarity with capsaicin, shows similar effects as capsaicin in terms of the above responses. Capsazepine itself inhibits the nicotinic acetylcholine receptor in rat trigeminal ganglial cells (Liu and Simon, 1997) and voltage-sensitive Ca²⁺ channels in dorsal root ganglion neurons (Docherty et al., 1997). These results are interesting because rat trigeminal ganglial cells and dorsal root ganglion cells are already known to express VR1 receptors. As shown in Fig. 8, capsazepine exhibited a lower IC₅₀ (~10 µM) than capsaicin (25 µM) in PC12 cells. It is noticeable that this pattern is very similar to the one reported for rat trigeminal ganglial cells (Liu and Simon, 1997) and dorsal root ganglion neurons (Docherty et al., 1997). Although it is uncertain whether a novel type of capsaicin receptor exists in these cells or whether capsazepine directly acts on the target molecules, the results suggest that a non-VR1-related mechanism could be activated by capsazepine.

Although little is known about the inhibitory mechanism on SOCE, it has been reported that protein kinase C inhibits SOCE in many types of cells, including HL-60 cells (Montero et al., 1993), human neutrophils (Montero et al., 1994), and X. laevis oocytes (Petersen and Berridge, 1994). Recently, we reported that protein kinases C and A have opposing effects on thapsigargin-sensitive SOCE (Lee et al., 1997; Song et al., 1998). There is a possibility that capsaicin may penetrate the membrane, activate protein kinase C, and so inhibit thapsigargin-sensitive SOCE, but our data do not support this possibility. Even at the maximal concentration of PMA, capsaicin produced additive inhibition. Moreover, pretreatment with GF109203X reversed the inhibitory effect of PMA but could not reverse the capsaicin effect. On the other hand, it has been reported that several drugs, including neomycin (Sipma et al., 1996) and SK&F96365 (Merritt et al., 1990), can directly inhibit SOCE without activation of any cytosolic components. It is also possible that capsaicin directly interacts with membrane proteins or channels in eliciting its effect. This suggestion is supported by a recent report in which Caterina et al. (1997) demonstrated that vanilloid receptor VR1 has a domain with homology to the Drosophila Trp, although VR1 is not a rodent counterpart of Trp. Capsaicin is already known to interact with VR1, so it is possible that capsaicin acts on the homologous domain of the Ca²⁺ release-activated channel.

Since Putney and Bird (1993) introduced the original idea, there is growing awareness of SOCE. The widespread interest in SOCE stems not only from its unique mechanism of activation but also from its physiological importance, which encompasses such things as the maintenance of Ca²⁺ oscillation, refilling of the intracellular Ca²⁺ stores, and modulation of secretion. The effect of capsaicin on thapsigargin-sensitive SOCE could serve as a tool for better understanding the modulation of SOCE- and PLC-mediated neurotransmitter secretion.