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Am J Physiol Cell Physiol 291: C424-C432, 2006. First published April 5, 2006; doi:10.1152/ajpcell.00553.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Thermosensitive TRP ion channels mediate cytosolic calcium response in human synoviocytes

Mikhail Y. Kochukov,1 Terry A. McNearney,1,2,3 Yibing Fu,1 and Karin N. Westlund1

Departments of 1Neuroscience and Cell Biology, 2Internal Medicine, and 3Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas

Submitted 28 October 2005 ; accepted in final form 17 March 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transient receptor potential (TRP) channels are important membrane sensors, responding to thermal, chemical, osmotic, or mechanical stimuli by activation of calcium and sodium fluxes. In this study, three distinct TRP channels were detected and their role established in mediating cytosolic free calcium concentration ([Ca2+]cyt) response in tumor-derived SW982 synoviocytes and primary cultures of human synovial cells from patients with inflammatory arthropathies. As shown by fura-2 ratio measurements while cells were incubated in a temperature-regulated chamber, significant [Ca2+]cyt elevation was elicited by rapid changes in bath temperature, application of TRPV1 receptor agonists capsaicin and resiniferatoxin, or a cold receptor stimulator, icilin. Temperature thresholds for calcium response were determined to be 12 ± 1°C for cold and 28 ± 2°C for heat activation. Temperature increases or decreases beyond these thresholds resulted in a significant rise in the magnitude of [Ca2+]cyt spikes. Observed changes in [Ca2+]cyt were completely abolished in calcium-free medium and thus resulted from direct calcium entry through TRP channels rather then by activation of voltage-dependent calcium channels. Two heat sensitive channels, TRPV1 and TRPV4, and a cold-sensitive channel, TRPA1, were detected by RT-PCR. Minimal mRNA for TRPV3 or TRPM8 was amplified. The RT-PCR results support the data obtained with the [Ca2+]cyt measurements. We propose that the TRP channels are functionally expressed in human synoviocytes and may play a critical role in adaptive or pathological changes in articular surfaces during arthritic inflammation.

transient receptor potential channels; vanilloid receptors; arthritis

CELLS IN SYNOVIAL COMPARTMENTS can be exposed to low pH conditions after inflammation, infection, or injury. An acid-sensing G protein-coupled receptor has been identified on synovial cells that are responsive to low pH (pH 5.5–7.0) in calcium-free media except in the presence of Cu2+ (6). A response profile in low pH (pH 7.0–7.2) in the presence of Cu2+ was also noted, consistent with transient receptor potential (TRP)V1 activation. The present study was designed to characterize TRP receptors on target human synovial cells. The presence of TRP receptors on target peripheral tissue would be an adaptive mechanism for activating intracellular responses outside the physiological range.

The TRP ion channels are important membrane sensors, responding to thermal, chemical, osmotic, or mechanical stimuli by activation of calcium and sodium fluxes. Currently, the mammalian TRP family consists of 28 unique channels, in 6 main subfamilies (43). Recent studies demonstrated that several members of TRP V, M, and A subfamilies act as the thermal sensation receptors, responding to moderate or noxious changes in the external temperature. These channels are called "heat" or "cold" receptors, depending on the temperature range required for their activation. Heat receptors include TRPV3 and TRPV4, activated by "warm" temperature (34–38 and 27–35°C, respectively) and "noxious heat" receptors TRPV1 and TRPV2, with a thermal activation threshold as high as 43 and 52°C, respectively (4, 5, 14, 20, 30, 36, 43, 50). The activation temperatures for two known cold receptors, TRPA1 and TRPM8, are 17 and 25–28°C, respectively (3, 19, 20, 23, 24, 30, 31, 40, 43, 50). Many of the thermoreceptor channels display significant ligand promiscuity and can be activated by additional modalities, such as hypotonicity and mechanical stretch [TRPV2 (28), TRPV4 (11, 28, 41)], extracellular acidification [TRPV1, TRPV4 (41)], numerous exo- and endogenous chemical ligands [TRPV1: vanilloids and cannabinoids (35, 51); TRPV4: arachidonic acid metabolites (47) and menthol, icilin, and bradykinin for cold receptors (2, 3, 19, 23, 31, 40)]. The polymodality of activation signals promotes an expansion in the TRP repertoire to potentiate activation in pain sensation, inflammatory response, or cellular/tissue adaptation to a variety of external stressors (29, 43, 45). Studies report a wide expression and activation of TRP channels on nonneuronal tissues that might further direct or accelerate the cellular response to physicochemical changes in the extracellular environment (38, 43, 45).

Recent studies have suggested that neuronally expressed TRPV1 is involved in the chronic inflammation and pain associated with arthritis (1, 16, 42). Synovial fibroblasts are key cells in the joint synovial tissue, pivotal in both joint maintenance and integrity and in the inflammatory response of arthritis (27, 33). In the present study we demonstrate the functional expression of four thermosensitive TRP channels in established and primary synoviocyte cultures (TRPV1, TRPV4, TRPM8, and TRPA1). We used fura-2 cytosolic free calcium concentration ([Ca2+]cyt) measurement to demonstrate the direct involvement of TRP channels in human synoviocyte calcium response elicited by temperature changes and agonist stimulation.


   EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell lines and cultures. The SW982 cells derived from a human synovial sarcoma are fibroblast-like synovial cells (12) obtained through the American Type Culture Collection (Bethesda, MD). The cells were maintained in Leibovitz's L-15 medium (GIBCO, Grand Island, NY), 10% heated fetal bovine serum (FBS, Gemini Bio-Products, Woodland, CA), 2 mM L-glutamine, 1,000 U/ml penicillin G (GIBCO), and 1,000 pg/ml streptomycin (GIBCO). Cells were incubated in a humidified cell incubator (37°C, ambient CO2) and used in passages 4–21. Primary cultures of surface-adherent synoviocytes were established from synovial fluid (15, 25) derived from the discarded sample of a clinical arthrocentesis procedure performed on patients with acute pseudogout or rheumatoid arthritis, based on American College of Rheumatology criteria (21) and in accordance with the guidelines and approved protocol of the University Institutional Review Board. Cells were maintained in DMEM, 10% heated FBS, 2 mM L-glutamine, and penicillin-streptomycin. Primary cultures were incubated in a humidified cell incubator (37°C with 5% CO2 atmosphere) for 2 wk before testing. Cells were split with 0.25% trypsin-0.02% EDTA disruption and plated on 15-mm circular quartz glass coverslips at a density of 200,000 cells/mm3.

Immunofluorescent staining of TRPV1 was performed with a rabbit polyclonal TRPV1 antibody (1:1,000; Neuromics, Northfield, MN) and a secondary anti-rabbit tagged with Texas red (Molecular Probes, Eugene, OR). Negative control stains included deletion of the primary antibody or normal rabbit serum (Sigma, St. Louis, MO) at the same dilution. A nuclear counterstain was applied in the coverslip medium (Vectashield Hardset Mounting medium with DAPI; Vector Labs, Burlingame, CA). Fluorescent images were acquired with a Nikon Eclipse E1000 microscope linked to a Metaview Imaging System.

Solutions and recording conditions. In the 24–48 h before experiments, cells were harvested and plated onto 15-mm quartz glass coverslips. A small coverslip chip containing cells was placed in the temperature-regulated chamber (0.8-ml volume), which was continuously perfused by gravity flow at a rate of 2 ml/min with solution prewarmed or cooled to the desired temperature. Standard bath solution consisted of (in mM) 150 NaCl, 5.5 KCl, 1 MgCl2, 4 CaCl2, 5 glucose, and 10 HEPES, adjusted to pH 7.4 at a room temperature with NaOH and an osmolarity of 330 mosM. For experiments in which cells were exposed to rapid changes in external solution temperature, the pH of the bath saline was adjusted to 7.4 at the desired temperature (5–46°C). Strict temperature control and rapid (1–2 °C/s) changes were achieved with a CL-100 bipolar temperature controller equipped with a SC-20 inline solution heater/cooler (Warner Instruments, Hamden, CT). Application of chemicals onto individual cells was achieved by gravity flow from a large-bore pipette placed 300–400 µm from the cell. Experiments were repeated a minimum of four times each.

Cytosolic free calcium measurement. On the day of the experiment, the cells were loaded with the calcium-sensitive fluorescent dye fura-2 (2 µM for 1 h at a room temperature), after which they were washed in a physiological bath solution and allowed to stay for 30 min to 2 h at room temperature before recording. The chamber with the coverslip was placed on the stage of a Nikon Diaphot microscope, and the cells were viewed with an x60 water immersion lens. Fura-2-loaded cells were excited at 340 and 380 nm with a Polychrome II monochromator (Till Photonics, Munich, Germany) controlled by X-chart software (HEKA, Heidelberg, Germany) and with an ITC-18 computer interface (Instrutech, Port Washington, NY). The resulting 510-nm emissions were detected with a Hamamatsu R928 photomultiplier. The 340-to-380 ratios were acquired from single cells, or occasionally two or three neighboring cells, every 200 ms. Ratios were converted into [Ca2+]cyt with the formula (13) [Ca2+] = Kd[(R – Rmin)/(Rmax – R)](Sf2/Sb2), in which fluorescence ratios at zero free Ca2+ (Rmin) and saturation free Ca2+ (Rmax), as well as fluorescence intensity of Ca2+-free (Sf2) and Ca2+-bound (Sb2) dye, excited at 380 nm, were measured experimentally and fura-2 Kd values for Ca2+ at different temperatures were taken from Shuttleworth and Thompson estimations (34). Igor Pro (WaveMetrics, Lake Oswego, OR) and Sigma Plot scientific software (SPSS, Chicago, IL) were used for conversion and analysis of acquired data. Data are reported as means ± SE.

PCR primer design. Primers were designed based on the sequence for the receptors obtained from the National Center for Biotechnology Information GenBank. Primers designed from published sequences of human origin were synthesized by Sigma-Genosys (The Woodlands, TX). Areas to be amplified were based on unique regions for the given receptor: TRPV1 accession no. NM_080706, TRPV1 5'-CTCCTACAACAGCCTGTAC (nt 2405), TRPV1 3'-AAGGCCTTCCTCATGCACT (nt 2689); TRPV3 accession no. NM_145068, TRPV3 5'-TCGAGGAATTCCCGGAAACCT (nt 2667), TRPV3 3'-AGTCACAGCAGAAGAGATGGT (nt 3141); TRPV4 accession no. NM_021625, TRPV4 5'-AACTGAACAAGAACTCGAACCCG (nt 2581), TRPV4 3'-ATGCAGCTCAGGCGCAGGCGT (nt 3107); TRPM8 accession no. NM_024080, TRPM8 5'-TGAAGCTTCTGCTGGAGTGGAA (nt 1319), TRPM8 3'-AGTCTTCAGAAGCTTGCTGGCT (nt 1840); and TRPA1 accession no. AY403101, TRPA1 5'-TGGTGCACAAATAGACCCAGT, TRPA1 (nt 783), TRPA1 3'-TGGGCACCTTTAGAGAGTAGC (nt 1100). Human brain mRNA (Ambion, catalog no. 7963) was used as a second tissue source of human origin to serve as a positive control for the primers. The RT-PCR experiments were repeated three times.

Synoviocyte mRNA isolation, RT-PCR, and nucleotide sequencing. Total mRNA was extracted from low-passage SW982 synovial cells grown to confluence in T-75 flasks with the manufacturer's reagents and protocol (RNAqueous 4PCR, Ambion, Austin, TX). RT-PCR was set up according to the following protocol with Invitrogen Platinum Taq polymerase after superscript first-strand synthesis (Invitrogen, Carlsbad, CA); 0.18 µg of mRNA was used per tube for the initial reaction per the manufacturer's protocol. For PCR reactions double-strand cDNA for PCR were synthesized from the purified mRNA. The protocol provided by the company was followed except for the use of a final concentration of 2% dimethyl sulfoxide. Final concentration of the primers was 0.2 µM. The PCR cycle protocol was as follows: 94 x 60 s, 94 x 15 s, 60 x 20 s, and 72 x 45 s, in succession for 35 cycles of amplification. In addition to the cycling reactions, extension at 72 for 5 min was also performed with a thermocycler (GeneAmp PCR system 2400, Perkin Elmer, Wellesley, MA). The DNAs amplified were assessed with 1% agarose gel electrophoresis. For primer controls, independent reactions were set up in parallel without templates. In addition, human brain tissue mRNA extract (Ambion, catalog no. 7963) was used as a second human tissue source in independent experiments. For nucleotide sequencing the cDNA bands were isolated from low-melt agarose gels and sequenced by the dideoxy chain termination method in an automated DNA autosequencer in the University of Texas Medical Branch Sequencing Core Laboratory.

Chemicals. Thapsigargin was obtained from Calbiochem (La Jolla, CA). Fura-2 AM was obtained from Molecular Probes, and L-menthol was obtained from Aldrich (Milwaukee, WI). Capsaicin, capsazepine, ionomycin, and nifedipine were obtained from Tocris (Ellisville, MO).


   RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
[Ca2+]cyt response in synoviocytes is elicited by thermal stimuli. To test whether synovial cells express functional thermosensitive ion channels, the response of synoviocytes to rapid changes in bath solution temperature was examined by continuous measurement of [Ca2+]cyt. SW982 cells were maintained in a continuously perfused chamber at a baseline temperature of 20°C. The cells were repeatedly exposed to rapid, short (30–60 s) changes in bath solution temperatures, in ranges of heating to 24–46°C or cooling to 5–16 °C. All monitored cells responded to thermal stimuli by rapid, nondelayed [Ca2+]cyt elevation and typically returned to baseline on temperature reversal (Fig. 1, A and B). Temperature thresholds for calcium responses were determined to be 12 ± 1°C (n = 10) for cold and 28 ± 2°C (n = 9) for heat activation. Temperature increases or decreases, respectively, beyond these thresholds resulted in a significant rise in the magnitude of [Ca2+]cyt spikes (Fig. 1D). Changing the baseline chamber temperature within a range of 16–24°C had no impact on the thermal activation threshold and did not result in significant changes in calcium spike amplitude. Prolonged exposure (2–5 min) to heat or cold stimuli resulted in a sustained elevation of [Ca2+]cyt of the cells, with only a slight decrease toward baseline values that was time dependent (Fig. 1C).


Figure 1
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  		Fig. 1. Increase in cytosolic free calcium concentration ([Ca2+]cyt) in SW982 synoviocytes caused by heat and cold stimuli. A: rise of bath solution temperature from 20 to 28.5°C (bottom) results in a [Ca2+]cyt elevation in synoviocytes (top). Traces show [Ca2+]cyt measured in a single cell, with (solid line) and without (dotted line) correction for the temperature-dependent fura-2 Kd changes. All subsequent graphs show only corrected measurements. B: rapid bath pulses of cold solution result in temperature-dependent calcium spikes in synoviocytes. [Ca2+]cyt was simultaneously recorded from 3 neighboring cells. Low-temperature bath solutions were applied for 30 s with 10-min intervals. C: trace showing that [Ca2+]cyt elevation in a synoviocyte is caused by stepwise elevation in bath temperature.

 
Changes in [Ca2+]cyt in synoviocytes evoked by pharmacological modulators of TRP channels. Specific chemical activators and blockers were used to identify which specific thermosensitive TRP channels are functionally expressed on human synoviocytes. Capsaicin, an agonist of TRPV1 noxious heat receptor, produced modest [Ca2+]cyt increase only in 2 of 11 tested cells even at high concentrations (up to 10 µM) under standard recording conditions at pH 7.4. However, extracellular acidification dramatically potentiated the cell responsiveness to capsaicin. At pH 7.2 1 µM capsaicin evoked a calcium response in 7 of 12 cells, and pH 7.1 evoked a response in 12 of 16 cells (Fig. 2A). Increasing the capsaicin concentration to 5 or 10 µM at pH 7.2 or 7.0 did not significantly affect the responsive-to-nonresponsive cell ratio and had no significant effect on calcium spike amplitude. Further decreases in extracellular pH were avoided because extracellular acidification below pH 7.0 itself produced significant changes in [Ca2+]cyt in synoviocytes due to the activation of an acid-sensitive G protein-coupled receptor we characterized previously (6). The changes in [Ca2+]cyt evoked by capsaicin differed from the heat-induced calcium response in that they produced a delayed, transient response of 15 s to 1 min. [Ca2+]cyt returned to baseline in 30–60 s despite the continuous presence of capsaicin in the bath. The amplitudes of capsaicin-evoked calcium spikes were significantly reduced after repeated agonist application, as seen in Fig. 2A. The activating effects of capsaicin, unlike heat-induced [Ca2+]cyt changes, were completely abolished by the application of capsazepine, an antagonist of TRPV1 and structurally related TRPM8 channels (49), and were restored on washout (Fig. 2C).


Figure 2
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  		Fig. 2. Pharmacology of temperature-sensitive receptors in SW982 synovial cells. A: gradually desensitizing calcium spikes are elicited by repeated stimulation with capsaicin (1 µM) at pH 7.1. B: calcium responses are evoked by cold and 1 µM icilin. Trace shows no response to 100 µM menthol, although menthol occasionally produced [Ca2+]cyt changes in some cells. C: capsazepine abolishes a calcium response to capsaicin but not temperature-induced changes in [Ca2+]cyt. Bars indicate application of 1 µM capsaicin (caps) and 5 µM capsazepine (cpsz) and bath solution heating to 36°C. D: capsazepine significantly reduces calcium response to cold stimulation, suggesting the presence of a functional transient receptor potential (TRP)M8 channel.

 
Icilin and menthol, two stimulators of cold receptors, had dramatically different effects on the synoviocytes. Icilin (1 µM), a potent agonist of TRPA1 and TRPM8 cold receptors, produced a prominent calcium spike in all cells tested. Menthol (100 µM), which stimulates TRPM8 only, elicited changes in [Ca2+]cyt in only 3 of 20 tested cells. In all cases, synoviocytes that were menthol unresponsive demonstrated a salient calcium response to either icilin or 8°C cold solution (Fig. 2B). The application of capsazepine only occasionally blocked a calcium response to cold (in 4 of 14 tested cells) or icilin (in 2 of 9 cells) stimulation (Fig. 2D).

The observed response of synoviocytes to capsaicin strongly suggests the presence of TRPV1 channels. The low heat activation threshold (28°C) and cellular unresponsiveness to capsazepine during heat-activated calcium response support the functional expression of the warm receptor, TRPV4. The characteristic 12°C cold activation threshold suggests that the majority of SW982 synoviocytes possess TRPA1, known as the "noxious cold" receptor. However, the sensitivity of some cells to menthol and abolishing of cold-induced calcium changes with capsazepine are strong evidence for functional expression of the TRPM8 channel, the "moderate cold" receptor, in a small subset of synoviocytes.

Mechanism of TRP channel-mediated changes in [Ca2+]cyt. The changes in [Ca2+]cyt induced by thermal triggers and capsaicin or icilin application were completely abolished when the calcium ions were omitted from the bath solution and restored on reintroduction of calcium-containing normal saline to the bath (Fig. 3). A 30-min preincubation with 1 µM thapsigargin to empty the intracellular calcium stores had no effect on the calcium responses to bath temperature, capsaicin, or icilin. Thus the influx of extracellular calcium is the primary source of [Ca2+]cyt rise caused by thermal receptor or agonist activation in cultured human synoviocytes. In initial experiments, we determined that SW982 synoviocytes possess voltage-dependent calcium channels, mainly L type. This was established by observation of dramatic [Ca2+]cyt elevations caused by membrane depolarization with 50–150 mM KCl bath solution (data not shown). This depolarization-induced calcium response was completely abolished by the specific L-type voltage-gated calcium channel blocker nifedipine (1–10 mM). However, nifedipine had no effect on calcium [Ca2+]cyt changes evoked by capsaicin, icilin, or thermal stimulation. We thus conclude that the observed calcium response directly results from opening of calcium-permeable TRP channels rather then secondary activation of voltage-dependent calcium channels induced by membrane depolarization.


Figure 3
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  		Fig. 3. Temperature receptor-activated [Ca2+]cyt increases in synoviocytes show absolute dependence on extracellular calcium. A: absence of heat-evoked calcium response was observed in calcium-free medium. B: capsaicin does not cause [Ca2+]cyt changes in the absence of extracellular calcium, but its ability is restored after solution switching to a calcium-containing medium.

 
Expression of TRP channel mRNA in synoviocytes. RT-PCR with primers to unique sequence regions of five different TRP genes (Fig. 4A) were used to confirm which particular thermosensitive channels are expressed in SW982 synovial cells. Electrophoresis of PCR products revealed strong intensity bands corresponding to TRPV1, TRPV4, and TRPA1 sequences and minimal-intensity bands for TRPV3 and TRPM8 (Fig. 4B). The identity of amplification products was confirmed by sequence analysis. These data correlate with the results of physiological experiments and support the expression of thermosensitive TRP channels on synoviocytes.


Figure 4
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  		Fig. 4. Detection of thermosensitive TRP channel mRNA in SW982 synoviocytes. RT-PCR was carried out with specific primers recognizing unique sequences in TRPA1, TRPM8, TRPV1, TRPV3, and TRPV4. A: unique regions from published human sequences were used for primer design. B: amplification products and DNA-free controls were separated by 1% agarose gel electrophoresis. Representative results from 3 independent experiments are shown. This figure represents a combination of 2 photographs: lines depicting TRPV3 and TRPM8 were exposed twice as long as the other lines to facilitate enhancement of their bands.

 
Immunofluorescent localization of TRPV1 in clonal and primary synoviocytes. Immunofluorescent staining revealed TRPV1 on both primary and clonal synovial cells. Figure 5, A and B, illustrates immunofluorescent staining for TRPV1 on SW982 clonal synoviocytes. In Fig. 5B, TRP localization at high power reveals membrane and vesicular intracellular localization. Primary synovial cells derived from a patient with active rheumatoid arthritis also stained for TRPV1 (Fig. 5, C and D).


Figure 5
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  		Fig. 5. Immunofluorescent imaging of TRPV1 localization on human SW982 and primary synoviocytes. TRPV1 staining is noted on membrane and cytoplasmic regions in primary and clonal synoviocytes (red). Cells are counterstained with DAPI for nuclear enhancement (blue). A and B: human SW982 synoviocytes. A: negative control staining with normal rabbit serum (x60). B: TRPV1 membrane and vesicular cytoplasmic localization on human SW982 synoviocytes (x60). C and D: primary synovial cells derived from a male patient with rheumatoid arthritis. C: negative control staining with normal rabbit serum (x20). D: immunofluorescent localization of TRPV1 (x20).

 
TRP channel activators elicit [Ca2+]cyt rise in human primary synovial cells. Primary human synoviocytes derived from a patient with acute pseudogout were assessed for their ability to respond to capsaicin (1 µM), icilin (1 µM), or temperature changes. As in SW982 cells, primary human synoviocytes responded to TRP channel activators capsaicin and icilin and thermal stimuli by rapid significant changes in [Ca2+]cyt (Fig. 6). The activating temperature range appeared to be very similar to that of SW982 cells: 13 ± 3°C (n = 4) and 27 ± 3°C (n = 4). Likewise, the activation produced by temperature and TRP agonist was abolished in Ca2+-free medium. Capsazepine blocked the capsaicin-induced changes in [Ca2+]cyt in the primary synoviocytes. The results provide strong evidence for the expression of heat- and cold-sensitive TRP channels in primary human synovial cells. Sensitivity to capsaicin and the characteristic temperature threshold provide strong evidence for the abundant expression of TRPV1, TRPV4, and TRPA1 thermosensitive channels in primary human synovial cells. Further studies are needed to detect whether these cells express TRPM8 and TRPV3 like SW982.


Figure 6
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  		Fig. 6. Functional thermosensitive TRP receptors in primary human synovial cell cultures. A: calcium responses are elicited from primary synoviocyte cultures with serial applications of capsaicin (1 µM) at pH 7.1. B: increases in [Ca2+]cyt are elicited by noxious cold.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we demonstrated the functional expression of temperature-sensitive TRP channels in human synoviocytes. The [Ca2+]cyt measurement studies demonstrate rapid calcium response in synoviocytes, activated by changes in extracellular temperature as well as agonist-dependent opening of TRP channels. Observed changes of [Ca2+]cyt directly result from the opening of calcium-permeable TRP channels. The TRP channel-mediated calcium response is independent of intracellular stores or concurrent activation of voltage-dependent calcium channels. In a previous study we demonstrated (6) that SW982 synoviocytes do not have ryanodine-sensitive calcium stores. Therefore, calcium-induced calcium release also does not contribute to calcium signaling in synovial cells.

These data provide solid physiological evidence for the presence of four TRP channels belonging to three distinct subfamilies, TRPV1, TRPV4, TRPM8, and TRPA1. Further confirmation of their presence was demonstrated by RT-PCR amplification and nucleotide sequence analysis. Amplification of trace amounts of TRPV3 cDNA was noted, but evidence for the functional expression of a TRPV3 channel was not detected by our activation protocols. The TRPV3 channel has significant overlap in temperature sensitivity with TRPV4, and no specific activators are known to date (7, 32, 50). One important distinction between TRPV3 and TRPV4 is that TRPV3 sensitizes whereas TRPV4 desensitizes with constant or repetitive stimulation (32, 48, 50). In our experiments, when the bath temperature was elevated for 1 min, [Ca2+]cyt usually remained steadily elevated or slowly decreased over time. This observation and the temperature activation threshold of 28°C that is characteristic for TRPV4 suggest that the TRPV4-mediated response prevails over TRPV3 in synoviocytes, although future studies using more precise electrophysiological techniques and prolonged activation strategies are needed.

Although all cells consistently responded to heat or cold stimulation with [Ca2+]cyt increases, at pH 7.4 most of the synoviocytes did not respond to the well-established TRP channel agonist capsaicin, even at high concentrations. However, slight acidification of the extracellular medium (pH 7.2 or 7.1) sensitized the cells to capsaicin, although 25% of the cells remained unresponsive. It is unclear whether the unresponsive cells lack TRPV1 receptors or require a specific pH range to potentiate activation. Previous studies indicate that the pH range in inflamed knee joints is below 7.0, suggesting that TRP channels would be active in pathological conditions (44, 46). The capsaicin-evoked calcium spike shows a rapidly transient profile, and the response to subsequent capsaicin stimulation could be partially restored within 1 min (Fig. 2A). This can be explained by TRPV1 receptor turnover on the plasma membrane, or possibly by rapid switching of the receptor from active to inactive functional states. The requirement for lowered pH for capsaicin activation may implicate the necessity for receptor coupling and coactivation of the G protein-coupled receptor we have reported for synovial cells (6) to generate selective physiological events. Acid-sensing ion channels were not found on these cells.

Experiments with icilin, menthol, and low-temperature stimulation reveal heterogeneity in the cold TRP channel-mediated response in synoviocytes. The TRPA1 and TRPM8 channels do not appear to coexist in these cells. The majority of synoviocytes possess TRPA1 and cannot be activated by menthol or prevented from cold activation by capsazepine. The functional expression of TRPA1 in synovial cells is not surprising because this receptor was first cloned from mammalian fibroblasts (17). In a small subset of cells responding to menthol, activation of the receptor by low temperature could be abolished by capsazepine, suggestive of the exclusive expression of TRPM8. Future immunocytochemical or molecular visualization studies are needed to observe the expression and possible inverse distribution of cold-sensitive TRP channels in what may be subpopulations of synoviocytes in the cultures.

The biological significance of the functional heterogeneity regarding the TRP channels on synovial cells is not known. In our previous study (6) characterizing an acid-sensitive G protein-coupled receptor on synoviocytes, nonuniform synoviocyte responses to lower extracellular pH were also observed. These cell-to-cell distinctions could reflect a clonal heterogeneity in the cell line or possibly reflect cell cycle or microenvironment changes. Further studies are planned to elucidate these differences in receptor distribution and activation to additional environmental stressors and pharmacological agents.

It may be important to note that the cold activation threshold in synoviocytes (12°C) is close to the reported mean threshold temperature in dorsal root ganglion neurons (37) but lower than that reported to be expressed in TRPA1-transfected CHO cells [17°C (40)] Both studies reported dramatic variability in threshold from cell to cell. Such differences suggest that the temperature gating mechanism in the TRPA1 cold-sensitive receptor is influenced by additional cellular mechanisms not yet elucidated.

The TRP receptors on peripheral sensory neuronal endings of primary afferent neurons are considered to be important effectors in low-pH-induced pain and hyperalgesia. The TRP channels are widely, but not uniformly, distributed in tissues (16, 38, 43), although the dominant focus of attention has been on TRP expression and functional significance in neurons. Their expression and activation in various nonneuronal cells suggest possible potentiating and coordinating functions with neural activity, or perhaps unique functions in many adaptive or pathological conditions. For example, Mitchell et al. (26) found upregulation of TRPV1 receptors in airway smooth muscle cells [which also possess functionally active TRPV4 receptors (18)] in patients with chronic cough. Some authors reported TRPV1 channel mediated proapoptotic, antitumor (8, 22), as well as vasoactive (1, 10) effects of cannabinoids. Stein et al. (39) demonstrated abundant expression and discussed possible clinical significance of TRPV1 and TRPM8 in the bladder and male genital tract. TRPV1 is also found in keratinocytes (9, 38), prostate cells (18), and other epithelial lining cells (38). The literature suggests that TRP channel expression may be extremely important in cells that interface with the external environment and therefore face dramatic changes induced by thermal or mechanical stress, acidity, anisosmolarity, foreign chemicals, and mediators. It is possible that TRP channels in nonneural tissues provide distinct and coordinated activating functions important to neural responses and specific to the distinct environmental forces that are routinely encountered for that tissue. Synoviocytes may be easily exposed to all the above stimuli, especially acidotic conditions and large ranges of thermal stress in the course of routine joint function or in acute or chronic inflammatory states. It is unclear how these peripheral TRP receptor responses impact physiological responses in the joint capsule. They may function alone or in concert with nervous system signals generated through activation of neuronal TRP or other acid-sensitive channels, such as the acid-sensing ion channels. Thus TRP channels may play an important role as the receptors mediating Ca2+-associated proliferative and secretory responses of synoviocytes, playing a critical role in joint inflammation and attracting interest as a potential therapeutic target.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by The Dana Foundation and National Institute of Neurological Disorders and Stroke Grant P01-NS-011255-31.


   ACKNOWLEDGMENTS
 
The authors thank Yinghong Ma for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Westlund High, Dept. of Neuroscience and Cell Biology, Univ. of Texas Medical Branch, Medical Research Bldg., Rm. 10.138D, 301 University Blvd., Galveston, TX 77555-1043 (kwhigh{at}utmb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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