RESUMEN
Endocannabinoids (eCBs) are lipid-signaling molecules involved in the regulation of numerous behaviors and physiological functions. Released by postsynaptic neurons, eCBs mediate retrograde modulation of synaptic transmission and plasticity by activating presynaptic cannabinoid receptors. While the cellular mechanisms by which eCBs control synaptic function have been well characterized, the mechanisms controlling their retrograde synaptic transport remain unknown. Here, we demonstrate that fatty-acid-binding protein 5 (FABP5), a canonical intracellular carrier of eCBs, is indispensable for retrograde eCB transport in the dorsal raphe nucleus (DRn). Thus, pharmacological inhibition or genetic deletion of FABP5 abolishes both phasic and tonic eCB-mediated control of excitatory synaptic transmission in the DRn. The blockade of retrograde eCB signaling induced by FABP5 inhibition is not mediated by impaired cannabinoid receptor function or reduced eCB synthesis. These findings indicate that FABP5 is essential for retrograde eCB signaling and may serve as a synaptic carrier of eCBs at central synapses.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Endocannabinoides/farmacología , Proteínas de Unión a Ácidos Grasos/fisiología , Ácido Glutámico/metabolismo , Glicéridos/metabolismo , Proteínas de Neoplasias/fisiología , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos , Animales , Células Cultivadas , Endocannabinoides/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/metabolismo , Sinapsis/efectos de los fármacosRESUMEN
The dorsal horn of the spinal cord represents the first relay station in the pain pathway where primary nociceptive inputs are modulated by local circuits and by descending signals before being relayed to supraspinal nuclei. To determine whether dopamine can modulate primary nociceptive Aδ- and C-fiber signals, the effects of dopamine were tested on the excitatory postsynaptic currents (EPSCs) recorded from large lamina I neurons and from retrograde-labeled spinoparabrachial lamina I neurons upon stimulation of the L4/L5 dorsal root in horizontal spinal cord slices in vitro Dopamine inhibited the EPSCs in a dose-dependent manner, with substantial inhibition (33%) at 1 µm and maximum inhibition (â¼70%) at 10-20 µm Dopamine reduced the frequency of miniature EPSCs recorded from large lamina I neurons, increased the paired pulse depression ratio of paired EPSCs, and induced similar inhibition of EPSCs after dialysis of large lamina I neurons with GDP-ß-S, consistent with actions at presynaptic sites. Pharmacological experiments suggested that the inhibitory effects of dopamine were largely mediated by D4 receptors (53%). Similar inhibition (66%) by dopamine was observed on EPSCs recorded from ipsilateral large lamina I neurons 6 d after injection of complete Freund's adjuvant in the hindpaw, suggesting that dopamine downregulates primary nociceptive inputs to lamina I neurons during chronic inflammatory pain. We propose that presynaptic inhibition of primary nociceptive inputs to lamina I projection neurons is a mechanism whereby dopamine can inhibit incoming noxious stimuli to the dorsal horn of the spinal cord.SIGNIFICANCE STATEMENT Lamina I projection neurons represent the main output for the pain signals from the dorsal horn of the spinal cord to brainstem and thalamic nuclei. We found that dopamine inhibits the nociceptive Aδ- and C-fiber synaptic inputs to lamina I projection neurons via presynaptic actions. Similar inhibitory effects of dopamine on the EPSCs were observed in rats subjected to complete Freund's adjuvant to induce peripheral inflammation, suggesting that dopamine inhibits the synaptic inputs to lamina I neurons in the setting of injury. A better understanding of how primary nociceptive inputs to the dorsal horn of the spinal cord are modulated by descending monoaminergic signals may help in the development of new pharmacological strategies to selectively downregulate the output from lamina I projection neurons.
Asunto(s)
Dopamina/fisiología , Inhibición Neural , Nocicepción/fisiología , Células del Asta Posterior/fisiología , Terminales Presinápticos/fisiología , Animales , Dopamina/administración & dosificación , Potenciales Postsinápticos Excitadores , Femenino , Ganglios Espinales/fisiología , Masculino , Potenciales Postsinápticos Miniatura , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Amielínicas/fisiología , Ratas Sprague-Dawley , Receptores de Dopamina D3/fisiología , Receptores de Dopamina D4/fisiologíaRESUMEN
Fatty acid-binding proteins (FABPs) are intracellular lipid carriers that regulate inflammation, and pharmacological inhibition of FABP5 reduces inflammation and pain. The mechanism(s) underlying the anti-inflammatory effects associated with FABP5 inhibition is poorly understood. Herein, we identify a novel mechanism through which FABP5 modulates inflammation. In mice, intraplantar injection of carrageenan induces acute inflammation that is accompanied by edema, enhanced pain sensitivity, and elevations in proinflammatory cytokines and prostaglandin E2 (PGE2). Inhibition of FABP5 reduced pain, edema, cytokine, and PGE2 levels. PGE2 is a major eicosanoid that enhances pain in the setting of inflammation, and we focused on the mechanism(s) through which FABP5 modulates PGE2 production. Cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1) are enzymes up-regulated at the site of inflammation and account for the bulk of PGE2 biosynthesis. Pharmacological or genetic FABP5 inhibition suppressed the induction of mPGES-1 but not COX-2 in carrageenan-injected paws, which occurred predominantly in macrophages. The cytokine interleukin 1ß (IL-1ß) is a major inducer of mPGES-1 during inflammation. Using A549 cells that express FABP5, IL-1ß stimulation up-regulated mPGES-1 expression, and mPGES-1 induction was attenuated in A549 cells bearing a knockdown of FABP5. IL-1ß up-regulates mPGES-1 via NF-κB, which activates the mPGES-1 promoter. Knockdown of FABP5 reduced the activation and nuclear translocation of NF-κB and attenuated mPGES-1 promoter activity. Deletion of NF-κB-binding sites within the mPGES-1 promoter abrogated the ability of FABP5 to inhibit mPGES-1 promoter activation. Collectively, these results position FABP5 as a novel regulator of mPGES-1 induction and PGE2 biosynthesis during inflammation.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Prostaglandina-E Sintasas/metabolismo , Células A549 , Animales , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células THP-1 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Background Fatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors. Results Immunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund's adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects. Conclusions This study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.
Asunto(s)
Analgésicos/uso terapéutico , Sistema Nervioso Central/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Dolor/tratamiento farmacológico , Nervios Periféricos/metabolismo , Analgésicos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Ciclobutanos/uso terapéutico , Ácidos Dicarboxílicos/uso terapéutico , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Adyuvante de Freund/toxicidad , Ganglios Espinales/metabolismo , Hiperalgesia/etiología , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Dolor/complicaciones , Dolor/etiología , Umbral del Dolor/efectos de los fármacos , Nervios Periféricos/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción GenéticaRESUMEN
The endocannabinoid anandamide (AEA) produces antinociceptive effects by activating cannabinoid receptor 1 (CB1). However, AEA also serves as an agonist at transient receptor potential vanilloid receptor 1 (TRPV1) in nociceptive sensory neurons, which may exacerbate pain. This potential functional duality is highlighted by the failure of an inhibitor of the AEA catabolic enzyme fatty acid amide hydrolase (FAAH) to afford pain relief in a clinical trial. Consequently, it remains to be determined whether elevating AEA levels in nociceptors leads to antinociceptive or pro-nociceptive effects. Fatty acid binding protein 5 (FABP5) is an intracellular carrier that mediates AEA transport to FAAH for inactivation. Leveraging the abundant expression of FABP5 in TRPV1+ nociceptors, we employed a conditional knockout strategy to demonstrate that FABP5 deletion in nociceptors augments AEA levels, resulting in the emergence of antinociceptive effects mediated by CB1. Mechanistically, FABP5 deletion suppresses inflammation- and nerve growth factor-mediated TRPV1 sensitization via CB1, an effect mediated by calcineurin. Unexpectedly, inhibition of FAAH failed to blunt TRPV1 sensitization, uncovering functionally distinct outputs resulting from FABP5 and FAAH inhibition. Collectively, our results demonstrate that FABP5 serves a key role in governing endocannabinoid signaling in nociceptors to disrupt TRPV1 sensitization and pain, and position FABP5 as a therapeutic target for the development of analgesics.
Asunto(s)
Endocannabinoides , Nociceptores , Analgésicos/uso terapéutico , Endocannabinoides/metabolismo , Proteínas de Unión a Ácidos Grasos , Humanos , Nociceptores/metabolismo , Dolor/tratamiento farmacológico , Manejo del Dolor , Canales Catiónicos TRPV/metabolismoRESUMEN
Total knee arthroplasty (TKA) is the final treatment option for patients with advanced knee osteoarthritis (OA). Unfortunately, TKA surgery is accompanied by acute postoperative pain that is more severe than arthroplasty performed in other joints. Elucidating the molecular mechanisms specific to post-TKA pain necessitates an animal model that replicates clinical TKA procedures, induces acute postoperative pain, and leads to complete functional recovery. Here, we present a new preclinical TKA model in rats and report on functional and behavioral outcomes indicative of pain, analgesic efficacy, serum cytokine levels, and dorsal root ganglia (DRG) transcriptomes during the acute postoperative period. Following TKA, rats exhibited marked deficits in weight bearing that persisted for 28 days. Home cage locomotion, rearing, and gait were similarly impacted and recovered by day 14. Cytokine levels were elevated on postoperative days one and/or two. Treatment with morphine, ketorolac, or their combination improved weight bearing while gabapentin lacked efficacy. When TKA was performed in rats with OA, similar functional deficits and comparable recovery time courses were observed. Analysis of DRG transcriptomes revealed upregulation of transcripts linked to multiple molecular pathways including inflammation, MAPK signaling, and cytokine signaling and production. In summary, we developed a clinically relevant rat TKA model characterized by resolution of pain and functional recovery within five weeks and with pain-associated behavioral deficits that are partially alleviated by clinically administered analgesics, mirroring the postoperative experience of TKA patients.
Asunto(s)
Artroplastia de Reemplazo de Rodilla , Ratas , Animales , Artroplastia de Reemplazo de Rodilla/efectos adversos , Ganglios Espinales , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/genética , Citocinas/genéticaRESUMEN
Sexual receptivity in the female rat is dependent on dose and duration of estradiol exposure. A 2 µg dose of estradiol benzoate (EB) primes reproductive behavior circuits without facilitating lordosis. However, 50 µg EB facilitates lordosis after 48 hours. Both EB doses activate membrane estrogen receptor-α (mERα) that complexes with and signals through metabotropic glutamate receptor-1a (mGluR1a). This mERα-mGluR1a signaling activates a multisynaptic lordosis-inhibiting circuit in the arcuate nucleus (ARH) that releases ß-endorphin in the medial preoptic nucleus (MPN), activating µ-opioid receptors (MOP). MPN MOP activation is maintained, inhibiting lordosis for 48 hours by 2 µg EB, whereas 50 µg EB at 48 hours deactivates MPN MOP, facilitating lordosis. We hypothesized that 50 µg EB down-regulates ERα and mERα-mGluR1a complexes in the ARH to remove mERα-mGluR1a signaling. In experiment I, 48 hours after 2 µg or 50 µg EB, the number of ARH ERα-immunopositive cells was reduced compared with controls. In experiment II, compared with oil controls, total ARH ERα protein was decreased 48 hours after 50 µg EB, but the 2 µg dose was not. These results indicate that both EB doses reduced the total number of cells expressing ERα, but 2 µg EB may have maintained or increased ERα expressed per cell, whereas 50 µg EB appeared to reduce total ERα per cell. In experiment III, coimmunoprecipitation and Western blot revealed that total mERα and coimmunoprecipitated mERα with mGluR1a were greater 48 hours after 2 µg EB treatment vs rats receiving 50 µg EB. These results indicate 2 µg EB maintains but 50 µg EB down-regulates mERα-mGluR1a to regulate the lordosis circuit activity.