Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization
2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês
10.1074/jbc.m707865200
ISSN1083-351X
AutoresIrina Vetter, Wei Cheng, Madusha Peiris, Bruce Wyse, Sarah J. Roberts‐Thomson, Jie Zheng, Gregory R. Monteith, Peter J. Cabot,
Tópico(s)Herbal Medicine Research Studies
ResumoTRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the μ opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the μ opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the μ opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for finetuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes. TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the μ opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the μ opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the μ opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for finetuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes. The ability to sensitize or desensitize painful stimuli is fundamental for survival. In inflammation, sensitization of peripheral nociception contributes to the development of hyperalgesia. Pro-inflammatory mediators including prostaglandins mediate an increase in cellular cAMP levels, which in turn leads to sensitization of nociception as a result of activation of cAMP-dependent protein kinase (PKA) 2The abbreviations used are: PKA, cAMP-dependent kinase; TRPV1, transient receptor potential vanilloid 1; HEK, human embryonic kidney cells; FRET, fluorescence resonance energy transfer; MOP, μ opioid receptor; RTX, resiniferatoxin; PSS, physiological salt solution; FSK, forskolin; DRG, dorsal root ganglion; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ANOVA, analysis of variance; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. (1Taiwo Y.O. Bjerknes L.K. Goetzl E.J. Levine J.D. Neuroscience. 1989; 32: 577-580Crossref PubMed Scopus (204) Google Scholar). Such sensitization may involve the transient receptor potential vanilloid 1 (TRPV1) (2Rathee P.K. Distler C. Obreja O. Neuhuber W. Wang G.K. Wang S.Y. Nau C. Kress M. J. Neurosci. 2002; 22: 4740-4745Crossref PubMed Google Scholar). TRPV1 is a calcium-permeable ion channel that is activated by the prototypical agonist capsaicin, the component that conveys the sensation of "heat" to chili peppers (3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7303) Google Scholar). Endogenous TRPV1 activators include lipophilic arachidonic acid metabolites such as N-arachidonoyl-dopamine and 12-hydroperoxyeicosatetraenoic acid (4Pomonis J.D. Harrison J.E. Mark L. Bristol D.R. Valenzano K.J. Walker K. J. Pharmacol. Exp. Ther. 2003; 306: 387-393Crossref PubMed Scopus (246) Google Scholar, 5Walker K.M. Urban L. Medhurst S.J. Patel S. Panesar M. Fox A.J. McIntyre P. J. Pharmacol. Exp. Ther. 2003; 304: 56-62Crossref PubMed Scopus (325) Google Scholar), but also heat and protons (3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7303) Google Scholar). TRPV1 and endovanilloid signaling are implicated in various inflammatory hyperalgesic conditions. The contribution of TRPV1 to inflammatory hyperalgesia has been established through observations that TRPV1 antagonists can dose-dependently reverse both thermal and mechanical inflammatory hyperalgesia (4Pomonis J.D. Harrison J.E. Mark L. Bristol D.R. Valenzano K.J. Walker K. J. Pharmacol. Exp. Ther. 2003; 306: 387-393Crossref PubMed Scopus (246) Google Scholar, 5Walker K.M. Urban L. Medhurst S.J. Patel S. Panesar M. Fox A.J. McIntyre P. J. Pharmacol. Exp. Ther. 2003; 304: 56-62Crossref PubMed Scopus (325) Google Scholar). In addition, thermal inflammatory hyperalgesia is significantly reduced in TRPV1 knock-out mice (6Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Science. 2000; 288: 306-313Crossref PubMed Scopus (2989) Google Scholar). Several pro-inflammatory mediators and cytokines, including prostaglandin E2, sensitize TRPV1 responses under inflammatory conditions (7Hu H.J. Bhave G. Gereau R.T. J. Neurosci. 2002; 22: 7444-7452Crossref PubMed Google Scholar). Moreover, the TRPV1 is sensitized directly by cAMP-dependent PKA (8Bhave G. Zhu W. Wang H. Brasier D. Oxford G. Gereau R.T. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), making the TRPV1 a direct molecular target for the development of inflammatory hyperalgesia (2Rathee P.K. Distler C. Obreja O. Neuhuber W. Wang G.K. Wang S.Y. Nau C. Kress M. J. Neurosci. 2002; 22: 4740-4745Crossref PubMed Google Scholar, 9Planells-Cases R. Garcia-Sanz N. Morenilla-Palao C. Ferrer-Montiel A. Pflugers Arch. 2005; 451: 151-159Crossref PubMed Scopus (130) Google Scholar). Although potentiation of TRPV1 responses by cAMP-dependent PKA appears crucial in the development of inflammatory hyperalgesia, the mechanisms underlying this potentiation are not fully understood. Peripheral sensitization of nociceptive responses represents a cellular process to encourage rest and recovery after injury. The ability to modify responses rapidly, without having to rely on altering expression levels of nociceptive receptors, is essential for rapid adaption to external circumstances. This system is exemplified by the release of endogenous opioids from immune cells, which affects analgesia in inflamed tissues (10Cabot P.J. Carter L. Gaiddon C. Zhang Q. Schäfer M. Loeffler J.P. Stein C. J. Clin. Investig. 1997; 100: 142-148Crossref PubMed Scopus (277) Google Scholar), and the increased effectiveness of exogenous opioids in inflammation (11Schafer M. Imai Y. Uhl G.R. Stein C. Eur. J. Pharmacol. 1995; 279: 165-169Crossref PubMed Scopus (112) Google Scholar). The capacity for rapid fine-tuning and modulation of sensitized nociception is further demonstrated by the ability of opioids to inhibit TRPV1-mediated capsaicin responses potentiated by cAMP-dependent PKA (12Vetter I. Wyse B.D. Monteith G.R. Roberts-Thomson S.J. Cabot P.J. Mol. Pain. 2006; 2: 22Crossref PubMed Scopus (95) Google Scholar). To date, no mechanism has been proposed to explain how TRPV1 sensitization by cAMP-dependent PKA can be achieved in a manner that provides for rapid potentiation while maintaining the capacity for dynamic modulation. Here, we present evidence for a model that describes how, as a consequence of activation of adenylate cyclase, an intracellular pool of inactive TRPV1 monomers is transported to the plasma membrane where they function as TRPV1 multimers. This mechanism incorporates 2-fold regulation involving both altered multimerization and trafficking. It provides the basis for rapid modulation of nociceptive TRPV1 responses and finetuning of nociception. Modification of this pathway by the opioid receptor agonist morphine can rapidly alter TRPV1 potentiation and thus allows dynamic regulation of nociceptive TRPV1 responses. Microplate Reader Measurement of Intracellular Ca2+ Responses in TRPV1/MOP HEK Cells—Double stable HEK293 cells expressing the μ opioid receptor (MOP) and TRPV1 (TRPV1/MOP HEK cells) were generated as described (12Vetter I. Wyse B.D. Monteith G.R. Roberts-Thomson S.J. Cabot P.J. Mol. Pain. 2006; 2: 22Crossref PubMed Scopus (95) Google Scholar). TRPV1/MOP HEK cells were plated on PDL-coated 96-well plates and loaded with the fluorescent calcium probe Fluo-3-AM (6 μm) as described (12Vetter I. Wyse B.D. Monteith G.R. Roberts-Thomson S.J. Cabot P.J. Mol. Pain. 2006; 2: 22Crossref PubMed Scopus (95) Google Scholar). Cells were washed 2–3 times with physiological salt solution (PSS; pH 7.4, composition KCl 5.9 mm, MgCl2 1.5 mm, NaH2PO4 1.2 mm, NaHCO3 5.0 mm, NaCl 140.0 mm, glucose 11.5 mm, CaCl2 1.8 mm, and HEPES 10.0 mm) and incubated for 15–30 min with pretreatments as appropriate. Preincubation steps and Ca2+ measurement were carried out at 29 °C to avoid dye sequestration. Changes in fluorescence after addition of capsaicin were measured using a fluorescent multi-well plate reader (NOVOstar, BMG Labtechnologies, Victoria, Australia) with the excitation wavelength set at 485 nm and emission recorded at 520 nm. As previously described, calcium responses were represented as ΔF/F values (12Vetter I. Wyse B.D. Monteith G.R. Roberts-Thomson S.J. Cabot P.J. Mol. Pain. 2006; 2: 22Crossref PubMed Scopus (95) Google Scholar). Maximum ΔF/F values were used to fit a 4-parameter logistic Hill equation to the data using GraphPad Prism (San Diego, CA, Version 4.03) to generate dose-response curves. All experiments were designed to include control experiments on the same plate as treated cells. For calcium measurements using the FLIPRTETRA fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA), TRPV1/MOP HEK cells were plated on PDL-coated, black-walled 96-well plates (Corning, Lindfield, NSW, Australia). After loading with Fluo-3 AM (6 μm), cells were washed three times with PSS or nominal calcium-free PSS (composition: KCl 5.9 mm, MgCl2 1.5 mm, NaH2PO4 1.2 mm, NaHCO3 5.0 mm, NaCl 140.0 mm, glucose 11.5 mm, and HEPES 10.0 mm) as appropriate. Capsaicin and other reagents were injected from 3–4× concentrated stock solutions prepared in PSS or nominal calcium-free PSS with maximum final ethanol concentrations not exceeding 0.003%. Fluo-3-loaded cells were excited at 470–495 nm, and emission at 515–575 nm was recorded every second using a cooled CCD camera. For experiments requiring no extracellular calcium, BAPTA (final concentration 100 μm) was added 30 s prior to addition of capsaicin. Whole Cell cAMP Accumulation Assay—TRPV1/MOP HEK cells were harvested with Versene (Invitrogen, Mount Waverley, Victoria, Australia) to avoid internalization of receptors. Cells were resuspended with anti-cAMP acceptor bead mix yielding final concentrations of 40,000 cells/well and 1 unit/well of anti-cAMP acceptor beads. Cells were incubated for 15–30 min with agonist in triplicate on 384-well plates (OptiPlate-384; PerkinElmer Life Sciences, Rowville, Victoria, Australia) and the reaction terminated by addition of lysis buffer containing Streptavidin Donor beads (1 unit/well) and biotinylated cAMP (1 unit/well). The plate was incubated under low light conditions for 16 h, and the bioluminescence reaction measured using the Envision Multilabel Plate Reader (PerkinElmer Life Sciences). SDS-PAGE and Western Blotting—To assess monomeric and multimeric TRPV1 species, total cell protein was isolated and separated by SDS-PAGE essentially as previously described (13Kedei N. Szabo T. Lile J.D. Treanor J.J. Olah Z. Iadarola M.J. Blumberg P.M. J. Biol. Chem. 2001; 276: 28613-28619Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Cells were grown to ∼80% confluency and incubated for 25–30 min at 37 °C with the appropriate treatments in PSS. Cells were then immediately placed on ice, washed with ice-cold phosphate-buffered saline (PBS; composition in mm: NaCl 137, KCl 2.7, NaH2PO4 10, KH2PO4 1.8) and dislodged with ice-cold PBS. After centrifugation at 3,000 × g for 10 min, cells were resuspended in ice-cold PBS containing protease inhibitors (Roche Applied Science, Castle Hill, NSW, Australia) and sonicated briefly on ice. The supernatant was collected after centrifugation at 1,000 × g for 10 min at 4 °C and protein estimation carried out using the Bio-Rad protein estimation kit. Protein samples were analyzed on precast 4–20% iGel gradient gels without SDS (Life Therapeutics, Frenchs Forest, NSW, Australia). Total cell protein (20 μg) was mixed with 5× loading buffer (composition: 365 mm Tris-HCl, pH 6.8, 37.5% glycerol, 0.02% bromphenol blue, and 10% SDS to yield SDS end concentrations of 2%) and denatured at 65 °C for 10 min. Samples were separated at 150 V for ∼60 min and transferred to nitrocellulose membrane for 1 h at 300 mA on ice in transfer buffer (composition: 25 mm Tris, 192 mm glycine, 15% methanol, pH 8.3). The nitrocellulose membrane was blocked overnight at 4 °C in blocking buffer (composition: 130 mm NaCl, 2.7 mm KCl, 10 mm NaH2PO4, 1.8 mm KH2PO4, 0.1% Tween-20, and 10% low-fat skim milk powder) and incubated for 1 h at room temperature with rabbit anti-rat TRPV1 antibody (1:5,000, Santa Cruz Biotechnology). A monoclonal anti-β-actin antibody (1:20,000, clone AC-15, Sigma Aldrich) was also included for visualization of β-actin to serve as a loading control. After washing in blocking buffer, the membrane was incubated for 1 h with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies (both 1:5,000; Zymed Laboratories Inc., Mount Waverley, Victoria, Australia). Blots were developed using ECL Plus (GE Life Sciences, Rydalmere, NSW, Australia) for visualization of chemiluminescence by exposure to ECL Hyperfilm (GE Life Sciences). The optical density of bands was determined using MetaMorph Imaging Software (Version 6.2R5; Universal Imaging, Downington, PA). [3H]Resiniferatoxin Binding—For preparation of total cell membranes to assess binding of [3H]resiniferatoxin to the TRPV1, cells were incubated at 37 °C in a 5% humidified CO2 incubator with the appropriate treatment for 20–30 min, subsequently placed on ice, and washed 2–3 times with ice-cold PBS. Cells were dislodged with a cell scraper and collected by centrifugation at 3,000 × g for 5 min. The cells were resuspended in 1 ml of assay buffer (composition: 5 mm KCl, 5.8 mm NaCl, 2 mm MgCl2, 320 mm sucrose, 10 mm HEPES; pH 7.4) and sonicated briefly. After centrifugation at 1,000 × g for 10 min, the supernatant was collected, and after protein estimation using a Bio-Rad protein assay kit, diluted to 20 μg of protein/200 μl with assay buffer containing an additional 0.25 mg/ml BSA. Plasma membrane samples for [3H]resiniferatoxin binding were prepared according to the manufacturer's instructions with a Plasma Membrane Protein Isolation kit (MBL International Corp, Woburn, MA) based on an aqueous two-phase polymer system of dextran-polyethylene glycol, which isolates plasma membrane proteins specifically with minimal contamination from other intracellular membrane fractions (14Morre D.J. Morre D.M. BioTechniques. 1989; 7 (950-954, 956-958): 946-948PubMed Google Scholar, 15Hong S. Wiley J.W. J. Biol. Chem. 2005; 280: 618-627Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) (supplemental Fig. S1). TRPV1/MOP HEK cells were incubated with the appropriate treatments in PSS at 37 °C for 20–30 min. Cells were placed on ice immediately, washed with ice-cold PBS, and 5–10 × 107 cells collected by centrifugation. The cell pellet was resuspended with 1 ml of the homogenization buffer included in the kit and briefly sonicated. The resulting homogenate was centrifuged at 700 × g for 10 min at 4 °C, and the resulting supernatant collected for protein estimation. For each treatment group, an identical amount of supernatant protein was utilized to isolate purified plasma membrane fractions. The plasma membrane protein pellet was collected by centrifugation at 44,800 × g for 30 min at 4 °C and resuspended in PBS for protein estimation. For [3H]resiniferatoxin binding to the TRPV1, the plasma membrane protein was diluted to 5 μg of plasma membrane protein/200 μl with assay buffer containing 0.25 mg/ml BSA and used fresh. Quantification of TRPV1 binding was performed by homologous competitive binding (16Matsuura B. Dong M. Naik S. Miller L.J. Onji M. J. Biol. Chem. 2006; 281: 12390-12396Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17von Wronski M.A. Raju N. Pillai R. Bogdan N.J. Marinelli E.R. Nanjappan P. Ramalingam K. Arunachalam T. Eaton S. Linder K.E. Yan F. Pochon S. Tweedle M.F. Nunn A.D. J. Biol. Chem. 2006; 281: 5702-5710Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Ross R.A. Gibson T.M. Brockie H.C. Leslie M. Pashmi G. Craib S.J. Di Marzo V. Pertwee R.G. Br. J. Pharmacol. 2001; 132: 631-640Crossref PubMed Scopus (211) Google Scholar, 19Dutertre S. Croker D. Daly N.L. Andersson A. Muttenthaler M. Lumsden N.G. Craik D.J. Alewood P.F. Guillon G. Lewis R.J. J. Biol. Chem. 2008; 283: 7100-7108Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) in the presence of 50 pm [3H]resiniferatoxin (PerkinElmer Life Sciences, 39.8 Ci/mmol) and varying concentrations of unlabeled resiniferatoxin. Nonspecific binding was defined as occurring in the presence of 1 μm non-radioactive resiniferatoxin. For total cell binding, 20 μg of protein was used, while for plasma membrane binding, 5 μg of purified plasma membrane fractions were sufficient. Using this protocol, nonspecific binding was generally below 10–20%, as previously reported (18Ross R.A. Gibson T.M. Brockie H.C. Leslie M. Pashmi G. Craib S.J. Di Marzo V. Pertwee R.G. Br. J. Pharmacol. 2001; 132: 631-640Crossref PubMed Scopus (211) Google Scholar). Binding reactions were carried out by incubation at 37 °C for 1 h and terminated by placing the assay mixtures on ice. Bound and free fractions were separated with a cell harvester on Whatman GF/B filters that had been soaked in filter buffer (composition: 50 mm Tris-HCl, 0.1% BSA, 0.5% polyethyleneimine; pH 7.4) at 4 °C for at least 1 h. Samples were read on a liquid scintillation counter after incubation of the filter papers with ∼3 ml of liquid scintillant (OptiPhase HiSafe 3, PerkinElmer Life Sciences) at room temperature for 10–14 h. The analysis of radioligand binding data was performed using GraphPad Prism homologous competitive binding analysis with one class of binding sites. Determination of Cell Viability by MTS Assay—The viability of TRPV1/MOP HEK cells was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Annandale, NSW, Australia) according to the manufacturer's specifications. In brief, cells were grown to ∼80–90% confluency on 96-well plates and treated with varying concentrations of dansylcadaverine in PSS for 30 min. After a wash with PSS, cells were incubated with the MTS reagent for another 2 h at 37 °C in 5% CO2 and absorbance measured at 490 nm with a Bio-Rad model 550 microplate reader. Fluorescence Resonance Energy Transfer (FRET) Measurements—FRET signals from murine homomultimeric TRPV1 channels were determined as previously described (20Cheng W. Yang F. Takanishi C.L. Zheng J. J. Gen. Physiol. 2007; 129: 191-207Crossref PubMed Scopus (155) Google Scholar). In brief, HEK293 cells were transiently transfected with a C-terminal Cerulean fluorescence protein-tagged TRPV1 construct (TRPV1-CFP) and a C-terminal enhanced yellow fluorescence protein-tagged TRPV1 construct (TRPV1-YFP) using Lipofectamine 2000 (Invitrogen). Fluorescence imaging was carried out at room temperature 1–2 days after transfection using a fully automated inverted fluorescence microscope (Olympus IX-81). For spectroscopic FRET measurements from the plasma membrane, a spectrograph (Acton SpectraPro 2150i) was used in conjunction with a Hamamatsu HQ CCD camera. Spectroscopic emission data specifically from the plasma membrane or the cytoplasm was collected by recording fluorescence intensity from the spectrograph slit location corresponding to each cellular structure as previously described (21Takanishi C.L. Bykova E.A. Cheng W. Zheng J. Brain Res. 2006; 1091: 132-139Crossref PubMed Scopus (90) Google Scholar). FRET efficiency was plotted as a function of the Cerulean to YFP fluorescence intensity ratio (Fc/Fy). Best fit FRET models were fitted to the data as previously described (20Cheng W. Yang F. Takanishi C.L. Zheng J. J. Gen. Physiol. 2007; 129: 191-207Crossref PubMed Scopus (155) Google Scholar) and were represented as solid or dotted lines. Immunofluorescence—TRPV1/MOP HEK cells were plated on 25-mm PDL-coated glass coverslips and grown to ∼80–90% confluence. Cells were treated for 30 min at 37 °C with the appropriate reagents in PSS and placed immediately on ice to minimize receptor internalization. Cells were washed twice in ice-cold PBS before a light fix with methanol/acetone (1:1) at -20 °C for 30 min. After permeabilization with 0.2% Triton X in PBS for 10 min, cells were blocked by immersion in PBS with 3% BSA for 30 min. Rabbit anti-rat TRPV1 primary antibody (1:100, Santa Cruz Biotechnology) was prepared in PBS containing 3% BSA. After 1 h of incubation at room temperature, cells were incubated with anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (1:300) under low light for 1 h. Cells were mounted using SlowFade AntiFade (Molecular Probes). Randomly selected images were viewed on a Nikon Eclipse TE 300 inverted fluorescent microscope (excitation 488 nm, emission 520 nm) and recorded with MetaFluor (Molecular Devices) imaging software. Corel Photo-Paint (Corel, Ottawa, Ontario, Canada) was used for processing of images. TRPV1 Expression in Calcium-rich Stores—As previously described (22Csordas G. Hajnoczky G. Cell Calcium. 2001; 29: 249-262Crossref PubMed Scopus (87) Google Scholar), to assess TRPV1 expression in calcium-rich stores, the low affinity calcium probe Fura-FF was sequestered into intracellular stores by loading TRPV1/MOP HEK cells with Fura-FF-AM (12 μm) for 2 h at 37 °C, followed by washing with PSS for 15 min at 37 °C. Coverslips were transferred to the recording chamber of an inverted Nikon Eclipse TE 300 fluorescent microscope and viewed under a Nikon 40×/1.3 oil immersion objective lens. Fluorescence signals from single cells were ratio-imaged by recording emission intensity at 510 nm from excitation at 340 and 380 nm. Ratios of F340/F380 were depicted as pseudocolor images. Cells were then lightly fixed with 4% paraformaldehyde for 20 min and stained for TRPV1 expression as described above. Assessment of TRPV1 Multimers and Immunofluorescence in DRG from Animals with Peripheral Inflammation—Ethical approval was obtained from the University of Queensland Animal Ethics Committee and experiments carried out in accordance with guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain. Adult male Wistar rats (250–300 g) were kept in a controlled environment at a temperature of 22 ± 0.5 °C, relative humidity of 40–60%, and a 12 h (6:30 AM to 6:30 PM) light-dark cycle with free access to standard lab chow and tap water. To induce peripheral inflammation, 100 μl of Freund's Complete Adjuvant (FCA, Sigma) was injected subcutaneously into the right hind paw under light isoflurane anesthesia. L5 DRGs ipsilateral to the inflamed paw were isolated 6–7 days after induction of inflammation. For analysis of TRPV1 multimerization in DRG neurons, individual L5 DRG were placed immediately in ice-cold PBS containing protease inhibitors (Roche Applied Sciences), minced with surgical scissors and sonicated on ice. Protein samples were collected after centrifugation at 1,000 × g for 10 min and 20 μg of protein analyzed by SDS-PAGE and Western blotting as described above. For immunofluorescence studies of plasma membrane TRPV1 expression, L5 DRG contralateral and ipsilateral to the inflamed paw were harvested and immediately fixed for 2–3 h in ice-cold paraformaldehyde (4%). DRGs were then transferred to 0.32 m sucrose and cryoprotected at 4 °C for at least 24 h. Tissues were embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek, Torrance, CA), cut to 10-μm cryostat sections, and mounted on SuperFrost Ultra Plus® tissue adhesion slides (Menzel GmbH Co KG, Braunschweig, Germany) before immunofluorescence labeling as described above. Data Analysis—Unless otherwise states, all graphs are representative of at least two to three independent experiments with minimum n values for each treatment group being n = 3. Unless individual data points are shown, data are presented as means ± S.E. of the mean (S.E.). As mentioned above, statistical analysis and fitting of 4-parameter Hill equations were carried out using GraphPad Prism Version 4.03. Statistical significance was defined as p < 0.05 and determined using an unpaired, two-tailed Student's t test where appropriate. Calcium traces were analyzed by statistical comparison of maximum ΔF/F values using an unpaired, two-tailed Student's t test with statistical significance defined as p < 0.05. Capsaicin Responses Potentiated by cAMP-dependent PKA Are Inhibited by Morphine—Potentiation of TRPV1-mediated Ca2+ responses to addition of varying concentrations of capsaicin was assessed after a short (15 min) incubation with 0.1% DMSO (control) or forskolin (50 μm, Fig. 1A). Treatment with the adenylate cyclase activator forskolin leads to an increase in cellular cAMP levels and resultant activation of cAMP-dependent PKA. PKA-mediated phosphorylation of TRPV1 in turn causes sensitization of TRPV1 responses (23Mohapatra D.P. Nau C. J. Biol. Chem. 2003; 278: 50080-50090Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). While cAMP analogues acting directly at PKA lead to a similar sensitization, opioids, by virtue of their antihyperalgesic activity resulting from adenylate cyclase inhibition, are unable to prevent TRPV1 sensitization by direct PKA activators (12Vetter I. Wyse B.D. Monteith G.R. Roberts-Thomson S.J. Cabot P.J. Mol. Pain. 2006; 2: 22Crossref PubMed Scopus (95) Google Scholar). Thus, to assess the opioid-sensitive mechanisms involved in TRPV1 sensitization by cAMP-dependent PKA, forskolin was used to potentiate TRPV1 responses. Consistent with previously described studies (12Vetter I. Wyse B.D. Monteith G.R. Roberts-Thomson S.J. Cabot P.J. Mol. Pain. 2006; 2: 22Crossref PubMed Scopus (95) Google Scholar), treatment with forskolin significantly increased TRPV1-mediated capsaicin responses (Fig. 1, A and B, p < 0.001). The (MOP) agonist morphine inhibited this potentiation (Fig. 1B) through inhibition of forskolin-stimulated cAMP production, which was maximal at 1 μm morphine, although reversal of forskolin-stimulated cAMP accumulation by morphine was not complete (Fig. 1B, inset). We then focused on assessing the mechanism of potentiation of TRPV1 by forskolin. Treatment of TRPV1/MOP HEK Cells with the PKA Activator Forskolin Increases TRPV1 Multimerization—Augmentation of TRPV1 responses by forskolin (Fig. 1A) appears consistent with an increase in functional TRPV1 channels. However, it is unclear how forskolin could achieve an increase in functional TRPV1 channels in a way that would be consistent with rapid and dynamic regulation of nociceptive TRPV1 signaling. As functional TRPV1 channels are proposed to exist in homomultimeric form (14Morre D.J. Morre D.M. BioTechniques. 1989; 7 (950-954, 956-958): 946-948PubMed Google Scholar, 19Dutertre S. Croker D. Daly N.L. Andersson A. Muttenthaler M. Lumsden N.G. Craik D.J. Alewood P.F. Guillon G. Lewis R.J. J. Biol. Chem. 2008; 283: 7100-7108Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Cheng W. Yang F. Takanishi C.L. Zheng J. J. Gen. Physiol. 2007; 129: 191-207Crossref PubMed Scopus (155) Google Scholar, 21Takanishi C.L. Bykova E.A. Cheng W. Zheng J. Brain Res. 2006; 1091: 132-139Crossref PubMed Scopus (90) Google Scholar, 22Csordas G. Hajnoczky G. Cell Calcium. 2001; 29: 249-262Crossref PubMed Scopus (87) G
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