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 .