A-kinase anchoring protein 79/150 coordinates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor sensitization in sensory neurons.

Changes in sensory afferent activity contribute to the transition from acute to chronic pain. However, it is unlikely that a single sensory receptor is entirely responsible for persistent pain. It is more probable that extended changes to multiple receptor proteins expressed by afferent neurons support persistent pain. A-Kinase Anchoring Protein 79/150 (AKAP) is an intracellular scaffolding protein expressed in sensory neurons that spatially and temporally coordinates signaling events. Since AKAP scaffolds biochemical modifications of multiple TRP receptors linked to pain phenotypes, we probed for other ionotropic receptors that may be mediated by AKAP and contribute to persistent pain. Here, we identify a role for AKAP modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor (AMPA-R) functionality in sensory neurons. Pharmacological manipulation of distinct AMPA-R subunits significantly reduces persistent mechanical hypersensitivity observed during hyperalgesic priming. Stimulation of both protein kinases C and A (PKC, PKA, respectively) modulate AMPA-R subunit GluR1 and GluR2 phosphorylation and surface expression in an AKAP-dependent manner in primary cultures of DRG neurons. Furthermore, AKAP knock out reduces sensitized AMPA-R responsivity in DRG neurons. Collectively, these data indicate that AKAP scaffolds AMPA-R subunit organization in DRG neurons that may contribute to the transition from acute-to-chronic pain.

Raf kinase inhibitory protein reduces bradykinin receptor desensitization.

Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin-stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G-protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down-regulation increases BK receptor desensitization in real-time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2-sensitive G-protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.

Comparison of the Accuracy of Maxillary Positioning With Interim Splints Versus Patient-Specific Guides and Plates in Executing a Virtual Bimaxillary Surgical Plan.

PURPOSE: An extension of digital technology is to provide patient-specific hardware to reposition the first jaw in a bimaxillary case without the use of an intermediate splint. The purpose of our study was to determine if there were significant differences in maxillary repositioning using interim splints versus patient-specific guides and implants (PSIs) in executing a bimaxillary virtual surgical plan (VSP). MATERIALS AND METHODS: This is a retrospective cohort study of patients who underwent bimaxillary orthognathic surgery with interim splints or PSIs planned with VSP at our institution. The difference in maxillary positions from the VSP to the postoperative cone-beam computed tomography (CBCT) was evaluated in both groups. The primary predictor variable was the method by which the maxilla was repositioned (interim splint vs PSI). The primary outcome variable was the postoperative 3D position of the maxillary incisors and right and left first molars in the anteroposterior, transverse, and vertical dimensions. Differences in the planned and postoperative positions of the above landmarks in all three planes of space between the two groups were statistically analyzed. RESULTS: A total of 82 patients were included. 13 patients had their maxillae repositioned with an interim splint between the unoperated mandible and the mobile maxilla, and 69 patients had their maxilla repositioned using custom drill/cutting guides and a PSI. The mean difference between the planned and actual position of the maxilla in the PSI group was smaller than in the splint group. In the PSI group alone, vertical changes were accurate whether the maxilla was being superiorly or inferiorly repositioned. CONCLUSION: The use of a PSI provides more accurate maxillary repositioning during bimaxillary surgery than the use of an interim splint.

Sensitization of small-diameter sensory neurons is controlled by TRPV1 and TRPA1 association.

Unique features of sensory neuron subtypes are manifest by their distinct physiological and pathophysiological functions. Using patch-clamp electrophysiology, Ca2+ imaging, calcitonin gene-related peptide release assay from tissues, protein biochemistry approaches, and behavioral physiology on pain models, this study demonstrates the diversity of sensory neuron pathophysiology is due in part to subtype-dependent sensitization of TRPV1 and TRPA1. Differential sensitization is influenced by distinct expression of inflammatory mediators, such as prostaglandin E2 (PGE2), bradykinin (BK), and nerve growth factor (NGF) as well as multiple kinases, including protein kinase A (PKA) and C (PKC). However, the co-expression and interaction of TRPA1 with TRPV1 proved to be the most critical for differential sensitization of sensory neurons. We identified N- and C-terminal domains on TRPV1 responsible for TRPA1-TRPV1 (A1-V1) complex formation. Ablation of A1-V1 complex with dominant-negative peptides against these domains substantially reduced the sensitization of TRPA1, as well as BK- and CFA-induced hypersensitivity. These data indicate that often occurring TRP channel complexes regulate diversity in neuronal sensitization and may provide a therapeutic target for many neuroinflammatory pain conditions.

Dynamic Opioid Receptor Regulation in the Periphery.

Opioids serve a vital role in the current analgesic array of treatment options. They are useful in acute instances involving severe pain associated with trauma, surgery, and terminal diseases such as cancer. In the past three decades, multiple receptor isoforms and conformations have been reported throughout literature. Most of these studies conducted systemic analyses of opioid receptor function, often generalizing findings from receptor systems in central nervous tissue or exogenously expressing immortalized cell lines as common mechanisms throughout physiology. However, a culmination of innovative experimental data indicates that opioid receptor systems are differentially modulated depending on their anatomic expression profile. Importantly, opioid receptors expressed in the peripheral nervous system undergo regulation uncommon to similar receptors expressed in central nervous system tissues. This distinctive characteristic begs one to question whether peripheral opioid receptors maintain anatomically unique roles, and whether they may serve an analgesic advantage in providing pain relief without promoting addiction.

Identification of a signaling cascade that maintains constitutive δ-opioid receptor incompetence in peripheral sensory neurons.

µ-Opioid receptor (MOR) agonists are often used to treat severe pain but can result in adverse side effects. To circumvent systemic side effects, targeting peripheral opioid receptors is an attractive alternative treatment for severe pain. Activation of the δ-opioid receptor (DOR) produces similar analgesia with reduced side effects. However, until primed by inflammation, peripheral DOR is analgesically incompetent, raising interest in the mechanism. We recently identified a novel role for G-protein-coupled receptor kinase 2 (GRK2) that renders DOR analgesically incompetent at the plasma membrane. However, the mechanism that maintains constitutive GRK2 association with DOR is unknown. Protein kinase A (PKA) phosphorylation of GRK2 at Ser-685 targets it to the plasma membrane. Protein kinase A-anchoring protein 79/150 (AKAP), residing at the plasma membrane in neurons, scaffolds PKA to target proteins to mediate downstream signal. Therefore, we sought to determine whether GRK2-mediated DOR desensitization is directed by PKA via AKAP scaffolding. Membrane fractions from cultured rat sensory neurons following AKAP siRNA transfection and from AKAP-knock-out mice had less PKA activity, GRK2 Ser-685 phosphorylation, and GRK2 plasma membrane targeting than controls. Site-directed mutagenesis revealed that GRK2 Ser-685 phosphorylation drives the association of GRK2 with plasma membrane-associated DOR. Moreover, overexpression studies with AKAP mutants indicated that impaired AKAP-mediated PKA scaffolding significantly reduces DOR-GRK2 association at the plasma membrane and consequently increases DOR activity in sensory neurons without a priming event. These findings suggest that AKAP scaffolds PKA to increase plasma membrane targeting and phosphorylation of GRK2 to maintain DOR analgesic incompetence in peripheral sensory neurons.

Persistent Nociception Triggered by Nerve Growth Factor (NGF) Is Mediated by TRPV1 and Oxidative Mechanisms.

Nerve growth factor (NGF) is elevated in certain chronic pain conditions and is a sufficient stimulus to cause lasting pain in humans, but the actual mechanisms underlying the persistent effects of NGF remain incompletely understood. We developed a rat model of NGF-induced persistent thermal hyperalgesia and mechanical allodynia to determine the role of transient receptor potential vanilloid 1 (TRPV1) and oxidative mechanisms in the persistent effects of NGF. Persistent thermal hypersensitivity and mechanical allodynia require de novo protein translation and are mediated by TRPV1 and oxidative mechanisms. By comparing effects after systemic (subcutaneous), spinal (intrathecal) or hindpaw (intraplantar) injections of test compounds, we determined that TRPV1 and oxidation mediate persistent thermal hypersensitivity via peripheral and spinal sites of action and mechanical allodynia via only a spinal site of action. Therefore, NGF-evoked thermal and mechanical allodynia are mediated by spatially distinct mechanisms. NGF treatment evoked sustained increases in peripheral and central TRPV1 activity, as demonstrated by increased capsaicin-evoked nocifensive responses, increased calcitonin gene-related peptide release from hindpaw skin biopsies, and increased capsaicin-evoked inward current and membrane expression of TRPV1 protein in dorsal root ganglia neurons. Finally, we showed that NGF treatment increased concentrations of linoleic and arachidonic-acid-derived oxidized TRPV1 agonists in spinal cord and skin biopsies. Furthermore, increases in oxidized TRPV1-active lipids were reduced by peripheral and spinal injections of compounds that completely blocked persistent nociception. Collectively, these data indicate that NGF evokes a persistent nociceptive state mediated by increased TRPV1 activity and oxidative mechanisms, including increased production of oxidized lipid TRPV1 agonists.

ß-arrestin-2-biased agonism of delta opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1) in primary sensory neurons.

Despite advances in understanding the signaling mechanisms involved in the development and maintenance of chronic pain, the pharmacologic treatment of chronic pain has seen little advancement. Agonists at the mu opioid receptor (MOPr) continue to be vital in the treatment of many forms of chronic pain, but side-effects limit their clinical utility and range from relatively mild, such as constipation, to major, such as addiction and dependence. Additionally, chronic activation of MOPr results in pain hypersensitivity known as opioid-induced hyperalgesia (OIH), and we have shown recently that recruitment of ß-arrestin2 to MOPr, away from transient potential vanilloid eceptor type 1 (TRPV1) in primary sensory neurons contributes to this phenomenon. The delta opioid receptor (DOPr) has become a promising target for the treatment of chronic pain, but little is known about the effects of chronic activation of DOPr on nociceptor sensitivity and OIH. Here we report that chronic activation of DOPr by the DOPr-selective agonist, SNC80, results in the sensitization of TRPV1 and behavioral signs of OIH via ß-arrestin2 recruitment to DOPr and away from TRPV1. Conversely, chronic treatment with ARM390, a DOPr-selective agonist that does not recruit ß-arrestin2, neither sensitized TRPV1 nor produced OIH. Interestingly, the effect of SNC80 to sensitize TRPV1 is species-dependent, as rats developed OIH but mice did not. Taken together, the reported data identify a novel side-effect of chronic administration of ß-arrestin2-biased DOPr agonists and highlight the importance of potential species-specific effects of DOPr agonists.

Phosphorylation regulates TRPV1 association with ß-arrestin-2.

Post-translational modifications in TRPV1 (transient receptor potential vanilloid 1) play a critical role in channel activity. Phosphorylation of serine/threonine residues within the N- and C-termini of TRPV1 are implicated in receptor sensitization and activation. Conversely, TRPV1 desensitization occurs via a calcium-dependent mechanism and leads to receptor de-phosphorylation. Importantly, we recently demonstrated that TRPV1 association with ß-arrestin-2 is critical to receptor desensitization via its ability to scaffold the phosphodiesterase PDE4D5 to the receptor, regulating TRPV1 phosphorylation. In the present study, we demonstrate that phosphorylation of TRPV1 and ß-arrestin-2 regulates this association at the membrane. Under serum-free media conditions, we observed a significant decrease in TRPV1 and ß-arrestin-2 association in transfected CHO (Chinese-hamster ovary) cells. Pharmacological activation of the kinases PKA (protein kinase A) and PKC (protein kinase C) led to a robust increase in TRPV1 and ß-arrestin-2 association, whereas inhibition of PKA and PKC decreased association. Previously, we identified potential PKA residues (Ser(116), Thr(370)) in the N-terminus of TRPV1 modulated by ß-arrestin-2. In the present study we reveal that the phosphorylation status of Thr(370) dictates the ß-arrestin-2 and TRPV1 association. Furthermore, we demonstrate that CK2 (casein kinase 2)-mediated phosphorylation of ß-arrestin-2 at Thr(382) is critical for its association with TRPV1. Taken together, the findings of the present study suggest that phosphorylation controls the association of TRPV1 with ß-arrestin-2.

ß-Arrestin-2 desensitizes the transient receptor potential vanilloid 1 (TRPV1) channel.

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel activated by multiple stimuli and is implicated in a variety of pain disorders. Dynamic sensitization of TRPV1 activity by A-kinase anchoring protein 150 demonstrates a critical role for scaffolding proteins in nociception, yet few studies have investigated scaffolding proteins capable of mediating receptor desensitization. In this study, we identify ß-arrestin-2 as a scaffolding protein that regulates TRPV1 receptor activity. We report ß-arrestin-2 association with TRPV1 in multiple cell models. Moreover, siRNA-mediated knockdown of ß-arrestin-2 in primary cultures resulted in a significant increase in both initial and repeated responses to capsaicin. Electrophysiological analysis further revealed significant deficits in TRPV1 desensitization in primary cultures from ß-arrestin-2 knock-out mice compared with wild type. In addition, we found that ß-arrestin-2 scaffolding of phosphodiesterase PDE4D5 to the plasma membrane was required for TRPV1 desensitization. Importantly, inhibition of PDE4D5 activity reversed ß-arrestin-2 desensitization of TRPV1. Together, these results identify a new endogenous scaffolding mechanism that regulates TRPV1 ligand binding and activation.

A-kinase anchoring protein 150 mediates transient receptor potential family V type 1 sensitivity to phosphatidylinositol-4,5-bisphosphate.

A-kinase anchoring protein 150 (AKAP150) is a scaffolding protein that controls protein kinase A- and C-mediated phosphorylation of the transient receptor potential family V type 1 (TRPV1), dictating receptor response to nociceptive stimuli. The phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) anchors AKAP150 to the plasma membrane in naive conditions and also affects TRPV1 activity. In the present study, we sought to determine whether the effects of PIP(2) on TRPV1 are mediated through AKAP150. In trigeminal neurons and CHO cells, the manipulation of cellular PIP(2) led to significant changes in the association of AKAP150 and TRPV1. Following PIP(2) degradation, increased TRPV1:AKAP150 coimmunoprecipitation was observed, resulting in increased receptor response to capsaicin treatment. Phospholipase C activation in neurons isolated from AKAP150(-/-) animals indicated that PIP(2)-mediated inhibition of TRPV1 in the whole-cell environment requires expression of the scaffolding protein. Furthermore, the addition of PIP(2) to neurons isolated from AKAP150 wild-type mice reduced PKA sensitization of TRPV1 compared with isolated neurons from AKAP150(-/-) mice. These findings suggest that PIP(2) degradation increases AKAP150 association with TRPV1 in the whole-cell environment, leading to sensitization of the receptor to nociceptive stimuli.

Paroxetine increases delta opioid responsiveness in sensory neurons.

There are currently no Food and Drug Administration (FDA)-approved delta opioid receptor (DOR)-selective agonists, despite having fewer side effects in rodents and non-human primates compared to traditional mu opioid receptor (MOR) therapeutics (Vanderah, 2010). Targeting peripheral receptors is an attractive strategy to reduce abuse potential. However, peripheral opioid receptors do not readily respond to agonists unless primed by inflammation, which would limit their efficacy in non-inflammatory pain patients (Stein et al., 1989). It was recently identified that G protein-coupled receptor kinase 2 (GRK2) maintains DOR incompetence in non-inflamed nociceptors (Brackley et al., 2016; Brackley et al., 2017). Here, we report that paroxetine, a selective serotonin reuptake inhibitor and potent GRK2 inhibitor (Thal et al., 2012), reduces chronic GRK2 association with membrane DOR, thereby enhancing peripheral DOR-mediated analgesic competence in the absence of inflammation. Interestingly, paroxetine's effects on GRK2 in vivo are limited to peripheral tissues in the male rat. The effects of paroxetine on DOR competence are notably antagonized by GRK2 overexpression. This is the first study to suggest that paroxetine induces peripheral DOR analgesic competence through a GRK2-dependent mechanism, improving analgesic efficacy in non-inflamed tissue. Because paroxetine targets the protein that governs peripheral opioid receptor responsiveness, and does so in the absence of inflammation, we propose that paroxetine may be suitable as a co-therapy with peripherally-restrictive doses of opioids to improve analgesic efficacy in non-inflammatory pain conditions.Significance StatementOpioids that target MOR represent the gold-standard for analgesic healthcare, despite widespread abuse potential and the ongoing opioid-epidemic. Work herein uncovers the therapeutic potential of targeting peripheral DOR for analgesic utility with an FDA-approved GRK2 inhibitor paroxetine to boost efficacy and reduce side effect profiles. Analgesic pain management targeting DOR with increased efficacy through adjuvant paroxetine treatment could reduce over-reliance on MOR agonist opioids for pain relief and usher in new options for analgesia.

Outcomes of total joint alloplastic reconstruction in TMJ ankylosis.

OBJECTIVE: The purpose of this study was to evaluate subjective and objective outcomes in patients with temporomandibular joint (TMJ) ankylosis treated with TMJ alloplastic reconstruction (TMJR). STUDY DESIGN: All patients diagnosed with TMJ ankylosis that underwent TMJR at our institution between 2010 and 2019 were retrospectively reviewed. Patients were divided into 2 cohorts: bony and fibrous ankylosis. Subjective variables assessed were facial pain and headaches, TMJ pain, jaw function, diet, and disability. Objective variables assessed were maximum interincisal opening and lateral excursions. The Mann-Whitney test was employed to analyze subjective variables and an unpaired t-test was used to analyze the objective variables. P < .05 was considered statistically significant. RESULTS: Twenty-eight patients met the inclusion criteria (21 female, 7 male). The mean age at the time of surgery was 42 years, and the mean number of prior TMJ surgeries was 3. A total of 52 TMJRs were performed in the 28 patients, and the mean follow-up time was 46 months. All subjective variables were significantly improved, and the mean maximum interincisal opening increased from 16.9 mm to 37.25 mm. CONCLUSIONS: The results of the study demonstrate that TMJR is an effective and reliable method for the management of both fibrous and bony TMJ ankylosis.

Contribution of TRPV1-TRPA1 interaction to the single channel properties of the TRPA1 channel.

Several lines of evidence suggest that TRPA1 and TRPV1 mutually control the transduction of inflammation-induced noxious stimuli in sensory neurons. It was recently shown that certain TRPA1 properties are modulated by TRPV1. However, direct interaction between TRPA1 and TRPV1 as well as regulation of TRPA1 intrinsic characteristics by the TRPV1 channel have not been examined. To address these questions, we have studied a complex formation between TRPA1 and TRPV1 and characterized the influence of TRPV1 on single channel TRPA1-mediated currents. Co-immunoprecipitation analysis revealed direct interactions between TRPA1 and TRPV1 in an expression system as well as in sensory neurons. Data generated with total internal reflection fluorescence-based fluorescence resonance energy transfer indicate that a TRPA1-TRPV1 complex can be formed on the plasma membrane. The fluorescence resonance energy transfer interaction between TRPA1 and TRPV1 channels is as effective as for TRPV1 or TRPA1 homomers. Single channel analysis in a heterologous expression system and in sensory neurons of wild type and TRPV1 knock-out mice demonstrated that co-expression of TRPV1 with TRPA1 results in outward rectification of single channel mustard oil (I(MO)) current-voltage relationships (I-V) and substantial modulation of the open probability at negative holding potentials. TRPV1 also does not influence the characteristics of single channel I(MO) in Ca(2+)-free extracellular solution. However, association of TRPA1 with TRPV1 was not affected in Ca(2+)-free media. To assess a role of intracellular Ca(2+) in TRPV1-dependent modulation of TRPA1 modulation, the TRPA1-mediated single channel WIN55,212-2-gated current (I(WIN)) was recorded in inside-out configuration. Our data indicate that single channel properties of TRPA1 are regulated by TRPV1 independently of intracellular Ca(2+). In summary, our results support the hypothesis that TRPV1 and TRPA1 form a complex and that TRPV1 influences intrinsic characteristics of the TRPA1 channel.

AKAP150-mediated TRPV1 sensitization is disrupted by calcium/calmodulin.

BACKGROUND: The transient receptor potential vanilloid type1 (TRPV1) is expressed in nociceptive sensory neurons and is sensitive to phosphorylation. A-Kinase Anchoring Protein 79/150 (AKAP150) mediates phosphorylation of TRPV1 by Protein Kinases A and C, modulating channel activity. However, few studies have focused on the regulatory mechanisms that control AKAP150 association with TRPV1. In the present study, we identify a role for calcium/calmodulin in controlling AKAP150 association with, and sensitization of, TRPV1. RESULTS: In trigeminal neurons, intracellular accumulation of calcium reduced AKAP150 association with TRPV1 in a manner sensitive to calmodulin antagonism. This was also observed in transfected Chinese hamster ovary (CHO) cells, providing a model for conducting molecular analysis of the association. In CHO cells, the deletion of the C-terminal calmodulin-binding site of TRPV1 resulted in greater association with AKAP150, and increased channel activity. Furthermore, the co-expression of wild-type calmodulin in CHOs significantly reduced TRPV1 association with AKAP150, as evidenced by total internal reflective fluorescence-fluorescence resonance energy transfer (TIRF-FRET) analysis and electrophysiology. Finally, dominant-negative calmodulin co-expression increased TRPV1 association with AKAP150 and increased basal and PKA-sensitized channel activity. CONCLUSIONS: the results from these studies indicate that calcium/calmodulin interferes with the association of AKAP150 with TRPV1, potentially extending resensitization of the channel.

PP2B/calcineurin-mediated desensitization of TRPV1 does not require AKAP150.

Activation of protein kinases and phosphatases at the plasma membrane often initiates agonist-dependent signalling events. In sensory neurons, AKAP150 (A-kinase-anchoring protein 150) orientates PKA (protein kinase A), PKC (protein kinase C) and the Ca2+/calmodulin-dependent PP2B (protein phosphatase 2B, also known as calcineurin) towards membrane-associated substrates. Recent evidence indicates that AKAP150-anchored PKA and PKC phosphorylate and sensitize the TRPV1 (transient receptor potential subfamily V type 1 channel, also known as the capsaicin receptor). In the present study, we explore the hypothesis that an AKAP150-associated pool of PP2B catalyses the dephosphorylation and desensitization of TRPV1. Biochemical, electrophysiological and cell-based experiments indicate that PP2B associates with AKAP150 and TRPV1 in cultured TG (trigeminal ganglia) neurons. Gene silencing of AKAP150 reduces basal phosphorylation of TRPV1. However, functional studies in neurons isolated from AKAP150-/- mice indicate that the anchoring protein is not required for pharmacological desensitization of TRPV1. Behavioural analysis of AKAP150-/- mice further support this notion, demonstrating that agonist-stimulated desensitization of TRPV1 is sensitive to PP2B inhibition and does not rely on AKAP150. These findings allow us to conclude that pharmacological desensitization of TRPV1 by PP2B may involve additional regulatory components.

Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice.

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.

Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia.

Despite the wealth of information on cannabinoid-induced peripheral antihyperalgesic and antinociceptive effects in many pain models, the molecular mechanism(s) for these actions remains unknown. Although metabotropic cannabinoid receptors have important roles in many pharmacological actions of cannabinoids, recent studies have led to the recognition of a family of at least five ionotropic cannabinoid receptors (ICRs). The known ICRs are members of the family of transient receptor potential (TRP) channels and include TRPV1, TRPV2, TRPV4, TRPM8 and TRPA1. Cannabinoid activation of ICRs can result in desensitization of the TRPA1 and TRPV1 channel activities, inhibition of nociceptors and antihyperalgesia and antinociception in certain pain models. Thus, cannabinoids activate both metabotropic and ionotropic mechanisms to produce peripheral analgesic effects. Here, we provide an overview of the pharmacology of TRP channels as ICRs.

Roles of transient receptor potential channels in pain.

Pain perception begins with the activation of primary sensory nociceptors. Over the past decade, flourishing research has revealed that members of the Transient Receptor Potential (TRP) ion channel family are fundamental molecules that detect noxious stimuli and transduce a diverse range of physical and chemical energy into action potentials in somatosensory nociceptors. Here we highlight the roles of TRP vanilloid 1 (TRPV1), TRP melastatin 8 (TRPM8) and TRP ankyrin 1 (TRPA1) in the activation of nociceptors by heat and cold environmental stimuli, mechanical force, and by chemicals including exogenous plant and environmental compounds as well as endogenous inflammatory molecules. The contribution of these channels to pain and somatosensation is discussed at levels ranging from whole animal behavior to molecular modulation by intracellular signaling proteins. An emerging theme is that TRP channels are not simple ion channel transducers of one or two stimuli, but instead serve multidimensional roles in signaling sensory stimuli that are exceptionally diverse in modality and in their environmental milieu.

GRK2 Dictates a Functional Switch of the Peripheral Mu-Opioid Receptor.

The peripheral mu-opioid receptor (MOR) has been recognized as a potential target to provide safer analgesia with reduced central side effects. Although analgesic incompetence of the peripheral MOR in the absence of inflammation was initially identified more than a decade ago, there has been very limited investigation into the underlying signaling mechanisms. Here we identify that G protein-coupled receptor kinase 2 (GRK2) constitutively interacts with the MOR in peripheral sensory neurons to suppress peripheral MOR activity. Brief exposure to bradykinin (BK) causes uncoupling of GRK2 from the MOR and subsequent restoration of MOR functionality in dorsal root ganglion (DRG) neurons. Interestingly, prolonged BK treatment induces constitutive activation of the MOR through a mechanism that involves protein kinase C (PKC) activation. After silencing Raf kinase inhibitory protein (RKIP) by RNA interference, BK-induced constitutive MOR activation is completely abrogated, which agrees with previous findings that BK activates PKC signaling to initiate GRK2 sequestration by RKIP. Furthermore, we demonstrate that constitutive, peripheral MOR activity requires GRK2 uncoupling and that the FDA-approved SSRI paroxetine promotes this state of uncoupling. Collectively, these results indicate that GRK2 tightly regulates MOR functional states and controls constitutive MOR activity in peripheral sensory neurons, supporting the potential for targeting the kinase to provide safer analgesia.