The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway
© Vetter et al; licensee BioMed Central Ltd. 2006
Received: 03 April 2006
Accepted: 16 July 2006
Published: 16 July 2006
The vanilloid receptor 1 (TRPV1) is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA) pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described.
In the present studies we investigated the hypothesis that the μ opioid receptor (MOP) agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+) responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine.
Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1.
The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.
Peripheral sensitization can contribute to the development of persistent pain and involves numerous cellular processes. An important receptor in this process is the vanilloid receptor 1 or TRPV1. The TRPV1 is a nociceptive calcium (Ca2+) channel that is activated by capsaicin, the pungent constituent of chilli peppers, as well as protons and heat . The TRPV1 is mainly expressed on nociceptive peripheral neurones and appears to be critical in the development of inflammatory hyperalgesia [1, 2], as axonal transport of TRPV1 mRNA as well as TRPV1 protein expression are significantly increased in inflamed tissues [3, 4], while mice lacking the TRPV1 develop less thermal hyperalgesia  and TRPV1 antagonists reversed thermal hyperalgesia in inflammation .
Phosphorylation of the TRPV1 by numerous kinases, including cAMP-dependent protein kinase A (PKA), can regulate function of the receptor [6–8]. cAMP levels are elevated in inflamed tissues [9, 10] and the cAMP/PKA pathway appears to be essential in sensitizing inflammatory nociception and contributes to the development of inflammatory hyperalgesia induced by proinflammatory mediators such as prostaglandin E2 (PGE2) [9, 11]. In line with reported sensitization of the TRPV1 in such inflammatory conditions [3, 4], PKA-mediated phosphorylation of the TRPV1 occurs in neurones as well as heterologous expression systems treated with adenylate cyclase activators such as forskolin, or direct PKA activators such as 8-Br-cAMP; while PKA is also involved in PGE2- and anandamide-mediated TRPV1 sensitization as well as potentiation of capsaicin responses through the metabotropic glutamate receptor 5 [6, 7, 12–17]. Although sensitization of TRPV1 responses is important, the concept of preventing sensitization may also be important in modulating signalling through the TRPV1. However, few studies have investigated pathways that interact with the TRPV1 to prevent sensitization [12, 14].
As a prototypical G-protein coupled receptor that produces analgesic effects upon activation , one such potential modulator is the μ opioid receptor or MOP. Direct G-protein coupled effects of opioid receptors include activation of inwardly rectifying potassium channels, inhibition of voltage-dependent Ca2+ channels as well as inhibition of adenylate cyclase . The present study describes the potential role of opioids, in particular the MOP agonist morphine, as modulators of TRPV1 responses by utilizing a HEK293 (human embryonic kidney) cell line stably expressing both TRPV1 and MOP and assesses the possible role of PKA in this modulation. Morphine did not affect unpotentiated TRPV1 responses nor TRPV1 responses potentiated by the direct PKA activator 8-Br-cAMP or the protein kinase C (PKC) activator Phorbol-12-myristate-13-acetate. However, TRPV1 responses potentiated by forskolin were modulated by morphine, highlighting the potential for endogenous opioid modulation of TRPV1 responses potentiated by the PKA pathway in inflammation.
FLAG-MOP/TRPV1 double stable HEK293 cells express functional TRPV1 and MOP
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 [19–21]. 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.
Morphine inhibits FSK-stimulated cAMP production in stable FLAG-MOP/TRPV1 HEK293 cell lines
Morphine reduces TRPV1-mediated capsaicin-induced Ca2+ in the presence of FSK through activation of MOP
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).
Morphine does not inhibit capsaicin responses potentiated by 8-Br-cAMP
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.
Morphine does not inhibit capsaicin responses potentiated by PMA
Several papers have reported potentiation of TRPV1 responses through PKC-mediated pathways [16, 22, 23], and PKC-potentiated TRPV1 responses have been shown to contribute to diabetic neuropathy . 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).
TRPV1 and MOP are co-expressed in cultured DRG neurones
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.
The results reported in this study provide the first evidence for a functional interaction between the MOP and the TRPV1 and indicate that this interaction likely occurs via modulation of PKA-augmented TRPV1 responses. These results may have implications in inflammatory pain.
For both the MOP and TRPV1, transfected HEK-293 cells are a widely used model for assessing receptor functions as well as interactions with other receptors [12, 16, 28–31]. Using a heterologous expression system, we showed that morphine does not inhibit capsaicin-induced TRPV1 responses under basal conditions. However, morphine inhibits FSK-potentiated TRPV1 responses through action at the MOP receptor, as indicated by reversal with the opioid receptor antagonist naloxone. This inhibition is likely dependent upon modulation of cAMP-dependent PKA, as morphine does not affect capsaicin responses potentiated by the direct PKA activator 8-Br-cAMP or the PKC activator PMA.
The antinociceptive actions of opioids are more efficacious in inflammation; and MOP as well as TRPV1 expression are increased in inflamed tissues, along with increased cAMP levels in these tissues [9, 32, 33]. While binding affinities of MOP agonists remain unchanged, the efficacy of G-protein coupling is increased during inflammation . In addition, opioid receptor expression is markedly upregulated as early as one day after induction of inflammation, and this increase in expression is paralleled by an increase in inhibition of neuronal Ca2+ responses [34, 35]. Due to disruption of the perineurium in inflammation, opioids have improved access to peripheral nerve endings on these inflamed tissues . These changes highlight not only the importance of inflammatory sensitization in altering nociceptive receptor function but also the importance of sensitization in possibly increasing the ability of MOP to inhibit these sensitized inflammatory nociceptive responses. However, molecular pathways involved in this increased efficacy of opioids in inflammation have not been systematically assessed to date.
In peripheral sites of inflammation, opioids have been shown to inhibit neurogenic inflammation by decreasing the release of substance P from peripheral afferent terminals . Notably, activation of the TRPV1 causes SP release and neurogenic inflammation  and opioids have been shown to inhibit capsaicin-evoked substance P release .
The specific MOP agonist endomorphine as well as the clinically useful MOP agonist morphine have been shown to produce anti-inflammatory effects in animal models and human studies when injected directly into inflamed tissues [40–44]. Importantly, activation of the TRPV1 by capsaicin induces hyperalgesia that can, dose-dependently and in a naloxone-sensitive manner, be inhibited by peripherally applied μ- and κ-opioid agonists [45–47], although the mechanisms involved in opioid-inhibition of TRPV1 responses have not been elucidated to date. Our results present a novel molecular basis for the anti-inflammatory action of peripheral opioids through a functional interaction of the MOP and PKA-sensitized TRPV1.
Several receptors have been reported to functionally interact with the TRPV1 [6, 12, 16, 17, 28, 48]. Most of these receptors effect potentiation of TRPV1 responses through activation of second messenger systems, resulting in activation of PKC and cAMP-dependent PKA pathways [6, 12, 16, 17, 28, 48]. Specifically, Insulin Growth Factor-1, insulin, bradykinin, nerve growth factor and prostaglandins can sensitize the TRPV1 through PKC-mediated phosphorylation, while PKA is involved in PGE2- and anandamide-mediated TRPV1 sensitization as well as potentiation of capsaicin responses through the metabotropic glutamate receptor 5 [6, 16, 17, 28, 48]. The PKA pathway has been proposed to be involved in the development of inflammatory hyperalgesia [9, 11]. cAMP levels are elevated in inflamed tissues [9, 10] and PKA or adenylate cyclase activators lower nociceptive thresholds while PKA inhibitors can be anti-hyperalgesic . The cAMP/PKA pathway appears to be essential in sensitizing inflammatory nociception and contributes to the development of inflammatory hyperalgesia induced by proinflammatory mediators such as prostaglandin E2 (PGE2) [9, 11].
Activation of peripheral group II metabotropic glutamate receptors inhibits potentiation of capsaicin responses by both PGE2 and FSK, but not potentiation by the direct PKA activator 8-Br-cAMP , consistent with our findings with MOP inhibiting TRPV1 in the presence of FSK. Although there are no previous studies of opioid receptors altering PKA-sensitization of capsaicin responses, the G-protein coupled cannabinoid receptor 1 (CB1) inhibits FSK-potentiated capsaicin responses in a PKA-dependent manner [12, 50]. Importantly, while anandamide and other CB1 agonist can directly activate the TRPV1 under basal conditions and cause TRPV1-mediated Ca2+ influx [12, 50], our results showed that opioids did not affect capsaicin-induced TRPV1 responses under basal conditions. Our results suggest that activation of opioid receptors could be utilized to prevent sensitization of TRPV1 receptors by cAMP-dependent PKA in inflammation without the potential to activate unpotentiated TRPV1.
Activation of opioid receptors decreases cAMP levels and inhibition of NMDA currents by opioids occurs in a PKA-dependent manner through inhibition of adenylate cyclase . Opioid modulation of TRPV1 responses may thus occur in inflamed tissues, where cAMP levels are elevated  through inhibition of adenylate cyclase and a subsequent inhibition of potentiation of TRPV1 responses by cAMP-dependent PKA. The results presented here support the hypothesis that the MOP and TRPV1 can functionally interact through a cAMP-dependent PKA pathway, as morphine inhibited FSK-potentiated capsaicin responses but not capsaicin responses in the absence of FSK. This is the first demonstration of a functional interaction between the TRPV1 and the MOP at the molecular level, and the interaction demonstrated here may be of significance in the treatment of inflammatory pain. In inflammation, when signalling through the TRPV1 is enhanced through increased PKA activity, activation of peripheral MOP could be utilised to prevent or modify TRPV1 sensitization. Moreover, while PKA pathways have been recognised to contribute to the development of inflammatory hyperalgesia, PKC pathways and in particular PKC-mediated phosphorylation have been postulated to contribute to the development of neuropathic hyperalgesia and allodynia [24, 52]. We did not observe morphine modulation of PKC-potentiated TRPV1 responses; thus, the differential involvement of PKA and PKC in neuropathic and inflammatory pain may provide a mechanistic explanation for the predominant efficacy of morphine in inflammatory but not neuropathic pain states.
The time-dependency of morphine-inhibition of FSK-potentiated capsaicin responses supports the notion that, via activation of the MOP, and subsequent negative coupling to adenylate cyclase through G proteins , morphine can prevent sensitization of capsaicin responses through the cAMP/PKA pathway. cAMP assays performed on our FLAG-MOP/TRPV1 cell line showed that while incubation with morphine significantly reduced FSK-stimulated cAMP production, morphine at the concentrations used here, did not reverse cAMP levels to unstimulated levels. Accordingly, morphine did not completely inhibit FSK-potentiation of TRPV1-mediated Ca2+ responses.
To rule out the possibility that morphine modulates FSK-potentiated capsaicin responses through action on phosphodiesterase or PKA and other downstream effectors, we used 8-Br-cAMP, a direct PKA activator, to potentiate capsaicin responses. While 8-Br-cAMP potentiated capsaicin responses similar to FSK, morphine did not modulate capsaicin responses potentiated by 8-Br-cAMP.
As the MOP is coupled negatively to adenylate cyclase and thus indirectly to PKA [18, 51], direct activation of PKA and thus potentiation of capsaicin responses would negate the inhibitory effect of opioids on adenylate cyclase. These results indicate that morphine modulates FSK-potentiated capsaicin responses by inhibiting adenylate cyclase.
Several studies show expression of the TRPV1 and MOP on peripheral neurones as well as DRG [53, 54]. More specifically, expression of TRPV1 has been reported in a small subset of trk-A and IB4-positive DRG neurons [1, 2, 55]. In addition, the number of TRPV1-expressing neurons increased after inflammation , with NGF reportedly regulating TRPV1 expression in trk-A-positive neurons .
While the DRG culture conditions utilised in our study included NGF and may therefore represent increased expression of TRPV1 in trk-A-expressing fibres, our findings are nonetheless relevant as increased NGF levels occur physiologically during inflammation . Our finding of significant co-expression of TRPV1 and MOP in cultured DRG neurons was further confirmed in a recently published study assessing TRPV1 and MOP expression in both L4 and L5 dorsal root ganglia as well as lumbar spinal cord of rats . While the proportion of neurons expressing TRPV1 and MOP was not quantified, similar to our results most neurons appeared to co-express TRPV1 and MOP with only relatively few cells expressing only one of the two receptors . In addition, comparable to the percentage of neurons co-expressing TRPV1 and MOP in our study, Sanderson Nydahl et al reported 83% of L4 and L5 DRGs to co-express the MOP agonist endomorphin and TRPV1 , This co-expression of TRPV1 and MOP in the majority cultured DRG neurones may provide the anatomical basis for functional interaction of the nociceptive TRPV1 and antinociceptive MOP receptors, thus allowing for local regulation and subsequent modification of nociception.
However, inhibition of capsaicin-induced hyperalgesia in vivo could rely not only on PKA mediated pathways but may also include inhibition of Ca2+ channels or the inhibition of substance P release .
Our studies demonstrate for the first time that in a heterologous expression system where MOP and TRPV1 are stably co-expressed, morphine can inhibit capsaicin responses when the cAMP pathway is activated and this occurs through opioid-modulation of adenylate cyclase and thus indirectly PKA-mediated TRPV1 sensitization. As TRPV1 is expressed peripherally [3, 4] and PKA-mediated sensitization occurs in these peripheral nociceptors via inflammatory mediators , targeting of non-central opioid receptors in inflammatory conditions may prevent peripheral sensitization and thus contribute to analgesia. Moreover, increased MOP expression and function during inflammation may contribute to local regulation of sensitization of nociceptors. This interaction may present the molecular basis for endogenous anti-inflammatory effects of opioids.
Materials and methods
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.
Construction of the pcDNA3.1 Hygro(+)-TRPV1 plasmid
The pcDNA3 plasmid containing the cDNA of the rat TRPV1 was a kind gift from David Julius (University of California, San Francisco) . 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.
Generation of a HEK293 cell line stably expressing MOP and TRPV1
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).
SDS-PAGE and Western Blotting
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.
cAMP accumulation assay
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.
Microplate reader measurement of intracellular Ca2+ responses in FLAG-MOP/TRPV1 HEK293 cells
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 [58–60]. 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.
Dorsal Root Ganglion (DRG) cell culture
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.
TRPV1 and MOP receptor immunoreactivity in cultured DRG neurones and FLAG-MOP/TRPV1 transfected HEK293 cells
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  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.
8-bromoadenosine 3'5'-cyclic monophosphate
bovine serum albumin
- Ca2+ :
Dulbecco's modified eagles medium
dorsal root ganglion
- Fluo-3 AM:
human embryonic kidney cells
μ opioid receptor
nerve growth factor
phosphate buffered solution
- PGE2 :
protein kinase A
protein kinase C
physiological salt solution
transient receptor potential vanilloid receptor 1.
This work was supported by a National Health and Medical Research Council NHMRC Dora Lush Scholarship.
- Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997, 389: 816–824. 10.1038/39807PubMedView Article
- Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D: Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000, 288: 306–313. 10.1126/science.288.5464.306PubMedView Article
- Amaya F, Oh-hashi K, Naruse Y, Iijima N, Ueda M, Shimosato G, Tominaga M, Tanaka Y, Tanaka M: Local inflammation increases vanilloid receptor 1 expression within distinct subgroups of DRG neurons. Brain Res 2003, 963: 190–196. 10.1016/S0006-8993(02)03972-0PubMedView Article
- Tohda C, Sasaki M, Konemura T, Sasamura T, Itoh M, Kuraishi Y: Axonal transport of VR1 capsaicin receptor mRNA in primary afferents and its participation in inflammation-induced increase in capsaicin sensitivity. J Neurochem 2001, 76: 1628–1635. 10.1046/j.1471-4159.2001.00193.xPubMedView Article
- Pomonis JD, Harrison JE, Mark L, Bristol DR, Valenzano KJ, Walker K: N-(4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: II. in vivo characterization in rat models of inflammatory and neuropathic pain. J Pharmacol Exp Ther 2003, 306: 387–393. 10.1124/jpet.102.046268PubMedView Article
- De Petrocellis L, Harrison S, Bisogno T, Tognetto M, Brandi I, Smith GD, Creminon C, Davis JB, Geppetti P, Di Marzo V: The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase. J Neurochem 2001, 77: 1660–1663. 10.1046/j.1471-4159.2001.00406.xPubMedView Article
- Lopshire JC, Nicol GD: The cAMP transduction cascade mediates the prostaglandin E2 enhancement of the capsaicin-elicited current in rat sensory neurons: whole-cell and single-channel studies. J Neurosci 1998, 18: 6081–6092.PubMed
- Mohapatra DP, Nau C: Desensitization of capsaicin-activated currents in the vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein kinase pathway. J Biol Chem 2003, 278: 50080–50090. 10.1074/jbc.M306619200PubMedView Article
- Malmberg AB, Brandon EP, Idzerda RL, Liu H, McKnight GS, Basbaum AI: Diminished inflammation and nociceptive pain with preservation of neuropathic pain in mice with a targeted mutation of the type I regulatory subunit of cAMP-dependent protein kinase. J Neurosci 1997, 17: 7462–7470.PubMed
- Elattar TM, Lin HS: The relationship between inflammation and cAMP level in human gingiva. J Dent Res 1981, 60: 674–676.PubMedView Article
- Naik SR: Increased cyclic AMP-phosphodiesterase activity during inflammation and its inhibition by anti-inflammatory drugs. Eur J Pharmacol 1984, 104: 253–259. 10.1016/0014-2999(84)90400-XPubMedView Article
- Hermann H, De Petrocellis L, Bisogno T, Schiano Moriello A, Lutz B, Di Marzo V: Dual effect of cannabinoid CB1 receptor stimulation on a vanilloid VR1 receptor-mediated response. Cell Mol Life Sci 2003, 60: 607–616. 10.1007/s000180300052PubMedView Article
- Mohapatra DP, Nau C: Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase. J Biol Chem 2005, 280: 13424–13432. 10.1074/jbc.M410917200PubMedView Article
- Yang D, Gereau RW: Peripheral group II metabotropic glutamate receptors (mGluR2/3) regulate prostaglandin E2-mediated sensitization of capsaicin responses and thermal nociception. J Neurosci 2002, 22: 6388–6393.PubMed
- Rathee PK, Distler C, Obreja O, Neuhuber W, Wang GK, Wang SY, Nau C, Kress M: PKA/AKAP/VR-1 module: A common link of Gs-mediated signaling to thermal hyperalgesia. J Neurosci 2002, 22: 4740–4745.PubMed
- Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, Tominaga T, Narumiya S, Tominaga M: Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain 2005, 1: 3. 10.1186/1744-8069-1-3PubMed CentralPubMedView Article
- Hu HJ, Bhave G, Gereau RW: Prostaglandin and protein kinase A-dependent modulation of vanilloid receptor function by metabotropic glutamate receptor 5: potential mechanism for thermal hyperalgesia. J Neurosci 2002, 22: 7444–7452.PubMed
- Connor M, Christie MD: Opioid receptor signalling mechanisms. Clin Exp Pharmacol Physiol 1999, 26: 493–499. 10.1046/j.1440-1681.1999.03049.xPubMedView Article
- Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De Petrocellis L, Fezza F, Tognetto M, Petros TJ, Krey JF, Chu CJ, Miller JD, Davies SN, Geppetti P, Walker JM, Di Marzo V: An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc Natl Acad Sci U S A 2002, 99: 8400–8405. 10.1073/pnas.122196999PubMed CentralPubMedView Article
- Wisnoskey BJ, Sinkins WG, Schilling WP: Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry. Biochem J 2003, 372: 517–528. 10.1042/BJ20021574PubMed CentralPubMedView Article
- Witte DG, Cassar SC, Masters JN, Esbenshade T, Hancock AA: Use of a fluorescent imaging plate reader--based calcium assay to assess pharmacological differences between the human and rat vanilloid receptor. J Biomol Screen 2002, 7: 466–475. 10.1177/108705702237679PubMedView Article
- Bhave G, Hu HJ, Glauner KS, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW: Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Proc Natl Acad Sci U S A 2003, 100: 12480–12485. 10.1073/pnas.2032100100PubMed CentralPubMedView Article
- Wang Y KNWMWQJHATATRBPM: Interaction between PKCmu and the vanilloid receptor type1. J Biol Chem 2004, [Epub ahead of print].
- Hong S, Wiley JW: Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1. J Biol Chem 2005, 280: 618–627.PubMedView Article
- Liu M, Liu MC, Magoulas C, Priestley JV, Willmott NJ: Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons. J Biol Chem 2003, 278: 5462–5472. 10.1074/jbc.M209111200PubMedView Article
- Arvidsson U, Riedl M, Chakrabarti S, Lee JH, Nakano AH, Dado RJ, Loh HH, Law PY, Wessendorf MW, Elde R: Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 1995, 15: 3328–3341.PubMed
- Belanger S, Ma W, Chabot JG, Quirion R: Expression of calcitonin gene-related peptide, substance P and protein kinase C in cultured dorsal root ganglion neurons following chronic exposure to mu, delta and kappa opiates. Neuroscience 2002, 115: 441–453. 10.1016/S0306-4522(02)00452-9PubMedView Article
- Van Buren JJ, Bhat S, Rotello R, Pauza ME, Premkumar LS: Sensitization and translocation of TRPV1 by insulin and IGF-I. Mol Pain 2005, 1: 17. 10.1186/1744-8069-1-17PubMed CentralPubMedView Article
- Jordan BA, Trapaidze N, Gomes I, Nivarthi R, Devi LA: Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proc Natl Acad Sci U S A 2001, 98: 343–348. 10.1073/pnas.011384898PubMed CentralPubMed
- Raynor K, Kong H, Chen Y, Yasuda K, Yu L, Bell GI, Reisine T: Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors. Mol Pharmacol 1994, 45: 330–334.PubMed
- Gharagozlou P, Demirci H, David Clark J, Lameh J: Activity of opioid ligands in cells expressing cloned mu opioid receptors. BMC Pharmacol 2003, 3: 1. 10.1186/1471-2210-3-1PubMed CentralPubMedView Article
- Pol O, Puig MM: Expression of opioid receptors during peripheral inflammation. Curr Top Med Chem 2004, 4: 51–61. 10.2174/1568026043451519PubMedView Article
- Schafer M, Imai Y, Uhl GR, Stein C: Inflammation enhances peripheral mu-opioid receptor-mediated analgesia, but not mu-opioid receptor transcription in dorsal root ganglia. Eur J Pharmacol 1995, 279: 165–169. 10.1016/0014-2999(95)00150-JPubMedView Article
- Zollner C, Shaqura MA, Bopaiah CP, Mousa S, Stein C, Schafer M: Painful inflammation-induced increase in mu-opioid receptor binding and G-protein coupling in primary afferent neurons. Mol Pharmacol 2003, 64: 202–210. 10.1124/mol.64.2.202PubMedView Article
- Ji RR, Zhang Q, Law PY, Low HH, Elde R, Hokfelt T: Expression of mu-, delta-, and kappa-opioid receptor-like immunoreactivities in rat dorsal root ganglia after carrageenan-induced inflammation. J Neurosci 1995, 15: 8156–8166.PubMed
- Tong XJ, Hirai K, Shimada H, Mizutani Y, Izumi T, Toda N, Yu P: Sciatic nerve regeneration navigated by laminin-fibronectin double coated biodegradable collagen grafts in rats. Brain Res 1994, 663: 155–162. 10.1016/0006-8993(94)90473-1PubMedView Article
- Jin S, Lei L, Wang Y, Da D, Zhao Z: Endomorphin-1 reduces carrageenan-induced fos expression in the rat spinal dorsal horn. Neuropeptides 1999, 33: 281–284. 10.1054/npep.1999.0040PubMedView Article
- Rigoni M, Trevisani M, Gazzieri D, Nadaletto R, Tognetto M, Creminon C, Davis JB, Campi B, Amadesi S, Geppetti P, Harrison S: Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin. Br J Pharmacol 2003, 138: 977–985. 10.1038/sj.bjp.0705110PubMed CentralPubMedView Article
- Aimone LD, Yaksh TL: Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo. Peptides 1989, 10: 1127–1131. 10.1016/0196-9781(89)90003-XPubMedView Article
- Stein A, Yassouridis A, Szopko C, Helmke K, Stein C: Intraarticular morphine versus dexamethasone in chronic arthritis. Pain 1999, 83: 525–532. 10.1016/S0304-3959(99)00156-6PubMedView Article
- Gavalas A, Victoratos P, Yiangou M, Hadjipetrou-Kourounakis L, Rekka E, Kourounakis P: The anti-inflammatory effect of opioids. Int J Neurosci 1994, 74: 259–264.PubMedView Article
- Stein C, Machelska H, Schafer M: Peripheral analgesic and antiinflammatory effects of opioids. Z Rheumatol 2001, 60: 416–424. 10.1007/s003930170004PubMedView Article
- Binder W, Machelska H, Mousa S, Schmitt T, Riviere PJ, Junien JL, Stein C, Schafer M: Analgesic and antiinflammatory effects of two novel kappa-opioid peptides. Anesthesiology 2001, 94: 1034–1044. 10.1097/00000542-200106000-00018PubMedView Article
- Hong Y, Abbott FV: Peripheral opioid modulation of pain and inflammation in the formalin test. Eur J Pharmacol 1995, 277: 21–28. 10.1016/0014-2999(95)00045-MPubMedView Article
- Ko MC, Tuchman JE, Johnson MD, Wiesenauer K, Woods JH: Local administration of mu or kappa opioid agonists attenuates capsaicin-induced thermal hyperalgesia via peripheral opioid receptors in rats. Psychopharmacology (Berl) 2000, 148: 180–185. 10.1007/s002130050040View Article
- Barrett AC, Smith ES, Picker MJ: Capsaicin-induced hyperalgesia and mu-opioid-induced antihyperalgesia in male and female Fischer 344 rats. J Pharmacol Exp Ther 2003, 307: 237–245. 10.1124/jpet.103.054478PubMedView Article
- Wenk HN, Nannenga MN, Honda CN: Effect of morphine sulphate eye drops on hyperalgesia in the rat cornea. Pain 2003, 105: 455–465. 10.1016/S0304-3959(03)00260-4PubMedView Article
- Tang HB, Inoue A, Oshita K, Nakata Y: Sensitization of vanilloid receptor 1 induced by bradykinin via the activation of second messenger signaling cascades in rat primary afferent neurons. Eur J Pharmacol 2004, 498: 37–43. 10.1016/j.ejphar.2004.07.076PubMedView Article
- Taiwo YO, Levine JD: Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia. Neuroscience 1991, 44: 131–135. 10.1016/0306-4522(91)90255-MPubMedView Article
- Oshita K, Inoue A, Tang HB, Nakata Y, Kawamoto M, Yuge O: CB(1) cannabinoid receptor stimulation modulates transient receptor potential vanilloid receptor 1 activities in calcium influx and substance P Release in cultured rat dorsal root ganglion cells. J Pharmacol Sci 2005, 97: 377–385. 10.1254/jphs.FP0040872PubMedView Article
- Xie CW, Lewis DV: Involvement of cAMP-dependent protein kinase in mu-opioid modulation of NMDA-mediated synaptic currents. J Neurophysiol 1997, 78: 759–766.PubMed
- Yajima Y, Narita M, Shimamura M, Kubota C, Suzuki T: Differential involvement of spinal protein kinase C and protein kinase A in neuropathic and inflammatory pain in mice. Brain Res 2003, 992: 288–293. 10.1016/j.brainres.2003.08.042PubMedView Article
- Kulkarni-Narla A, Brown DR: Opioid, cannabinoid and vanilloid receptor localization on porcine cultured myenteric neurons. Neurosci Lett 2001, 308: 153–156. 10.1016/S0304-3940(01)01998-XPubMedView Article
- Guo A, Vulchanova L, Wang J, Li X, Elde R: Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. Eur J Neurosci 1999, 11: 946–958. 10.1046/j.1460-9568.1999.00503.xPubMedView Article
- Amaya F, Shimosato G, Nagano M, Ueda M, Hashimoto S, Tanaka Y, Suzuki H, Tanaka M: NGF and GDNF differentially regulate TRPV1 expression that contributes to development of inflammatory thermal hyperalgesia. Eur J Neurosci 2004, 20: 2303–2310. 10.1111/j.1460-9568.2004.03701.xPubMedView Article
- Chen SR, Pan HL: Loss of TRPV1-expressing sensory neurons reduces spinal mu opioid receptors but paradoxically potentiates opioid analgesia. J Neurophysiol 2006, 95: 3086–3096. 10.1152/jn.01343.2005PubMedView Article
- Sanderson Nydahl K, Skinner K, Julius D, Basbaum AI: Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat. Eur J Neurosci 2004, 19: 1789–1799. 10.1111/j.1460-9568.2004.03284.xPubMedView Article
- Grant ER, Dubin AE, Zhang SP, Zivin RA, Zhong Z: Simultaneous intracellular calcium and sodium flux imaging in human vanilloid receptor 1 (VR1)-transfected human embryonic kidney cells: a method to resolve ionic dependence of VR1-mediated cell death. J Pharmacol Exp Ther 2002, 300: 9–17. 10.1124/jpet.300.1.9PubMedView Article
- Tekkok S, Medina I, Krnjevic K: Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices. J Neurophysiol 1999, 81: 174–183.PubMed
- Takahashi A, Camacho P, Lechleiter JD, Herman B: Measurement of intracellular calcium. Physiol Rev 1999, 79: 1089–1125.PubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.