Modulation of G-protein activation, calcium currents and opioid receptor phosphorylation by the pH-dependent antinociceptive agonist NFEPP.

N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide is a newly-designed pain killer selectively activating G-protein-coupled mu-opioid receptors (MOR) in acidic injured tissues, and therefore devoid of central side effects which are typically elicited at normal pH values in healthy tissues. However, the neuronal mechanisms underlying NFEPP's antinociceptive effects were not examined in detail so far. Voltage-dependent Ca2+ channels (VDCCs) in nociceptive neurons play a major role in the generation and inhibition of pain. In this study, we focused on the effects of NFEPP on calcium currents in rat dorsal root ganglion (DRG) neurons. The inhibitory role of the G-protein subunits Gi/o and Gßγ on VDCCs was investigated using the blockers pertussis toxin and gallein, respectively. GTPγS binding, calcium signals and MOR phosphorylation were also investigated. All experiments were performed at acidic and normal pH values using NFEPP in comparison to the conventional opioid agonist fentanyl. At low pH, NFEPP produced more efficient G-protein activation in transfected HEK293 cells and significantly reduced VDCCs in depolarized DRG neurons. The latter effect was mediated by Gßγ subunits, and NFEPP-mediated MOR phosphorylation was pH-dependent. Fentanyl's responses were not affected by pH changes. Our data indicate that NFEPP-induced MOR signaling is more effective at low pH and that the inhibition of calcium channels in DRG neurons underlies NFEPP's antinociceptive actions.

Transient Receptor Potential Vanilloid 1 Signaling Is Independent on Protein Kinase A Phosphorylation of Ankyrin-Rich Membrane Spanning Protein.

The sensory ion channel transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in small to medium sized dorsal root ganglion neurons, which are involved in the transfer of acute noxious thermal and chemical stimuli. The Ankyrin-rich membrane spanning protein (ARMS) interaction with TRPV1 is modulated by protein kinase A (PKA) mediating sensitization. Here, we hypothesize that PKA phosphorylation sites of ARMS are crucial for the modulation of TRPV1 function, and that the phosphorylation of ARMS is facilitated by the A-kinase anchoring protein 79 (AKAP79). We used transfected HEK293 cells, immunoprecipitation, calcium flux, and patch clamp experiments to investigate potential PKA phosphorylation sites in ARMS and in ARMS-related peptides. Additionally, experiments were done to discriminate between PKA and protein kinase D (PKD) phosphorylation. We found different interaction ratios for TRPV1 and ARMS mutants lacking PKA phosphorylation sites. The degree of TRPV1 sensitization by ARMS mutants is independent on PKA phosphorylation. AKAP79 was also involved in the TRPV1/ARMS/PKA signaling complex. These data show that ARMS is a PKA substrate via AKAP79 in the TRPV1 signaling complex and that all four proteins interact physically, regulating TRPV1 sensitization in transfected HEK293 cells. To assess the physiological and/or therapeutic significance of these findings, similar investigations need to be performed in native neurons and/or in vivo.

Interleukin-4 Induces the Release of Opioid Peptides from M1 Macrophages in Pathological Pain.

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), ß-endorphin and dynorphin A 1-17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca2+ chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca2+-mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue.SIGNIFICANCE STATEMENT Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1ß, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca2+-dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4-opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects.

Patch Clamp Analysis of Opioid-Induced Kir3 Currents in Mouse Peripheral Sensory Neurons Following Nerve Injury.

Patch clamp is an electrophysiological technique that allows to analyze the activity of ion channels in neurons. In this chapter, we provide a detailed description of patch clamp protocol to measure the effect of a µ-opioid receptor agonist on the activity of G protein-coupled inwardly rectifying potassium (GIRK or Kir3) channels. This is performed in peripheral sensory neurons isolated from dorsal root ganglia (DRG) of mice without or with a chronic constriction injury (CCI) of the sciatic nerve, which models neuropathic pain. We describe the induction of the CCI , isolation and culture of DRG neurons, performance of the patch clamp recordings, and identification of opioid-responding neurons.

A low pKa ligand inhibits cancer-associated pain in mice by activating peripheral mu-opioid receptors.

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC50 of NFEPP to displace bound [3H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30-100 nmol/kg) or fentanyl (17-30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

Author Correction: pKa of opioid ligands as a discriminating factor for side effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

pKa of opioid ligands as a discriminating factor for side effects.

The non-selective activation of central and peripheral opioid receptors is a major shortcoming of currently available opioids. Targeting peripheral opioid receptors is a promising strategy to preclude side effects. Recently, we showed that fentanyl-derived µ-opioid receptor (MOR) agonists with reduced acid dissociation constants (pKa) due to introducing single fluorine atoms produced injury-restricted antinociception in rat models of inflammatory, postoperative and neuropathic pain. Here, we report that a new double-fluorinated compound (FF6) and fentanyl show similar pKa, MOR affinity and [35S]-GTPγS binding at low and physiological pH values. In vivo, FF6 produced antinociception in injured and non-injured tissue, and induced sedation and constipation. The comparison of several fentanyl derivatives revealed a correlation between pKa values and pH-dependent MOR activation, antinociception and side effects. An opioid ligand's pKa value may be used as discriminating factor to design safer analgesics.

Modulation of µ-opioid receptor activation by acidic pH is dependent on ligand structure and an ionizable amino acid residue.

BACKGROUND AND PURPOSE: Adverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on µ-opioid receptor binding and signalling induced by the µ-opioid receptor ligands fentanyl, DAMGO, and naloxone. EXPERIMENTAL APPROACH: HEK 293 cells stably transfected with µ-opioid receptors were used to study opioid ligand binding, [35 S]-GTPγS binding, and cAMP reduction at physiological and acidic pH. We used µ-opioid receptors mutated at H2976.52 to A (MOR-H2976.52 A) to delineate ligand-specific interactions with H2976.52 . KEY RESULTS: Low pH and the mutant receptor MOR-H2976.52 A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G-protein activation were decreased under these conditions. Fentanyl-induced signalling was not influenced by pH and largely independent of H2976.52 . CONCLUSIONS AND IMPLICATIONS: Our investigations indicate that low pH selectively impairs µ-opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52 . We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52 .