Receptor mRNA for both TRPV1 and FLAG-MOP was effectively translated to receptor protein as Western Blot analysis showed bands of the expected sizes for both receptors (Figure 1A and 1B). Two different colonies of FLAG-MOP/TRPV1 double stable HEK293 cells, colony 13 and colony 21, were selected for functional studies based on their expression of TRPV1 and FLAG-MOP protein (Figure 1A and 1B, lanes 1 and 2) and capsaicin responses (Figure 2). For both FLAG-MOP/TRPV1 colonies, the TRPV1 was present in two forms; a non-glycosylated form with a corresponding band at approximately 95 kDa, and a glycosylated form with a predicted molecular weight of 113 kDa (Figure 1A, lanes 1 and 2). Colony 13 (Figure 1A, lane 1) expressed slightly more TRPV1 than colony 21 (Figure 1A, lane 2) while no TRPV1 protein was detected in either untransfected HEK293 cells (Figure 1A, lane 3) or HEK293 cells that were transfected with FLAG-MOP only (Figure 1A, lane 4). Colonies 13 and 21 were selected for functional studies as they responded reproducibly to stimulation with capsaicin as described below (Figure 2). Untransfected HEK cells did not respond to stimulation with capsaicin (10 μM) (ΔF/F 0.010 ± 0.0015).

Colony 13 (Figure 1B, lane 1), colony 21 (Figure 1B, lane 2) as well as HEK293 cells transfected with FLAG-MOP only (Figure 2B, lane 3) showed a protein band at approximately 60 kDa, representative of numerous glycosylated forms of the MOP. Non-transfected HEK293 cells lacked this band (Figure 1B, lane 4). TRPV1 and MOP expression were also confirmed using immunohistochemistry with the overlay (Figure 1C, yellow) clearly demonstrating co-expression of the MOP (Figure 1D, green) and TRPV1 (Figure 1E, red). Non-specific binding is represented in Figures 1F and 1G for TRPV1 and MOP, respectively.

To assess functionality of the TRPV1 in the double stable cell lines, Ca²⁺ responses to capsaicin were recorded. Both colonies 13 and 21 responded with concentration-dependent increases in intracellular free Ca²⁺ to capsaicin (Figure 2). EC50s of approximately 34 nM and 40 nM were determined for colonies 13 and 21, respectively, similar to previous studies in TRPV1-transfected HEK293 cell lines 192021. The response to capsaicin was mediated through activation of the TRPV1, as the TRPV1 antagonist capsazepine (10 μM; Sigma) inhibited capsaicin responses to 6.4 ± 2.5% of the control response at 60 seconds.

Functionality of the FLAG-MOP was confirmed using a cAMP accumulation assay, which assesses functional coupling of the MOP to G_i proteins 18. Incubation with the phosphodiesterase inhibitor IBMX (100 μM) and the adenylate cyclase activator FSK (50 μM) significantly (p < 0.01) increased intracellular cAMP levels in both FLAG-MOP/TRPV1 colonies (Figure 3, vehicle) compared to untreated cells (Figure 3, no FSK). As expected, the MOP agonist morphine (1 μM) significantly (p < 0.01) inhibited the FSK-induced increased in cAMP production by 67.0 ± 4.6% for colony 13 and 79.2 ± 7.4% for colony 21, although cAMP levels were not reduced to unstimulated levels (Figure 3, morphine).

We examined whether morphine can inhibit TRPV1-mediated increases in intracellular free Ca²⁺ under basal conditions and with prior sensitization of TRPV1. To identify if cAMP is a potentiator of TRPV1 responses in our heterologous expression system, FLAG-MOP/TRPV1 transfected HEK293 cells were pre-incubated with varying concentrations of FSK as well as a constant concentration of IBMX (100 μM) and the Ca²⁺ response to 300 nM capsaicin was determined (Figure 4). FSK activates adenylate cyclase, which in turn increases cellular cAMP levels, thus, FSK indirectly activates cAMP-dependent PKA. Pre-incubation with vehicle (DMSO) had no effect on Ca²⁺ responses of colonies 13 (Figure 4A and 4B) and 21 (Figure 4C and 4D) to 300 nM capsaicin but increasing FSK concentrations potentiated capsaicin responses in a concentration-dependent manner (Figure 4A–D). Injection of FSK (50 μM) and IBMX (100 μM) caused a small (ΔF/F 0.0152 ± 0.0011) addition response that returned to baseline after approximately 3 s. This response was not affected (p > 0.05) by pre-incubation with morphine (1 μM) (p > 0.05; ΔF/F 0.0144 ± 0.0013).

Having identified that FSK potentiates TRPV1 responses in our heterologous expression system, we assessed whether morphine inhibits basal or FSK-potentiated TRPV1 responses to capsaicin. This was determined using Fluo-3 loaded FLAG-MOP/TRPV1 HEK293 colonies pre-incubated for 15 min with morphine (1 μM) for opioid-treated cells or PSS for non-opioid treated cells, followed by 15 min pre-incubation with FSK (50 μM) and IBMX (100 μM) for stimulated cells or PSS for unstimulated cells. Ca²⁺ responses after injection of various capsaicin concentrations were monitored for 45 s. Pre-incubation of FLAG-MOP/TRPV1 colonies with morphine concentrations shown to be effective at reducing cAMP accumulation (Figure 3) did not affect the capsaicin dose-response curve for colonies 13 and 21 for unstimulated responses (p > 0.05; Figure 5A,B,D and 5E). However, pre-incubation with morphine significantly altered the capsaicin dose-response curve in FSK/IBMX stimulated cells (p < 0.05; Figure 5).

The inhibition of FSK-stimulated capsaicin (300 nM) responses by morphine occurred through a MOP-specific pathway as the opioid receptor antagonist naloxone (50 μM) blocked inhibition by morphine (1 μM) (p < 0.05 compared to FSK+morphine and p > 0.05 compared to FSK; Figure 6A).

Since morphine-inhibition of FSK-stimulated capsaicin responses could be due to a reduction in cAMP level induced by treatment with opioid agonist, we assessed whether pre-incubation with morphine was required to achieve modulation of FSK-potentiated capsaicin responses. To do this, 300 nM capsaicin alone or 300 nM capsaicin with morphine (final well concentration of 1 μM) in cells were added to FLAG-MOP/TRPV1 HEK293 cells pre-incubated with FSK (50 μM) and IBMX (100 μM). Pre-incubation with morphine was required to achieve inhibition of FSK-stimulated capsaicin responses, as co-injection of morphine (1 μM) and capsaicin did not change the Ca²⁺ response of FLAG-MOP/TRPV1 cells to 300 nM capsaicin (p > 0.05; Figure 6B).

FSK is an indirect PKA activator, as it stimulates adenylate cyclase, which in turn leads to an increase in cellular cAMP levels, which activates cAMP-dependent PKA 13. Several papers have reported potentiation or sensitization of TRPV1 responses through PKA-dependent pathways, including by treatment with indirect PKA activators like FSK or direct PKA activators such as 8-Br-cAMP 6713. The MOP is negatively coupled to adenylate cyclase and can inhibit FSK-stimulated cAMP production but would not affect direct PKA activators such as 8-Br-cAMP. To determine whether morphine-inhibition of FSK-stimulated capsaicin response relies on indirect inhibition of PKA through a decrease of cAMP levels, FLAG-MOP/TRPV1 double stable HEK293 cells were pre-incubated for 15 min with varying concentrations of 8-Br-cAMP as well as constant IBMX concentrations (100 μM) and Ca²⁺ responses to 300 nM capsaicin were monitored (Figure 7A). Pre-incubation of FLAG-MOP/TRPV1 double expressants with 8-Br-cAMP (300 μM) and IBMX (100 μM) potentiated Ca²⁺ responses to 300 nM capsaicin (Figure 7A &7B) in a manner that was dependent on 8-Br-cAMP concentration, as pre-incubation with IBMX alone did not significantly affect capsaicin responses (ΔF/F capsaicin 0.116 ± 0.004; ΔF/F capsaicin +IBMX 0.125 ± 0.003; p > 0.05).

Pre-incubation for 15 min with morphine (1 μM) did not (p > 0.05) affect Ca²⁺ responses to injection of 300 nM capsaicin potentiated by 15 min pre-incubation with 8-Br-cAMP (300 μM) and IBMX (100 μM) (Figure 7B), suggesting that morphine inhibits FSK-potentiated capsaicin responses by indirectly inhibiting cAMP-dependent PKA.

Several papers have reported potentiation of TRPV1 responses through PKC-mediated pathways 162223, and PKC-potentiated TRPV1 responses have been shown to contribute to diabetic neuropathy 24. Thus, in order to determine whether morphine can inhibit TRPV1 responses potentiated by other kinase pathways, we assessed inhibition of capsaicin responses by the PKC activator PMA.

Treatment with PMA (100 nM) significantly (p < 0.05) potentiated TRPV1-mediated capsaicin responses. MOP function, assessed by morphine inhibition of cAMP accumulation, was preserved in the presence of PMA (data not shown). Injection of PMA (10 μM) did not cause a Ca²⁺ responses in FLAG-MOP/TRPV1 expressants (ΔF/F PSS 0.012 ± 0.004; ΔF/F PMA (10 μM) 0.016 ± 0.002; p > 0.05).

Pre-incubation for 15 min with morphine (1 μM) did not (p > 0.05) affect Ca²⁺ responses to injection of 30 nM capsaicin potentiated by 10 min pre-incubation with PMA (100 nM) (Figure 7C).

To determine if a population of DRG neurons co-expressed MOP and TRPV1, immunohistochemical studies were performed. Nuclear labelling with DAPI showed that TRPV1 and MOP antibodies only labelled cells with neuronal morphology, while many cells showing DAPI labelling, presumably accessory cell types such as glia and astrocytes, remained unlabelled for TRPV1 and MOP (Figure 8A–C). Staining patterns for TRPV1 and MOP were in accordance with published literature 25262728.

Of the 173 cultured DRG neurones which represented the subset of neurons that were labelled for TRPV1 and MOP, 88.4% were co-labelled for TRPV1 (Figure 8C, red) and MOP (Figure 8B, green) with the overlay (yellow) clearly showing co-expression of the receptors, although the level of expression was somewhat variable between cells. Eleven neurones (6.4%) were labelled for TRPV1 only, while a further 9 neurones (5.2%) expressed only MOP.

Capsaicin and capsazepine were purchased from Sigma Aldrich (Castle Hill, NSW, Australia) and prepared as stock solutions in ethanol. Maximum final ethanol concentrations did not exceed 0.001%. 3-Isobutyl-1-methyl-xanthine (IBMX), forskolin (FSK), 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and Phorbol 12-myristate 13-acetate(PMA) were from Sigma Aldrich and prepared as stocks in 100% dimethylsulfoxide (DMSO), with maximum DMSO concentrations not exceeding 0.1%. Morphine was obtained from David Bull Laboratories (Mulgrave, Victoria, Australia) and diluted in physiological salt solution (PSS). Geneticin and hygromycin selection antibiotics were from Invitrogen (Mulgrave, Victoria, Australia). QIAEX II agarose gel extraction kit was purchased from Qiagen (Doncaster, Victoria, Australia). Polyclonal rabbit anti-FLAG M2 antibody was purchased from Sigma. Polyclonal guinea-pig anti-MOP and polyclonal rabbit anti-TRPV1 antibodies were from Chemicon (Boronia, Victoria, Australia). Cy3-conjugated goat anti-mouse antibody was from Zymed Laboratories (San Francisco, CA) and FITC-conjugated goat anti-guinea pig as well as FITC-conjugated anti-mouse antibodies were purchased from Sigma. Slow-Fade Antifade was from Molecular Probes (Eugene, OR). Lipofectamine 2000 was purchased from Invitrogen (Mulgrave, Victoria, Australia). Protein estimation kit and Mini Trans-Blot^® Electrophoretic Transfer Cell were purchased from Bio-Rad (Hercules, CA). Enhanced chemiluminescence Plus (ECL Plus) was obtained from Amersham Biosciences (Buckinghamshire, England), and cAMP kits were supplied by Cayman Chemicals (Ann Arbor, MI). All other chemicals were purchased from Sigma.

The pcDNA3 plasmid containing the cDNA of the rat TRPV1 was a kind gift from David Julius (University of California, San Francisco) 1. The TRPV1 sequence was released from the pcDNA3 plasmid using KpnI and XbaI, isolated with QIAEX II agarose gel extraction kit and subcloned into the plasmid pcDNA3.1 Hygro (+) (Invitrogen), which contains a hygromycin resistance sequence, using the corresponding KpnI and XbaI sites. The nucleotide sequence of the subcloned TRPV1 was compared against GeneBank.

A plasmid containing the cDNA encoding the murine FLAG-epitope-tagged μ opioid receptor (FLAG-MOP) as well as a geneticin-resistance gene was a generous gift from Dr Selena Bartlett (Ernest Gallo Clinic and Research Centre, San Francisco, California).

In brief, HEK293 cells were transfected with pcDNA3 containing FLAG-MOP using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol and stable FLAG-MOP expressants were selected with geneticin (0.5 mg/ml). Colonies originating from a single cell were selected and FLAG-MOP expression verified using immunohistochemistry and Western Blotting and a single clone was selected for transfection of the TRPV1. In a similar manner the pcDNA3.1 Hygro (+)/TRPV1 was transfected into the stable FLAG-MOP expressant. In order to select cells stably expressing functional TRPV1, cells were selected using hygromycin (75 μg/ml) and colonies of approximately 500 cells were isolated, plated on 96 well plates and further screened for functional TRPV1 expression by measuring Ca²⁺ responses after injection of capsaicin (10 μM). TRPV1 expression was verified by Western blotting. Double stable FLAG-MOP/TRPV1 expressants were split every 4–6 days using trypsin/EDTA and maintained at 37°C in a 5% humidified CO₂ incubator in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine, pyridoxine and 110 mg/ml sodium pyruvate supplemented with geneticin (0.2 mg/ml) and hygromycin (50 μg/ml).

For detection of MOP and TRPV1 receptor proteins, stable FLAG-MOP/TRPV1 double expressants were plated on 10 cm dishes and grown to confluency before sample preparation with ice cold lysis buffer (NaCl 100 mM; Igepal 1%, Na deoxycholate 0.5%; Tris pH 8.0 50 mM) containing complete protease inhibitor mix (Roche, Castle Hill, NSW). Cells were removed from the culture dish with lysis buffer, homogenised by vortexing and incubated for 20 min on ice. After centrifugation at 6000 × g at 4°C for 20 min, protein estimation of the supernatant was performed according to manufacturer's instructions using a protein estimation kit (BioRad). Protein (20 μg) was precipitated by boiling at 90°C for 2 min and separated on a 7.5% SDS PAGE gel. Proteins were transferred to nitrocellulose membrane and blocked overnight at 4°C in blocking buffer (130 mM NaCl, 2.7 mM KCl, 10 mM NaH₂PO₄, 1.8 mM KH₂PO₄, 0.1% Tween-20 and 5% low-fat skim milk powder), followed by incubation for 1 h with anti-TRPV1 antibody (1:1000) for detection of TRPV1 protein or anti-FLAG M2 antibody (1:330) for detection of FLAG-MOP protein. For TRPV1 immunoblots, an anti-β-actin antibody (1:10 000) was also included for visualisation of β-actin to serve as a loading control (Data not shown). After a wash with blocking buffer, blots were incubated with appropriate secondary horseradish peroxidase (hrp)-conjugated antibody (1:5000; Zymed) in blocking buffer for 1 h and processed using ECL Plus for visualization by exposure to Eastman Kodak XAR film.

For the cAMP accumulation assay, FLAG-MOP/TRPV1 double expressants were plated on 12 well plates and grown to 90% confluency. Twenty-four hours prior to sample preparation, media was changed to DMEM supplemented with 0.5% heat-inactivated FBS. On the day of sample preparation, cells were washed with DMEM to remove serum and incubated with serum-free DMEM containing the phosphodiesterase inhibitor IBMX (100 μM) for 30 min, to which morphine (1 μM) was then added and cells incubated for a further 15 min. Following this, FSK (50 μM) was added to the wells and the cells incubated for 15 min to stimulate cAMP production. DMSO alone was used as a vehicle control. After incubation, reactions were terminated by aspiration of the medium and addition of 0.1 M HCl followed by 20 min incubation at room temperature. After centrifugation of the cell samples at 10,000 × g for 10 min, the protein content of the supernatant was assessed and the samples were diluted to protein concentrations of 20 μg/ml using enzyme immunoassay (EIA) buffer supplied with the cAMP EIA kit. Intracellular cAMP levels were measured with a competitive cAMP EIA kit in triplicate as per manufacturer's instructions.

Ca²⁺ responses of FLAG-MOP/TRPV1 HEK293 cells were assessed using the fluorescent Ca²⁺ probe Fluo-3. FLAG-MOP/TRPV1 expressants were plated on poly-D-lysine (PDL)-coated 96-well plates at a cell density of approximately 3–4 × 10⁵ cells/ml 3 to 5 days prior to experiments and used at 70–90% confluency. Cells were loaded with Fluo-3 AM (6 μM) in loading buffer (pH 7.4, composition as PSS plus 3 mg/ml bovine serum albumin (BSA)) for 20–30 min at 37°C. Wells containing FLAG-MOP/TRPV1 double expressants were then washed three times with 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 min with PSS or opioid followed by 15 min of incubation with PSS or kinase activators with opioid as appropriate. Pre-incubation steps and Ca²⁺ imaging was carried out at 29°C in order to avoid subcellular dye compartmentalization while maintaining constant experimental conditions. Capsaicin and other drugs were injected from a 10 × concentrated stock in order to achieve the required well concentration.

Fluo-3 loaded cells were excited at 420 nm and emission was recorded at 510 nm using a NOVOstar fluorescence microplate reader (BMG Victoria, Australia). Fluorescent emission readings were recorded every 0.5 s. Raw fluorescence data were corrected by subtracting the average fluorescence from 4 recordings just prior to addition of agonist from all subsequent time points. This value was then divided by the average fluorescence prior to addition of agonist to yield ΔF/F values 585960. For dose-response curves, maximum ΔF/F values from the response after addition of agonist were plotted against agonist concentration and a 4-parameter logistic Hill equation was fitted to the data using GraphPad Prism (San Diego, California). IBMX (100 μM) was contained in all experiments utilizing FSK and 8-Br-cAMP as well as in appropriate controls to exclude any effect of morphine on IBMX. All experiments were designed to include control experiments on the same plate as treated cells.

DRGs were collected from adult male Wistar rats (200–350 g). The animals were kept in a controlled environment at a temperature of 22 ± 0.5°C, relative humidity of 40–60% and a 12 h (6 am–6 pm) light-dark cycle with free access to standard lab chow and tap water. Animals were sedated by inhalation of 50% O₂/50% CO₂ and sacrificed by asphyxiation with 100% CO₂. DRGs were collected from the rat spinal cord under sterile conditions from level T12 to S2. Harvested DRGs were dissociated in media containing trypsin (0.5 mg/ml), collagenase (1 mg/ml) and DNAse (1 μg/ml) and incubated at 37°C and 5% CO₂ for 45 to 60 min. Trypsin-chymotrypsin inhibitor (0.1 mg/ml) was then added and cell suspension centrifuged at 200 rpm for 10 min. The cell pellet was resuspended in 2 ml of warm medium consisting of DMEM supplemented with glutamine (4 mM), 10% horseserum, 10% foetal bovine serum, penicillin streptomycin (2 mM) and NGF (nerve growth factor) (100 ng/ml). Cells were plated on 25 mm PDL-coated glass coverslips at a density of 75,000 to 200,000 cells per coverslip. After 1 h, the medium was changed to Neurobasal-A medium supplemented with glutamine (4 mM), NGF (100 ng/ml) and B27 (20 μl/ml). Media was replaced every 2 days and experiments were performed after 3–6 days in culture. Cell cultures were confirmed visually to have characteristic DRG neuronal morphology combined with positive staining for synaptophysin.

Cells plated on 25 mm coverslips were washed twice in PBS (mM: NaCl 137; KCl 2.7; NaH₂PO₄ 10; KH₂PO₄ 1.8) for 10 min before being fixed with 4% paraformaldehyde for 20–30 min at room temperature. Cells were permeabilized with 0.2% Triton X in PBS for 10 min and blocked by immersion in PBS with 3% BSA for 30 min. Cells were washed thoroughly with PBS between all incubation steps. All antibodies were prepared in PBS containing 3% BSA. For DRG neurones, guinea pig anti-MOP primary antibody (1:5000) was applied for 1 h, while for FLAG-MOP/TRPV1 transfected HEK cells, mouse anti-FLAG M2 primary antibody (1:500) was applied for 1 h, followed by FITC-labelled anti-guinea pig or FITC-labelled anti-mouse secondary antibody (1:300) as appropriate in low light for 30 min. Cells were then incubated with rabbit anti-rat TRPV1 primary antibody (1:4000) for 1 h, followed by incubation with anti-rabbit Cy3-conjugated secondary antibody (1:300) under low light for 30 min. Subsequent incubation of coverslips with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; 300 nM) for 1–5 min was utilized to visualize cellular DNA. Specificity of the TRPV1 antibody was previously demonstrated 54 and confirmed here through lack of non-specific binding in cultured DRG neurones as well as double stable HEK293 cells (Figure 1F and 1G). Specificity of the MOP antibody was demonstrated by dual labelling of stable FLAG-MOP expressants with anti-MOP and anti-FLAG M2 antibodies, which showed excellent overlay (data not shown). Cells were thoroughly rinsed with PBS and distilled water and mounted using SlowFade AntiFade before viewing randomly selected fields of view on a Nikon Eclipse TE 300 inverted fluorescent microscope (excitation 488 nm, emission 520 nm for FITC; excitation 540 nm, emission 570 nm for Cy3 and excitation 364 nm, emission 454 nm for DAPI). Pictures were recorded using MetaFluor (Molecular Devices, Downington, PA) imaging software and processed using Paintshop Pro for Windows (Microsoft).

Data are presented as mean ± standard error of the mean (SEM) of at least two to three independent experiments. Fitting of 4-parameter Hill equations and statistical analysis were carried out using GraphPad Prism Version 4 (San Diego, CA). Statistical significance was determined using an unpaired, two-tailed Student's t-test unless otherwise stated with statistical significance defined as p < 0.05.