Capsaicin Inhibits Phospholipase C-Mediated Ca 21 Increase by Blocking Thapsigargin-Sensitive Store-Operated Ca 21 Entry in PC12 Cells 1
SE-YOUNG CHOI and KYONG-TAI KIM
Department of Life Science, Pohang University of Science and Technology, Pohang, Republic of Korea Accepted for publication June 8, 1999 This paper is available online at http://www.jpet.org
ABSTRACT
Capsaicin has been shown to act through vanilloid receptors, which are temperature-sensitive cation channels. However, there also are indications that suggest the capsaicin effect is not mediated by the vanilloid receptor. We therefore investi- gated the effect of capsaicin on the phospholipase C-mediated Ca21rise in PC12 cells. Capsaicin caused a rapid decline in extracellular ATP- or bradykinin-induced calcium transients to the basal level without significant attenuation of the peak level.
However, capsaicin did not inhibit either ATP- or bradykinin- induced Ca21elevation in the absence of extracellular Ca21or inositol-1,4,5-trisphosphate production. Capsaicin also inhib- ited ATP-induced norepinephrine secretion. Capsaicin dramat- ically reduced the thapsigargin-induced sustained Ca21level, suggesting that capsaicin inhibits thapsigargin-sensitive store- operated Ca21entry (SOCE). Thapsigargin-induced Ba21and Mn21influx was also inhibited by capsaicin. Furthermore, cap-
saicin overlapped SK&F96365 in inhibiting thapsigargin-sensi- tive SOCE. Capsaicin-induced inhibition of SOCE also oc- curred in thapsigargin-treated Jurkat-T cells, which have a rather prominent SOCE. Resiniferatoxin, a vanilloid receptor agonist, did not mimic the effect of capsaicin. Ruthenium red and capsazepine, which are known to inhibit the vanilloid re- ceptor, did not affect this capsaicin effect. The results suggest that capsaicin does not mediate vanilloid receptor signaling when inhibiting the thapsigargin-sensitive SOCE. The capsaicin action was also not mediated by activation of protein kinase C because phorbol-12-myristate 13-acetate and capsaicin did not overlap each other’s effect and GF109203X did not reverse the inhibitory effect of capsaicin. The results suggest that cap- saicin negatively modulates thapsigargin-sensitive SOCE sub- sequent to phospholipase C activation.
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, capsa- icin 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 Ca21 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 pathologi- cal 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 recep- tors are expressed almost exclusively by primary sensory neurons involved in nociception and neurogenic inflamma- tion (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 H1 (Tominaga et al., 1998). Until now, at least two different receptors (C- and R-type) were known for
Received for publication January 11, 1999.
1This work was supported by the Korea Research Foundation and the Ministry of Science and Technology (98-J04-02-05-A-06). We are also grateful for the support from the Brain Research Program of the Ministry of Science and Technology.
ABBREVIATIONS:PLC, phospholipase C; SOCE, store-operated Ca21entry; InsP3, inositol-1,4,5-trisphosphate; fura-2/AM, fura-2 pentaace- toxymethyl ester; PMA, phorbol-12-myristate-13-acetate; SK&F96365, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydro- chloride.
THEJOURNAL OFPHARMACOLOGY ANDEXPERIMENTALTHERAPEUTICS Vol. 291, No. 1
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.
JPET 291:107–114, 1999
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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-medi- ated capsaicin effects. This hypothesis is due to the observa- tion that some capsaicin-mediated effects did not follow the typical features of vanilloid receptors, such as effective con- centration (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 stud- ied the effect of capsaicin on receptor-mediated phospho- lipase C (PLC) activation and intracellular Ca21elevation 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 non- selective 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 Ca21 in- crease by blocking the store-operated Ca21 entry (SOCE;
formerly referred to as capacitative Ca21entry) that occurs subsequent to PLC activation.
Experimental Procedures
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/pen- taacetoxymethyl 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 pur- chased 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 mg/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 humid- ified atmosphere of 95% air/5% CO2.
Measurement of [3H]Norepinephrine Secretion. Catechol- amine secretion by PC12 cells was measured by the method reported by Suh and Kim (1994). In brief, cells were loaded with [3H]norepi- nephrine (1mCi/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 mMd-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 solu- tion for 15 min to measure basal secretion. The cells were subse- quently 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% trichloro- acetic 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 [Ca21]i.[Ca21]iwas determined using the flu- orescent Ca21 indicator fura-2 as reported previously (Suh et al., 1995). Briefly, the cell suspension was incubated in Locke’s solution with 3mM fura-2/AM for 50 min at 37°C under continuous stirring.
The loaded cells were then washed twice with Locke’s solution.
Sulfinpyrazone (250mM) was added to all solutions to prevent dye leakage. For the fluorometric measurement of the [Ca21]i, 13106 cells/ml was placed into a quartz cuvette in a thermostatically con- trolled 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 [Ca21]i was performed as described by Grynkiewicz et al. (1985) using the following equation: [Ca21]i 5 KD[(R 2 Rmin)/(Rmax 2 R)](Sf2/Sb2), whereRis the ratio of fluorescence emitted after exci- tation at 340 and 380 nm, andSf2andSb2are the proportionality coefficients at 380 nm excitation of Ca21-free fura-2 and Ca21-satu- rated fura-2, respectively. To obtainRmin, the fluorescence ratios of the cell suspension were measured successively at a final concentra- tion of 4 mM EGTA, 30 mM Trizma base, and 0.1% Triton X-100.
Then, the cell suspension was treated with CaCl2to a final concen- tration of 4 mM Ca21, which is sufficient to saturate fura-2 with Ca21under these conditions, and the fluorescence ratios were mea- sured to obtainRmax.
Mn21Quenching of fura-2 Fluorescence. The Mn21quench assay was performed as described by Lee et al. (1997) to measure the influx of Ca21 from the extracellular space. Briefly, as described above; fura-2-loaded cells (53106cells/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 Ca21does not affect fura-2 fluores- cence and at which, therefore, any changes are caused by Mn21 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 MnCl2and the drugs to be tested.
Measurement of InsP3 Production. InsP3 mobilization was determined by competition assay using [3H]InsP3as described pre- viously (Lee et al., 1997). To determine InsP3production, 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% trichlo- roacetic 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 ml of the extract was added to 20ml of assay buffer (0.1 M Tris buffer contain- ing 4 mM EDTA) and 20ml of [3H]InsP3(100 nCi/ml). The mixture was incubated for 15 min on ice and then centrifuged at 2000gfor 10 min. Next, 100ml 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 Brad- ford method after sonication of cells.
Analysis of Data.All quantitative data are expressed as mean6 S.E. We calculated EC50and IC50values with the Microcal Origin for Windows program. Differences were determined by one-way ANOVA and considered to be significant only forP,.05.
Results
We studied the effect of capsaicin on PLC-mediated [Ca21]i
increase and norepinephrine secretion in PC12 cells. Figure 1A shows that 50 mM capsaicin markedly reduced the sus- tained [Ca21]ilevel elevated by extracellular ATP, whereas it had less of an effect on the peak [Ca21]ilevel. We had reason to assume that the difference in the inhibitory effect of cap-
saicin on the peak and the sustained [Ca21]ilevel might be due to differences in [Ca21]i-elevating pathways. Cytosolic Ca21increase triggered by the PLC pathway could be distin- guished as Ca21release from the intracellular Ca21reservoir and Ca21influx from the extracellular space. In the absence of extracellular Ca21, the ATP-induced [Ca21]irise was not inhibited by capsaicin (data not shown), suggesting that cap- saicin does not exert an inhibitory effect on the ATP-induced Ca21 release from Ca21 stores in the cell. The data also suggest that capsaicin has an inhibitory effect on the ATP- induced Ca21influx 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-medi- ated signaling, because the PC12 cells were cultured in a medium containing streptomycin (;34mM). We tested cap- saicin 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 re-
ceptor, we tested whether capsaicin had an effect on re- sponses to bradykinin, which is known to activate PLC in PC12 cells (Fig. 1B). Capsaicin inhibited the bradykinin- induced Ca21elevation without significant attenuation of the peak level, but capsaicin did not inhibit the bradykinin- induced [Ca21]irise in the absence of extracellular Ca21, as was the case for the extracellular ATP stimulation. Further- more, we tested the effect of capsaicin on the production of InsP3to determine whether capsaicin directly inhibited PLC.
As seen in Fig. 1C, there were no statistically significant differences in the extracellular ATP- or bradykinin-induced InsP3production in the presence or absence of capsaicin (P. .05). The result shows that capsaicin inhibited neither extra- cellular ATP- nor bradykinin-induced InsP3production. 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 [Ca21]i. Fig- ure 2 shows that capsaicin inhibited the extracellular ATP- induced norepinephrine secretion. The inhibition shows a concentration-dependent manner with an IC50 value of 42.967.7mM.
PLC activation can cause [Ca21]ielevation by InsP3-depen- dent Ca21release 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 Ca21increase after PLC-coupled receptor activation. We triggered SOCE by treatment with 1mM thapsigargin, which inhibits microsomal Ca21-ATPase. Figure 3 demonstrates that 10 to 100mM capsaicin markedly inhibited the thapsi- gargin-induced Ca21level when added during the sustained Ca21elevation. The effect was concentration-dependent, and the IC50value was 24.862.4mM. The thapsigargin-induced Ca21influx was confirmed by influx of Ba21and Mn21added to the extracellular space to monitor the influx of Ca21sep- arate from the release of Ca21. Capsaicin inhibited fluores- cence changes induced by the influx of Ba21(Fig. 4A). Cap- Fig. 1.Effect of capsaicin on ATP- and bradykinin-induced [Ca21]irise
and InsP3production in PC12 cells. Fura-2-loaded cells were challenged with 300mM ATP (A) or 3mM bradykinin (BK; B) in the presence (b) or absence (a) of 50mM capsaicin (Cap). Typical Ca21traces from more than three separate experiments are presented. The results were reproducible.
C, capsaicin effect on InsP3production was monitored in PC12 cells. Cells were preincubated with (hatched column) or without (open column) 50 mM capsaicin for 3 min and then treated with 300mM ATP or 3mM bradykinin for 15 s. The production of InsP3was measured as described inExperimental Procedures. Each result is the mean6S.E. of triplicate assays. The experiments were performed three times independently. and the results were reproducible.
Fig. 2.Effect of capsaicin on [3H]norepinephrine secretion and its inhib- itory effect on extracellular ATP-induced [3H]norepinephrine secretion by PC12 cells. [3H]Norepinephrine-loaded PC12 cells were treated with 300 mM 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 mean6S.E. The results were reproducible.
Capsaicin Inhibits Store-Operated Ca Entry 109
saicin also decreased the rate of fluorescence quenching, which indicates binding of fura-2 and Mn21(Fig. 4B). The results suggest that the target of the inhibitory action of capsaicin is Ca21 influx through calcium release-activated channels. Next, we confirmed the inhibition of the thapsigar- gin-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, 20mM SK&F96365 decreased the thap- sigargin-induced sustained Ca21elevation as did capsaicin;
however, the subsequent addition of capsaicin did not change the inhibition of the sustained Ca21elevation, and vice versa.
SOCE has been well studied, and it is prominent in Jur- kat-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 thapsigar- gin-induced sustained Ca21 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 6.9 mM, which
compares well with the IC50value for PC12 cells. The results suggest that the capsaicin-mediated inhibition of thapsigar- gin-sensitive SOCE may be a general phenomenon and not specific to neuronal cell type.
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). Res- iniferatoxin (1 mM), which is a concentration 100 times higher than that generally used, did not exhibit any inhibi- tory 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, 10mM ruthenium red and 30mM capsaz- epine 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 concen- tration-dependent (IC5059.660.3mM) and about 2.5 times more potent than that of capsaicin (Fig. 8).
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 Fig. 3.Effect of capsaicin on thapsigargin-induced SOCE in PC12 cells.
A, fura-2-loaded cells were treated with indicated concentrations of cap- saicin (Cap) after incubation with 1mM thapsigargin (TG). Stimuli were vehicle (a), 10mM capsaicin (b), 50mM capsaicin (c), and 100mM capsa- icin (d). B, concentration-dependent effect of capsaicin on thapsigargin- induced SOCE. Cells were treated with various concentrations of capsa- icin after incubation with 1mM thapsigargin. The net decreases in [Ca21]i
are expressed as the percentage of control (thapsigargin-induced Ca21 level without capsaicin treatment). Each point was obtained from tripli- cate experiments and is the mean6S.E. The results were reproducible.
Fig. 4.Effect of capsaicin on thapsigargin-induced Ba21and Mn21influx in PC12 cells. A, fura-2-loaded cells were stimulated with 1mM thapsi- gargin with or without capsaicin (Cap) in Ca21-free medium and then 5 mM Ba21was added: vehicle (a), 10mM capsaicin (b), 50mM capsaicin (c), and 100mM capsaicin (d). The experiments were independently carried out more than three times. The results were reproducible. B, Mn21- induced fura-2 fluorescence quenching was recorded in fura-2/AM-loaded cells incubated with 1 mM Mn21and drug addition at the indicated time (arrow). Stimuli were vehicle (a), 1mM thapsigargin with 100mM cap- saicin (b), and 1mM thapsigargin (c). The influx of Mn21was measured as described inExperimental Procedures. The data are depicted as fluo- rescence intensity at 360 nm (F360). The presented data are representa- tive of four independent experiments. The results were reproducible.
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 mM PMA reduced the sustained Ca21elevation caused by thapsigargin. However, a subsequent capsaicin treatment added to the reduction in Ca21, although 3mM PMA is the maximal concentration for inhibition of thapsigargin-induced SOCE. The result sug- gests that the inhibitory mechanisms of PMA and capsaicin are independent of each other. In addition, pretreatment with 10mM GF109203X did not reverse the capsaicin effect, whereas it completely blocked the inhibitory effect of PMA (Fig. 9B). Thus, we conclude that capsaicin inhibits thapsi- gargin-sensitive SOCE via a mechanism separate from the activation of protein kinase C.
Discussion
In this study, we demonstrate that capsaicin inhibits thap- sigargin-sensitive SOCE. We detected that capsaicin inhib- ited extracellular ATP-induced [Ca21]iincrease and norepi- nephrine secretion, even though capsaicin itself did not trigger any significant effects, but capsaicin slightly inhib- ited the peak level in the [Ca21]irise. In addition, the same was observed in the bradykinin response. This confirms the hypothesis and suggests that capsaicin does not inhibit Ca21 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-medi- ated signaling is therefore very important to the understand- ing of neurotransmission by neuronal cells. The activation of PLC results in the production of InsP3, the opening of the InsP3 receptors of intracellular Ca21 pools, and the subse- quent Ca21release. The depletion of the Ca21stores induces Ca21influx from the extracellular space to refill the emptied Ca21 stores (Putney and Bird, 1993; Fasolato et al., 1994;
Berridge, 1995). It has been suggested that the depletion of the intracellular Ca21pools produces a diffusible messenger, the Ca21-influx factor CIF (Randriamampita and Tsien, 1993; Parekh et al., 1995), although the nature of CIF is still obscure. Thapsigargin, an inhibitor of Ca21-ATPase in the Ca21 stores, is generally used to inhibit the pumping of cytosolic Ca21into 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 con- troversial, the depletion of a thapsigargin-sensitive Ca21 store is sufficient to induce SOCE.
We demonstrated that capsaicin inhibits SOCE that is activated subsequent to PLC activation and depletion of the Fig. 5.Effect of SK&F96365 on inhibition of the thapsigargin-induced
SOCE by capsaicin. A, fura-2-loaded PC12 cells were treated with 1mM thapsigargin (TG) and then challenged with 100mM capsaicin (Cap) in the presence of 20mM SK&F96365 (SKF). B, cells were treated with 1mM thapsigargin (TG) and then challenged with 20mM SK&F96365 (SKF) in the presence of 100mM capsaicin (Cap). The data are representative of more than four independent experiments. The results were reproducible.
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 concen- trations of capsaicin (Cap) after incubation with 1mM thapsigargin (TG).
Stimuli were vehicle (a), 10mM capsaicin (b), 50mM capsaicin (c), and 100 mM 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 1mM thapsi- gargin. The net decrease in [Ca21]iis depicted as percent of the control (thapsigargin-induced Ca21 level without capsaicin treatment). Each point was obtained from triplicate experiments and is the mean6S.E.
The results were reproducible.
Capsaicin Inhibits Store-Operated Ca Entry 111
thapsigargin-sensitive Ca21 stores. The evidence is as fol- lows: 1) capsaicin does not inhibit calcium release from in- ternal stores in the absence of external calcium, (2) capsaicin does not inhibit extracellular ATP- or bradykinin-induced InsP3production, (3) capsaicin inhibits thapsigargin-induced sustained Ca21elevation, (4) capsaicin also inhibits thapsi- gargin-induced Ba21influx and fluorescence quenching with Mn21influx, 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. Gen- erally, the effects mediated by vanilloid receptors have typi- cal characteristics. Capsaicin works at nanomolar concentra- tions and opens the cation channels dominantly. These effects are inhibited by vanilloid receptor antagonists (ruthe- nium red, capsazepine) and activated with agonists (resinif- eratoxin). Vanilloid receptors are exclusively localized in sen- sory 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 Fig. 7.Effects of resiniferatoxin, ruthenium red, and capsazepine on the
capsaicin-evoked inhibition of thapsigargin-induced [Ca21]irise in PC12 cells. A, fura-2-loaded cells were treated with 1mM resiniferatoxin (Res) after incubation with 1mM thapsigargin (TG). B, cells were treated with 1mM thapsigargin and then challenged with 100mM capsaicin (Cap) in the presence of 10mM ruthenium red (RR). C, after the pretreatment with 1mM thapsigargin, the cells were challenged with 100mM capsaicin in the presence (dashed trace) or absence (dotted trace) of 30mM capsaz- epine (Capz). All data presented are typical Ca21traces of more than five separate experiments. The results were reproducible.
Fig. 8. Capsazepine-induced inhibition of the thapsigargin-induced [Ca21]irise in PC12 cells. Fura-2-loaded cells were treated with indicated concentrations of capsazepine after incubation with 1mM thapsigargin.
The net decrease in [Ca21]iis expressed as a percentage of the control (thapsigargin-induced Ca21level without capsazepine treatment). Each point was obtained from triplicate experiments and is the mean6S.E.
The results were reproducible.
Fig. 9.GF109203X effect on PMA and capsaicin-evoked inhibition of the thapsigargin-induced [Ca21]i rise. A, fura-2-loaded PC12 cells were treated with 1mM thapsigargin (TG) and then challenged sequentially with 3mM PMA and 100mM capsaicin (Cap). The trace of the effect by capsaicin only (without PMA treatment) is the negative control (dotted trace). B, after treatment with 10mM GF109203X for 10 min, the cells were treated with 1mM thapsigargin and then challenged with 3mM PMA and 100mM capsaicin. The trace of the treatment with capsaicin only (without PMA treatment) is the negative control (dotted trace). All presented data are typical Ca21traces of more than four separate exper- iments. The results were reproducible.
the VR1-mediated responses; 2) the VR1 antagonist capsaz- epine exhibited a partial agonistic effect instead of an antag- onistic effect; 3) another VR1 antagonist, ruthenium red, did not inhibit the capsaicin effect; and 4) the VR1 agonist res- iniferatoxin did not mimic the capsaicin effect. It has been reported that capsaicin inhibits the acetylcholine- or 80 mM KCl-induced [Ca21]i rise at micromolar concentrations in PC12 cells (Nakazawa et al., 1994). In equine tracheal smooth muscle, capsaicin induced relaxation via the activa- tion of Ca21-sensitive K1channels at 100mM, 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 K1and Ca21currents 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 com- pared 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 dis- tinct from the vanilloid receptors or directly on unique, un- known 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 Ca21chan- nels 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 ex- hibited a lower IC50 (;10 mM) than capsaicin (25 mM) 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 capsaz- epine directly acts on the target molecules, the results sug- gest 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. laevisoocytes (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 thapsi- gargin-sensitive SOCE, but our data do not support this possibility. Even at the maximal concentration of PMA, cap- saicin 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 inter- acts 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 theDrosophila
Trp, although VR1 is not a rodent counterpart of Trp. Cap- saicin is already known to interact with VR1, so it is possible that capsaicin acts on the homologous domain of the Ca21 release-activated channel.
Since Putney and Bird (1993) introduced the original idea, there is growing awareness of SOCE. The widespread inter- est 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 Ca21oscil- lation, refilling of the intracellular Ca21stores, and modula- tion 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 neurotransmit- ter secretion.
Acknowledgments
We thank S. J. Kim for her helpful technical assistance and G.
Hoschek for editing this manuscript.
References
Acs G, Biro T, Acs P, Modarres S and Blumberg PM (1997) Differential activation and desensitization of sensory neurons by resiniferatoxin.J Neurosci17:5622–
5628.
Amann R and Maggi CA (1991) Ruthenium red as a capsaicin antagonist.Life Sci 49:849 – 856.
Berridge MJ (1995) Capacitative Ca21entry.Biochem J312:1–11.
Bevan S and Szolcsanyi J (1990) Sensory neuron-specific actions of capsaicin: Mech- anisms and applications.Trends Pharmacol Sci11:330 –333.
Bian JS, Zhang WM, Xia Q and Wong TM (1998) Phospholipase C inhibitors atten- uate arrhythmias induced by kappa-receptor stimulation in the isolated rat heart.
J Mol Cell Cardiol30:2103–2110.
Biro T, Maurer M, Modarres S, Lewin NE, Brodie C, Acs G, Acs P, Paus R and Blumberg PM (1998) Characterization of functional vanilloid receptors expressed by mast cells.Blood91:1332–1340.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD and Julius D (1997) The capsaicin receptor: A heat-activated ion channel in the pain pathway.
Nature (Lond)389:816 – 824.
Docherty RJ, Yeats JC and Piper AS (1997) Capsazepine block of voltage-activated calcium channels in adult rat dorsal root ganglion neurones in culture.Br J Pharmacol121:1461–1467.
Dray A (1992) Neuropharmacological mechanisms of capsaicin and related sub- stances.Biochem Pharmacol44:611– 615.
Gergawy M, Vollrath B and Cook D (1998) The mechanism by which aminoglycoside antibiotics cause vasodilation of canine cerebral arteries.Br J Pharmacol125:
1150 –1157.
Grynkiewicz G, Poenie M and Tsien RY (1985) A new generation of Ca21indicators with greatly improved fluorescence properties.J Biol Chem260:3440 –3450.
Janusz JM, Buckwalter BL, Young PA, LaHann TR, Farmer RW, Kasting GB, Loomans ME, Kerckaert GA, Maddin CS, Berman EF, Bohne RL, Cupps TL and Milstein JR (1993) Vanilloids. 1. Analogs of capsaicin with antinociceptive and antiinflammatory activity.J Med Chem36:2595–2604.
Kuenzi FM and Dale N (1996) Effect of capsaicin and analogues on potassium and calcium currents and vanilloid receptors inXenopusembryo spinal neurones.Br J Pharmacol119:81–90.
Lee H, Suh BC and Kim KT (1997) Feedback regulation of ATP-induced Ca21 signaling in HL-60 cells is mediated by protein kinase A- and C-mediated changes in capacitative Ca21entry.J Biol Chem272:21831–21838.
Liu L and Simon SA (1994) A rapid capsaicin-activated current in rat trigeminal ganglion neurons.Proc Natl Acad Sci USA91:738 –741.
Liu L and Simon SA (1996) Similarities and differences in the currents activated by capsaicin, piperine, and zingerone in rat trigeminal ganglion cells.J Neurophysiol 76:1858 –1869.
Liu L and Simon SA (1997) Capsazepine, a vanilloid receptor antagonist, inhibits nicotinic acetylcholine receptors in rat trigeminal ganglia.Neurosci Lett228:29 – 32.
Marsh SJ, Stansfeld CE, Brown DA, Davey R and McCarthy D (1987) The mecha- nism of action of capsaicin on sensory C-type neurons and their axonsin vitro.
Neuroscience23:275–289.
Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh BK, McCarthy SA, Moores KE and Rink TJ (1990) SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.Biochem J271:515–522.
Montero M, Garcia-Sancho J and Alvalez J (1993) Inhibition of the calcium store- operated calcium entry pathway by chemotactic peptide and by phorbol ester develops gradually and independently along differentiation of HL60 cells.J Biol Chem268:26911–26919.
Montero M, Garcia-Sancho J and Alvalez J (1994) Phosphorylation down-regulates the store-operated Ca21entry pathway of human neutrophils.J Biol Chem269:
3963–3967.
Nakazawa K, Inoue K, Koizumi S, Ikeda M and Inoue K (1994) Inhibitory effects of
Capsaicin Inhibits Store-Operated Ca Entry 113
capsaicin on acetylcholine-evoked responses in rat phaeochromocytoma cells.Br J Pharmacol113:296 –302.
Park TJ, Song SK and Kim KT (1997) A2Aadenosine receptors inhibits ATP-induced Ca21influx in PC12 cells by involving protein kinase A.J Neurochem68:2177–2185.
Petersen CCH and Berridge MJ (1994) The regulation of capacitative Ca21entry by Ca21and PKC inXenopusoocytes.J Biol Chem269:32246 –32253.
Putney JW Jr and Bird GStJ (1993) The signal for capacitative Ca21entry.Cell 75:199 –201.
Randriamampita C and Tsien RY (1993) Emptying of intracellular Ca21stores releases a novel small messenger that stimulates Ca21influx.Nature (Lond)364:809 – 814.
Schwertz DW, Kreisberg JI and Venkatachalam MA (1984) Effects of aminoglyco- sides on proximal tubule brush border membrane phosphatidylinositol-specific phospholipase C.J Pharmacol Exp Ther231:48 –55.
Sipma H, Zee LV, Hertog AD and Nelemans A (1996) Neomycin inhibits histamine and thapsigargin mediated Ca21entry in DDT1MF-2 cells independent of phos- pholipase C activation.Eur J Pharmacol305:207–212.
Song SK, Choi SY and Kim KT (1998) Opposing effects of protein kinase A and C on capacitative calcium entry into HL-60 promyelocytes.Biochem Pharmacol56:561–567.
Suh BC and Kim KT (1994) Inhibition by ethaverine of catecholamine secretion through blocking of L-type Ca21channel in PC12 cells.Biochem Pharmacol47:
1262–1266.
Suh BC, Lee CO and Kim KT (1995) Signal flows from two phospholipase C-linked receptors are independent in PC12 cells.J Neurochem64:1071–1079.
Szallasi A and Blumberg PM (1996) Vanilloid receptors: New insights enhance potential as a therapeutic target.Pain68:195–208.
Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Rau- mann BE, Basbaum AI and Julius D (1998) The cloned capsaicin receptor inte- grates multiple pain-producing stimuli.Neuron21:531–543.
Winter J, Dray A, Wood JN, Yeats JC and Bevan S (1990) Cellular mechanism of action of resiniferatoxin: A potent sensory neuron excitotoxin.Brain Res520:131–
140.
Wood JN, Winter J, James IF, Rang HP, Yeats J and Bevan S (1988) Capsaicin- induced ion fluxes in dorsal root ganglion cells in culture.J Neurosci8:3208 –3220.
Zhu FX, Zhang XY, Olszewski MA and Robinson NE (1997) Mechanism of capsaicin- induced relaxation in equine tracheal smooth muscle.Am J Physiol273:L997–
L1001.
Send reprint requests to:Kyong-Tai Kim, Ph.D., Department of Life Sci- ence, POSTECH, San 31, Hyoja Dong, Pohang, 790-784, Republic of Korea.
E-mail: [email protected]
